Genebanking of Vegetatively Propagated Medicinal Plants – Two Cases: Allium and Mentha E.R. Joachim Keller, Angelika Senula and Marion Dreiling Institute of Plant Genetics and Crop Plant Research (IPK) Department of Genebank Corrensstr. 3 D-06466 Gatersleben Germany Keywords: garlic, mint, in vitro genebank, virus elimination, cryopreservation Abstract Allium and Mentha represent two genera with important species of medicinal and aromatic plants. Large proportions of Allium (1000 of 3500) and Mentha accessions (160 of 220) are traditionally maintained vegetatively in field plots in the genebank of IPK Gatersleben. The main species used for medicinal purposes (A. sativum and M. x piperita) are seed-sterile. The field cultivation is endangered by risks (diseases, bad weather conditions). Studies were done on the field infection for LYSV, OYDV, GCLV, SLV and allexi viruses in garlic and some other Allium species. Significant differences were found between species without infection (A. saxatile), species infested by one virus mostly (A. obliquum with LYSV, A. globosum with SLV) and multiple infection (A. sativum). Meristem culture was performed on 100 garlic accessions resulting in virus free clones in 95 of them. Both medicinal plants were maintained in slow-growth culture cycles including storage at reduced temperature (2 or 10°C) on medium MS without hormones for 12 months (garlic) and 15 months (mint). Cryopreservation of garlic was successful with regrowth rates of 100% (clove explants) and 73-80% (bulbil explants). Lower success was achieved with material from in vitro culture. The vitrification technique and the droplet method were compared. INTRODUCTION Genebanks acting as living plant collections for the benefit of a large users’ community are always challenged by plants which can be only vegetatively propagated. Their maintenance requires much more efforts than the storage of seeds. The genebank at Gatersleben, Germany, is one of the largest living plant collections with an especially high degree of diversity. Medicinal and aromatic plants play an important role in this collection. The Allium species collection, amounting to 3500 accessions, has a high proportion of vegetatively propagated material (1000 accessions). A similar proportion, amounting to 1085 of 4385 accessions of other medicinal and aromatic plants, is also in field culture. These percentages of 29 and 25%, respectively, are much higher than those of the field reproduction of wheat (4%) or leguminous plants (7%) in our collection. Therefore, much attention is given to maintain Allium and other medicinal and aromatic plants, such as mint, by means of in vitro culture methods. The comparison of two main groups, namely Allium and Mentha, which are relatively homogeneous, should give some insight into the species-specific problems. Virus accumulation is usually found in material permanently maintained in field conditions. Studies of the field infection were performed on garlic and other Allium species. No investigations have been done so far in Mentha. The methods and results of earlier virus investigations have been published in Senula et al. (2000) and Keller and Senula (2001). MATERIALS AND METHODS The field collection of the genebank at Gatersleben was the source of explants for in vitro storage. Plant material was maintained in field plots with a rotation of five years. Proc. WOCMAP III, Vol 2: Conservation Cultivation & Sustainable Use of MAPs Eds.: A. Jatisatienr, T. Paratasilpin, S. Elliott, V. Anusarnsunthorn, D. Wedge, L.E. Craker and Z.E. Gardner 103 Acta Hort. 676, ISHS 2005 Bulb scale bases, bulbil explants or even proper meristems were used for Allium, shoot tips from potted plants grown in the greenhouse for mint. Once the culture was established, the slow growth followed a cycle of warm culture at 20°C for multiplication and cold storage at 2 or 10°C for the maintenance phase, all phases on hormone-free MS medium (Murashige and Skoog, 1962). Clove bases, bulbils and in vitro plantlets were used for cryopreservation of garlic. The method had been published by Makowska et al. (1999) and Keller (2002). Three repetitions of 10-20 explants each were used. Survival and regrowth were recorded. Survival is defined as percentage of living explants 2 weeks, regrowth is the percentage of sprouting explants 6-8 weeks after rewarming. Virus indexing was performed by ELISA (DAS, TAS) for leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), garlic common latent virus (GCLV), shallot latent virus (SLV) and allexi viruses. Statistical comparisons were made by means of 2 x 2 contingency tables using the χ² test for frequency results and t-tests for normally distributed values (Oehmisch and Klemm, 1971). RESULTS AND DISCUSSION Phytosanitary Conditions Garlic and the other Allium species, mentioned below, were tested for the field infection by several viruses. All viruses, so far indexed, were found in garlic, GCLV most abundant and present in garlic only. Accessions of wild species showed more differences: LYSV was found in A. albidum, A. angulosum, A. carolinianum, A. lineare, and A. obliquum, OYDV in A. hymenorrhizum and A. nutans. SLV was found in A. globosum, A. nutans, and A. obliquum. No viruses could be found in A. rubens, A. saxatile and A. senescens. Hybrids of A. cepa with several wild species seemed to inherit susceptibilities in some cases from the parents (Keller and Senula, 2003). Meristem culture was performed in 100 accessions of garlic, in order to produce virus free clones. It was successful in 95 accessions. Virus elimination depends on the size of the meristems used. A percentage of 26.7% virus free plants could be obtained with small explants possessing diameters of 0.3-0.5 mm using unripe garlic bulbils. Multiple infections with various viruses may persist in larger explants, especially from ripe bulbils. The most resistant virus was GCLV, which was present even with small explants, but even there, a slight reduction was obtained. Further reduction can be expected with additional thermo- or chemotherapy. Slow Growth Culture At present, 300 accessions of Allium and 159 accessions of mint are in slow growth storage. In garlic, a maintenance sample is represented by two independently treated units of 18 plantlets each (two plantlets per tube). This was the maximum storage capacity per accession that was not yet attained in most cases. The sample is maintained in cycles, two to four weeks cultivated in 20°C in 16-h day conditions (60-80 µmol cm-2 s-2) and then cold storage at 2°C or 10°C for 12 months. Mint is cultivated in glass jars in two independently treated sets of three glass jars. Each jar contains five explants. The culture cycle is similar to that of garlic with a four-week warm phase at 20°C for multiplication and recovery and cold storage at 2°C or 10°C for 15 months. The main Allium groups requiring vegetative maintenance have so far been garlic, shallot, and seed-sterile clones, which were obtained in a programme to produce interspecific hybrids between onion and wild species (Keller et al., 1996). A number of 37 garlic accessions were introduced to an in vitro storage experiment in 1998. Table 1 represents all accessions which were established there with more than five initial plantlets. The plantlets were stored without manipulations for one year and subsequently transferred onto new medium, cultivated at 20°C for one month, and the resulting plant numbers were then calculated. Data were collected in the three years, 1999 until 2001. 104 The survival rates (SR) do not significantly differ from year to year (between 83 and 88%). Media without hormones were used for slow growth storage. Therefore, only some accessions showed spontaneous multiplication that resulted in a moderate propagation factor (PF) of 1.2, 1.7 and 1.4, respectively, in the three years. The difference of the first two years was significant with p<0.05. The total maintenance rate (TMR) is defined as the quotient of the number of plantlets present in the respective year related to the initial number. It characterizes, therefore, the storability of the different accessions. Allium hybrids were also stored for one year. Results of storage comparisons between various species are given in Table 2, where F characterizes the storability, spontaneous multiplication in the storage phase and the recovery of the plantlets after storage in the subsequent warm phase at 20°C. The final result is given with the total maintenance rate (TMR). After storage at 2°C, the survival rate was higher than after 10°C. The resulting maintenance factor shows, that the lower temperature is better usable than the higher one. Only hybrid combinations represented by more than one clone and higher plantlet numbers were compared in detail (represented by bold letters). Hybrids with A. albidum and A. globosum survived better in lower temperature whereas hybrids with A. chevsuricum were better after 10°C. Comparing the resulting total maintenance rate, the hybrid with A. globosum showed the best results after storage at 2°C (170%). There was no significant difference between the storability at 2°C and that at 10°C in most accessions of mint. However, a small part (7% of the in vitro collection) of the accessions did not withstand cold at all. It has to be stored at 20°C. Their origins were from Tunisia (5), Italy (2), Cuba (1), Canada (1) and Germany (1). The southern origin of most of these accessions could explain their weaker vigour at 2°C. Three accessions were selected as standard material for long term studies, in order to explore whether the vigour of the in vitro material is declining with the time. The material consists of a tetraploid Mentha x villosa (MEN 148), octoploid Mentha x piperita (MEN 166) and a tetraploid Mentha x piperita (MEN 186). Storage periods of half years each were used for a comparative study (Fig.