Plant Cell Reports

Plant Cell Reports (1982) 1:135-138

© Springer-Verlag 1982

Cryopreservation of Periwinkle,

Catharanthus roseus

Cells Cultured in vitro*

K. K. Kartha, N. L. Leung, P. Gaudet-LaPrairie and F. Constabel Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Sask. S7N 0W9, Canada Received September 29, 1981 ; March 29, 1982

Abstract A procedure for prolonged cryogenic storage of periwinkle cell cultures is described. Cells derived from periwinkle, Catharanthus roseus (L.) G. Don, and subcultured as suspension in I-B5C n u t r i e n t medium have been frozen, stored in l i q u i d nitrogen (-196°C) for I I weeks, thawed and recultured. Maximal survival was achieved when 3-4 day-old c e l l s precultured for 24 h in n u t r i e n t medium with 5% DMSOwere frozen at slow cooling rates of 0.5 or l°C/min p r i o r to storage in l i q u i d nitrogen. The only loss in v i a b i l i t y of c e l l s occurred subsequent to treatment with DMSO. Abbreviations: DMSO, dimethylsulfoxide; 2,4-D, 2,4dichlorophenoxyacetic acid; TTC, t r i p h e n y l t e t r a z o l i u m chloride. Key Words: Catharanthus roseus, periwinkle, cell cultures, DMSO, cryopreservation, cryogenic storage. Introduction Plant c e l l s cultured in v i t r o have been shown to synthesize and accumulate a l k a l o i d s in various amounts (Zenk, 1978). The e x p l o i t a t i o n of the c e l l culture system for i n d u s t r i a l production of a l k a l o i d s , therefore, has been envisaged by a number of authors (Zenk, 1978; Constabel, 1981). One f a c t o r which would severely impede the u t i l i z a t i o n of plant c e l l s in industry is genetic i n s t a b i l i t y . Loss of a b i l i t y to synthesize a l k a l o i d s over a period of time has been found with cultured cells of several species (Dhoot and Henshaw, 1977; Barz and E l l i s , 1981). Various means of preserving biosynthetic capacity have been contemplated, and cryopreservation of desirable c e l l lines is regarded as the most effect i v e way, i f only plant cells would lend themselves to such treatment. However, successful cryopreservation of c e l l s cultured in v i t r o has been demonstrated f o r over a dozen species (for a review see Withers and Street, 1977; Kartha, 1981). Chemical analysis of c e l l lines derived from Catharan thus roseus led to the detection of material which produced corynanthe-, strychnos-, aspidosperma-, and iboga-type a l k a l o i d s (Kurz et al. 1980; Kutney et al. Ig80). However, one cell l i n e of C. roseus designated as #916 did not produce a l k a l o i d s in detectable amounts; but i t appeared to be p a r t i c u l a r l y amenable to attempt cryopreservation, because i t consisted of small c e l l s which were densely cytoplasmic without a big central vacuole. The present i n v e s t i g a t i o n was therefore undertaken in an attempt to cryopreserve * NRCC

No.

this cell l i n e in the hope, that i f this nonproducing l i n e can be frozen and stored in l i q u i d nitrogen by cryogenic methods then, possibly, the producing lines can be likewise. Materials and Methods Cell material employed consisted of serial sub-cultures of callus designated #916 i n i t i a t e d from anthers of periwinkle, Catharanthus roseus (L.) G. Don in August 1978 (Ku~ et al. ~ 8 0 ) . The callus was cultured for periods of 3-4 weeks on O.I-B5C medium, i . e . medium a f t e r Gamborg et al. (1968) supplemented with O.l mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and I g / l casein hydrolyzate (Humko-Sheffield, N.Y.) and s o l i d i f i e d with 0.6% Difco-agar. At i n t e r v a l s , callus material was suspended in l i q u i d I-B5C medium and subcultured as 50 ml batches in 250 ml Delong flasks on gyratory shakers (130 rpm) for periods of 4 days. All callus and suspension cultures were grown in continuous l i g h t of ca. lO ~Lm-L at 28°C. For cryopreservation I0 ml samples of 3-4 day-old cell cultures of l i n e 916 were precultured in 5% dimethylsulfoxide (DMSO) in a 50 ml erlenmeyer flask by gradually adding 5 ml of culture medium containing >~5% DMSOover a period of 15 min and placed on a gyratory shaker at 150 rpm f o r 24 h. At the end of the preculture period the flask containing the c e l l s as well as equal amounts of c e l l s in culture medium whic~ were not precultured in DMSOwere c h i l l e d in an ice bath for 2 h. In both cases, the DMSO concentration was brought to a f i n a l concentration of 7.5% by gradual addition of appropriate amounts of DMSO over a period of 15 min allowing 30 min for e q u i l i b r a t i o n and avoiding sudden plasmolysis shock. The c e l l suspensions were transferred to two 15 ml conical tubes and centrifuged at lO0 x g for 3 min. The f i n a l sample for each set was obtained by removing some of the supernatant and pooling the contents of the tubes to y i e l d one ml suspension for every 0.2 ml of packed cell volume. One ml aliquots were d i s t r i b u t e d into cryogenic glass ampoules ( I . 2 ml capacity, Wheaton), a l l ampoules flame-sealed except one, and arranged in the freezing chamber (CRYOMED 972 Freezer). The temperature probe for monitoring the cooling rates of the specimen and the chamber was introduced into the unsealed ampoule. The specimens were cooled at a rate of 0.5 to 5°C/min with a programmable CRYOMED lO00 c o n t r o l l e r to a terminal temperature of -40°C followed by immersion in l i q u i d nitrogen (-196°C). The controls included in the study consisted of:

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0721-7714/82/0001/135/$ 01.00

136 i ) c e l l s which were not precultured in DMS0 but frozen the same way as for precultured c e l l s , i i ) DMSO-treated c e l l s which were not frozen but maintained in an ice bath and, i i i ) c e l l s which were neither treated with DMS0 nor frozen. The ampoules stored in l i q u i d nitrogen for 1 h or longer (as long as I I weeks) were r a p i d l y thawed in a 37°C water bath for 90 sec and transferred immediately to an ice bath. The thawed cell suspensions were pipetted into 15 ml conical centrifuge tubes and d i luted with 6 volumes of c h i l l e d culture medium over a 30 min period and washed 3 times by c e n t r i f u g a t i o n . The c e l l s were plated onto solid n u t r i e n t medium containing 0.8% agar or cultured in l i q u i d medium in 10-20 ml batches in order to determine whether or not these c e l l s would resume growth. The rate of v i a b i l i t y of frozen-thawed c e l l s was compared with the controls by means of the t r i p h e n y l t e t r a z o l i u m chloride (TTC) reduction assay (Steponkus and Lanphear, 1967). The color value of TTC reduction r e s u l t i n g in the formation of formazan was spectrophotometrically measured at 530 nm (Steponkus and Lanphear, 1967; Ulrich et a l . 1979) and was expressed as per cent survival over the control = TTC value of frozen c e l l s TTC value of the Control X I00). No attempt has been made to correlate the results of regrowth of c e l l s e i t h e r in l i q u i d or on solid n u t r i e n t medium to the TTC assay. However, a l l c e l l cultures which reduced the TTC showed vigorous growth when plated (Fig. 4-7). Cells which were k i l l e d e i t h e r with absolute ethanol or heat f a i l e d to reduce TTC. The m i t o t i c index determination was carried out according to the method described in Gamborg and Wetter (1975) using carbolfuchsin stain. At least 2000 nuclei were scored f o r each sample of frozen and unfrozen c e l l s in order to calculate the m i t o t i c index. All experiments were repeated at least 3 times. Results and Discussion C r i t i c a l steps in cryopreserving plant c e l l s cultured in v i t r o are the time of sampling or period of growth phase, nature and type of cryoprotectants employed, treatment with cryoprotectants, cooling rates and the terminal freezing temperature before the specimen is stored in l i q u i d nitrogen (Kartha, 1981). Preliminary experiments with Catharanthus roseus l i n e #916 revealed that 3-4 day-old subcultures were most suitable for freezing, because c e l l s at that stage were small, had undergone active d i v i s i o n and displayed dense cytoplasm (Fig. 1 and 2) and, accordingly, in subsequent experiments cell cultures at this stage were used.

8

I

i

i

]

i

i C. roseus

I

;0o

D916

i

Fig. 2.

Cells of periwinkle l i n e #916 before DMS0 treatment and freezing (Nomarski optics X 370). Cryopreserved cells grown for one month in l i q u i d culture medium. Ilote the change in vacuolation (Nomarski optics X 370).

Fig. 3.

The TTC reduction obtained with untreated and unfrozen c e l l s was taken as 100% v i a b i l i t y (Table I ) . Cells which were precultured f o r one day in culture medium containing 5% DMS0 showed 66% s u r v i v a l , or a v i a b i l i t y loss of 34% occurred subsequent to preculture in DMS0. When such precultured c e l l s were then treated with 7.5% DMS0 for 30 min p r i o r to freezing the v i a b i l i t y was reduced to 50%, i . e . an addit i o n a l loss of 16% v i a b i l i t y occurred subsequent to f u r t h e r addition of DMS0 to the precultured c e l l s . Table I .

o,

~

' TIME

II

'

,o

(days) III

Effect of DMSO treatments on the v i a b i l i t ~ of Catharanthus roseus c e l l s of l i n e #916u

Treatments

% Survival

r~o preculture and no DMSOtreatment

% V i a b i l i t y loss

I00

0

24 h preculture in 5% DMSO

66

34

24 h preculture in 5% DMSO + 30 min in 7.5% DMSO

50

50

No preculture, 30 min in 7.5% DMSO

37

63

a. 5o ~

' I

Frequency of m i t o t i c c e l l s (MI) and level of ploidy of periwinkle cells #916 in I-B5C medium over 3 subculture periods of 4 days before (-o- 91600) and a f t e r l month ( - ~ - 00916) storage at -196°C. Shaded symbols (-o-, - & - ) represent the ploidy level.

No. 91600

~ - 00916

F-

Fig. I.

TRANSFERS

,2

Survival based on TTC reduction assay and represents average of 3 experiments.

When untreated c e l l s were gradually exposed d i r e c t l y to 7.5% DMSO, even f o r a period of 30 min, a v i a b i l i t y loss of 63% occurred. This drastic reduction in v i a b i l i t y may not be uncommon, since DMSO at high concentrations is known to a l t e r membrane permeabili t y and also to i n t e r f e r e with the RNA and protein synthesis.

137 Experiments using 5% DMSOas the f i n a l concentration in the freezing experiments resulted in very poor recovery (data not shown here) and, therefore, 7.5% had to be chosen. The e f f e c t of preculturing the c e l l s in medium supplemented with DMSOand of various cooling rates on survival of the c e l l s is shown in Table 2. Maximal v i a b i l i t y (I00%) was obtained when the c e l l s were frozen at cooling rates of 0.5 or l.O°C/min to -40°C followed by l h storage in l i q u i d nitrogen. The rate of survival shown in Table 2 should be compared with 2 sets of controls: i ) cells which were not treated with DMSO, and i i ) c e l l s t r e a t ed in the same manner as those used for freezing but not subjected to freezing. Analysing the results in such a perspective i t could be stated that c e l l s f r o zen at 0.5 to l.O°C/min did not suffer additional v i a b i l i t y loss once they have been precultured in DMSO medium for 24 h. In terms of net survival a v i a b i l i t y loss of 50% had occurred. The c e l l s which were not subjected to preculturing in 5% DMSO-supplemented medium showed survival of lesser magnitude. This could be explained on the basis of the results presented in Table l where c e l l s treated with 7.5% DMSO without p r i o r preculturing resulted in a v i a b i l i t y loss of 63%. Of the remaining 37% viable c e l l s , 90-85% could successfully be cryopreserved when frozen at cooling rates of 0.5 to l.O°C/min. As the cooling rates were increased to 5.0°C/min, c e l l s from precultured and nonprecultured groups showed reduced survival. Table 2.

0.5 l.O 2.0 3.0 4.0 5.0 a. b.

198o).

Effect of preculture and various cooling rates on the survival of periwinkle cell l i n e #916 frozen and stored in l i q u i d nitrogen for l ha .

Cooling rates (°C/min)

p r e f e r e n t i a l growth to e i t h e r di- or polyploid c e l l s . This observation is encouraging in l i g h t of e a r l i e r reports on retrieved c e l l s of carrot which had retained the a b i l i t y to produce embryos and p l a n t l e t s (~4ag and Street, 1973) and to accumulate anthocyanins (Dougall and Whitten, 1980). However, cryopreserved c e l l s of C. roseus have undergone a structural change due to treatment leading to cryopreservation, i . e . the vacuome of c e l l s of l i n e #916 c h a r a c t e r i s t i c a l l y consisting of numerous small vacuoles have now been replaced by one big central vacuole as is common with c e l l s of most other periwinkle cell lines (Fig. 3). At this stage we do not have any information at which stage of the cryopreservation process, the amalgamat i o n of vacuoles into one unit may have occurred. These findings are in contrast to e a r l i e r studies on the structure and u l t r a s t r u c t u r e of cryopreserved pea meristem c e l l s wherein no structural changes appeared to have occurred subsequent to the cryoprotectant treatment or the cryopreservation (Haskins and Kartha,

Cell survival (%)b with without preculture preculture lO0 lO0 42 35 25 15

(50) (50) (21) (17.5) (12.5) (7.5)

90 85 30 22 15 15

(33.3) (31.5) (ll.l) (8.1) (5.6) (5.6)

The c e l l s were precultured f o r 24 h in n u t r i e n t medium containing 5% DMSOand frozen employing 7.5% DMSO. Survival based on TTC reduction and compared against the appropriate DMSO-treated unfrozen control. Values given in parentheses represent the % survival as compared to the untreated and unfrozen contol. All figures represent averages of 3 experiments.

Samples of c e l l s from untreated and unfrozen controls, resumed growth within 6-7 days of p l a t i n g or transfer to l i q u i d n u t r i e n t medium (Fig. 4-7). The cells which were cooled at higher rates, 2°C/min or more, displayed a longer lag phase, i . e . as much as 3 weeks, before regrowth was discernible in part of the cell population. The c e l l s which were frozen at O.5°C/min and stored in l i q u i d nitrogen (-196°C) up to I I weeks retained the o r i g i n a l a b i l i t y to reduce TTC and resume growth. As shown in Fig. l , the frequency of mitosis and level of ploidy in the culture stored for i h at -196°C, retrieved and regrown f o r one month, were not s i g n i f i cantly d i f f e r e n t before and a f t e r cryopreservation. Treatment with DMSO, freezing at regulated slow cooling rates and thawing appeared not to have given

Fig. 4-7.

Regrowth and colony formation from C. roseus c e l l s # 916, 2 weeks a f t e r pTating. ~ ) Cells not subjected to DMSO-treatments and freezing. (5) Cells precultured for 24 h in culture medium supplemented with 5% DMSO. (6) Precultured c e l l s treated with 7.5% DMSOfor 30 min but not frozen. (7) Cells shown in Fig. 6 frozen at a cooling rate of O.5°C/min to -40°C followed by storage in l i q u i d nitrogen f o r l h.

The results presented here confirmed the f e a s i b i l i t y of storing C. roseus c e l l s cultured in v i t r o at the temperature of l i q u i d nitrogen. The protocol reported here for one cell l i n e may prove applicable to other cell cultures, in p a r t i c u l a r to periwinkle cell lines which displayed a l k a l o i d p r o f i l e s including the iboga-type a l k a l o i d catharanthine. The question whether the cryopreservation w i l l a f f e c t the potential for a l k a l o i d synthesis of a culture or whether alkal o i d accumulation affects the cryopreservation process w i l l s t i l l have to be answered.

138 References Barz IJ, E l l i s BE (1981) Ber. Deutsch. Bot. Ges. 94:1-26 Constabel F (1981) Proc.VIth Intern. Ferment. Symp. London/Ont. July 1980 Dhoot GK, Henshaw GG (1977) Ann.Bot. 41:943-949 Dougall DK, Whitten GH (1980) Planta Med. Suppl. 129-135 Gamborg OL, M i l l e r RA, Ojima K (1968) Exp.Cell Res. 58:151-158 Gamborg OL, Wetter LR (1975) In: Gamborg OL, Wetter LR (eds) Plant tissue culture methods, ~lational Research Council of Canada, Saskatoon, p 96 Haskins RH, Kartha KK (1980) Can.J.Bot. 58:833-840 Kartha KK (1981) In: Thorpe TA (ed) Plant tissue culture methods and applications, Academic Press, Inc, flew York, pp 181-211

Kurz WGW, Chatson KB, Constabel F, Kutney JP, Choi LSL, Kolodziejezyk P, Sleigh SK, Stuart KL, Worth BR (1980) Phytochemistry 19:2583-2587 Kutney JP, Choi LSL, Kolodiejezyk P, Sleigh SK, Stuart KL, Worth BR, Kurz WGW, Chatson KB, Constabel F (1980) Heterocycles 14:765-768 I~ag KK, Street HE (1973) Nature 245:270-272 Steponkus PL, Lanphear FO (1967) Plant Physiol. 42:1423-1426 Ulrich JM, Finkle BJ, Moore PH, Ginoza H (1979) Cryobiology 16:550-556 IJithers LA, Street HE (1977) In: Barz W, Reinhard E, Zenk MH (eds) Plant tissue culture and i t s biotechnical application, Springer-Verlag, BerlinHeidelberg-New York, pp 226-244 Zenk MH (1978) In: Thorpe TA (ed) Frontiers of plant tissue culture 1978, Int.Assoc. Plant Tissue Cult. Univ.of Calgary, pp 1-14