Cryopreservation and Micropropagation Methods for Conservation of Genetic Resources of Ulmus Laevis and Ulmus Glabra

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Article Cryopreservation and Micropropagation Methods for Conservation of Genetic Resources of Ulmus laevis and Ulmus glabra

Sakari Välimäki * , Mari Rusanen, Daniela Peˇcínková, Mikko Tikkinen and Tuija Aronen

Natural Resources Institute Finland (Luke), FI-57200 Savonlinna, Finland; mari.rusanen@luke.fi (M.R.); [email protected] (D.P.); mikko.tikkinen@luke.fi (M.T.); tuija.aronen@luke.fi (T.A.) * Correspondence: sakari.valimaki@luke.fi; Tel.: +358-29-532-2127

Abstract: Elms are threatened by Dutch elm disease, and conservation methods are needed to protect their genetic diversity. Cryopreservation of dormant buds allows large numbers of genotypes to be conserved with small space requirements and minimal upkeep. Cryopreservation through slow controlled cooling was tested for both elm species native to Finland, Ulmus glabra and Ulmus laevis. Regeneration of the thawed buds by micropropagation was studied on different basal media and using different growth regulators. Multiple surface sterilisation methods were tried out for bud explants. The multiplication of U. glabra was investigated with Driver and Kuniyuki walnut medium with either 0.5 mg/L meta-topolin or 0.5 mg/L 6-benzylaminopurine. Rooting with short indole-6- butyric acid induction in liquid medium and direct transplantation of the shoots to peat ex vitro after induction were tested. For initiation, either Murashige and Skoog or Driver and Kuniyuki walnut medium with 0.02 mg/L gibberellic acid 4 + 7 and 0.5 mg/L 6-benzylaminopurine were found to Citation: Välimäki, S.; Rusanen, M.; best promote shoot formation. Surface sterilisation remains the most challenging step. No signiﬁcant Peˇcínková,D.; Tikkinen, M.; Aronen, T. Cryopreservation and differences were found between the multiplication media in either shoot production or rooting Micropropagation Methods for success. Rooting by direct transplanting was achieved in both species, but further development is Conservation of Genetic Resources of required before application on a larger scale. With further improvements to sterilisation success Ulmus laevis and Ulmus glabra. Forests especially in U. glabra, the method can be applied to the conservation of genetic resources of both U. 2021, 12, 1121. https://doi.org/ laevis and U. glabra, and knowledge of regeneration success can be used to design the cryoconservation 10.3390/f12081121 plan and optimise the sampling.

Academic Editors: Fulvio Ducci, Keywords: wych elm; European white elm; Dutch elm disease; vegetative propagation; tissue culture Alberto Santini and Tore Skrøppa

Received: 30 June 2021 Accepted: 16 August 2021 1. Introduction Published: 21 August 2021 Elms are deciduous trees valued in silviculture and urban landscaping due to their

Publisher’s Note: MDPI stays neutral unique appearance and adaptability to city conditions [1]. Finland has two native elm with regard to jurisdictional claims in species, European white elm (Ulmus laevis Pall.) and wych elm (Ulmus glabra Huds.), both published maps and institutional afﬁl- at the northern edge of their distribution range [2,3]. The populations are mainly located iations. in Southern Finland, separated from each other and too small to allow effective in situ conservation [4]. The genetic resources of the two species are therefore being conserved in outdoor ex situ collections bringing together elms from multiple populations to produce seed with higher genetic diversity than natural populations. In addition to habitat loss due to agriculture, logging and water regulation [3,4], elms are globally threatened by Dutch elm Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. disease (DED), which is considered one of the most devastating known plant diseases [5]. This article is an open access article The ﬁrst DED pandemic occurred around 1910 when the fungal pathogen Ophiostoma distributed under the terms and ulmi Buisman spread throughout Europe, and later with infested timber imports into conditions of the Creative Commons North America and parts of Central and Southwest Asia [6]. The second and still ongoing Attribution (CC BY) license (https:// pandemic was recorded in the 1970s and is caused by the more aggressive Ophiostoma creativecommons.org/licenses/by/ novo-ulmi Brasier [7]. DED disrupts xylem water transfer and results in vascular wilt 4.0/). syndrome, visible as crown defoliation followed by the eventual death of the tree [8,9].

Forests 2021, 12, 1121. https://doi.org/10.3390/f12081121 https://www.mdpi.com/journal/forests Forests 2021, 12, 1121 2 of 13

No DED has yet been found in Finland [10], but the spread of DED to the genetic reserve collections could wipe out the valuable trees. The protecting factors are a less suitable cold climate for DED vectors, the genus Scolytus bark beetles [11], and the small size and fragmentation of Finnish elm populations. However, because of climate change, DED is expected to arrive in Finland, and is indeed already found, both in neighbouring Sweden and very close to the border with Russia [10]. As a way to use elms for urban landscaping, more DED-resistant elms have been bred using native material, or by crossing with more resistant elm species [5]. Asian elms are generally considered more resistant, but there is still high variation in how well the crossings perform [1]. Hybrids can also be susceptible to local pathogens [12], and the use of material of local origin is preferable because of the danger genetic pollution poses to natural populations [3]. In any case, elm genotypes adapted to northern conditions are needed for resistance breeding if such efforts are to take place in Finland. Because of global warming, elms may also become more attractive trees for landscaping in Finland. As part of the national conservation programme [13], cryopreservation and regeneration methods are being developed for U. laevis and U. glabra. The aim of genetic conservation is to ensure the survival of the elm species adapted to northern conditions. The advantages of cryopreservation are minimal labour requirements for collection upkeep and small space requirements [14]. The material is also protected from pathogens and pests. On the other hand, suitable facilities and staff with special skills are needed for plant regeneration. Cryopreservation provides a more genetically stable method of long-term ex situ conservation than continuous in vitro culture, which is prone to somaclonal variation [15]. Previous efforts to set up cryostorage gene banks for local elm species in Europe [16,17] provide a good starting point to develop the methods suitable for northern genotypes. In North America, efforts to store and propagate old Ulmus americana L. trees that have survived DED epidemics have taken place [18]. Cryopreservation of dormant winter buds is a space-effective and relatively easy way to conserve large amounts of genotypes. However, the methods must be tested and validated for the specific plant material used. The prerequisites for the cryopreservation of dormant buds by controlled rate cooling are cold-hardy plant material and access to a programmable freezer that can provide an adjustable steady decrease in temperature [19]. Controlled rate cooling aims to avoid lethal cryodamage caused by intracellular ice formation. As the sample is gradually cooled, extracellular ice forms first and draws water from the cells by osmotic pressure, thus preventing ice nucleation within the cells [20]. Precooled samples are then plunged into liquid nitrogen, in contact with which the cytoplasm of the cells vitrifies without ice crystal formation. The rewarming of samples is usually done rapidly to avoid crystal formation [21]. After thawing, a suitable micropropagation method is essential to regenerate the explants into ex vitro plants and for cryopreservation to be useful for genetic conservation. The aim of this study was to develop and test cryopreservation and micropropagation methods for U. glabra and U. laevis to function as a backup method for living genetic resource collections.

2. Materials and Methods 2.1. Plant Material A total of 36 U. laevis and 13 U. glabra genotypes from three living genetic reserve collections and from the Punkaharju research forest were used in the experiments, as described in detail in the Supplementary Tables S1–S3. For the initiation of the media experiment, fresh U. laevis buds from five trees (E11630-E11634) from the Punkaharju research forest were collected at the beginning of September 2016. The branches from which fresh control buds were taken were kept in water in a cold room (+2 ◦C) until preparation. Cryopreserved buds were frozen in October 2016 and prepared starting in November 2016. For the first cryopreservation experiment, U. laevis buds from 20 trees from living gene reserve collections 205 and 232 (Paimio/Preitilä) were collected in January 2017, frozen in February 2017, and the experiment was initiated in February 2017. Reduced light intensity and Driver and Kuniyuki walnut (DKW) media were first tested with buds Forests 2021, 12, 1121 3 of 13

from the same five trees from Punkaharju (E11630-E11634) as previously. The branches were collected in November 2017 and cryopreserved in December 2017. Cultures from fresh buds were initiated in November, and from cryopreserved buds in December. Shoots from these cultures were used in later rooting experiments. Buds were collected from the following collections for the 2020 experiments: Preitilä 205 and 232 (U. laevis, ten trees), Preitilä 184 (U. glabra, five trees), and Solböle 192 (U. glabra, five trees). U. laevis buds were collected in in February 2019 (six trees, cryo in March), or January 2017 (four trees, cryo in February), and U. glabra buds were collected in March 2019 (cryo in March). For more details see Supplementary Table S2. Fresh controls of both species were collected in January 2020, and the experiment started in January 2020. Material from these experiments and from various surface sterilisation tests (not presented in this article) were also used in later cytokinin and rooting experiments. For the 2021 experiments, cryopreserved buds from five trees in U. laevis collections 205 and 232 and seven trees in U. glabra collection 184 were used, which were collected and cryopreserved at the end of January 2021. The buds were thawed, starting 13 days after cryopreservation. Twigs with dormant buds from both species were transported and stored, both with and without snow in the bags (for details on plant material see Supplementary Table S3). Shoots from these cultures were used for the 2021 rooting experiments.

2.2. Cryopreservation For cryopreservation, elm branches or twigs with dormant buds were collected at varying times in different years between October and March (see Supplementary Tables) when the trees had been exposed to sub-zero temperatures. Twigs of 5 to 20 cm were placed in sealed plastic bags with snow or moistened tissue paper and transported with cold blocks in the container to sustain sub-zero temperature and dormancy. The branches collected from the nearby research forest in Punkaharju were up to 1 m long and placed in a bucket containing snow. All the branches were stored until preparation for cryopreservation in a cold room (–5 ◦C). The buds were cut with a small piece of the branch into 1.8 ml cryotubes (Sarstedt), and the tubes kept on ice overnight in a cold room (+2 ◦C). The buds were frozen the next day using a programmable liquid nitrogen freezer (Planer Kryo 10) 0.17 ◦C min−1 until they reached –38 ◦C[22], when they were submerged directly in liquid nitrogen. Thawing was carried out in a water bath (+38 ◦C, 2 min). Immediately after this, the tubes were placed on ice for at least 2 min.

2.3. Surface Sterilisation In the ﬁrst initiation and cryopreservation experiments, the buds were surface sterilised with 70% ethanol under agitation for 10 min. For the DKW experiment, the buds were kept in 10% H2O2 for 3 h before exposed to ethanol for 10 min. For the subsequent experiments, H2O2 was omitted, but disinfection time in ethanol was increased to 20 min. In 2021, sterilisation with H2O2 under agitation for 10 min was tested with four U. glabra and four U. laevis genotypes (for details on plant material see Supplementary Table S3) in the same way as for Norway spruce buds for explants [23]. The buds were kept on ﬁlter paper moistened with 100 mg/L polyvinylpyrrolidone after sterilisation until they were prepared for the initiation medium.

2.4. Initiation Both fresh and thawed buds (Figure1A) were prepared for culture under a stereo microscope by excising the outer bud scales and shaping the stem into a small wedge. The buds were placed into sterile De Wit polycarbonate tubes (130 × 27 mm, Duchefa Biochemie) containing 7 mL of initiation media. Modified Murashige and Skoog (MS) and modified woody plant medium (WPM) basal media (AppendixA), solidified with 6 g/L Plantagar S1000 (B&W, Parma, Italy) and with different hormone compositions were tested for U. laevis initiation: 0.1 mg/L gibberellic acid 4 + 7 (GA 4 + 7) (Ducheva Biochemie) and 0.5 mg/L 6-benzylaminopurine (BA) (Sigma Aldrich), 0.5 mg/L BA, 1 mg/L BA, Forests 2021, 12, x FOR PEER REVIEW 4 of 13

Forests 2021, 12, 1121 modified woody plant medium (WPM) basal media (Appendix A), solidified with4 of 6 13g/l Plantagar S1000 (B&W, Parma, Italy) and with different hormone compositions were tested for U. laevis initiation: 0.1 mg/l gibberellic acid 4 + 7 (GA 4 + 7) (Ducheva Biochemie) and 0.5 mg/l 6-benzylaminopurine (BA) (Sigma Aldrich), 0.5 mg/l BA, 1 mg/l BA, or 0.02 ormg/l 0.02 thidiazuron mg/L thidiazuron (TDZ) (TDZ)(Sigma (Sigma Aldrich) Aldrich) [24,25]. [24 In,25 later]. In experiments, later experiments, initiation initiation media mediawith DKW with DKWsalts (5.2 salts g pouches (5.2 g pouches for 1 l of for medium, 1 L of medium, Sigma Aldrich, Sigma Aldrich,appendix Appendix A) with 30A )g/l withsucrose 30 g/L and sucrose solidified and with solidiﬁed 6 g/l withPlantagar 6 g/L S1 Plantagar000 and S10000.1 mg/l and GA 0.1 4 mg/L + 7 and GA 0.5 4 +mg/l 7 and BA 0.5as mg/Lgrowth BA regulators as growth were regulators used for were both used U. forglabra both andU. U. glabra laevisand [26–28].U. laevis All[ 26media–28]. were All mediasupplemented were supplemented with varying with concentrations varying concentrations (see Appendix (see Appendix A) of myo-inositol,A) of myo-inositol, glycine, glycine,nicotinic nicotinic acid, pyridoxine-HCl, acid, pyridoxine-HCl, and thiamine-HCl. and thiamine-HCl. All media All were media adjusted were to adjusted a pH of to 5.8 ◦ aprior pH of to 5.8 autoclaving prior to autoclaving for 20 min at for 121 20 °C. min GA at 1214 + 7,C. TDZ, GA and 4 + meta-topolin 7, TDZ, and meta-topolin(Ducheva Bio- (Duchevachemie) (mT) Biochemie) were added (mT) werethrough added filter through sterilisation ﬁlter sterilisationafter autoclaving. after autoclaving.

Figure 1. A) Comparison of U. laevis (left) and U. glabra (right) unprepared buds. The scale bar Figure 1. (A) Comparison of U. laevis (left) and U. glabra (right) unprepared buds. The scale bar represents 0.1 cm. B) Multiplication result of U. glabra genotype 0107 01 by cutting the apical part representsof the shoot 0.1 and cm. transferring (B) Multiplication the callus result and of basalU. glabra part genotypeof the shoot 0107 onto 01 new by cutting media. the The apical scale partbar ofrepresents the shoot 1 and cm. transferringC) U. laevis genotype the callus 0357 and 06 basal shoot part grown of the from shoot an in onto vitro new bud media. excisedThe for sub- scale barculture. represents The scale 1 cm. ba (rC represents) U. laevis genotype1 cm. D) U. 0357 laevis 06 and shoot U. grownglabra shoots from an growingin vitro inbud peat excised 41 d after for subculture.transplantation. The scale The barscale represents bar represents 1 cm. 5 ( Dcm.) U. laevis and U. glabra shoots growing in peat 41 d after transplantation. The scale bar represents 5 cm. After initiation, the buds were grown in the culture room at around +25 °C under ◦ PhilipsAfter Master initiation, TL-D the 36W/840 buds were 5G fluorescent grown in thelights culture (Poland) room for at a around 16/8 light-dark +25 C under photo- Philipsperiod Master with light TL-D intensit 36W/840y of 5Gapproximately fluorescent lights150 µmol (Poland) m-2 s-1 for, with a 16/8 the light-darkfirst 2 to 3 pho- days µ −2 −1 toperiodcovered with with light veils intensity to reduce of approximatelyinitial light exposure. 150 mol In mthe laters , withexperiments the first 2(from to 3 daysDKW coveredexperiments with veilsin 2017 to reduceand onwards), initial light the veils exposure. were always In the laterkept experimentson, keeping light (from intensity DKW experiments in 2017 and onwards), the veils were always kept on, keeping light intensity under 50 µmol m-2 s-1. The outcome of the initiations was classified as shoot grown, dead, under 50 µmol m−2 s−1. The outcome of the initiations was classified as shoot grown, dead, or contaminated. If a shoot grew, but the culture was contaminated, it was not counted as or contaminated. If a shoot grew, but the culture was contaminated, it was not counted as a successful initiation. a successful initiation.

2.5.2.5. Multiplication Multiplication AfterAfter the the shoots shoots started started to to grow grow (1 (1 to to 2 2 weeks), weeks), they they were were moved moved into into Magenta Magenta glass glass jarsjars with with approximately approximately 25–30 25–30 mL ml of of multiplication multiplication media, media, which which was was the samethe same as initiation as initia- media,tion media, except except without without GA 4 + GA 7. Multiplication 4 + 7. Multiplication was carried was outcarried to produce out to produce enough shootsenough for rooting experiments. Subculture timing was determined based on the growth and visual appearance of the cultures, usually every three to four weeks. The subculture was mainly made by cutting off the apical part of the shoot and transferring both it and the

basal part of the shoot with some callus onto new media (Figure1B). With some genotypes, Forests 2021, 12, x FOR PEER REVIEW 5 of 13

Forests 2021, 12, 1121 shoots for rooting experiments. Subculture timing was determined based on the growth5 of 13 and visual appearance of the cultures, usually every three to four weeks. The subculture was mainly made by cutting off the apical part of the shoot and transferring both it and the basal part of the shoot with some callus onto new media (Figure 1B). With some gen- multiplicationotypes, multiplication of nodal segments of nodal ofsegments varying of length varying was length also possible was also (Figure possible1C). (Figure Until the 1C). 2020Until experiments, the 2020 experiments, the subculture the subculture was mainly was made mainly by cutting made by the cutting shoot into the shoot two or into more two segmentsor more segments and transferring and transferring them without them callus withou tissuet callus onto tissue fresh onto media. fresh The media. effect The of effect mT wasof testedmT was as tested an alternative as an alternative cytokinin cytokinin at 0.5 mg/L. at 0.5 The mg/l. tests wereThe tests carried were out carried with material out with U. glabra initiatedmaterial and initiated multiplied and multiplied with 0.5 mg/L with BA 0.5 as mg/l cytokinin, BA as cytokinin, using four using genotypes four ofgenotypes .of For the cytokinin experiment, material with an equal number of shoots, nodal segments, or U. glabra. For the cytokinin experiment, material with an equal number of shoots, nodal shoots with callus (Figure2) was transferred into paired jars with either mT or BA as plant segments, or shoots with callus (Figure 2) was transferred into paired jars with either mT growth regulator (PGR). The shoot production was then assessed after 21 d by counting or BA as plant growth regulator (PGR). The shoot production was then assessed after 21 the number of shoots growing in each jar. d by counting the number of shoots growing in each jar.

Figure 2. An example of a jar pair in the cytokinin experiment: U. glabra genotype 0109 04 on 0.5 Figure 2. An example of a jar pair in the cytokinin experiment: U. glabra genotype 0109 04 on mg/l mT (jar on the left) and on 0.5 mg/l BA (jar on the right) (A) 1 d, (B) 7 d, (C) 14 d and (D) 21 d 0.5 mg/L mT (jar on the left) and on 0.5 mg/L BA (jar on the right) (A) 1 d, (B) 7 d, (C) 14 d and after subculture, with an equal number of shoots from previous media with 0.5 mg/l BA. The scale (Dbar) 21 represents d after subculture, 2 cm. with an equal number of shoots from previous media with 0.5 mg/L BA. The scale bar represents 2 cm.

2.6.2.6. Rooting Rooting RootingRootingin in vitro vitro,, in in semi-solidsemi-solid media,media, was was tried tried out out with with Punkaharju Punkaharju originated originated ma- materialterial grown grown on on DKW DKW media. media. Rooting Rooting was was done done in half-strength in half-strength DKW DKW media, media, either either with with0.5 0.5mg/l mg/L indole-3-butyric indole-3-butyric (IBA), (IBA), no PGRs, no PGRs, or with or with3 d induction 3 d induction with with3 mg/l 3 mg/LIBA (Sigma IBA (SigmaAldrich), Aldrich), after which after which the shoots the shoots were were transfe transferredrred onto onto hormone-free hormone-free media. media. Liquid Liquid root rootinduction induction was was used used for both for bothU. glabraU. glabraand U.and laevisU. with laevis 2020with initiated 2020 initiated material materialaccording accordingto Micheli to et Micheli al. [29]. et Induction al. [29]. Induction was performed was performed in a 5 mg/l in IBA a 5 solution mg/L IBA with solution 15 g/l sucrose with 15and g/L wassucrose tested and for was six testedgenotypes for six of genotypes U. laevis and of U. six laevis genotypesand six genotypesof U. glabra of. TheU. glabra shoots. Thewithout shoots callus without tissue callus were tissue cut wereand put cut into and rooting put into liquid rooting and liquid kept and in the kept dark in the for dark 2 to 3 fordays. 2 to After 3 days. the Afterinduction the inductionperiod, the period, shoots thewere shoots directly were transplanted directly transplanted into growing into con- growingtainers containers(Pl 81f) filled (Pl with 81f) ﬁlledsemi-coarse with semi-coarse pre-fertilised pre-fertilised light sphagnum light sphagnum peat in a greenhouse peat in a greenhouseon 2 July in on 2020 2 July and in 20 2020 May and in 2021. 20 May The in large 2021. greenhouse The large greenhousehad been sown had with been Norway sown withspruce Norway container spruce seedlings, container and seedlings, the greenhouse and the greenhouseconditions were conditions maintained were maintainedin conditions infavourable conditions for favourable Norway for spruce Norway germination spruce germination [30,31]. Relative [30,31]. humidity Relative humiditywas kept was high kept(above high 80%), (above and 80%), the temperature and the temperature was above was+20 °C above for three +20 ◦ weeks,C for three after weeks,which the after hu- whichmidity the was humidity gradually was decreased gradually by decreased increasing by increasingventilation. ventilation. The survival The of survival the shoots of the was shootsevaluated was evaluated41 days (2020) 41 days (Figure (2020) 1D) (Figure or 21 1daD)ys or (2021) 21 days after (2021) transplantation. after transplantation. In the 2021 Inexperiment, the 2021 experiment, the largest the leaves largest of leavesthe cuttings of the were cuttings trimmed were trimmedto a smaller to a size smaller to reduce size toevaporation. reduce evaporation. The survival The survival of the shoots of the was shoots evaluated was evaluated on site onin site2020 in and 2020 from and fromphoto- photographsgraphs in 2021. in 2021. For the For rooting the rooting of shoots of shoots from fromthe multiplication the multiplication experiment, experiment, the shoots the shootswere wererooted rooted and transplanted and transplanted in the in same the sameway as way for as other for otherexperiments experiments but were but weregrown grownin the in culture the culture room room at 25 at°C 25 in◦ ventilatedC in ventilated mini minigreenhouses greenhouses and irrigated and irrigated manually. manually.

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Forests 2021, 12, 1121 6 of 13 2.7. Statistical Analysis Statistical analyses were conducted using SPSS Version 25 software. The differences in shoot production in the cytokinin experiment were investigated with independent sam- 2.7. Statistical Analysis ples using the Mann-Whitney U-test, because the data were not normally distributed. The differencesStatistical in shoot analyses formation were conducted and rooting using success SPSS Versionwere analysed 25 software. with Thea Chi-square differences test in inshoot all experiments. production in If the any cytokinin cells in experimentthe analysis were had investigatedan expectedwith value independent < 5, Fischer’s samples exact testusing was the used Mann-Whitney instead. A p- U-test,value < because0.05 was the considered data were significant. not normally The distributed.effect of location The insidedifferences the containers in shoot formation was ruled and out rooting with successlogistic wereregression analysed (not with shown) a Chi-square with row test and in columnall experiments. covariates, If anywhich cells explained in the analysis only 0.4% had of an correctly expected predicted value <5, cases Fischer’s after exact the spe- test cieswas variable. used instead. A p-value < 0.05 was considered significant. The effect of location inside the containers was ruled out with logistic regression (not shown) with row and column 3.covariates, Results which explained only 0.4% of correctly predicted cases after the species variable. 3.1.3. Results Cryopreservation and Initiation 3.1. CryopreservationThe different basal and Initiationinitiation media and hormone compositions were tried out for shootThe culture different initiation basal with initiation five genotypes media and collected hormone from compositions Punkaharju were (Figure tried 3). out Both for cryopreservedshoot culture initiationand fresh withbuds five were genotypes cultured wi collectedth GA 4 from + 7; other Punkaharju media were (Figure tested3). Both only forcryopreserved fresh buds. andNo significant fresh buds differences were cultured were with found GA 4on + MS 7; other with media 0.1 mg/l were GA tested 4 + 7 onlyand 0.5for mg/l fresh BA buds. between No significant cryopreserved differences and fresh were buds, found but on on MS WPM with with 0.1 mg/Lthe same GA PGRs, 4 + 7 andthe difference0.5 mg/L BAwas between significant cryopreserved (p < 0.001). and When fresh all buds, the initiations but on WPM were with compared the same regardless PGRs, the ofdifference the PGRs, was MS significant had a 45% (p success< 0.001). compared When all to the 28% initiations with WPM were (χ compared2 (1) = 16.947; regardless p < 0.001). of the PGRs, MS had a 45% success compared to 28% with WPM (χ2 (1) = 16.947; p < 0.001).

100 70 50 80 70

60 50 40 50 50 20 50 48 53 49 0 1 mg/lBA 1 mg/lBA % of buds producing producing shoots buds of % 0.5 mg/l BA 0.5 mg/l BA 0.02 mg/l TDZ 0.02 mg/l TDZ 0.1 mg/l GA4/7, 0.5 mg/l BA 0.1 mg/l GA4/7, 0.5 mg/l BA 0.1 mg/l GA4/7, 0.5 mg/l BA 0.1 mg/l GA4/7, 0.5 mg/l BA

cryo fresh cryo fresh

MS WPM

Figure 3. The effect of initiation media base and PGR concentrations, and cryopreservation on the Figure 3. The effect of initiation media base and PGR concentrations, and cryopreservation on the shoot regeneration of five genotypes of U. laevis collected from the Punkaharju research park. The shoot regeneration of five genotypes of U. laevis collected from the Punkaharju research park. The total number of fresh (8–12 per genotype) or cryopreserved (14 per genotype) buds prepared for each total number of fresh (8–12 per genotype) or cryopreserved (14 per genotype) buds prepared for eachmedia media is shown is shown on the on top the of top the of bar. the bar. Based on the results, MS with 0.1 mg/L GA 4 + 7 and 0.5 mg/L BA was selected for Based on the results, MS with 0.1 mg/l GA 4 + 7 and 0.5 mg/l BA was selected for further experiments, and further verified with 20 genotypes from Preitilä collections 205 further experiments, and further verified with 20 genotypes from Preitilä collections 205 and 232. A slightly higher proportion of prepared cryopreserved U. laevis buds produced a and 232. A slightly higher proportion of prepared cryopreserved U. laevis buds produced shoot (64%, n = 222, 10–12 per genotype) than fresh (57%, n = 200, 10 per genotype) U. laevis a shoot (64%, n = 222, 10–12 per genotype) than fresh (57%, n = 200, 10 per genotype) U. buds, but the difference was not significant (χ2 (1) = 2.138; p = 0.144). However, despite 2 laevisgood initiationbuds, but frequencies, the difference the was shoots not tended significant to die ( afterχ (1) moving = 2.138; onto p = 0.144). multiplication However, media. de- spiteDKW-based good initiation initiation frequencies, and multiplication the shoots media tended were to die therefore after moving tried out onto as multiplication an alternative for buds collected from five trees from Punkaharju. With reduced light intensity (from 150 to less than 50 µmol m−2 s−1), this resulted in an improved shoot formation with 82% (n = 28) success for fresh and 94% (n = 51) for cryopreserved buds. Two hours of H2O2 treatment

Forests 2021, 12, x FOR PEER REVIEW 7 of 13

media. DKW-based initiation and multiplication media were therefore tried out as an al- Forests 2021, 12, 1121 ternative for buds collected from five trees from Punkaharju. With reduced light intensity7 of 13 (from 150 to less than 50 µmol m-2 s-1), this resulted in an improved shoot formation with 82% (n = 28) success for fresh and 94% (n = 51) for cryopreserved buds. Two hours of H2O2 treatmentwas added was to added the surface to the sterilisation surface sterilisat protocol,ion protocol, because contaminationsbecause contaminations were a problem were a problemwith earlier with sets earlier of buds sets (data of buds not (data shown). not Noshown). significant No significant differences differences were found were in survival found inbetween survival cryopreserved between cryopreserved and fresh buds and fresh (Fischer’s buds exact (Fischer’s test; p exact= 0.099). test; Cryopreservationp = 0.099). Cryo- preservationexperiments experiments were conducted were again conducted in 2020 withagain a in larger 2020 set with of genotypes, a larger set and of withgenotypes, both U. andglabra withand bothU. laevisU. glabra(Figure and4A). U. Cryopreservedlaevis (Figure 4A).U. glabraCryopreservedbuds showed U. glabra poorer buds regeneration showed poorerthan fresh regeneration buds (χ2 (1)than = 22.806;fresh budsp < 0.001)(χ2 (1) or = cryopreserved22.806; p < 0.001)U. or laevis cryopreservedbuds (χ2 (1) U. = 51.598;laevis budsp < 0.001).(χ2 (1) No= 51.598; significant p < 0.001). differences No significant were found differen betweences were cryopreserved found between and fresh cryo-U. preservedlaevis buds. andU. fresh glabra U. budslaevis buds. had significantly U. glabra buds more had contaminations significantly more than contaminationsU. laevis buds than(χ2 (1)U. laevis = 32.072; budsp (<χ2 0.001).(1) = 32.072; Surface p < 0.001). sterilisation Surface was sterilisation done for was 20 min done in for 70% 20 ethanol.min in 70%Contaminations ethanol. Contaminations were more prevalentwere more than prevalent in the firstthan initiationin the first and initiation cryopreservation and cryo- preservationexperiments. experiments.U. glabra appeared U. glabra to suffer appeared from to cryopreservation suffer from cryopreservation more than U. laevis more. Shoots than U.were laevis also. Shoots lost after were transfer also lost to after a multiplication transfer to a multiplication medium. However, medium. based However, on the resultsbased onfrom the initiatedresults from fresh initiated buds, the fresh same buds, DKW the mediumsame DKW also medium appears also suitable appears for U.suitable glabra for, as U.nine glabra out, ofas tennine genotypes out of ten produced genotypes shoots produced fromfresh shoots buds. from Similar fresh buds. results Similar were obtained results werefor the obtained 2021 experiment for the 2021 with experiment only cryopreserved with only budscryopreserved with U. glabra budshaving with U. a significantlyglabra hav- inglower a significantly initiation success lower initiation rate (χ2 (1) success = 27.842; rate (pχ2< (1) 0.001) = 27.842; and higherp < 0.001) contamination and higher con- rate 2 tamination(χ (1) = 19.910; rate (χp2< (1) 0.001) = 19.910; than pU. < laevis0.001)(Figure than U.4 B).laevis (Figure 4B). 10 10 10 10 10 10 10 10 10 10 10 10 12 10 10 10 10 10 10 10 10 10 100 100 104 12

A) 100 10 10 10 10 10 10 10 9 80 10 10 10 98 10 10 10 60 10 % 40 10

20 9 0 K421 Total Total E3032 E3033 E3034 E3571 0139 02 0328 04 0357 01 0359 08 0360 03 0361 09 0511 03 0514 08 0515 05 0517 07 0107 01 0210 07 0212 09 0213 03 0331 02

U. laevis 2020 U. glabra 2020

contamination fresh shoots cryo shoots

10 10 10 100 9 B) 6 10 50 8 63 80 10 10 10

60 10 10 % 40 20 0 Total Total 0357 06 0359 11 0363 09 0512 08 0516 09 0106 02 0107 01 0109 04 0207 07 0210 07 0212 09 0213 03

U. laevis 2021 U. glabra 2021

contamination cryo shoots

Figure 4. Shoot production and contamination rates (i.e., both growing and non-growing explants Figure 4. Shoot production and contamination rates (i.e., both growing and non-growing explants showing contamination) of U. laevis and U. glabra buds of different genotypes in (A) 2020 and (B) in showing contamination) of U. laevis and U. glabra buds of different genotypes in A) 2020 and B) in 2021. The remaining portion of the 100% represents failed but non-contaminated initiations. In 2020, 2021. The remaining portion of the 100% represents failed but non-contaminated initiations. In 2020,both both fresh fresh and cryopreserved and cryopreserved buds werebuds used;were inus 2021,ed; in only2021, cryopreserved only cryopreserved ones. The ones. number The num- of buds berprepared of buds is prepared shown at is the shown top of at the the bar top for of the each bar genotype for each and genotype for the and total for number the total of number genotypes of in genotypesthat treatment. in that treatment. 3.2. Surface Sterilisation 3.2. Surface Sterilisation The sterilisation time in 70% ethanol was increased from 10 min to 20 min for buds prepared in 2020 and thereafter. Different sterilisation methods with PPM, antibiotics and sodium hypochlorite were tested in preliminary trials, but with not markedly better

and sometimes worse results than the ethanol-sterilised controls (data not shown). The Forests 2021, 12, x FOR PEER REVIEW 8 of 13

The sterilisation time in 70% ethanol was increased from 10 min to 20 min for buds

Forests 2021, 12, 1121 prepared in 2020 and thereafter. Different sterilisation methods with PPM, antibiotics and 8 of 13 sodium hypochlorite were tested in preliminary trials, but with not markedly better and sometimes worse results than the ethanol-sterilised controls (data not shown). The contamination rates from different experiments are not comparable, as different plant material wascontamination used. For the 2021 rates experiments, from different a surf experimentsace sterilisation are not method comparable, that works as differentfor Nor- plant way sprucematerial primordial was used. shoot For explants the 2021 according experiments, to Varis a surface et al. [23] sterilisation with H2O method2 was tested. that works This resultedfor Norway in significantly spruce primordial higher contamination shoot explants rates according than with to controls Varis et sterilised al. [23] withwith H2O2 ethanolwas for tested.both U. This laevis resulted (49% vs in 23%, significantly χ2 (1) = 6.084; higher p = contamination0.014) and U. glabra rates (97% than vs with 82%, controls 2 Fischer’ssterilised exact test; with p ethanol= 0.049). for No both significantU. laevis differences(49% vs 23%, in contaminationχ (1) = 6.084; p rate= 0.014) were andfoundU. glabra between(97% dry vs (no 82%, snow) Fischer’s and wet exact storage test; (with p = 0.049). snow Noor moistened significant tissue differences paper inin contaminationthe plastic bag)rate conditions were found after between collection dry until (no preparation snow) and wet for storagecryopreservation. (with snow or moistened tissue paper in the plastic bag) conditions after collection until preparation for cryopreservation. 3.3. Multiplication 3.3. Multiplication As an alternative to BA, mT, was tested as a cytokinin for U. glabra (Figure 5). Slightly As an alternative to BA, mT, was tested as a cytokinin for U. glabra (Figure5). Slightly more shoots were obtained from mT-media (66 vs 50 shoots in total), but the difference more shoots were obtained from mT-media (66 vs 50 shoots in total), but the difference was was not statistically significant. The rooting of shoots was also tested with liquid IBA in- not statistically significant. The rooting of shoots was also tested with liquid IBA induction, duction, and no differences were found between cytokinins used in the multiplication and no differences were found between cytokinins used in the multiplication stage. stage.

A) 8 B) 1 3 3 15 100 10

7 26 30 6 80 12

5 11 60 4 3 40 2 20 1 1

Number of shoots of Number 0 0 total 0331 02 0107 01 0210 07 0109 04 0331 02 0107 01 0210 07 0109 04 % of shoots and alive rooted % of shoots

mT BA

Figure 5. The effect of cytokinin type on multiplication and rooting success of four genotypes of Figure U.5. The glabra effect.(A) of Shoot cytokinin production type on from multiplication each jar-pair and with rooting a comparison success of with four similar genotypes starting of U. material. glabra. A(B)) Shoot Rooting production success of from shoots each from jar-pair both media, with a with comparison the number with of similar shoots forstarting rooting material. and transplanted B) Rooting success of shoots from both media, with the number of shoots for rooting and trans- shown on the top of each bar. planted shown on the top of each bar. During the shoot multiplication phase, the growth habit and multiplication rate of Duringdifferent the genotypes shoot multiplication varied substantially. phase, the Some growth genotypes, habit and especially multiplication in U. glabra rate, produced of differentexcessive genotypes amounts varied of substantially. callus with nodal Some segments,genotypes, and especially the subculture in U. glabra with, pro- them was duced unsuccessful.excessive amounts Sometimes of callus transferring with noda a wholel segments, shoot, and especially the subculture without awith callus, them resulted was unsuccessful.in withering Sometimes of the shoots. transferring However, a thiswhole was shoot, not always especially the case.without For a some callus, genotypes, resulted subculturesin withering were of the successful shoots. However, even with athis very was limited not always amount the of case. starting For material some geno- (Figure2 ). types, subcultures were successful even with a very limited amount of starting material (Figure3.4. 2). Rooting and Transplantation From the first rooting experiments with U. laevis on solidified half-strength DKW 3.4. Rootingmedium, and Transplantation the best results were obtained from 3 mg/L induction treatment, with 67% From(n = 30the) offirst the rooting shoots aliveexperiments and with with roots U. visible laevis in on the solidified medium twohalf-strength weeks after DKW induction. medium,With the constant best results 0.1 mg/Lwere obtained IBA rooted from and 3 mg/l alive induction were 45% treatment, (n = 31) with and with67% (n hormone- = 30) of freethe shoots media alive 7% (n and = 29). with Rooting roots visibl wase tested in the for medium a larger two set weeks of shoots after grown induction. from buds With constantprepared 0.1 in mg/l 2020 IBA and 2021,rooted for and both aliveU. were laevis 45%and (nU. = glabra 31) and. First, with a small-scalehormone-free test was mediadone 7% (n with = 29). both Rooting liquid was and tested agar for induction, a larger set with of shoots liquid mediagrown givingfrom buds better prepared results (data in 2020not and shown). 2021, for Therefore, both U. liquidlaevis inductionand U. glabra was. First, tested a in small-scale July 2020 ontest a largerwas done scale, with resulting both liquidin 18% and of agarU. laevis induction,shoots with and liquid 64% ofmediaU. glabra givingshoots better being results rooted (data andnot shown). alive 41 days after transplantation (Figures6A and1D ). Rooting was significantly more successful for U. glabra than for U. laevis (χ2 (1) = 47.645; p < 0.001). Similar results were obtained U. laevis from the May 2021 experiment, with 20% of shoots surviving (Figure6B). However, for U. glabra, the results were markedly worse than in 2020, with only 9% survival. Forests 2021, 12, x FOR PEER REVIEW 9 of 13

Therefore, liquid induction was tested in July 2020 on a larger scale, resulting in 18% of U. laevis shoots and 64% of U. glabra shoots being rooted and alive 41 days after transplantation (Figure 6A and 1D). Rooting was significantly more successful for U. glabra than for Forests 2021, 12, 1121 U. laevis (χ2 (1) = 47.645; p < 0.001). Similar results were obtained U. laevis from the May 9 of 13 2021 experiment, with 20% of shoots surviving (Figure 6B). However, for U. glabra, the results were markedly worse than in 2020, with only 9% survival. A) B) 100 100 17 5 80 74 80 31 7 60 12 2 60 38 40 40 97 9 164 12 148 16 83 16 7 20 15 12 20 15 34 15 11 0 0 % of shoots 21 d alive at % of shoots % of shoots 41 d alive at % of shoots total total total total K421 E3032 E3571 E3033 E3034 0357 06 0357 08 0512 09 0516 11 0359 01 0107 04 0109 07 0207 E11633 E11630 0357 01 0357 02 0360 08 0359 02 0328 0210 07 0210

U. laevis U. glabra U. laevis U. glabra

Figure Figure6. A) Rooting 6. (A) Rootingsuccess of success micro-propagated of micro-propagated elm shoots elm following shoots followingroot induction root inductionin liquid 41 in liquid d from 41transplantation d from transplantation in July 2020. in B July) Rooting 2020. (successB) Rooting of micro-prop success ofagated micro-propagated shoots following shoots root following inductionroot in induction liquid 21 d in after liquid transplantation 21 d after transplantation in May 2021. inThe May number 2021. at The the numbertop of the at bar the repre- top of the bar sents therepresents total number the total of shoots number transpla of shootsnted transplanted from each genotype from each or genotypespecies. or species.

4. Discussion4. Discussion The establishmentThe establishment of in vitro of in cultures vitro cultures and re andgeneration regeneration of explants of explants into intoplants plants capa- capable ble of survivingof surviving ex exvitro vitro from from cryopreserved cryopreserved material material is is a amulti-stage multi-stage process process with with many many steps steps requiringrequiring optimisation. optimisation. However, However, many many protocols protocols for for micropropagation and and cryo- cryopreser- preservationvation of plant material have been developed and function as an excellent starting point. point. MultipleMultiple protocolsprotocols forfor elmselms cancan alsoalso bebe foundfound [ 17[17,28,32].,28,32]. However,However, the the protocols protocols can be can beimproved improved upon upon and and must must always always be be tested tested for for the the specific specific plant plant materials materials in in question. ques- For tion. Forcryopreservation cryopreservation to serveto serve as as a backup a backup for for the the collections collections of of living living trees, trees, the the methods meth- need U. laevis ods needto beto be validated validated and and proven proven reliable. reliable. It It was was possible possible toto regenerateregenerate U. laevis equallyequally from both fresh and cryopreserved dormant buds. This is consistent with results from other from both fresh and cryopreserved dormant buds. This is consistent with results from deciduous trees, e.g., Betula pendula Roth [22] and hybrid aspen Populus tremula L.× Populus other deciduous trees, e.g., Betula pendula Roth [22] and hybrid aspen Populus tremula L.× tremuloides Michx. [33]. The first results indicated slightly better regeneration with cryopre- Populus tremuloides Michx. [33]. The first results indicated slightly better regeneration with served than with fresh buds, which is in agreement with the results of Harvengt et al. [17]. cryopreserved than with fresh buds, which is in agreement with the results of Harvengt However, cryopreservation can decrease viability, for example, with Malus species [34,35]. et al. [17]. However, cryopreservation can decrease viability, for example, with Malus spe- This was the case with U. glabra, as in vitro cultures from cryopreserved buds were harder cies [34,35]. This was the case with U. glabra, as in vitro cultures from cryopreserved buds to establish than from fresh buds. According to Harvengt et al., micrografting can be used were harder to establish than from fresh buds. According to Harvengt et al., micrografting to successfully regenerate cryopreserved U. glabra buds but is unnecessary for U. laevis and can be used to successfully regenerate cryopreserved U. glabra buds but is unnecessary Ulmus minor Mill. Compared to U. laevis, U. glabra buds were larger (see Figure1A), which for U. laevis and Ulmus minor Mill. Compared to U. laevis, U. glabra buds were larger (see could lead to a more uneven temperature change within the tissues and subsequent tissue Figuredamage. 1A), which With couldPrunus lead persica to a more(L.), unev tissueen cracking temperature of the change connecting within tissue the tissues between and the bud subsequentand the tissue stem damage. has been With reported Prunus for persica cryopreserved (L.), tissue material, cracking resulting of the connecting in their separation tissue betweenand bud the falling bud and off the [36 ].stem A similar has been phenomenon reported for was cryopreserved occasionally material, observed resulting with U. glabra. in theirThis separation could possibly and bud be falling ameliorated off [36] by. A a similar slower coolingphenomenon rate or was desiccation occasionally to around ob- 25% servedto with 30% U. water glabra content. This could prior possibly to freezing, be ameliorated as is often done by a withslower dormant cooling bud rate cryopreserva- or des- iccationtion to [around19,36]. Adding25% to GA30% towater initiation content media prior improved to freezing, the initialas is often shoot done formation with of U. dormantlaevis bud. However,cryopreservation GA can [19,36]. also have Adding negative GA effectsto initiation on tissue media cultures improved and the was ini- omitted tial shootafter formation the first subculture of U. laevis [.27 However,]. Based on GA these can resultsalso have with negative Punkaharju effectsU. on laevis tissuematerial, culturesGA and 4 + was 7 was omitted used forafter all the the first subsequent subculture initiations. [27]. Based GA on 3 has these been results used with for U. Pun- americana kaharjuproliferation U. laevis material, [18], and GA GA 4 + 47 +was 7 substantially used for all the improved subsequentU. laevis initiations.initiation GA frequencies.3 has been usedWhen for the U. pooramericana survival proliferation after successful [18], and shoot GA induction 4 + 7 substantially on multiplication improved medium U. laevis in 2017 experiments was observed, several actions to improve the protocol were taken. First, the basal media was switched from MS to DKW following the recommendation of Fenning et al. [28]. Successful micropropagation of both U. laevis and U. glabra on MS has been reported [17,32]. However, DKW basal medium has been reported to suit U. americana [18] and Ulmus procera Salisb., which does not establish on MS [28]. Second, the light intensity used (more than 150 µmol m−2 s−1) was found to be excessive compared with published elm protocols and was reduced to less than 50 µmol m−2 s−1 found suitable for several elm Forests 2021, 12, 1121 10 of 13

species [17,28,37]. As both adjustments were made simultaneously, it is not certain if the better shoot survival in later experiments was due to one of the factors or their interaction. However, based on our results, DKW basal medium suits both U. glabra and U. laevis. High contamination rates were the biggest challenge, especially with U. glabra. Contamination reduces regeneration reliability from cryostorage and currently presents a major obstacle for the successful application of cryopreservation in U. glabra genetic resources conservation. Compared with U. laevis buds, U. glabra have larger buds and hairier bud scales. This could make it more difficult for sterilising agents to achieve the potential contamination sources. Multiple surface sterilisation methods and anti-microbial agents were tested for different sets of buds, but without better results than with controls sterilised with 70% ethanol (data not shown). The sterilisation method used initially was very light, with only a 10-min exposure to ethanol, and close to no contaminations were encountered in the first experiments. This was based on working sterilisation methods for aspen and birch [22,33], for which even lighter methods are used successfully. However, generally more extreme, and often chlorite-based methods are reported for elm species due to contaminations often presenting challenges [17,28,38]. Multiple methods were unsuccessfully tested on a small scale, with controls sometimes outperforming treatments. Antibiotics were also tested, with a decrease in bacterial but an increase in fungal contaminations (data not shown). U. laevis cultures were generally easier to multiply than U. glabra, although there were differences between genotypes. The subsequent rooting experiments therefore had highly varying numbers of shoots for testing from different genotypes, and almost twice the number of U. laevis shoots were obtained than from U. glabra. Alternative cytokinin, mT was tested for U. glabra cultures to improve shoot growth. MT has been shown to improve U. glabra shoot multiplication [37] and reduce physiological disorders like shoot tip necrosis [38]. No differences were found between the two cytokinins in this study. However, the culture time on mT media was only 21 d, and BA residues could be present in the plants. To obtain a more comprehensive view, the two cytokinins should be compared from the outset. MT is reported to cause less rooting inhibition than BA [39], but no differences were found between rooting from either culture. IBA or 1-naphthaleneacetic acid is used in elm rooting in most protocols [17,28], either as a continuous low concentration or as higher induction concentration, after which the shoots are transferred to hormone-free media. Auxin was needed for U. laevis rooting, as only 7% of shoots on hormone-free media survived rooting and produced roots, in contrast with 67% on the IBA induction media. However, some of the shoots with in vitro grown roots were lost after transplanting, resulting in greenhouse plant loss. Rooting in peat directly after liquid induction was more successful with U. glabra than with U. laevis in 2020, but not in 2021. Direct transplanting may be more sensitive to edaphic and ambient conditions and may require more advanced climate control than present in the large commercial greenhouse where the tests were conducted. However, the rooting of U. glabra for the multiplication experiment gave better results, suggesting advantages in using mini greenhouses. Rooting of the shoots directly after induction was done to eliminate damage caused to fragile in vitro roots when they were removed from agar. This also reduced labour because no solidified media was needed for rooting. Liquid induction still needs to be tested for U. laevis in mini greenhouses to see if this results in better survival. Alternatively, rooting induction should be further tested on semi-solid media. If either rooting success or multiplication rate are poor, they can be compensated with longer multiplication with more subcultures and with multiple rounds of rooting. Therefore, the initiation and establishment of vigorous and contamination-free in vitro cultures constitute the main limiting step for successful regeneration. Based on our results with the described method, approximately 40% of U. glabra and 80% of U. laevis genotypes could be regenerated. Although there are genotypical differences, few genotypes appear completely recalcitrant to regeneration. However, with the high contamination rates of U. glabra, this is difficult to estimate. Forests 2021, 12, 1121 11 of 13

5. Conclusions The aim of this study was to create reliable backup storage for living elm genetic reserve collections in the face of the potential DED threat in Finland. The micropropagation protocol works for both U. laevis and U. glabra, and the regeneration of most U. laevis genotypes from cryostorage is successful. Rooting without any agar media works for U. glabra but needs to be further tested for U. laevis. This can majorly reduce labour for micropropagation especially with potentially multiple rounds of rooting. However, contaminations in bud explants need to be reduced, especially in U. glabra, and the cryo-protocol then re-evaluated for U. glabra to improve the initiation frequency. After these improvements, the method can be applied to the conservation of elm genetic resources, and knowledge of regeneration success can be used to design the cryoconservation plan and optimise the sampling. Considering the approach of DED, this is an important complementary option for conservation of elm genotypes adapted to northern latitudes.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/f12081121/s1, Table S1: Information on the plant material (bud explants per treatment) used in 2017 experiments. Table S2: Information on the plant material (bud explants per treatment) used in 2017 experiments. Table S3: Information on the plant material used in 2021 experiments. Author Contributions: Conceptualisation, M.R. and T.A.; methodology, S.V., D.P., M.T., and T.A.; formal analysis, S.V.; investigation, S.V. and D.P.; resources, M.R. and T.A.; data curation, S.V.; writing—original draft preparation, S.V.; writing—review and editing, S.V., M.R., D.P., M.T., and T.A.; visualisation, S.V.; supervision, T.A.; project administration, M.R. and T.A.; funding acquisition, S.V., M.R., and T.A. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Finnish Ministry of Agriculture and Forestry and the Finnish Cultural Foundation, South Savo Regional fund. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We wish to thank the technical staff at Luke Finland involved in bud collection, laboratory and greenhouse work, and Trevor Fenning for invaluable advice on the various steps of the micropropagation process. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Appendix A

Table A1. Different basal media used in the experiments (mM). Both MS and WPM were modiﬁed media with stocks prepared in-house, whereas DKW was bought from Sigma Aldrich (Merck) as ready salt mix. Organic components were in line with instructions from Dr T. Fenning.

DKW MS WPM

H4NO3 17.70 10.30 5.00 KNO3 9.40 KH2PO4 1.95 0.62 1.25 MgSO4 3.00 MgSO4·7H2O 3.00 1.50 CaCl2 1.01 CaCl2·2H2O 1.50 0.65 Ca(NO3)2 8.33 Ca(NO3)2·4H2O 2.35 K2SO4 8.95 5.68 MnSO4·H2O 0.20 0.26 0.13 ZnSO4·7H2O 0.06 0.03 CuSO4 x 5H2O 0.001 0.002 0.001 Forests 2021, 12, 1121 12 of 13

Table A1. Cont.

DKW MS WPM KI 0.005 CoCl2·6H2O 0.0001 Zn(NO3)2 0.09 NiSO4·6H2O 0.00002 H3BO3 0.08 0.10 0.10 Na2MoO4·2H2O 0.002 0.001 0.001 Na2-EDTA 0.12 FeSO4·7H2O 0.12 NaFe-EDTA 0.05 0.11 Myo-Inositol 5.55 0.56 0.56 Glycine 0.027 0.027 0.027 Nicotinic acid 0.008 0.004 0.004 Pyridoxine-HCl 0.010 0.002 0.002 Thiamine-HCl 0.006 0.0003 0.003

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