Art_16353.indd Journal of Applied Botany and Food Quality 94, 206 - 212 (2021), DOI:10.5073/JABFQ.2021.094.025 1Julius Kühn Institute – Federal Research Centre for Cultivated Plants, Institute for Breeding Research on Horticultural Crops, Quedlinburg, Germany 2 Kühne Jungpflanzen, Claus Kühne, Dresden, Germany 3 Department of Horticultural Plant Systems, Faculty of Life Sciences, Humboldt University Berlin, Germany Spontaneous polyploidisation of interspecific and intersectional Pelargonium hybrids during embryo rescue Plaschil, S.1*, Budahn, H.1, Klocke, E.1, Wiedemann, M.2, Olbricht, K.3 (Submitted: August 17, 2021; Accepted: November 15, 2021) * Corresponding author Summary Modern Pelargonium crispum hybrids (section Pelargonium) show low genetic and phenotypic variation due to the domestication effect. Species of the sections Cortusina, Ligularia, and Pelargonium are potential breeding partners at the diploid level (2n = 2x = 22). Five P. × crispum cultivars were used as seed parents and pollinated with one genotype of P. grandiflorum  (section Pelargonium) and three genotypes of P. fulgidum (section Ligularia). In both combinations, embryo rescue was necessary. Embryos were rescued and cultured on Murashige & Skoog medium supplemented with phytohormones. After callus and adventitious shoot regeneration 15 viable inter- specific hybrids were obtained from crossbreeding with P. grandi- florum and 11 intersectional hybrids from crossings with P. fulgi- dum, respectively. The hybrids were cultivated in the greenhouse until flowering. Their hybrid character was evident due to the inter- mediate morphological traits. Molecular investigations using dp- RAPD analysis confirmed this. Within the F1 population P. × crispum with P. grandiflorum three hybrids and after crossing with P. fulgi- dum one hybrid possessed larger flowers and fully developed anthers, respectively. Their ploidy level was confirmed as tetraploid using flow cytometry. Therefore, a spontaneous polyploidisation occurred during in vitro regeneration. The tetraploid F1 hybrids are fertile and could be used for further breeding. Key words: Genome doubling, flow cytometry, Ligularia, Pelar- gonium crispum, Pelargonium fulgidum, Pelargonium grandiflorum, ploidy level, somaclonal variation Introduction Angel or pansy pelargoniums (P. crispum (P.J. Bergius) L’Hér. hybrids) (section Pelargonium (DC.) Harv.) are popular ornamental plants already for centuries. Their percentage on the production of bedding plants increases continuously. In the 1920s, the docu- mented breeding of Angel pelargoniums started in England done by Langley-Smith (Brawner, 2003). However, it is believed that the interspecific hybridisation with P. crispum began much earlier soon after the import of different Pelargonium species to Europe during the 17th century (Brawner, 2003). Modern P. crispum hybrids, which should be referred as P. × crispum (OlBricht, 2013), show low genetic (Plaschil et al., 2017) and phenotypic variation due to crossings within a very narrow gene pool during domestication (Brawner, 2003). Increase of genetic variability by crosses with wild species could overcome this genetic bottleneck (Plaschil et al., 2012, 2015; OlBricht, 2013). The genus Pelargonium L’Hér. with its about 280 species (Bakker et al., 1998, 2004, 2005; weng et al., 2012) in 16 sections (Bakker et al., 1999a, b, 2000, 2004, 2005; röschenBleck et al., 2014) com- prises species of different sections such as Cortusina (DC.) Harv., Ligularia (Sweet) Harv. and Pelargonium, which are potential breed- ing partners for P. × crispum at the diploid level (2n = 2x = 22). A study of genetic distances of several species of the working col- lection at the Julius Kühn Institute, Quedlinburg provides additional valuable information on potential crossing partners (Plaschil et al., 2017). Natural diploid hybrids of the section Pelargonium are known, e.g. P. cucullatum (L.) L’Hér. × P. betulinum (L.) L’Hér. and P. scabrum (L.) L’Hér. × different diploid species (alBers and van der walt, 1984; van der walt et al., 1990; Bakker et al., 2004, 2005). In addition, experimental hybrids were created several times. Thus P. grandiflorum (Andr.) Willd. was used as pollen parent for crosses with P. fruticosum (Cav.) Willd, P. crispum and P. cucullatum and as seed parent for crosses with P. cucullatum, P. glutinosum (Jacq.) L’Hér., and P. cordifolium (Cav.) Curtis (hOrn, 1994). Yu (1985) suc- cessfully hybridised the species P. crispum with P. grandiflorum and OlBricht (2013) P. grandiflorum with P. × crispum, respectively. Pelargonium fulgidum (L.) L’Hér. (section Ligularia), with its very attractive bright red flower colour, was one of the most popular spe- cies used for crossings in the early 19th century (alBers et al., 1992). Together with P. × domesticum L.H. Bailey, P. fulgidum is reputed to be an ancestor of the cultivar group Uniques, but the pedigrees are not well documented (Brawner, 2003). To a small extent Uniques are propagated for satisfying the wishes of few enthusiasts in Europe and America. However, the extraordinary flower colour of P. fulgi- dum is still missing in modern P. × crispum assortments. Based on these facts one genotype of P.  grandiflorum  (section Pelargonium) and three genotypes of P. fulgidum were chosen as pollen donors for crossings with P. × crispum cultivars to enhance the genetic variability. Materials and methods Plant material Five P. × crispum cultivars were used as seed parents (Tab. 1). Pelargonium grandiflorum and three genotypes of P. fulgidum (11, 123, 48) were the pollen donors. All genotypes, maintained as a clone with at least three plants, were cultivated under greenhouse conditions at the Julius Kühn Institute. The internal standards for flow cytometric investigations, radish (Raphanus sativus L.), tomato (Solanum lycopersicum L.) ‘Stupické’ (Doležel et al., 1992) and cauliflower (Brassica oleracea L. subsp. capitata convar. botrytis var. botrytis L.) ‘Korso’ (Plaschil et al., 2020) were cultivated in vitro on Murashige and skOOg (MS) medium (1962) with 1.07 μM 1-naphtalene acetic acid at 25 °C at 16 h photoperiod. Cross Combinations and Embryo Rescue Each P. × crispum cultivar was crossed with each pollen donor (Tab. 1). For that, flower buds of the seed parents were emasculated Polyploidisation of Pelargonium hybrids 207 and isolated. The stigmas, when ripen, were pollinated with pollen from at least one anther of the pollen donor. Reciprocal crosses were not carried out, because of the low flower number of P. grandiflorum and P. fulgidum. Fourteen to thirty-one days after pollination unripe fruits were collected and surface sterilised in a sodium hypochlorite solution (3% active chlorine) (Carl Roth, Karlsruhe, Germany) followed by threefold rinsing with autoclaved distilled water. Embryos were dissected under sterile conditions and cultivated on MS medium (Duchefa, Haarlem, The Netherlands) supplemented with 2.28 μM zeatin (Duchefa), 1.14 μM indole-3-acetic acid (Duchefa), and 1.44 mM gibberellic acid 3 (Duchefa) in petri dishes (ø 6 cm) at 24 °C in the dark. One week later, a light exposure of 16 h (108 μmol/m-2s-1, provided by cool white fluorescent lamps, Philips F17T8/TL741 Alto, 17 W, U.S.A.) alternated by 8 h darkness per day was applied. When the cultivated embryos formed adventitious shoots, the shoots were separated, multiplied, and rooted on MS without phytohormones. Rooted microplants were transferred into Fruhstorfer® sowing and cutting substrate and later potted in a mixture of Stender® substrate C420 and sand (1:1). Firstly, they were cultivated in a climate cham- ber. After hardening cultivation took place in the greenhouse until flowering. Molecular hybrid identification Total DNA was isolated using the protocol of POreBski et al. (1997) with some modifications: 100 mg fresh leaf material were disrupted in a 1.5 mL tube with 400 μL preheated extraction buffer by a stain- less steel grinding ball using a mixer mill MM300 (Retsch, Haan, Germany) two times for 5 minutes with a frequency of 30 s-1. Finally, the DNA was resuspended in 50 μL TE buffer. Concentration and purity of the DNA were spectrophotometrically determined to ad- just all samples to 10 ng μL-1 DNA. RAPD analysis was performed according to williaMs et al. (1990) but using a mixture of two de- camer primers (dpRAPD, Carl Roth, Karlsruhe, Germany) (Budahn et al., 2009) to get more and smaller DNA fragments. The amplifica- tion products were separated on 1% agarose gels and stained with ethidium bromide solution. Size determination was realised by com- parison to a 100 bp DNA ladder (Fischer Scientific, Carlsbad, CA, USA). Flow cytometric and statistical analysis Three to seven biological replications of each genotype were analysed with one or two of the three internal standards. Radish (2C = 1.11 pg; Doležel et al., 1992), tomato ‘Stupické’ (2C = 1.96 pg; Doležel et al., 1992) and cauliflower ‘Korso’ (2C = 1.31 pg; Plaschil et al., 2020) were used as internal standards to estimate the DNA content and ploidy level of the investigated genotypes. Sample preparation, calculation of the 2C values and data analysis were performed as described by Plaschil et al. (2020). Results Fertilisation, embryo rescue and plant regeneration Regarding the pollen donor P. grandiflorum 77 flowers of the five P. × crispum cultivars, which were pollinated, resulted in 45 fruits and 88 embryos. Between four flowers (‘Harlekin’) and twenty-seven flowers (‘Soft Pink’) were pollinated per cultivar. Within the cross- ing group P. × crispum × P. fulgidum, 319 flowers were pollinated, 173 fruits were harvested and 227 embryos rescued. Per cross combination between five flowers (‘Harlekin’ × P. fulgidum 11) and 41 flowers (‘Merlot’ × P. fulgidum 123) were pollinated. The detailed results of all twenty cross combinations are shown in Tab. 2 and 3, respectively. The in vivo embryo development was insufficient so that the hybrid embryos started to abort about 14 days after pollination. At that time, we start to take the embryos in vitro. After a successful fertilisation, 1 - 3 embryos could be dissected per fruit. The hybrid Pelargonium embryos underwent in vitro a callus stage with soft yellow callus fol- lowed by formation of adventitious shoots eight to twelve weeks later. On average, 17% of rescued embryos from the crossings P. × crispum × P. grandiflorum and 5% from P. × crispum × P. fulgidum resulted in viable plants, respectively. In both crossing groups the percentage of fruit set per pollinated flowers was nearly similar (58.4% versus 54.2%). Nevertheless the cross combinations have a strong influence on the embryo develop- ment (Tab. 2, 3). A difference in the number of developed em- bryos per fruits was noticed. Whereas in the cross combination P. × crispum × P. grandiflorum predominantly more than one em- bryo could be dissected this was rarely the case in the crossing group with P. fulgidum. The further development of the embryos was sig- Tab. 1: Pelargonium genotypes used for crossing Seed parents (short names) Pollen donors* P. × crispum ‘Piccola™ Harlekin’ (‘Harlekin’) P. grandiflorum (DEU648PELAR038) P. × crispum ‘Piccola™ Merlot’ (‘Merlot’) P. fulgidum 11 (DEU648PELAR034) P. × crispum ‘Piccola™ Soft Pink’ (‘Soft Pink’) P. fulgidum 123 (DEU648PELAR036) P. × crispum ‘Piccola™ Pink Picotee’ (‘Pink Picotee’) P. fulgidum 48 (DEU648PELAR035) P. × crispum ‘Angeleyes® Randy’ (‘Randy’) * https://www.bundessortenamt.de/apps6/genbank_zierpfl/public/de Tab. 2: Regeneration success of hybrids after embryo rescue from crossings P. × crispum × P. grandiflorum Explants with In vitro Greenhouse adventitious shoots microplants plants pollinated Number of rescued Seed parents flowers fruits embryos total %1 total %1 total %1 ‘Harlekin’ 4 3 7 4 57 4 57 4 57 ‘Merlot’ 23 6 9 0 0 0 0 0 0 ‘Soft Pink’ 27 22 42 14 33 12 29 8 19 ‘Pink Picotee’ 10 6 9 3 33 2 22 1 11 ‘Randy’ 13 8 21 9 43 5 24 2 10 Crossing group Σ 77 45 88 30 34 23 26 15 17 1Percentage relating to the number of cultivated embryos, rounded to whole numbers 208 Plaschil, S., Budahn, H., Klocke, E., Wiedemann, M., Olbricht, K. nificantly better in the cross combination with P. grandiflorum. Concerning the fertilisation and regeneration success, there were also big differences according to the cross combination. In the cross- ing group P. × crispum × P. grandiflorum, ‘Harlekin’ and ‘Soft Pink’ were the best seed parents. Seventy-five percent of the pollinated flowers of ‘Harlekin’ and 81.5% of ‘Soft Pink’ showed fruits and cultivable embryos giving four and eight viable plants in the green- house. On the other hand, with ‘Merlot’, despite the fruit set, embryo development during in vitro culture was completely absent. In the crossing group with P. fulgidum embryos with seed parents ‘Soft Pink’ and ‘Pink Picotee’ did not grow further. In the combina- tion ‘Harlekin’ × 48 one rescued embryo could be cultivated to a microplant but died later on. Only five of fifteen combinations exclu- sively with the seed parents ‘Merlot’ and ‘Randy‘ resulted in viable F1 hybrids. Regarding the three pollen donors, the highest number of plants originated from P. fulgidum 123 (seven F1 hybrids what cor- responds to 9.6% of pollinated flowers with ‘Merlot’ and ‘Randy‘) followed by P. fulgidum 48 (three F1 hybrids, 5.4% of the pollinated flowers) and P. fulgidum 11 (one F1 hybrid, 1.5% of the pollinated flowers). Hybrid identification and characters For all regenerated hybrids at least three clone plants were cultivated in the greenhouse until flowering. Their hybrid character was evi- dent because of the intermediate morphological traits in flower, leaf, and habit (Fig. 1 and 2). F1 hybrids of P. × crispum × P. grandiflorum had single rose flowers of intermediate size with fully developed anthers. They always possessed darker marks at the upper petals like P. grandiflorum and in some cases additional marks at the lower petals. The intermediate sized leaves were different lobated, the typi- cal zone from the pollen donor P. grandiflorum was lacking (Fig. 1). Longitudinal growth was also intermediate. The plants were clearly Tab. 3: Regeneration success of hybrids after embryo rescue from crossings P. × crispum × P. fulgidum 11, 123 and 48 Explants with In vitro Greenhouse adventitious shoots microplants plants pollinated Number of rescued Crossings flowers fruits embryos total (%)1 total (%)1 total (%)1 ‘Harlekin’ × 11 6 6 6 0 0 0 0 0 0 ‘Merlot’ × 11 40 18 28 2 7 2 7 0 0 ‘Soft Pink’ × 11 23 10 6 0 0 0 0 0 0 ‘Pink Picotee’ × 11 17 11 5 0 0 0 0 0 0 ‘Randy × 11 28 22 31 2 6 1 3 1 3 Pollen donor 11 Σ 114 67 76 4 5 3 4 1 1 ‘Harlekin’ × 123 5 3 1 0 0 0 0 0 0 ‘Merlot’ × 123 41 15 24 7 29 7 29 5 21 ‘Soft Pink’ × 123 16 10 10 0 0 0 0 0 0 ‘Pink Picotee’ × 123 15 7 8 0 0 0 0 0 0 ‘Randy’ × 123 32 25 33 2 6 2 6 2 6 Pollen donor 123 Σ 109 62 80 9 11 9 11 7 9 ‘Harlekin’ × 48 6 4 5 1 20 1 20 0 0 ‘Merlot’ × 48 36 10 25 5 20 4 16 1 4 ‘Soft Pink’ × 48 19 11 20 0 0 0 0 0 0 ‘Pink Picotee’ × 48 9 3 1 0 0 0 0 0 0 ‘Randy’ × 48 20 16 20 5 25 5 25 2 10 Pollen donor 48 Σ 96 44 71 11 16 10 14 3 4 Crossing group Σ 319 173 227 24 11 22 10 11 5 1Percentage relating to the number of cultivated embryos, rounded to whole numbers Fig. 1: Flower and leaf of P. × crispum ‘Soft Pink’, the hybrids SG2-3.1 and SG2-3.2 and P. grandiflorum (from left to right). Fig. 2: Flower and leaf of P. × crispum ‘Merlot’, the hybrid MF4-2 and P. fulgidum 123 (from left to right). 1 cm 1 cm Polyploidisation of Pelargonium hybrids 209 more vigorous than those of P. × crispum, but with a poorer branch- ing. Three out of eight hybrids from the cross ‘Soft Pink’ × P. gran- diflorum showed distinct larger flowers than their siblings had. In the greenhouse, two of these hybrid clones (SG2-3, SG3-1) consisted of plants with medium (SG2-3.1, SG3-1.1) and plants with large sized flowers (SG2-3.2, SG3-1.2) (Fig. 1). F1 hybrids of P. × crispum × P. fulgidum inherited the red flower colour from the pollen donor and the marks from the seed parent (Fig. 2). They were male sterile with no or rudimentary anthers. The growth was weaker and plants tended to become succulent. Among the five different F1 hybrids of ‘Merlot’ × P. fulgidum 123, one geno- type possessed larger flowers with fully developed anthers and fruit set after open pollination. In addition, dpRAPD analysis confirmed the hybrid character of all regenerated F1 plants. Specific DNA bands of the seed and pollen parents were detected in the F1 plants (Fig. 3 and 4). Flow cytometry and ploidy levels Six genotypes of the crossing group ‘Soft Pink’ × P. grandiflorum and five genotypes of P. × crispum × P. fulgidum 123, including the assumed tetraploid plants, were proven together with the seed and pollen parents by flow cytometric analysis. The 2C values, calculated with a corresponding internal standard, are given in Tab. 4. It was shown, that the assumed tetraploid F1 hybrids of both cross- ing groups have nearly a doubled 2C value compared to the di- ploid hybrids. As expected from different 2C values of the pollen donors of both crossing groups, the mean 2C value of the diploid and tetraploid F1 hybrids, originating from P. fulgidum, was high- er than mean 2C value of the diploid and tetraploid F1 hybrids, originating from P. grandiflorum. Overall, in the crossing group P. × crispum × P. grandiflorum, spontaneous polyploidisation oc- curred with a frequency of about 20% and within the crossings P. × crispum × P. fulgidum of about 9%, respectively. Fig. 4: dpRAPD analysis of four F1 hybrids of the crossing ‘Merlot’ × P. fulgidum 123 (MF). Specific band of ‘Merlot’ at about 280 bp (left arrow) and P. fulgidum 123 at about 100, 600 and 1350 bp (right arrows), respectively (M = 100 bp DNA ladder). Fig. 3: dpRAPD analysis of eight F1 hybrids of ‘Soft Pink’ × P. grandiflorum (SG). Specific bands of ‘Soft Pink’ at about 350 and 960 bp (left arrows) and P. grandiflorum at about 490, 590 and 1010 bp (right arrows), respectively (M = two different 100 bp DNA ladders). 210 Plaschil, S., Budahn, H., Klocke, E., Wiedemann, M., Olbricht, K. Discussion In the genus Pelargonium, only intra- and intersubgeneric crossings between genotypes of the same basic chromosome number and ploi- dy level give a viable, fertile progeny (Yu, 1985; hOrn, 1994). The knowledge of phylogeny, genetic distance, variability, ploidy level, and fertility can support plant breeding and interspecific crosses. Although after polyploidisation of P. × crispum the variability was already widened by crossings with P. × domesticum at the tetraploid ploidy level (Plaschil et al., 2012, 2015), further interspecific and intersectional hybridisation at the diploid level (2n = 2x = 22) is de- sirable. Reciprocal crosses were omitted, because of fewer flowers of the species compared to the cultivars and the not significant effect on successful fertilisation in Pelargonium (Yu, 1985). Yu (1985) reported about successful interspecific crosses using P. crispum as seed parent and P. grandiflorum as pollen parent. Six F1 hybrids regenerated without embryo rescue. It is not known, whether these six hybrids were used for further breeding work. Furthermore, OlBricht (2013) succeeded in crossing P. grandiflorum with a breeding clone of P. × crispum and backcrosses with P. × crispum until the F3 generation. Intersectional hybridisation in Pelargonium was stated several times (Yu, 1985; hOrn, 1994). Yu (1985) de- scribed viable hybrids between P. fulgidum and diploid Regal pelar- goniums (P. × domesticum, (sect. Pelargonium) resulting in cultivars (Brawner, 2003) whereas crosses between P. fulgidum and P. ra- paceum (L.) L’Her. (section Hoarea (Sweet) De Candolle) were not successful. Regarding P. × crispum, the successful intersectional hybridisation with P. fulgidum we reported for the first time, because the breeding pedigree of Uniques was not documented (Brawner, 2003). Even though, it is assumed, that the cultivar pac® Angeleyes® ‘Orange’, which underwent several hybridisation steps, descended from P. fulgidum. One parent of pac® Angeleyes® ‘Orange’ is the cultivar ‘Erin’ (breeder kaPac, 1997, USA) and ‘Erin’ itself should be origi- nated from crosses with P. fulgidum (hOfMann & OBlricht, pers. comm.; OlBricht, 2013). Due to insufficient embryo development after hybridisation, embryo rescue was necessary for both crossing groups as described for other ornamentals (van tuYl et al., 1991; winkelMann et al., 2010; arOs et al., 2019). The overall plant regeneration was possible on a low scale of about 3-57% of cultivated embryos. As expected, the rege- neration of interspecific P. × crispum × P. grandiflorum hybrids was more successful than that of intersectional P. × crispum × P. fulgi- dum hybrids, because of the lower genetic distance to the seed parent P. × crispum (Plaschil et al., 2017). The regeneration could probably be improved by further investi- gations concerning the optimal time for fruit harvesting. So far, sceMaMa and raquin (1990) only succeeded in direct embryo cul- ture of a diploid P. × hortorum Bailey, when embryos were removed at least 13 days after pollination, whereas they achieved embryo development and germination as early as 5-6 days after pollination using the ovary culture followed by cultivation of the contained em- bryos with their scarified seed coat. The authors achieved best results with ovary culture 11 days after pollination combined with the em- bryo culture five to seven days later. In addition, a more subtle adapt- ed mixture of the in vitro medium due to the different genotypes may increase the percentage of viable regenerants (Bentvelsen et al., 1990; denis-PeixOtO et al., 1997; kaMlah et al., 2019). Through embryo rescue we obtained spontaneous tetraploid F1 hy- brids from both, interspecific cross P. × crispum × P. grandiflorum and the intersectional cross P. × crispum × P. fulgidum showing that the in vitro stage is important for the embryo development but it is also an appropriate tool to restore the hybrid fertility and an impor- tant prerequisite for further breeding. Tab. 4: Results of the flow cytometric analysis: Mean 2C values of the seed parents, pollen donors and the F1 hybrids of the cross combinations ‘Soft Pink’ × P. grandiflorum and P. × crispum × P. fulgidum 123 Genotype Internal standard n 2C value (pg)1 SD (±) Ploidy level Seed parents ‘Merlot’# ‘Korso‘/‘Stupické’ 6 1.00a 0.05 2x ‘Soft Pink’# ‘Korso‘/‘Stupické’ 7 1.01a 0.01 2x ‘Randy’# ‘Korso‘/‘Stupické’ 7 1.01a 0.02 2x Pollen donors and their hybrids P. grandiflorum ‘Korso’ 3 0.99a 0.01 2x SG2-3.1* ‘Korso’ 3 0.98a 0.02 2x SG2-3.2* ‘Korso’ 3 2.07d 0.02 4x SG3-1.1* ‘Korso’ 4 0.97a 0.01 2x SG3-1.2* ‘Korso’ 3 2.09e 0.02 4x SG3-4* ‘Korso’ 3 1.01a 0.02 2x SG3-5* ‘Korso’ 3 2.14e 0.02 4x P. fulgidum 11 Raphanus 3 1.56c 0.09 2x P. fulgidum 123 Raphanus 3 1.52c 0.09 2x P. fulgidum 48 Raphanus 3 1.55c 0.05 2x MF2-1** ‘Stupické’ 4 1.23b 0.03 2x MF3-2** ‘Stupické’ 3 2.59f 0.01 4x MF4-2** ‘Stupické’ 3 1.19b 0.01 2x RF4-2** ‘Stupické’ 3 1.22b 0.02 2x RF7-1** ‘Stupické’ 3 1.19b 0.04 2x # Data taken from Plaschil et al., 2020; *Cross combination: ‘Soft Pink’ × P. grandiflorum (SG); ** Seed parents: M = ‘Merlot’, R = ‘Randy’ and pollen donor F = P. fulgidum 123; n = number of biological replications analysed per genotype; SD = standard deviation; 1 different letters indicate significant differences, Tukey’s b-test, α = 5% Polyploidisation of Pelargonium hybrids 211 Chromosomal disturbances up to a whole genome doubling of in vitro plant tissues are a common phenomenon. Firstly larkin and scOwcrOft (1981) summarised these changes under the term soma- clonal variation. Spontaneous polyploidisation was often reported in in vitro haploid techniques such as microspore, anther or ovule culture, respectively (ahMadi and eBrahiMzadeh, 2020; BOerMan et al., 2020; Yuan et al., 2015; caMPiOn et al., 1995). The sponta- neous chromosome doubling is an important factor for the produc- tion of homozygous plants because it is a desirable replacement for colchicine treatment. This phenomenon is described in almost all in vitro techniques such as long term cultures (ziauddin and kasha, 1990), in vitro shoot regeneration from leaves (MeYer et al., 2009) or from embryogenic calli (ishigaki et al., 2014). As far as we know, no polyploidisation of Pelargonium during in vitro embryo rescue has been described, yet. The influencing factors on somaclonal variation such as exogenous phytohormones or other chemicals in the nutrient medium, the de- velopmental stage of the explants, and the duration of the in vitro culture were comprehensively investigated. Nevertheless, questions remain open for better use of the process of spontaneous chromo- some doubling. Obviously, the genetics of the cultivated plants plays a role in induction of the chromosome doubling during the tissue culture. The frequency and regularity of spontaneous polyploidisa- tion during embryo rescue of Pelargonium needs further explora- tions. Already larkin and scOwcrOft (1981) mentioned in vitro Pelargonium plants as an example for the presence of somaclonal variations after various in vitro cultures such as genetic instabilities and changes of the ploidy. cassells et al. (1995, 1997) confirmed these findings with other Pelargonium accessions applying mo- lecular methods. It could be assumed, that the tendency to genetic changes during the in vitro phase also made the duplication of chro- mosomes during Pelargonium embryo rescue possible. Considering the morphological traits of the hybrids, the bright red flower colour of P. fulgidum inherited in crossings with P. × crispum is a promising new feature, but the vigour of the F1 hybrids was reduced. All diploid hybrids were male sterile with further unfavour- able morphological traits. Their value for further breeding as seed parent has to be proved. Probably, the fertility must be restored by polyploidisation. Only the fertile tetraploid hybrid is an appropriate crossing partner. In any case, further backcrosses with P. × crispum are necessary to establish cultivars. Hybridisation between P. × crispum and P. grandiflorum is supposed to be a promising enhancement of genetic variation in the very nar- row gene pool of P. × crispum cultivars. The primary diploid and tetraploid F1 hybrids were vigorous, had attractive flowers with marks and fertile anthers. Hence, they could be used for further breeding at two different ploidy levels, e.g. for backcrosses with P. × crispum or crossings with P. × domesticum. The hybridisation success could be increased using embryo rescue. The current results encourage the usage of Pelargonium species in breeding programs of P. × crispum combined with the application of embryo rescue. Acknowledgement The authors thank Dagmar Franke for the embryo rescue, Martina Fuß and Anika Kunze for molecular analysis, Simone Abel for flow cyto- metric measurements as well as Annette Benecke, Eveline Kummer and Denise Brocka for the horticultural assistance. Moreover, we thank Kühne-Jungpflanzen, Claus Kühne GbR, Dresden and Günter Schumann for supporting this project. Conflict of interest No potential conflict of interest was reported by the authors. 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DOI: 10.1007/BF00023662 ORCID Sylvia Plaschil https://orcid.org/0000-0001-6689-4113 Holger Budahn https://orcid.org/0000-0003-1639-8036 Evelyn Klocke https://orcid.org/0000-0002-3153-1280 Klaus Olbricht https://orcid.org/0000-0003-1124-2585 Address of the corresponding author: Julius Kühn Institute – Federal Research Centre for Cultivated Plants, In- stitute for Breeding Research on Horticultural Crops, Erwin-Baur-Straße 27, 06484 Quedlinburg, Germany E-Mail: sylvia.plaschil@julius-kuehn.de © The Author(s) 2021. 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