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. 1). The number of newly formed leaves is a measure for the number of nodes which would be available upon maximum propagation of the samples. So far no vigour decline has been found after 3.5 years. Rather there is the tendency of increasing of the leaf numbers with the time from the first steps until a stable value after this initial period. The same standard clones were used for transfer studies to soil conditions. Usually 100% of the plantlets survived the transfer after a transient phase with high humidity (plastic covers over the trays with the plantlets).

Cryopreservation The vitrification method using the cryoprotectant mixture PVS 3 (Nishizawa et al., 1993) was used (Makowska et al., 1999) for cryopreservation of clove explants. It has been published earlier, that the vitrification and the droplet methods gave the same regrowth responses, independently on the explant type. Differences were found, however, between the explant types: Explants taken from in vivo material (bulbils) gave 73 and 80% regrowth, respectively, for both methods. In vitro explants developed to 13% in case of vitrification and 15-20% using the droplet method (Keller, 2002). The first four clones were introduced into routine long-term storage in three independent repetitions each: All 290, All 508, All 525, All 844, all possessing large bulbils. They were used already for some experiments published in previous reports (Makowska et al., 1999; Keller, 2002; Keller and Senula, 2003). Survival and regrowth rates of these accessions are shown in Fig. 2. Differences between the repetitions within one accession are similar to differences between repetitions of various accessions. This marks clearly, that any deviations are not statistically significant.

CONCLUSION Garlic and mint are both important medicinal and aromatic plants which can only

105 be maintained in vegetative constitution. The examples presented here show, that in vitro culture and cryopreservation are suitable means to improve the maintenance of these plants in genebanks. It is, however, necessary to improve the technology and to find special conditions to extend these measures to the entire collections of these species. Comparison of both genera resulted in the conclusion, that in vitro systems are better manageable with mint than with garlic. Garlic is currently subject of a strategic research line beginning with meristem culture, proof of the virus-free state through micropropagation of a virus-free nucleus until cryopreservation of the virus-free material. Success was achieved in several steps of this line: meristem culture, virus-free state for the five mentioned viruses, micropropagation, and slow growth culture.

ACKNOWLEDGEMENTS The authors thank Mrs. Carola Bollmann, Mrs. Doris Büchner, Mrs. Gabriele Matzig and several students for their technical work to maintain the garlic and mint germplasm.

Literature Cited Keller, E.R.J. 2002. Cryopreservation of Allium sativum L. (Garlic). p.37-47. In: L.E. Towill and Y.P.S. Bajaj (eds.), Biotechnology in Agriculture and Forestry, Vol. 50, Cryopreservation of Plant Germplasm II, Springer, Heidelberg. Keller, E.R.J., Schubert, I., Fuchs, J. and Meister, A. 1996. Interspecific crosses of onion with distant Allium species and characterization of the presumed hybrids by means of flow cytometry, karyotype analysis and genomic in situ hybridization. Theor. Appl. Genet. 92:417-424. Keller, E.R.J. and Senula, A. 2001. Progress in structuring and maintaining the garlic (Allium sativum) diversity for the European GenRes Project. Acta Hort. 555:189-193. Keller, E.R.J. and Senula, A. 2003. Germplasm preservation in Allium species - an integrated approach to store morphologically characterized virus-free plant material via cryopreservation. Acta Hort. 623:201-208. Makowska, Z., Keller, J. and Engelmann, F. 1999. Cryopreservation of apices isolated from garlic (Allium sativum L.) bulbils and cloves. Cryo-Lett. 20:175-182. Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15:473-487. Nishizawa, S., Sakai, A., Amano, Y. and Matsuzawa, T. 1993. Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification. Plant Science 91:67-73. Oehmisch, W. and Klemm, P. 1971. Biostatistische Methoden für den Arzt. Volk & Gesundheit, Berlin. 104p. Senula, A., Keller, E.R.J. and Lesemann, D.E. 2000. Elimination of viruses through meristem culture and thermotherapy for the establishment of an in vitro collection of garlic (Allium sativum). Acta Hort. 530:121-128.

106

Tables

Table 1. Maintenance behaviour in 13 garlic accessions during three years from the introduction into one year cold storage at 10°C.

SR3 PF4 TMR5 Acc. A1 B2 1999 2000 2001 1999 2000 2001 1999 2000 2001 All 0100 10 28 90 88 92 1.0 2.6 2.3 90 180 280 All 0291 14 12 100 57 82 1.1 1.3 1.3 107 71 86 All 0511 6 10 100 100 100 1.0 1.9 1.3 100 217 167 All 0655 9 21 78 78 75 1.3 3.3 1.8 100 256 233 All 0657 19 14 84 100 82 1.3 1.0 1.0 110 79 74 All 0784 20 41 75 89 100 1.8 3.4 1.6 135 275 205 All 0786 18 29 100 100 100 1.1 1.1 1.5 111 122 161 All 0813 14 7 100 80 50 1.2 1.1 1.2 121 93 50 All 0873 7 2 43 100 67 1.0 1.3 1.0 43 57 29 All 1278 9 22 78 89 60 1.1 2.1 1.8 89 189 244 All 1301 8 6 100 89 67 1.3 1.0 1.0 125 100 75 All 1311 7 6 86 67 100 1.3 1.0 1.5 114 57 86 Tax 0452 7 9 100 89 100 1.3 1.5 1.0 129 171 129 Sum / Average 148 207 88 87 83 1.2*6 1.7* 1.4 110 145 140 1 initial plantlet number 2 final plantlet number after three years 3 survival rate (%) after cold storage 4 propagation factor in the warm phase 5 TMR rate of grown plantlets (%) related to A 6 This difference marked by asterisk is significant with p<0,05.

107

Table 2. Results of one year storage of in vitro plantlets of various Allium hybrids at two different temperatures.

Hybrid with 2°C 10°C (mother A. cepa) A1 F2 TMR3 A1 F2 TMR3 A. albidum 4,5 43 40 93 48 13 27 A. altaicum 18 0 0 21 0 0 A. altyncolicum 20 20 100 18 18 100 A. angulosum 16 22 138 53 32 60 A. chevsuricum 4,6 90 67 74 7 64 69 108 A. globosum 4,6 46 78 170 45 28 62 A. hymenorrhizum 4 52 52 100 66 63 95 A. karelinii 22 7 32 17 8 47 A. lineare 20 21 105 20 23 115 A. senescens 20 0 0 18 0 0 A. sphaerocephalon 19 0 0 22 0 0 A. saxatile 4,5 187 133 71 220 154 70 Sum/Average 8 553 440 80 612 408 67 1 initial plantlet number 2 plantlet number after one year 3 TMR rate of grown plantlets (%) related to A 4 species, represented in bold letters (more than one accession/higher plant numbers) were included in statistical comparisons with χ² test (p<0,05) 5 significantly weaker than the medium species A. hymenorrhizum 6 significantly better than the medium species A. hymenorrhizum 7 figure pairs with significant differences are underlined in the species-specific lines 8 all summarized temperature-dependent differences significant (p<0,05 at least)

108 Figures

25

20 04 / 1999 10 / 1999 15 04 / 2000 04 / 2001 10 10 / 2001

leaf number 04 / 2002 5 10 / 2002

0 MEN 148 MEN 166 MEN 186

Fig. 1. Example for long-term storage of Mentha standard clones introduced in October 1998 and transferred twice the year to new media (in the year 2000 only one data record). Numbers of leaves larger than 2 mm in diameter per plantlet are recorded. Number of plantlets per sample year 1: 25-100, other years: 100-250, last year: 112-118. Bars mark confidence intervals.

100

80

60 % 40

20

0 All290 All508 All525 All844

Fig. 2. Cryopreservation of garlic accessions. Three independent control sets (10-20 explants each) per accession. Source organs: large bulbils. Method: vitrification with PVS3, 120 min dehydration before cooling. Grey columns: survival (% of rewarmed explants); white columns: regrowth (% of rewarmed explants).

109