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Cryopreservation of chrysanthemum shoot tips
ScientiaHorticulturae, 45 (1990) 167-174 Elsevier Science Publishers B.V., Amsterdam
167
Cryopreservation of chrysanthemum shoot tips Seiichi Fukai Osaka Agricultural Research Center, Habikino, Osaka 583 (Japan) (Acceptedfor publication28 December 1989)
ABSTRACT Fukai, S., 1990. Cryopreservationof chrysanthemumshoot tips. Scientia Hortic., 45:167-174. Shoot tips of chrysanthemum (Dendranthema grandiflorum (Ramt.) Kitam.) werepreculturedon Murashige and Skoog (MS) medium supplementedwith 0. I mg 1-1 6-benzylaminopurine(BA), 1 mg 1-1 1-naphthaleneaceticacid (NAA), 2% sucrose and 5% dimethyl sulphoxide (DMSO) for 2 days, slowlycooled with a cryoprotectantsolution (10% DMSO and 3% glucose) at a rate of 0.2°C rain-1 from 0 to -40°C and then immersed and stored in liquid nitrogen. After thawing in warm water, more than 87% of the shoot tips survivedand 47% of them regeneratedshoots. Keywords:chrysanthemum;cryopreservation;shoot tips. Abbreviations: BA=6-benzylaminopurine; DMSO=dimethyl sulphoxide; LN2=liquid nitrogen; MS= Murashige and Skoog;NAA= 1-naphthaleneaceticacid.
INTRODUCTION T h e c h r y s a n t h e m u m is one o f the m o s t i m p o r t a n t o r n a m e n t a l plants. All o v e r the wo r ld m a n y new cultivars c o m e o n t o the c o m m e r c i a l m a r k e t annually. H o w e v e r , for breeders a n d nurseries with a restricted supply o f l abour a n d lan d it is difficult to m a i n t a i n m a n y cultivars. An effective m e t h o d o f in vitro storage for the c h r y s a n t h e m u m w oul d allow breeders and nurseries to m a i n t a i n a greater n u m b e r o f cultivars. T h e r e are two ways to store plants in vitro; one e m p l o y s slow-growth techniques, the ot her uses c r y o p r e s e r v a t i o n t e c h n iq u es ( I n t e r n a t i o n a l B o a r d for Plant G e n e t i c Resources, 1982). Successful slow-growth t echni ques in the c h r y s a n t h e m u m are k n o w n (Preil a n d H o f f m a n n , 1985; Bajaj, 1986; Fukai et at., 1988). T h e c r y o p r e s e r v a t i o n o f callus culture ( B a n n i e r a n d Steponkus, 1976) was report ed, but a prot ocol for an easy an d r e p r o d u c i b l e c r y o p r e s e r v a t i o n o f shoot tips in the chrysanthem u m has n o t yet be e n described. This study was d o n e to investigate the cryop r e s e r v a t i o n o f shoot tips o f c h r y s a n t h e m u m .
0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
16 8
s. FUKAI
MATERIALS AND METHODS
T o x i c i t y o f D M S O . - To estimate the toxicity of dimethyl sulphoxide (DMSO) for chrysanthemum tissue, 2 × 2 m m caUi derived from leaf segment cultures were soaked in various concentrations of DMSO solution for various periods of time at 20 ° C. The calli were then cultured on Murashige and Skoog (MS) m e d i u m (Murashige and Skoog, 1962) supplemented with 1 mg 1-1 6-benzylaminopurine (BA), 2 mg 1-1 1-naphthaleneacetic acid (NAA), 40 g 1sucrose and 8 g 1-~ agar under a constant cool white fluorescent light at 19 /tmol m -2 s -~ and 25°C for a period of 60 days. P l a n t m a t e r i a l s . - The m o t h e r plants g r a n d i f l o r u m (Ramt.) Kitam. cultivar
of chrysanthemum ( D e n d r a n t h e m a 'Shuhounotikara') were grown in a greenhouse without heating and were maintained in the vegetative phase by night break lighting ( 22.00-02.00 h, 1.3 #mol m - 2 s - ~, cool white fluorescent light). All experiments were done during September 1988-May 1989.
Shoot tips (ca. 0.5-0.7 m m ) excised from the mother plants were precultured on the shoot tip culture m e d i u m (MS supplemented with 0.1 mg 1-~ BA, 1.0 mg 1-1 NAA, 2 0 g 1-1 sucrose and 8 g 1-1 agar). Half of the shoot tips were precultured on the culture m e d i u m with and the other half without 5% DMSO ( v / v ) . The shoot tips were placed under a constant cool white fluorescent light at 19 #mol m -2 s -1 and 25°C for various periods of time ranging from 2 to 14 days. Preculture. -
After preculture, the shoot tips were put into 0.5-ml plastic straws (bull semen straws manufactured by Fujihira Industry Co. Ltd., Tokyo, 3 m m ~ × 125 m m ) with a cryoprotectant solution ( 10% DMSO ( v / v ) and 3% glucose ( w / v ) ) . After heat sealing, the straws were incubated for 1 h in ice water. Then, the straws were slowly cooled at a rate of 0.1-1.0 °C rain-1 from 0 to-- 40 ° C and ice seeding was automatically carried out at - 3.5 oC with the program-freezer FFP-190 (Osaka Sanso Industry Co. Ltd., Osaka). Finally, the straws were immersed in liqid nitrogen (LN2) ( - 196 oC). Freezing. -
T h a w i n g a n d reculture. - After storage in L N 2 for 15 to 30 min, the frozen straws were rapidly thawed in a water bath at 25-30°C. The shoot tips were r i n s e d three times with sterilized water and recultured on the shoot-tip culture m e d i u m under identical conditions to that of the preculture. Viability of the shoot tips was determined by noting the ability of the shoot tips to turn green and to show signs of growth and shoot regeneration within 60 days of culture. Each experiment, comprising 20 shoot tips, was replicated two to three times.
CRYOPRESERVATIONOF CHRYSANTHEMUMSHOOT TIPS
169
RESULTS AND DISCUSSION
Toxicity o f D M S O f o r c h r y s a n t h e m u m tissue. - DMSO is known to exhibit varying degrees of cytotoxicity which depend on the species and the duration of exposure (Kartha, 1985 ). The effects of DMSO concentration on survival rate and fresh weight of calli of chrysanthemum are shown in Table 1. As the concentration of DMSO increased, survival rate and fresh weight of calli declined sharply. The data suggest that the concentration of DMSO in a process of cryopreservation should be less than 10%. Effect o f preculture d u r a t i o n a n d D M S O in a precultural m e d i u m on survival o f the shoot tips cooled d o w n to - 4 0 ° C or - 196 o C. - Shoot tips were precul-
tured on the shoot-tip culture m e d i u m with or without 5% DMSO for various days. The shoot tips were cooled at a rate of 0.2°C m i n -1 down to - 4 0 ° C and then half of t h e m were immersed into LN2 before thawing. During the preculture, the shoot tips showed depressed growth and morphogenesis if cultured on the m e d i u m containing 5% DMSO compared with those cultured on the m e d i u m without DMSO (Fig. 1 ). After freezing and thawing, viable shoot tips turned visibly brown and then TABLE1 Toxicity of DMSO for chrysanthemum callus tissue. Survival rate and fresh weight after 60 days of culture. Average of two replicates of nine calli Dipping treatment o f DMSO
Survival rate (%)
Fresh weight (mg)
10 30 60 10 10 30 60 15 10 30 60 20 10 30 60 Control (non-treatment)
100 100 100 88.9 83.4 100 61.1 77.8 55.6 44.5 11.1 11.1 100
100.9 87.9 81.4 35.5 24.9 36.0 22.2 18.0 15.4 15.6 4.6 10.8 113.8
F-test significance Conc Time Conc. × time
** n.s. n.s.
** n.s. n.s.
Conc. (%)
Time (min)
5
**Significant at 1% level; n.s., not significant.
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Fig. 1. Effect o f D M S O i n the m e d i u m o n growth o f c h r y s a n t h e u m shoot tips. T h e shoot tips were cultured o n MS m e d i u m s u p p l e m e n t e d with 0.1 m g 1- ~ BA, 1.0 m g 1- ~ N A A a n d 20 g l sucrose with ( • ) or w i t h o u t 5% D M S O ( O ). I n t h e figure each b a r represents SE o f d i a m e t e r o f shoot tips a n d the n u m b e r s show the s h o o t regeneration rate ( % ) . TABLE 2 Effect of preculture duration and DMSO in the precultural medium on the survival rate of the shoot tips after cooling down to - 4 0 ° C without and - 196°C with LN2 treatment Duration of preculture (days)
DMSO conc. in precultural medium (%)
Survival rate of shoot tips (%) _40°C
-- 196°C (LN2)
0 2 2 7 7 14 14
5 0 5 0 5 0
0 94.4 52.4 83.9 97.4 85.5 86.5 n.s.
0 79.4 50.0 72.6 72.5 83.1 69.4 n.s.*
*No significant difference by F-test.
regreened within 10 days of reculture. No survival was observed when the shoot tips were frozen without preculture. A preculture of 2 days on the DMSO precultural medium already induced a high survival rate of the shoot tips (Table 2 ). High survival rates of the shoot tips were also observed with 7 and 14 clays of preculture, regardless of the presence of DMSO in the precultural medium. The survival rate of the shoot tips dropped slightly when they were immersed into LN2 before thawing. Effect o f cooling rate. - After 2 days of preculture on the shoot-tip culture medium with 5% DMSO, the shoot tips were cooled at various cooling rates down to - 40 ° C and subsequently immersed into LN2. The survival and shoot regeneration rate of the shoot tips increased as the cooling rate decreased from 1 to 0.2°C min -~ but decreased again at 0.1 °C
CRYOPRESERVATION OF CHRYSANTHEMUM SHOOT TIPS
171
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40
,~
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0.5 rate
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Fig. 2. Effect of the cooling rate on survival and shoot regeneration of chrysanthemum shoot tips. The shoot tips were cooled at various rates down to -40°C after 2 days of preculture on the shoot-tip culture medium with 5% DMSO and were then immersed into LN2. The results represent an average of two replicates of 20 shoot tips. m i n - 1 (Fig. 2). O p t i m a l cooling rates clearly differed interspecifically, e.g. 0 . 8 4 ° C m i n -1 for strawberry ( K a r t h a et al., 1980), 0.8°C m i n -1 for sugarbeet (Braun, 1988 ), 0.6 ° C m i n - ~ for pea (Kartha et al., 1979 ) and 0.5-1 ° C min-~ for carnation ( U e m u r a and Sakai, 1980). The differences in optimal cooling rates for each species d e p e n d on the relative speed o f cell dehydration during the cooling process.
Effect o f terminal temperature o f slow cooling on survival and shoot regeneration of shoot tips. - After 2 days o f p r e c u l t u r e on the shoot-tip culture m e d i u m with 5% D M S O , the shoot tips were cooled at a rate o f 0.2 °C m i n - 1 d o w n to various temperatures and then i m m e r s e d into LN2. All shoot tips cooled d o w n to - 10 and - 20 ° C before immersion into LN2 were killed (Fig. 3 ). Survival rate and shoot regeneration rate o f the shoot tips increased as the terminal temperature was lowered to between - 2 5 and - 40 ° C. To establish a protocol o f cryopreservation, the interrelationship between the cooling rate, terminal temperature o f slow cooling and the subsequent survival o f shoot tips after thawing and return to reculture should be taken into account. A too-slow cooling rate can be damaging due to excessive dehydration o f shoot tips and this can be a v o i d e d b y termination o f the slow cooling at a relatively high subzero t e m p e r a t u r e (Withers, 1986 ). In the chry-
172
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Fig. 3. Effect o f various t e r m i n a l t e m p e r a t u r e s o f slow cooling o n t h e survival a n d shoot regeneration o f c h r y s a n t h e m u m s h o o t tips. T h e shoot tips were cooled at a rate o f 0.2 ° C m i n - 1 d o w n to i n d i c a t e d t e r m i n a l t e m p e r a t u r e s after 2 days o f preculture o n the shoot-tip culture m e d i u m with 5% D M S O a n d t h e n i m m e r s i o n into LN2. T h e results represent a n average o h w o replicates o f 20 s h o o t tips. TABLE3 Variation in response to cryopreservation by sources of shoot tips Cultivar
Growing condition of mother plant
Portion
Survival rate (%)
Shoot regeneration rate (%)
Shuhounotikara
G A A
ST ST NS
87.1 80.0 23.8 23.2
47.9 10.0 0 34.5
LSD (5%)
G = greenhouse; A = aseptic cultural condition; ST = shoot tip; NS = nodal segment with an axillary bud.
santhemum, the maximum survival and shoot regeneration rate was observed when the method combined a relatively slow cooling rate (0.2 ° C m i n - 1) and a low terminal temperature ( - 4 0 °C). These data suggest that the dehydration of chrysanthemum shoot tips is slow compared with other species. Effect o f source o f shoot tips on response to cryopreservation. - Shoot tips excised from plants growing in a greenhouse and shoot tips and nodal segments (approx. 0.5 m m l o n g with one axillary bud) excised from plants growing in aseptic cultural conditions were compared. Again samples were cooled at a
CRYOPRESERVATION OF CHRYSANTHEMUM SHOOT TIPS
173
Fig. 4. Shoot regeneration from a cryopreserved shoot tip (right) and an unfrozen shoot tip (left). Unfrozen shoot tips usually grow up into monoshoot plantlets with a small basal callus, but some of the cryopreserved shoot tips regenerate small plantlets with a large callus.
rate of0.2°C min -1 down to - 4 0 ° C after 2 days of preculture on the shoottip culture medium with 5% DMSO and then immersed into LN2. Shoot regenerations for the shoot tips taken from an aseptic condition were lower than the rate for shoot tips from greenhouse plants, especially nodal segments (Table 3). The shoot tips of aseptic plants are, however, a more suitable material for cryopreservation because of prevention of microbial contamination. The present study shows that the regeneration of plantlets from chrysanthem u m shoot tips stored in LN2 can be achieved routinely and reproducibly. Moreover, in a later experiment chrysanthemum shoot tips have shown viability after storage in LN2 for up to 1 month (Fukai, unpublished result ). The surviving shoot tips, (Fig. 4) however, did not always regenerate shoots, but tended to form calli. Callus formation in the frozen-thawed shoot tips was reported in many other species (Kartha et al., 1982; Henshaw et al., 1985; Kuo and Lineberger, 1985; Reed and Lagerstedt, 1987). The regenerated shoots via calli are less genetically stable; therefore, further study is required to find ways to prevent calli formation and to minimize the risk of genetic change in the shoot tips during cryopreservation. ACKNOWLEDGEMENTS
Thanks are expressed to K. Nishimura for his invaluable suggestions for the cryopreservation techniques. I also thank Prof. Dr. H. Imanishi for his critical reading of the manuscript. REFERENCES Bajaj, Y.P.S., 1986. In vitro preservation of genetic resources. In: Nuclear Techniques and in vitro Culture for Plant Improvement. Proc. Int. Syrup., 19-23 August 1985, Intemational Atomic Energy Agency, Vienna, pp. 43-57.
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Bannier, L.J. and Steponkus, P.L., 1976. Cold acclimation of chrysanthemum callus cultures. J. Am. Soc. Hortic. Sci., 101 (4) 490-512. Braun, A., 1988. Cryopreservation of sugarbeet germplasm. Plant Cell, Tissue and Organ Culture, 14: 161-168. Fukai, S., Morii, M. and Oe, M., 1988. Storage of chrysanthemum (Dendranthemagandiflorum (Ramat.) Kitamura) plantlets in vitro. Plant Tissue Culture Letter, 5 ( 1 ): 20-25 (in Japanese with English summary). Henshaw, G.G., O'Hara, J.F. and Stamp, J.A., 1985. Cryopreservation of potato meristems. In: K.K. Kartha (Editor), Cryopreservation of Plant Cells and Organs. CRC Press, BocaRaton, FL, pp. 159-170. -. . . . . International Board for Plant Genetic Resources, 1983. IBPGR Advisory Committee on in vitro Storage. Report of the First Meeting, IBPGR, Rome, pp. 1-11. Kartha, K.K., 1985. Meristemculture and germplasm preservation. In: K.K. Kartha (Editor), Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, FL, pp. 115-134. Kartha, K.K., Leung, N.L. and Gamborg, O.L., 1979. Freeze-preservation of pea mefistems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett., 15: 7-16. Kartha, K.K., Leung, N.L. and Phal, K., 1980. Cryopreservation of strawberry meristems and mass propagation ofplanflets. J. Am. Soc. Hortic. Sci., 105: 481-484. Kartha, K.K., Leung, N.L. and Mroginski, L.A., 1982. In vitro responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz). Z. Pflanzenphysiol., 107: 133-140. Kuo, C.-C and Lineberger, R.D., 1985. Survival of in vitro cultured tissue of'Jonathan' apples exposed to - 196 ° C. HortScience, 20 (4): 764-767. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Preil, W. and Hoffmann, M., 1985. In vitro storage in chrysanthemum breeding and propagation. In: A. Sch~ifer-Menuhr (Editor), In Vitro Techniques. Martinus Nijhoff, Dordrecht, pp. 161-165. Reed, B.M. and Lagerstedt, H.B., 1987. Freeze preservation of apical mefistems of Rubus in liquid nitrogen. HortScience, 22 (2): 302-303. Uemura, M. and Sakai, A., 1980. Survival of carnation (Dianthus caryophyllusL. ) shoot apices frozen to the temperature of liquid nitrogen. Plant Cell Physiol., 21 ( 1 ): 85-94. Withers, L.A., 1986. Cryopreservation and genebanks. In: M.M. Yoeman (Editor), Plant Cell Culture Technology. BlackweU, Oxford, pp. 96-140.
167
Cryopreservation of chrysanthemum shoot tips Seiichi Fukai Osaka Agricultural Research Center, Habikino, Osaka 583 (Japan) (Acceptedfor publication28 December 1989)
ABSTRACT Fukai, S., 1990. Cryopreservationof chrysanthemumshoot tips. Scientia Hortic., 45:167-174. Shoot tips of chrysanthemum (Dendranthema grandiflorum (Ramt.) Kitam.) werepreculturedon Murashige and Skoog (MS) medium supplementedwith 0. I mg 1-1 6-benzylaminopurine(BA), 1 mg 1-1 1-naphthaleneaceticacid (NAA), 2% sucrose and 5% dimethyl sulphoxide (DMSO) for 2 days, slowlycooled with a cryoprotectantsolution (10% DMSO and 3% glucose) at a rate of 0.2°C rain-1 from 0 to -40°C and then immersed and stored in liquid nitrogen. After thawing in warm water, more than 87% of the shoot tips survivedand 47% of them regeneratedshoots. Keywords:chrysanthemum;cryopreservation;shoot tips. Abbreviations: BA=6-benzylaminopurine; DMSO=dimethyl sulphoxide; LN2=liquid nitrogen; MS= Murashige and Skoog;NAA= 1-naphthaleneaceticacid.
INTRODUCTION T h e c h r y s a n t h e m u m is one o f the m o s t i m p o r t a n t o r n a m e n t a l plants. All o v e r the wo r ld m a n y new cultivars c o m e o n t o the c o m m e r c i a l m a r k e t annually. H o w e v e r , for breeders a n d nurseries with a restricted supply o f l abour a n d lan d it is difficult to m a i n t a i n m a n y cultivars. An effective m e t h o d o f in vitro storage for the c h r y s a n t h e m u m w oul d allow breeders and nurseries to m a i n t a i n a greater n u m b e r o f cultivars. T h e r e are two ways to store plants in vitro; one e m p l o y s slow-growth techniques, the ot her uses c r y o p r e s e r v a t i o n t e c h n iq u es ( I n t e r n a t i o n a l B o a r d for Plant G e n e t i c Resources, 1982). Successful slow-growth t echni ques in the c h r y s a n t h e m u m are k n o w n (Preil a n d H o f f m a n n , 1985; Bajaj, 1986; Fukai et at., 1988). T h e c r y o p r e s e r v a t i o n o f callus culture ( B a n n i e r a n d Steponkus, 1976) was report ed, but a prot ocol for an easy an d r e p r o d u c i b l e c r y o p r e s e r v a t i o n o f shoot tips in the chrysanthem u m has n o t yet be e n described. This study was d o n e to investigate the cryop r e s e r v a t i o n o f shoot tips o f c h r y s a n t h e m u m .
0304-4238/90/$03.50
© 1990 - - Elsevier Science Publishers B.V.
16 8
s. FUKAI
MATERIALS AND METHODS
T o x i c i t y o f D M S O . - To estimate the toxicity of dimethyl sulphoxide (DMSO) for chrysanthemum tissue, 2 × 2 m m caUi derived from leaf segment cultures were soaked in various concentrations of DMSO solution for various periods of time at 20 ° C. The calli were then cultured on Murashige and Skoog (MS) m e d i u m (Murashige and Skoog, 1962) supplemented with 1 mg 1-1 6-benzylaminopurine (BA), 2 mg 1-1 1-naphthaleneacetic acid (NAA), 40 g 1sucrose and 8 g 1-~ agar under a constant cool white fluorescent light at 19 /tmol m -2 s -~ and 25°C for a period of 60 days. P l a n t m a t e r i a l s . - The m o t h e r plants g r a n d i f l o r u m (Ramt.) Kitam. cultivar
of chrysanthemum ( D e n d r a n t h e m a 'Shuhounotikara') were grown in a greenhouse without heating and were maintained in the vegetative phase by night break lighting ( 22.00-02.00 h, 1.3 #mol m - 2 s - ~, cool white fluorescent light). All experiments were done during September 1988-May 1989.
Shoot tips (ca. 0.5-0.7 m m ) excised from the mother plants were precultured on the shoot tip culture m e d i u m (MS supplemented with 0.1 mg 1-~ BA, 1.0 mg 1-1 NAA, 2 0 g 1-1 sucrose and 8 g 1-1 agar). Half of the shoot tips were precultured on the culture m e d i u m with and the other half without 5% DMSO ( v / v ) . The shoot tips were placed under a constant cool white fluorescent light at 19 #mol m -2 s -1 and 25°C for various periods of time ranging from 2 to 14 days. Preculture. -
After preculture, the shoot tips were put into 0.5-ml plastic straws (bull semen straws manufactured by Fujihira Industry Co. Ltd., Tokyo, 3 m m ~ × 125 m m ) with a cryoprotectant solution ( 10% DMSO ( v / v ) and 3% glucose ( w / v ) ) . After heat sealing, the straws were incubated for 1 h in ice water. Then, the straws were slowly cooled at a rate of 0.1-1.0 °C rain-1 from 0 to-- 40 ° C and ice seeding was automatically carried out at - 3.5 oC with the program-freezer FFP-190 (Osaka Sanso Industry Co. Ltd., Osaka). Finally, the straws were immersed in liqid nitrogen (LN2) ( - 196 oC). Freezing. -
T h a w i n g a n d reculture. - After storage in L N 2 for 15 to 30 min, the frozen straws were rapidly thawed in a water bath at 25-30°C. The shoot tips were r i n s e d three times with sterilized water and recultured on the shoot-tip culture m e d i u m under identical conditions to that of the preculture. Viability of the shoot tips was determined by noting the ability of the shoot tips to turn green and to show signs of growth and shoot regeneration within 60 days of culture. Each experiment, comprising 20 shoot tips, was replicated two to three times.
CRYOPRESERVATIONOF CHRYSANTHEMUMSHOOT TIPS
169
RESULTS AND DISCUSSION
Toxicity o f D M S O f o r c h r y s a n t h e m u m tissue. - DMSO is known to exhibit varying degrees of cytotoxicity which depend on the species and the duration of exposure (Kartha, 1985 ). The effects of DMSO concentration on survival rate and fresh weight of calli of chrysanthemum are shown in Table 1. As the concentration of DMSO increased, survival rate and fresh weight of calli declined sharply. The data suggest that the concentration of DMSO in a process of cryopreservation should be less than 10%. Effect o f preculture d u r a t i o n a n d D M S O in a precultural m e d i u m on survival o f the shoot tips cooled d o w n to - 4 0 ° C or - 196 o C. - Shoot tips were precul-
tured on the shoot-tip culture m e d i u m with or without 5% DMSO for various days. The shoot tips were cooled at a rate of 0.2°C m i n -1 down to - 4 0 ° C and then half of t h e m were immersed into LN2 before thawing. During the preculture, the shoot tips showed depressed growth and morphogenesis if cultured on the m e d i u m containing 5% DMSO compared with those cultured on the m e d i u m without DMSO (Fig. 1 ). After freezing and thawing, viable shoot tips turned visibly brown and then TABLE1 Toxicity of DMSO for chrysanthemum callus tissue. Survival rate and fresh weight after 60 days of culture. Average of two replicates of nine calli Dipping treatment o f DMSO
Survival rate (%)
Fresh weight (mg)
10 30 60 10 10 30 60 15 10 30 60 20 10 30 60 Control (non-treatment)
100 100 100 88.9 83.4 100 61.1 77.8 55.6 44.5 11.1 11.1 100
100.9 87.9 81.4 35.5 24.9 36.0 22.2 18.0 15.4 15.6 4.6 10.8 113.8
F-test significance Conc Time Conc. × time
** n.s. n.s.
** n.s. n.s.
Conc. (%)
Time (min)
5
**Significant at 1% level; n.s., not significant.
170
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Fig. 1. Effect o f D M S O i n the m e d i u m o n growth o f c h r y s a n t h e u m shoot tips. T h e shoot tips were cultured o n MS m e d i u m s u p p l e m e n t e d with 0.1 m g 1- ~ BA, 1.0 m g 1- ~ N A A a n d 20 g l sucrose with ( • ) or w i t h o u t 5% D M S O ( O ). I n t h e figure each b a r represents SE o f d i a m e t e r o f shoot tips a n d the n u m b e r s show the s h o o t regeneration rate ( % ) . TABLE 2 Effect of preculture duration and DMSO in the precultural medium on the survival rate of the shoot tips after cooling down to - 4 0 ° C without and - 196°C with LN2 treatment Duration of preculture (days)
DMSO conc. in precultural medium (%)
Survival rate of shoot tips (%) _40°C
-- 196°C (LN2)
0 2 2 7 7 14 14
5 0 5 0 5 0
0 94.4 52.4 83.9 97.4 85.5 86.5 n.s.
0 79.4 50.0 72.6 72.5 83.1 69.4 n.s.*
*No significant difference by F-test.
regreened within 10 days of reculture. No survival was observed when the shoot tips were frozen without preculture. A preculture of 2 days on the DMSO precultural medium already induced a high survival rate of the shoot tips (Table 2 ). High survival rates of the shoot tips were also observed with 7 and 14 clays of preculture, regardless of the presence of DMSO in the precultural medium. The survival rate of the shoot tips dropped slightly when they were immersed into LN2 before thawing. Effect o f cooling rate. - After 2 days of preculture on the shoot-tip culture medium with 5% DMSO, the shoot tips were cooled at various cooling rates down to - 40 ° C and subsequently immersed into LN2. The survival and shoot regeneration rate of the shoot tips increased as the cooling rate decreased from 1 to 0.2°C min -~ but decreased again at 0.1 °C
CRYOPRESERVATION OF CHRYSANTHEMUM SHOOT TIPS
171
v
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I
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Fig. 2. Effect of the cooling rate on survival and shoot regeneration of chrysanthemum shoot tips. The shoot tips were cooled at various rates down to -40°C after 2 days of preculture on the shoot-tip culture medium with 5% DMSO and were then immersed into LN2. The results represent an average of two replicates of 20 shoot tips. m i n - 1 (Fig. 2). O p t i m a l cooling rates clearly differed interspecifically, e.g. 0 . 8 4 ° C m i n -1 for strawberry ( K a r t h a et al., 1980), 0.8°C m i n -1 for sugarbeet (Braun, 1988 ), 0.6 ° C m i n - ~ for pea (Kartha et al., 1979 ) and 0.5-1 ° C min-~ for carnation ( U e m u r a and Sakai, 1980). The differences in optimal cooling rates for each species d e p e n d on the relative speed o f cell dehydration during the cooling process.
Effect o f terminal temperature o f slow cooling on survival and shoot regeneration of shoot tips. - After 2 days o f p r e c u l t u r e on the shoot-tip culture m e d i u m with 5% D M S O , the shoot tips were cooled at a rate o f 0.2 °C m i n - 1 d o w n to various temperatures and then i m m e r s e d into LN2. All shoot tips cooled d o w n to - 10 and - 20 ° C before immersion into LN2 were killed (Fig. 3 ). Survival rate and shoot regeneration rate o f the shoot tips increased as the terminal temperature was lowered to between - 2 5 and - 40 ° C. To establish a protocol o f cryopreservation, the interrelationship between the cooling rate, terminal temperature o f slow cooling and the subsequent survival o f shoot tips after thawing and return to reculture should be taken into account. A too-slow cooling rate can be damaging due to excessive dehydration o f shoot tips and this can be a v o i d e d b y termination o f the slow cooling at a relatively high subzero t e m p e r a t u r e (Withers, 1986 ). In the chry-
172
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Fig. 3. Effect o f various t e r m i n a l t e m p e r a t u r e s o f slow cooling o n t h e survival a n d shoot regeneration o f c h r y s a n t h e m u m s h o o t tips. T h e shoot tips were cooled at a rate o f 0.2 ° C m i n - 1 d o w n to i n d i c a t e d t e r m i n a l t e m p e r a t u r e s after 2 days o f preculture o n the shoot-tip culture m e d i u m with 5% D M S O a n d t h e n i m m e r s i o n into LN2. T h e results represent a n average o h w o replicates o f 20 s h o o t tips. TABLE3 Variation in response to cryopreservation by sources of shoot tips Cultivar
Growing condition of mother plant
Portion
Survival rate (%)
Shoot regeneration rate (%)
Shuhounotikara
G A A
ST ST NS
87.1 80.0 23.8 23.2
47.9 10.0 0 34.5
LSD (5%)
G = greenhouse; A = aseptic cultural condition; ST = shoot tip; NS = nodal segment with an axillary bud.
santhemum, the maximum survival and shoot regeneration rate was observed when the method combined a relatively slow cooling rate (0.2 ° C m i n - 1) and a low terminal temperature ( - 4 0 °C). These data suggest that the dehydration of chrysanthemum shoot tips is slow compared with other species. Effect o f source o f shoot tips on response to cryopreservation. - Shoot tips excised from plants growing in a greenhouse and shoot tips and nodal segments (approx. 0.5 m m l o n g with one axillary bud) excised from plants growing in aseptic cultural conditions were compared. Again samples were cooled at a
CRYOPRESERVATION OF CHRYSANTHEMUM SHOOT TIPS
173
Fig. 4. Shoot regeneration from a cryopreserved shoot tip (right) and an unfrozen shoot tip (left). Unfrozen shoot tips usually grow up into monoshoot plantlets with a small basal callus, but some of the cryopreserved shoot tips regenerate small plantlets with a large callus.
rate of0.2°C min -1 down to - 4 0 ° C after 2 days of preculture on the shoottip culture medium with 5% DMSO and then immersed into LN2. Shoot regenerations for the shoot tips taken from an aseptic condition were lower than the rate for shoot tips from greenhouse plants, especially nodal segments (Table 3). The shoot tips of aseptic plants are, however, a more suitable material for cryopreservation because of prevention of microbial contamination. The present study shows that the regeneration of plantlets from chrysanthem u m shoot tips stored in LN2 can be achieved routinely and reproducibly. Moreover, in a later experiment chrysanthemum shoot tips have shown viability after storage in LN2 for up to 1 month (Fukai, unpublished result ). The surviving shoot tips, (Fig. 4) however, did not always regenerate shoots, but tended to form calli. Callus formation in the frozen-thawed shoot tips was reported in many other species (Kartha et al., 1982; Henshaw et al., 1985; Kuo and Lineberger, 1985; Reed and Lagerstedt, 1987). The regenerated shoots via calli are less genetically stable; therefore, further study is required to find ways to prevent calli formation and to minimize the risk of genetic change in the shoot tips during cryopreservation. ACKNOWLEDGEMENTS
Thanks are expressed to K. Nishimura for his invaluable suggestions for the cryopreservation techniques. I also thank Prof. Dr. H. Imanishi for his critical reading of the manuscript. REFERENCES Bajaj, Y.P.S., 1986. In vitro preservation of genetic resources. In: Nuclear Techniques and in vitro Culture for Plant Improvement. Proc. Int. Syrup., 19-23 August 1985, Intemational Atomic Energy Agency, Vienna, pp. 43-57.
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Bannier, L.J. and Steponkus, P.L., 1976. Cold acclimation of chrysanthemum callus cultures. J. Am. Soc. Hortic. Sci., 101 (4) 490-512. Braun, A., 1988. Cryopreservation of sugarbeet germplasm. Plant Cell, Tissue and Organ Culture, 14: 161-168. Fukai, S., Morii, M. and Oe, M., 1988. Storage of chrysanthemum (Dendranthemagandiflorum (Ramat.) Kitamura) plantlets in vitro. Plant Tissue Culture Letter, 5 ( 1 ): 20-25 (in Japanese with English summary). Henshaw, G.G., O'Hara, J.F. and Stamp, J.A., 1985. Cryopreservation of potato meristems. In: K.K. Kartha (Editor), Cryopreservation of Plant Cells and Organs. CRC Press, BocaRaton, FL, pp. 159-170. -. . . . . International Board for Plant Genetic Resources, 1983. IBPGR Advisory Committee on in vitro Storage. Report of the First Meeting, IBPGR, Rome, pp. 1-11. Kartha, K.K., 1985. Meristemculture and germplasm preservation. In: K.K. Kartha (Editor), Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton, FL, pp. 115-134. Kartha, K.K., Leung, N.L. and Gamborg, O.L., 1979. Freeze-preservation of pea mefistems in liquid nitrogen and subsequent plant regeneration. Plant Sci. Lett., 15: 7-16. Kartha, K.K., Leung, N.L. and Phal, K., 1980. Cryopreservation of strawberry meristems and mass propagation ofplanflets. J. Am. Soc. Hortic. Sci., 105: 481-484. Kartha, K.K., Leung, N.L. and Mroginski, L.A., 1982. In vitro responses and plant regeneration from cryopreserved meristems of cassava (Manihot esculenta Crantz). Z. Pflanzenphysiol., 107: 133-140. Kuo, C.-C and Lineberger, R.D., 1985. Survival of in vitro cultured tissue of'Jonathan' apples exposed to - 196 ° C. HortScience, 20 (4): 764-767. Murashige, T. and Skoog, F., 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant., 15: 473-497. Preil, W. and Hoffmann, M., 1985. In vitro storage in chrysanthemum breeding and propagation. In: A. Sch~ifer-Menuhr (Editor), In Vitro Techniques. Martinus Nijhoff, Dordrecht, pp. 161-165. Reed, B.M. and Lagerstedt, H.B., 1987. Freeze preservation of apical mefistems of Rubus in liquid nitrogen. HortScience, 22 (2): 302-303. Uemura, M. and Sakai, A., 1980. Survival of carnation (Dianthus caryophyllusL. ) shoot apices frozen to the temperature of liquid nitrogen. Plant Cell Physiol., 21 ( 1 ): 85-94. Withers, L.A., 1986. Cryopreservation and genebanks. In: M.M. Yoeman (Editor), Plant Cell Culture Technology. BlackweU, Oxford, pp. 96-140.