Conservation of potato (Solanum tuberosum L.) cultivars by cryopreservation

The shoot-tips of plants of 10 potato cultivars maintained by micropropagation were frozen (ultra rapid freezing) and cryopreserved in liquid nitrogen for three months. The survival rate of the thawed specimens exceeded 50%, and even reached 100% in the case of the cultivar Fénix. The average regeneration rate was between 2.5% and 22.0%, depending on the cultivar and the presence of extra plant hormones in the regeneration medium. Cytogenetic stability was confirmed by flow cytometry: no polyploid plants were found. The regenerated plants showed vegetative development characteristics of their respective cultivars.


Introduction
For several years, the Basque Institute of Agricultural Research and Development [BIARD]-NEIKER (Instituto Vasco de Investigación y Desarrollo Agrario-NEIKER) has maintained a partially characterised potato (Solanum tuberosum L.) germplasm bank (http://www.neiker.net/patdb). The collection contains material from 412 cultivars, 332 of which are commercial and the rest either local forms or cultivars developed at the Institute. The bank is a source of material for the genetic improvement of the species and the creation of new varieties (Ruiz de Galarreta et al., 2000).
Potato germplasm is normally maintained using in vitro culture techniques employing media that retard growth and controlled conditions of light and temperature. This works well for short and medium term preservation since material is always ready for use. However, the necessary periodic subculturing of plantlets in MS medium (Murashige and Skoog, 1962) has the disadvantage that infections may develop. There are also the risks of culture chamber breakdown and the appearance of somaclonal variation (Harding, 1991).
For the long term conservation of material, cryopreservation offers several advantages, such as reduced storage space, low maintenance (periodic refilling of the freezer tanks with liquid nitrogen is all that is needed), and the independence of survival on the duration of freezing (Bajaj, 1981).
Palabras clave: meristemos, germoplasma, congelación, regeneración. (Fabre and Dereuddre, 1990;Bouafia et al., 1996) have all been investigated as methods of freezing the meristems or shoot apices of selected Solanum genotypes. Some of these methods are very laborious, complicated, time consuming, and only offer low regeneration rates (Benson et al., 1989). The aim of the present work was to improve and simplify the cryopreservation (ultrarapid freezing technique) of different potato cultivars, for the conservation and long term storage of their germplasm.
Meristem samples 2-3 mm long and 0.5-1.0 mm thick (depending on the cultivar) were removed from 10-12 cm long, in vitro micropropagated plantlets. These were incubated at 23ºC for 24 h in liquid MSTo medium (Towill, 1983) containing the salts and vitamins provided by MS medium plus 0.2 mg L -1 gibberellic acid, 0.5 mg L -1 indole acetic acid, and 0.5 mg L -1 zeatin riboside.
Aluminium laminae (0.7 cm × 2.0 cm and 0.03 mm thick) were cut and five drops of DMSO 10% (w/v) placed on top of each. One meristem was then placed in each drop. The laminae were introduced into cryotubes (two per tube), which were then only partially closed to allow the entry of liquid nitrogen from the freezer tank. Specimens were kept frozen for three months. A modified version of the ultrarapid freezing method of Schäfer-Menhur et al. (1997) was used. Table 2 shows the number of meristems frozen per cultivar.
To thaw the meristems, the aluminium laminae were removed from their cryotubes and placed in Petri dishes containing liquid MS medium at room temperature. The meristems thawed immediately and floated to the top of the medium.
Half the meristems of each cultivar were used in two different regeneration experiments. In experiment 1, five drops of MS medium containing 1% (w/v) low melting point agarose (Duchefa) were added to a Pe-tri dish, and a meristem transferred to each drop before solidification. Each Petri dish was then filled with liquid MSTo medium (i.e., with hormones: 0.5 mg L -1 zeatin riboside, 0.2 mg L -1 gibberellic acid, and 0.5 mg L -1 indole acetic acid); the solidified MS/agarose solid drops containing the meristems therefore appeared as 'islands' within the MSTo medium. The Petri dishes were then sealed and incubated for three months at 23ºC. A 16 h photoperiod was established.
In experiment 2, exactly the same procedure was followed, except that the MS/agarose droplets also contained plant hormones (0.5 mg L -1 zeatin riboside, 0.2 mg L -1 gibberellic acid, and 0.5 mg L -1 indole acetic acid).
After 14 days the meristems were examined using a microscope and callous structures could be seen. If no plantlets had regenerated from the meristem after 3 months, these calluses were transferred to modified callus medium (CM) (Keil et al., 1989) (Table 1) to ensure plantlet regeneration. The plantlets obtained from both calluses and meristems were then transferred to solid MS medium.
Survival rate was determined by counting the number of meristems considered to be alive (by examining their colour and appearance) after thawing. The regeneration rate was calculated from the number of plants regenerated (at the end of the three month study period) from the initial number of frozen meristems. The ploidy level of the plants was estimated by flow cytometry using a PA analyser (Partec), quantifying the DNA in the leaf cells of the regenerated plants. Nontreated, original cultivar representatives of the same physiological age were used as controls.   Table 2 shows the survival rate, the number of plants regenerated per cultivar and the number of meristems initially frozen.

Results
The cultivar Fénix had the highest meristem survival rate (100%). Cazona, Ganade, Gorbea, Isola and Kennebec showed rates of around 50%. The remaining cultivars showed rates of around 80%. Significant differences were seen in regeneration rates depending on whether the regeneration medium contained extra plant hormones (i.e, hormones in the MS/agarose drop): 13.2% with extra hormones compared to 7.8% with hormones in the MSTo medium only (P = 0.05; least significant difference test). High survival rates clearly did not guarantee high regeneration rates (correlation rate r = 0.08) (Fig. 1).
Great differences were seen in the regeneration rates of the different cultivars. Isola and Fénix had the highest rates (over 20%) in the presence of extra plant hormones, but less than half this value when hormones were only present in the MSTo medium. The Galician local cultivars Cazona and Ganade showed very different values (17.5% compared to 7% in the me-  dium with extra hormones). Of the NEIKER cultivars, Gorbea showed a 17% regeneration rate in the presence of extra plant hormones, falling to 13% when these were present only in the MSTo medium. Figure 2 shows the ploidy levels recorded by flow cytometry analysis of Isola plants. No differences were seen between regenerated plants and controls. In fact, no polyploid genotype was seen for any of the plants evaluated, and all the regenerated plants had phenotypes characteristic of their cultivar.

Discussion
The post-thaw survival rate of the meristems was generally high: half the cultivars showed values of over 80%. These figure are in agreement with those obtained by Schäfer-Menuhr et al. (1996), in which all the cultivars used showed survival rates of > 50% (though notable differences were seen).
With respect to the regeneration rate, Mix-Wagner (1999), who used the cultivars Hydra and Isola, obtained values of 9%-16%, somewhat less than those obtained in the present study. However, Schäfer-Menuhr et al. (1996), achieved better survival rates with Kennebec than those reported here. Regeneration rates were greater in the presence of extra hormones, perhaps due to an increase in their local concentration. However, more work is necessary to confirm these preliminary findings, using growth regulators at different concentrations.
The difference between the survival and regeneration rates shows that cryopreservation involves two processes that need to be improved separately: freezing and regeneration. The survival rate was high, sug-gesting that the process is already optimised. Better regeneration rates were achieved when plant hormones were added to the MS/agarose medium -nonetheless they need to be further improved. The regeneration rate was much more influenced by the genotype of the plant than the survival rate (Fig. 1). If improvements could be achieved, less material would need to be cryopreserved. Schäfer-Menuhr et al. (1996) indicated that the longer it takes for a meristem to regenerate, the lower the vigour of the plant. This agrees with that observed in the present study; three months after the meristems were placed in the regeneration medium, their vigour gradually declined (results not shown).
In this type of culture procedure, there may be uncertainty about whether the regenerated plant has come from the meristem or the leaf that covered it. Plants that develop from callus, which is easily produced by the leaf, can show somaclonal variation. Those plants that regenerate first are more likely to have come from the meristem and are less likely to show such variation. In the present work, some cultivars, e.g., Zorba and Isola, were quicker to regenerate, but no molecular tests were performed to determine whether they had developed from the meristem or other tissue.
Mix-Wagner (1999) used RFLP and microsatellites as molecular markers to detect somaclonal variation, but found no anomalies in their regenerated plants. In the present study, no polyploids were detected by flow cytometry, even when regenerated from meristem callus cells. All the plants were phenotypically characteristic of their respective cultivars.
The present results, plus those of other authors, show that cryopreservation is a viable alternative in the long term conservation of potato germplasm. Many genotypes can be preserved at a reduced cost, and the risks involved in manipulation and periodic subculturing eliminated. However, for experiments or genetic improvement programmes involving the periodic use of potato cultivars, in vitro maintenance is recommended alongside cryopreservation.