CRYOPRESERVATION OF POTATO CULTIVARS - DESIGN OF A METHOD FOR ROUTINE APPLICATION IN GENEBANKS Angelika Schafer-Menuhr and H. Martin Schumacher Gunda Mix-Wagner DSMZ-Deutsche Sammlung von Mikroorganismen Institut flr Pflanzenbau, und Zellkulturen GmbH, Bundesforschungsanstalt fir Mascheroder Weg 1B, Landwirtschaft Braunschweig D-38124 Braunschweig, Vélkenrode (FAL), Germany Bundesallee 50, D-38116 Braunschweig, Germany Keywords: Solanum tuberosum L., germplasm preservation, apical meristems, shoot tips, ultra rapid freezing, flow cytometry, DNA fingerprinting Abstract To improve germplasm preservation of potato cultivars many attempts have been made to freeze potato meristems in the past. Slow freezing, ultra rapid freezing, vitrification as well as encapsulation/dehydration have been successfully applied for cryopreservation. Nevertheless all methods have only been applied to a single or a few cultivars and under conditions of academic research. Our aim was to design a method suitable for routine genebank work, which provides high sample throughput, low costs, fitting into a working days time schedule, low susceptibility to errors and applicability to a broad range of different potato cultivars. Working at suboptimum conditions for a specific cultivar was accepted as long as plants could be regenerated. The method developed is based on ultra rapid freezing. Apical shoot tips were incubated in DMSO, transferred into 2,5 ml droplets placed on small leaflets of aluminium foil and immersed directly into liquid nitrogen. Under these conditions the droplets stick to the aluminium foil and the leaflets can be stored in cryovials. Tests to compare different methods for the pregrowth of mother plants, type and size of meristems, methods for cryoprotection and pregrowth stimulation after thawing have been carried out. The method could be applied routinely without adaptations to more than 150 different cultivars up to now. Genetic stability was tested by phenotypical inspection of regrown plants and tubers, flow cytometry and DNA. fingerprinting. Only one polyploid was found. From 161 tested samples no polyploids or abnormal banding patterns have been found. The developed method is applicable for routine genebank work and provides a safe cryopreservation method for potato cultivars 1. Introduction A lot of efforts have been made to develop methods for the preservation of potato germplasm by deep freezing of apical or axillary meristems. The first method successfully applied was rapid or ultra rapid freezing (Grout, ef al. 1978). Later the completely different approach of slow freezing has been used by different authors (Towill 1981, Benson, ef al. 1989), Recently vitrification (Schnabel-Preikstas, ef al. 1992) and the encapsulation/dehydration method (Fabré ef al, 1990) were also applied for the cryopreservation of potato meristems. Although all different methods have been successful most of the work-has been done only with a few selected genotypes of Solanum tuberosum. A broader spectrum was only published by Towill (1981, 1984). On this basis the aim of our work was to refine the technology for practical application in a genebank. For practical use in a genebank the applicability of a method to a broad range of different cultivars is essential because limitations by staff and time make an adjustment of the methodology to each specific genotype impossible. Additionally a routine method should Hon.Biotech. In Vitro Cult, and Breeding 477 Eds, A.Altman, MZiv ‘Acta Hon. 447, ISHS 1997provide a high sample throughput, work at low costs, fit into a normal working days time schedule and have a low susceptibility to errors, To achieve these prerequisites we designed a method by the combination of ultra rapid freezing with the droplet method developed by Kartha, et al. (1982). nm . Material and methods 2.1, Plant material and cultivation methods New in vitro were plantlets initiated from noda! segments and cultivated in twist off jars (2=7em, h=12cm, 10 - 15 plants per jar). Holes in the lid (O=1cm) closed by cotton plugs provided aeration. The plantlets were cultured at 20 - 23°C and at 4 kix light (12 hr.) from the top. At a height of about 10 cm plants were used for further propagation or for freezing. All culture media were based on the MS medium of Murashige, e/ al. (1962). The pH value of all media was 5.6 - 5.8. MSI medium: MS medium supplemented with 20 g/l sucrose (solidified with 8 g/l agar. MSTo medium (Towill 1983): MS medium containing 0.5 mg/l zeatinriboside, 0.2 mg/l gibberellic acid 3 (GA3), 0.5 mg/l indoleacetic acid (IAA), 6 g/l sucrose. MSB medium: MS medium containing 0.1 mg/l zeatinriboside, 0.04 mg/l gibberellic acid 3 (GAj), 6 g/l sucrose. The medium was filter sterilized, Well developed in vitro plants - recovered from freezing or control plants - were potted (O=18 cm pots) and grown in a growth chamber. For the first two weeks the plants were covered with translucent plastic cups. Tubers were harvested and replanted. During growth and after harvest photographs were taken for documentation. 2.2. Freezing of shoot tips and recovery of planis For routine work shoot tips of 2 - 3 mm length and 0.1 - 0.5 mm thickness consisting of the apical dome, the leaf primordia and adjacent leaf material were dissected with hypodermic needles under a stereo microscope. The shoot tips were placed on filter paper in Petri dishes (@=5 cm) wetted with MSTo medium and incubated at 23°C over night. 100 - 200 shoot tips were prepared for one experiment. On the next day the shoot tips were transferred to filter paper soaked with cryoprotectant (10 % dimethylsulfoxid in MSTo medium, freshly prepared, filter sterilized) and incubated at room temperature (23°C) for 2 hr. 2.5 il droplets of cryoprotectant were pipetted on heat sterilized pieces of aluminium foil (0.7 x 2 cm , 0.03 mm thick, 6 droplets per foil), With hypodermic needles one shoot tip is placed into each droplet. The foils were then dropped directly into liquid nitrogen. After freezing 2 foils were transferred into precooled 2 ml cryovials. Frozen aluminium foils were taken out of the cryovials and dropped in the medium Each thawed shoot tip is placed in a Petri dish (= 3 cm) and covered with a drop of low melting agarose (1 % agarose in MS1 medium, added at 40°C). After 30 min 1 - 1.5 ml MSTo medium is added. The Petri dishes were sealed with Parafilm and incubated at 23 °C and 2 kix (12 hr.) light in a translucent plastic box containing 2 Petri dishes filled with water. Shoots of about 1 cm length were transferred to 6 cm (@) Petri dishes containing solid MSI medium. Ata height of 3 cm shoots were planted in larger vessels, 2.3. Flow cytometry and DNA fingerprinting For flow cytometry leaves were chopped with a razor blade in ‘isolation and staining buffer’ (Partec, containing DAPI (4'-6'-diamidino-2-phenyl-indole)) as described by de Laat, et al. (1987). The crude extract was filtered through 100 im filters (Partec) and stored over night on ice. Measurement was performed the next day with a FAC Star flow cytometer (Becton-Dickinson) at the Institute of Plant Genetics and Crop Plant Research, Gatersleben. The detector was calibrated for DAPI. DNA isolation is based on protocols of Schweizer (1990). Samples containing 20 pg DNA dissolved in TE buffer (10 mM Tris-HCl, pH 8.0) were digested over night with 478DRA I Boehringer). Fragments were separated on 0.9% agarose gels in NEB buffer (100 mM Tris, 12.5 mM sodium acetate, | mM acetate, ImM EDTA, pH 8.1). The gels were blotted onto neutral nylon membranes (Amersham). Hybridization was carried out with digoxigenin labeled oligonucleotides ((GACA),, (GATA),, Fresenius) according to the recommendations of the supplier. Detection was carried out with the DIG Nucleic Acid Detection Kid (Boehringer). 3. Results 3.1. Freezing experiments with 150 different cultivars Before the routine application to different cultivars many experiments have been carried out for the optimization of the standard method. Very small meristems or explants consisting only of the meristematic dome and 2 or 3 leaf primordia performed less good during recovery growth. Best results were obtained when the basis and parts of the covering leaves were left on the explant. With four different cultivars (Amigo, Hansa, Pommerat, Ulster Glade) it has-been tried to use also nodal segments for freezing. The plant regeneration rate of apical meristems was for all the cultivars tested at least 5 times higher than with nodal segments. The optimum duration for the preincubation with the cryoprotectant DMSO has been tested with the cultivars Adagin, Gatcinskii, MPI 50.140/5, R-10, Rys and Ulster Glade. For periods from 1 - 3 hours of incubation the specific percentage of survival (callus + plant formation) for each cultivar did not change after freezing (mean values: 1h/70%, 2h/76%, 3h/81%, 4h/55%). Shorter or longer incubation periods decreased the survival rate (mean values: 10min/7%, 4h/55%, 17h/13%) for all cultivars tested. The cultivars CF-69, MPI BH17, Paarsput, Peconic, Rys, Ulster Glade and Varmas have been used to test whether changed regrowth procedures may enhance plant formation. All the cultivars showed an increased rate of plant regeneration after freezing when MSTo medium was exchanged 2 weeks after thawing against MSB medium. With the standard procedure described above freezing experiments with more than 150 different potato cultivars have been performed. The cultivars have not been selected by any criterion and the freezing procedure has been carried out without any adaptations for a specific cultivar. Also after thawing plants did not get any special attention. As expected cultivars reacted differently. Success has been measured either as ‘% survival’, which means the percentage of frozen shoot tips that developed callus or plants or both after thawing or as ‘% plant regeneration’ which means the percentage of shoot tips that developed plants during recovery growth. Most of the cultivars showed 90 -100 % survival, whereas the mean % of plant regeneration was lower (see Figure 1). The comparison of the results of different cultivars shows that there is only a very low if any correlation between survival (callus + plant regeneration) and plant regeneration. Figure 2 lists the survival and plant regeneration rate for 10 cultivars showing very high and 10 cultivars showing very low survival rates. Although the plant regeneration rate is low for some cultivars there was not a single cultivar which did not yield plants after thawing (Schéifer-Menuhr, et al. 1994). 3.2. Evaluation of geneti¢ stability Since often cultivars develop callus together with regrown plants it was difficult to decide whether plants did regrow directly from the meristem or from primary callus. The latter case could result in genetically altered plants. Therefor the plants regenerated from up to now 98 cultivars have been planted out and the phenotype of tubers and plants compared to that of unfrozen controls. In nearly all cases the regrown plants could not be distinguished from the control. Only three fully developed plants differed from the controls (one plant of the cultivar Granola, two plants of the cultivar Rys). Even though the phenotype of plants and tubers did not show changes we looked for ‘hidden’ genetic 479changes by flow cytometry and DNA fingerprinting. Although it is doubtful whether our flow cytometric measurements could detect aneuploidy it could be demonstrated that none of the 20 cultivars tested was polyploid. A single polyploid plant had already been detected earlier. The same 20 cultivars were also analyzed by DNA fingerprinting. Although the method applied is sensitive enough to distinguish between all 20 cultivars tested, we in fact observed no changed banding pattern from a total of 161 plants tested. Only minor differences were detected for 3 plants, which are considered to be artifacts, 4. Discussion The method described fulfills all requirements that have been stated for a routine method for genebank application. More than 150 cultivars could be frozen by the procedure although regeneration rates are sometimes low. Different individual plants of a vegetatively propagated crop like potato are clones and do not represent a genetic diversity which has to be preserved. Therefore it is justified to work at low plant regeneration rates. Nevertheless genetic changes might occur due to the regeneration of genetically altered plants from callus. The fact that we observed phenotypical changes only in some very rare cases seems to indicate that the method works safe enough. Whether flow cytometric analysis or DNA fingerprinting is more sensitive for the detection of genetic changes than phenotypical inspection in our case is doubtful. In two cases were an altered phenotype was observed (cultivar Rys) DNA fingerprinting did not show a changed banding pattern. Further improvement of plant regeneration rates may be possible by an improvement of the regeneration process for example by the use of specific regeneration media. Nevertheless it has to be taken into account that plant regeneration after prolonged culture periods or after the exchange of auxin containing for auxin free media might lead to a higher percentage of plants regenerated from callus and thereby an increased rate of somaclonal variation. It is estimated that one technician is able to freeze up to 50 cultivars per year using our method routinely. A cost analysis based on salaries and prices in Germany revealed that 90% of the costs for freezing one cultivar is due to labor costs. We recommend the cryopreservation methodology for genebanks mainly to establish a safety or base collection: Acknowledgments The work described has been funded by IPGRI-International Plant Genetic Resources Institute, Rome. We are thankful to Dr. Meister, Institute for Plant Genetics and Crop Plant Research (IPK), Gatersleben for performing the flow cytometric measurements and Drs. L. Schilde-Rentschler and V. Campos, University of Tiibingen for their advice how to perform the DNA fingerprints. References Benson, E.E., K. Harding and H. Smith 1989, Variation in recovery of cryopreserved shoot-tips of Solanum tuberosum exposed to different pre- and post-freeze light regimes. Cryoletters 10: 367-372. de Laat, A.M.M., W. Géhde and M.J.D.C. Vogelzang, 1987. Determination of ploidy of single plants and plant populations by flow cytometry. Plant Breeding 99: 303-307. Fabré, J. and J. Dereuddre, 1990. Encapsulation-dehydration: a new approach to cryopreservation of Solanum shoot-tips. Cryoletters 11: 413-426. Grout, B.W. and G.G. Henshaw, 1978. Structural observations on the growth of potato shoot-tip cultures after thawing from liquid nitrogen. Annals of Botany 46: 1227-1229. Kartha, K.K., N.L. Leung and L.A. Mroginsky, 1982. In vitro growth responses and plant regeneration from cryopreserved meristems of cassawa (Manihot esculentum Crantz). Zeitschschrift fir Pflanzenphysiologie 107: 133-140. 480Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiologia plantarum 15: 473-497. Schafer-Menuhr, A., Schumacher H.M. and G. Mix-Wagner, 1994. Langzeitlagerung alter Kartoffelsorten durch Kryokonservierung der Meristeme. Landbauforschung Vélkenrode 44: 301-131. Schnabel-Preikstas, B., E.D. Earle and P. Steponkus, 1992. Cryopreservation of potato shoot tips by vitrification. Abstracts of the 29th Annual Meeting of the Society for Cryobiology, p. 48. Schweizer, G., 1990. Charakterisierung und RFLP Analyse spezifischer Genom- komponenten bei Solanum und ihr Einsatz zur Identifikation somatischer Hybride von Solanum tuberosum Zuchtlinien. PhD-thesis University of Tubingen, Germany. Towill, L.E., 1981. Survival at low temperatures of shoot-tips from cultivars of Solanum tuberosum group tuberosum. Cryoletters 2: 373-382. Towill, L.E., 1984. Survival of ultra-low temperatures of shoot tips from Solanum tuberosum groups andigena, phureja, stenotomum, tuberosum and other tuber bearing Solanum species. Cryoletters 5: 319-326.90-100% a E:T EEE EEELELEE CEE LECECEE LETS i " oO 9 18 27 36 45 Range Number of cultivars V2 Total survival {ITNT Plant regeneration Figure | Distribution of cultivars according to the range of total survival and plant regeneration Superior ~ czar Vanmas — (220 | RedLaSode — Priwal Planeta —222sAmaraat ae — MPI67 1.04/16 ~2ezazcccerz EIEIO Linzer Starke —zazmzzé@ Lama ——_——<_<—=————— Ackersegen ~ 22 reetale ———= Freja ~ ean ——— Ema = Eleisa — zanna —— Desiree ~taamenmma—— Debora — zai Creata ~ jamaznmmzaanies — Cosima azz —_— SO — Capella — zanna Blanik — zzz 0 20 40 60 80 100% (Total survival (callus + plant regeneration) (ZZ Plant regeneration rate (plant regeneration only) Figure 2% total survival and plant regeneration of 10 cultivars showing very high and 10 cultivars showing very low survival rates 482