Biochem. Physiol. Pflanzen 186, 63-67 (1990) VEB Gustav Fischer Verlag lena

Cryopreservation of Chamomilla recutita Shoot Tips

B. DIETTRICH, P. DONATH, A. S. Popov l), R. O. BUTENKOl), and M. LUCKNER

Martin-Luther-University Halle-Wittenberg, Section of Pharmacy, Halle (Saale), GDR l) Academy of Sciences of the USSR, Timirjazev Institute of Plant Physiology, Moscow, USSR

Key Term Index: Meristems, shoot tips, cryopreservation, freezing; Asteraceae, Chamomilla recutita

Summary

Shoots of Chamomilla recutita (L.) RAuscHERT (chamomile) were hardened by cultivation at 4°C for 8 weeks. Then the shoot tips were explanted and treated for a period of 16h with a cryoprotector solution containing 0.5 mol DMSO, 1.0mol glycerol, and 0.2mol sucrose I-I. The shoot tips were placed in ampoules which were cooled with 0.5Kmin- 1 to -40°C (slow cooling), or they were directly submerged in liquid nitrogen (ultrarapid cooling) and then transferred into precooled ampoules. The ampoules with the frozen shoot tips were stored in liquid nitrogen. The slowly cooled shoot tips were thawed by rewarming the ampoules in a water bath of 40°C. The ultrarapidly frozen shoot tips were thawed by submerging in liquid nutrient medium at room temperature. For regrowth the shoot tips were plated on the surface of nutrient agar. About 65 % of the slowly cooled shoot tips survived freezing and thawing, and about 40 % regenerated shoots. Similar rates were obtained with the ultrarapidly cooled shoot tips. The survival rates did not decrease when the shoot tips were stored for 3 years in liquid nitrogen. Normal C. recutita plants were grown from the shoot tips obtained after freezing and thawing.

Introduction

The micropropagation of Chamomilla recutita (L.) RAUSCHERT (chamomile) by shoot tip

culture has been described recently (DON ATH et al. 1990). It allows the cloning of elite

genotypes with the growth of healthy daughter plants which may be used in the rationalization

of C. recutita breeding. For the breeding process it is necessary to keep interesting genotypes

for a long period of time. The preservation of shoot tips at ultralow temperature (cryopreserva­

tion) is one of the most valuable methods in this respect (KARTHA 1985; NITZSCHE 1983;

WITHERS 1985 and 1988). In the following a protocol for the cryopreservation of C. recutita shoot tips is given. It follows the procedure established for shoot tips of Digitalis lanata (DIETTRICH et al. 1987).

Materials and Methods

Nutrient medium

All values in mmoll- I : sucrose 100, NH4N03 20, KN03 20, KH2P045, CaCI2 3, MgS04 1.5, FeS04

0.2, Na2EDTA 0.1, MnS04 0.1, H3B03 0.1, ZnS04 0.03, KI 0.005, Na2Mo04 0.001. Ifnecessary, 109 agar I-I were added for solidification.

Preparation of shoot tips

Axenic shoots of C. recutita (L.) RAUSCHERT were grown for 8 weeks at 4 °C on a solidified nutrient medium containing 0.12 f,lmol benzyladenine I-I upon illumination with fluorescent lamps

BPP 186 (1990) 1 63

LS 40 day light and LS lumoflor 80 1 : 1 (7 - 9 W m - 2, 16 h light, 8 h dark; spectral composition of the emitted light s. SCHEIBNER et a1. 1987). Shoot tips (length 0.2-1.0mm) were explanted and placed on filter paper strips wetted with nutrient medium. These filter paper strips were put in Petri dishes (diameter 50mm) containing 1.25 ml cryoprotector solution (0.5 mol DMSO, 1.0mol glycerol, and 0.2mol sucrose I-I in water) for 18h.

Ultra rapid freezing and thawing

The strips carrying the shoot tips were submerged in liquid nitrogen under aseptical conditions. They were then transferred into plastic ampoules precooled with liquid nitrogen. The ampoules were stored in liquid nitrogen. Before recultivation the filter paper strips were removed from the ampoules and the shoot tips were thawed within 2 - 3 s by submerging the strips in the nutrient medium (1. 25 ml in Petri dishes with a diameter of 50mm).

Slow freezing and rapid thawing

The filter paper strips carrying the shoot tips were folded and transferred into plastic ampoules containing 1 ml cryoprotector solution. The strips were located in the ampoules in such a way that their lower edge dipped into the solution. The ampoules were cooled to b °C in an ice bath and put into the precooled chamber of the freezing machine, i.e., either the electronic freezer SP 00.00.00 of the Institute of Cryobiology and Cryomedicine of the Ukrainian Academy of Sciences, Charkov, USSR, or the remodelled Ultracryostat MK 70, Priifgeratewerk Medingen, GDR (DIETTRICH et a1. 1984). The ampoules were cooled with 0.5 K min -I. At - 7 °C they were dipped with their lower part into liquid nitrogen for about I s and kept at - 7 °C for 20 min. After cooling to - 40°C the ampoules were transferred to and stored in liquid nitrogen. Thawing was carried out by submerging the ampoules in water of 40 °C till the ice was melted (necessary period of time 1-2 min). They were then placed in ice water to reduce toxic effects of the cryoprotectors, e.g., of DMSO.

Recultivation

The filter paper strips with the ultrarapidly frozen and thawed shoot tips or the slowly frozen shoot tips were spread on the surface of solidified nutrient medium containing 0.08 !-lmol benzyl adenine 1-1. They were cultivated at 25°C in the light (s. above). After 7 days the rate of survival and after 3 weeks the rate of shoot formation were examined.

Results and Discussion

The procedure for cryopreservation of C. recutita shoot tips described in this paper includes the following steps:

Precultivation

The tips of shoots grown in vitro under the normal conditions of shoots multiplication showed rather low rates of survival after freezing and thawing. The rates were increased considerably, however, if the C. recutita shoots were precultivated for 8 weeks at 4 °C (cold hardening; cf. SEIBERT and WETHERBEE 1977) before explantation of the tips. Under suitable conditions (see below) up to 50 % of the hardened shoot tips survived freezing and thawing, and half of them regenerated normal shoots (Fig. 1). Cold hardening had a similar effect on other plant species also, e.g. carnation (SEIBERT and WETHERBEE 1977) and Digitalis lanata (DIETTRICH et al. 1984), but was shown to be unsuitable for the shoot tips of the tropical plant Rauwolfia serpentina (unpublished results).

64 BPP 186 (1990) I

Vitality ['I,]

60

50

40

30

20

10

4

--~~J1 Rate of greening

I Rate of shoot

regeneration

6 10 12

[ weeks]

Vitality ('I, 1 100,--------------------------------------,

80

Rate of greeni ng

60

Rate of shoot regeneration

0.25 0.5 5 10 75

Rate of cooling [K min-1 ]

Fig. I. Influence of cold hardening of C. recutita shoots on the survival of shoot tips after freezing and thawing. Shoots were kept at 4 DC for various periods of time. Than the shoot tips were explanted, treated with cryoprotector solution and placed in ampoules. The ampoules were cooled with a rate of 0.5 K min -) to -40 DC and then were transferred into liquid nitrogen. After 20 h the ampoules were rewarmed by submerging in water of 40 DC. The thawed shoot tips were cultivated on solidified nutrient medium. After 7 days the rates of surviving shoot tips and after 3 weeks the rates of shoots tips regenerating shoots were determined.

Fig. 2. Influence of the rate of cooling on the survival of C. recutita shoot tips after freezing and thawing. Tips of cold hardened shoots were treated with cryoprotector solution, and placed in ampoules. The ampoules were cooled slowly with the rates given to -40DC. At -7"C crystallization was initiated by seeding. After 20 h the ampoules containing the shoot tips were rewarmed in a water bath of 40 DC and the shoot tips were cultivated on solidified nutrient medium.

Treatment with cryoprotectors

A prerequisite for the recovery of the shoot tips after freezing and thawing was the treatment of the hardened shoot tips with cryoprotectors. Satisfactory results were obtained with mixtures of DMSO and osmotically active compounds, like sucrose (Table 1, experiment No.5) or sucrose and glycerol (Table 1, experiments Nos. 12-15). The highest rates of survival and shoot regeneration yielded a cryoprotector mixture containing 0.5mol DMSO, l.Omol glycerol, and 0.2 mol sucrose I-I (Table 1, experiment No. 14). The treatment with the cryoprotector sulutiun must last at least 18 h. Shorter periods resulted in decreased survival rates.

Cooling and Rewarming

The rate of cooling was of special influence on the survival of the shoot tips (Fig. 2). Optimum results were obtained with low cooling rates (0.25Kmin- l and 0.5 K min-I), which allowed a complete dehydration of the cells by the osmotically active cryoprotectors.

5 BPP 186 (1990) I 65

Table 1. Influence of different cryoprotectors Oil survival and shoot regeneration after freezing and thawing.

Expt. DMSO Glycerol Sucrose Total concen- Rates of Shoot No. [moll-I] [moll-I] [moll-I] tration of Survival regen-

cryoprotectors [%] eration [moll-I] [%]

1 0 0 0 0 0 0 2 0 0.5 0.2 0.7 0 0 3 0 0.5 0.5 1.0 0 0 4 0 1.0 0.2 1.2 0 0 5 0.5 0 0.2 0.7 26 8 6 0.5 0 0.5 1.0 0 0 7 1.0 0 0.2 1.2 14 0 8 0.5 0.5 0 1.0 20 0 9 0.5 1.0 0 1.5 34 0

10 1.0 1.0 0 2.0 0 0 11 0.5 0.25 0.2 0.95 0 0 12 0.5 0.5 0.2 1.2 38 16 13 0.5 0.75 0.1 1.35 50 16

114 0.5 1.0 0.2 1.7 52 18

15 1.0 0.5 0.2 1.7 46 16 16 1.0 0.75 0.2 1.95 10 0 17 1.0 1.0 0.2 2.2 0 0

Tips of cold hardened C. recutita shoots were treated with the cryoprotector solutions given and cooled with a rate of 0.5 K to -40°C. At -7°C crystallization was initiated by seeding. The ampoules containing the shoot tips were rewarmed in a water bath of 40 °C and the shoot tips were cultivated on solidified nutrient medium.

Crystallization in the cryoprotector solution surrounding the shoot tips was initiated artificially by seeding at -7°C. Seeding increased the reproducibility of the experiments by the prevention of supercooling. The ampoules containing the shoot tips were stored in liquid nitrogen. During a period of 3 years they showed no reduction in viability and in the rate of shoot regeneration.

Rates of survival matching those obtained after slow cooling were found also after ultrarapid cooling (rate ca. 4000Kmin-'). For ultrarapid cooling the shoot tips were submerged directly in liquid nitrogen. This method caused vitrification of the shoot tips. The vitrified tips were transferred to precooled containers which were stored in liquid nitrogen.

The uitrarapidly cooled shoot tips had to be rewarmed very fast before further cultivation to prevent crystallization of the cell water. They therefore were placed in the nutrient medium directly. The transfer of the shoot tips to and from the ampoules used for storage caused, however, the risk of an uncontrolled rise of the temperature which resulted in crystallization of the vitrified cell water. Because the formation of intracellular ice crystals killed the cells ultrarapid cooling gave reproducible rates of surviving only if handled by skilled persons, and was not suitable for routine work.

66 BPP 186 (1990) I

Recultivation

For recultivation the frozen and thawed shoot tips were placed on nutrient agar and kept in the light. Within 7 days the surviving shoot tips turned green. About 50 % of the surviving shoot tips developed into shoots. In the not regenerating shoot tips the morphologically potent cells obviously were destroyed (cf. WITHERS 1985). Healthy plants were grown from the shoots formed. These plants showed no deviation in the phenotype and the composition of the essential oil if compared with control plants. Also the rate of shoot multiplication during micropropagation was similar to that of the control shoots. There was no decrease in the rates of survival and shoot regeneration if the shoot tips were stored in liquid nitrogen up to 3 years. This indicates that cryopreservation of shoot tips is a suitable method for the establishment of a gene bank which may be used in C. recutita breeding.

References

DIETTRICH, B., WOLF, T., BORMANN, A., Popov, A. S., BUTENKO, R. G., and LUCKNER, M.: Cryopreservation of Digitalis lanata shoot tips Planta Medica 53, 359-363 (1987).

DIETTRICH, B., SCHINDLER, U., WENZEL, U., and LUCKNER, M.: Umriistungdes Ultrakryostaten N 180 fUr das kontrollierte Tieffrieren von biologischem Material. Z. med. Labor-Diagn. 25, 182 -184 (1984).

DONATH, P., DIETTRICH, B., HANNIG, H.-.I., and LUCKNER, M.: In vitro Vermehrung von Chamomilla recutita-Hochleistungspflanzen durch SproBspitzenkultur. Drogen-Report, in press (1990).

KARTHA, K. K.: Cryopreservation of Plant Cells and Organs. CRC Press, Boca Raton 1985. NiTZSCHE, W.: Germplasm preservation. In: Handbook of Plant Cell Culture, Vol. I: Techniques for

Propagation and Breeding (Eds. EVANS, D. A., SHARP, W. R., AMMIRATO, P. V., and YAMADA, Y.), pp. 782-805. MacMillan, New York 1983.

SCHEIBNER, H., BJORK, L., SCHULZ, U., DIETTRICH, B., and LUCKNER, M.: Influence of light on cardenolide accumulation in somatic embryos of Digitalis lanata. J. Plant Physiol. 130,211-219 (1987).

SEIBERT, M., and WETHERBEE, P . .I.: Increased survival and differentiation of frozen herbaceous plant organ cultures through cold treatment. Plant Physiol. 59, 1043-1046 (1977).

WiTHERS, L. A.: Cryopreservation of cultured cells and meristems. In: Cell Culture and Somatic Cell Genetics of Plants, Vol. 2: Cell Growth, Nutrition, Cytodifferentiation and Cryopreservation (Ed. VASIL, I. K.), pp. 253-316. Academic Press, Orlando 1985.

WITHERS, L. A.: Germplasm preservation. In: Applications of Plant Cell and Tissue Culture (Ed. YAMADA, Y.), pp. 163-177 . .1. Wiley & Sons, Chichester 1988.

Received August 23, 1989; accepted August 31, 1989

Authors'addresses: Dr. B. DIETTRICH, Dr. P. DONATH, Prof. Dr. M. LUCKNER, Martin-Luther­Universitat, Sektion Pharmazie, Weinbergweg 15, Halle (Saale), DDR-4050; Dr. A. S. PoPOv and Prof. R. G. BUTENKO, Academy of Sciences of USSR, Timirjazev Institute of Plant Physiology, Botanicheskaya ul. 35, Moscow 127 273, U,SSR.

5* BPP 186 (1990) I 67