In Vitro Induction and Primary Evaluation of Octoploid Plants in Saskatoon Berry (Amelanchier alnifolia Nutt.)

Kucharska, Danuta; Podwyszyńska, Małgorzata; Trzewik, Aleksandra; Marasek-Ciołakowska, Agnieszka; Pluta, Stanisław; Seliga, Łukasz

doi:10.3390/agronomy12051215

Open AccessArticle

In Vitro Induction and Primary Evaluation of Octoploid Plants in Saskatoon Berry (Amelanchier alnifolia Nutt.)

by

Danuta Kucharska

^1,*

,

Małgorzata Podwyszyńska

¹

,

Aleksandra Trzewik

¹

,

Agnieszka Marasek-Ciołakowska

¹

,

Stanisław Pluta

²

and

Łukasz Seliga

²

¹

Department of Applied Biology, The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3 Street, 96-100 Skierniewice, Poland

²

Department of Breeding, The National Institute of Horticultural Research, Konstytucji 3 Maja 1/3 Street, 96-100 Skierniewice, Poland

^*

Author to whom correspondence should be addressed.

Agronomy 2022, 12(5), 1215; https://doi.org/10.3390/agronomy12051215

Submission received: 25 April 2022 / Revised: 16 May 2022 / Accepted: 17 May 2022 / Published: 18 May 2022

(This article belongs to the Special Issue New Polyploids of Crop Plants—Application in Breeding, Phenotypic and Genetic Evaluation)

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Abstract

:

Polyploids of many species of horticultural plants are widely used in breeding programs since they are characterized by vigorous growth, larger organs, and greater resistance to biotic and abiotic stress. Saskatoon berry (Amelanchier alnifolia Nutt.) is in a form of diploid (2n = 2x = 34) and tetraploid. So far, no reports on induction of polyploidy in this species have been found. The aim of the study was to obtain Saskatoon berry octoploids with the use of in vitro shoot cultures. Shoots of the tetraploid of Canadian cultivars ‘Martin’ and ‘Smoky’ derived from in vitro cultures were used for induction of chromosome doubling. The shoot explants were incubated in a multiplication medium with the addition of one of the following antimitotics: colchicine, trifluralin, oryzalin or amiprophos methyl (APM) for two weeks (six days in the darkness and then eight days under a 16-h photoperiod). Then, the shoots were transferred to the multiplication medium without antimitotics and propagated in vitro by two subcultures. Octoploids were selected based on flow cytometry. Trifluralin showed the most phytotoxic effect. Six octoploids were obtained, four for ‘Martin’ after treatment with 250 mg L⁻¹ of colchicine and two for ‘Smoky’ following the treatment with 5 mg L⁻¹ of APM. All obtained octoploid individuals were multiplied and the level of ploidy was re-examined by flow cytometry and chromosome counting, which confirmed their octoploid genotype (2n = 8x = 136). They are probably the first octoploids of the genus Amelanchier in the world. The newly obtained octoploids were rooted in vitro, acclimatized to ex vitro conditions and grown in a greenhouse. Octoploids grew very slowly and showed a tendency to premature dormancy, which was manifested by inhibition of growth. In octoploid plants, the stomata were significantly larger and the number of stomata per 1 mm² of leaf area was lower, compared to the tetraploid counterparts.

Keywords:

Amelanchier alnifolia; chromosome doubling; octoploids; colchicine; oryzalin; trifluralin; amiprophos methyl

1. Introduction

The Saskatoon berry (Amelanchier alnifolia Nutt.) is a fruit species grown in Canada and the northern regions of the USA for its valuable fruits [1]. This species has been grown in home gardens and on commercial plantations for many years. The Saskatoon berry is a long-lived shrub that adapts relatively well to soil and climate and can be cultivated in various environmental conditions. Currently, Polish producers are interested in growing this species, so there is the potential for development of fruit production of this crop in our country. Saskatoon berry shrubs usually bear fruits in the two to three years after planting, and their full fruiting occurs in four to five years. The fruits ripen in Poland at the end of June or the beginning of July depending on year and weather conditions. They can be harvested both by hand and with harvesters used to pick the fruits of currants, gooseberries and aronia (Aronia melanocarpa). For this reason, it can be planted as alternative or complementary to these crops [2,3].

The valuable fruits can be used in a variety of ways in the processing and freezing industries, in bakery and as dessert fruits. The nutritional and health-promoting benefits of the fresh fruits and its products is due to the high content of phenols, flavonoids, anthocyanins, fibre, B vitamins, minerals, especially potassium, and microelements such as cobalt and copper [1,4,5,6,7,8]. For this reason, fruits could be considered as an attractive material for the production of functional food or dietary supplements [9]. In addition, Saskatoon berry fruits have better nutritional and health properties and are richer in minerals than blueberries (Vaccinium corymbosum L.) [10]. The great advantage of Saskatoon berry is a low susceptibility to diseases and pests, and high frost hardiness [3,11].

Polyploidization is one of the important sources of the genetic variability used in plant breeding [12,13,14]. Polyploidization not only causes the multiplication of the same genes, including resistance-related genes, but is also the source of other types of genetic changes, such as chromosomal and point mutations, and alteration in DNA methylation pattern [13,15,16,17]. In the case of the Saskatoon berry, taxa of this species occur in their diploid (2n = 34), triploid and tetraploid (2n = 68) forms [18,19]. In our preliminary study, based on flow cytometry, we found that the cultivar ‘Martin’ was tetraploids, not diploid as it had been thought before. In the case of ‘Smoky’, its tetraploidy was confirmed by Burges et al. [19]. These authors indicated a high polyploid and apomictic complexity within the genus Amelanchier. So far, no reports have been found in the world literature on the induction of polyploids, including octoploids of the Saskatoon berry (A. alnifolia). The formation of polyploids with favourable features was confirmed by our previous research, in which autotetraploid daylilies (Hemerocallis) with much larger flowers and apple (Malus domestica) autotetraploids with increased resistance the apple to scab (Venturia inaequalis) and fire blight (Erwinia amylovora) were obtained [20].

The aim of the studies was to develop a method of in vitro mitotic chromosome doubling of Amelanchier alnifolia to obtain octoploids for the breeding purposes.

2. Material and Methods

2.1. Induction of Octoploids

Saskatoon berry tetraploids of Canadian cultivars ‘Smoky’ and ‘Martin’ were used for the research. As explants for polyploidy induction, the in vitro propagated auxiliary shoots were used. The multiplication medium contained MS [21] salts and vitamins (Duchefa, Haarlem, The Netherlands) supplemented with 325 mg L⁻¹ CaCl₂, 175 mg L⁻¹ MgSO₄, 1.0 mg L⁻¹ 1 benzyladenine (BA) (Duchefa), 1.0 mg L⁻¹ gibberellic acid (GA₃) (Duchefa), 0.1 mg L⁻¹ indole-3-acetic acid (IAA) (Duchefa), 30 g L⁻¹ sucrose, 6 g L⁻¹ plant agar (Duchefa), pH 5.6. Micropropagated well-formed, 1–2 cm long shoots, from four-week- subculture were used for inducing polyploids. There were 16 shoots in each antimitotic treatment. Shoots were incubated on the multiplication medium supplemented with one of the following antimitotics: colchicine (125 and 250 mg L⁻¹) (Merck, Darmstadt, Germany), trifluralin (50 and 100 mg L⁻¹) (Merck), oryzalin (5 and 10 mg L⁻¹) (Sigma-Aldrich, Saint Louis, MI, USA) and APM (5 and 10 mg L⁻¹) (Duchefa). Colchicine was dissolved in distilled water and filtered through microporous filter membrane of 0.22 μm (NalgeneTM Sterile Syringe Filters, Thermo Fisher Scientific, Waltham, MA, USA), and the remaining antimitotics were dissolved in 99.8% ethanol and directly added to the medium. The antimitotics were included to the media after autoclaving. In control, the multiplication medium did not contain antimitotics. Shoots were treated with antimitotics for two weeks (6 days in the darkness and then 8 days under a 16 h photoperiod, 16 h light/8 h darkness). Then, the shoots were transferred to the multiplication medium described above but without antimitotics. The phytotoxicity of antimitotics was assessed by observation of shoot survival and the number of shoots per explant four weeks after antimitotic treatment (at the end of the four-week subculture) and after another four weeks on the multiplication medium (second subculture). Experiments were carried out in a growth room with a temperature of 22–23 °C and 16 h photoperiod (fluorescent lamps OSRAM L 36 W/77 Fluora, Monachium, Germany) with 30 μmol m⁻² s⁻¹ photosynthetic photon flux density (PPFD).

2.2. Detection of Octoploids

Octoploids were detected by cytometric evaluation of the ploidy level of all of the plants regenerated at the end of the second subculture (eight weeks after antimitotic treatment). Cytometric analysis (FCM-DAPI) was performed using leaf samples taken from regenerants. Samples (two leaves from the base of the shoot) were taken from all of the regenerated shoots. Plant tissue was chopped in a Petri dish that contained 0.5 mL nuclear isolation Partec buffer (0.1 M Tris, 2.5 mM MgCl₂ 6H₂O, 85 mM NaCl, 0.15 (v/v) Triton X-100, pH 7) made by ourselves according to Śliwinska [22], with 1% polyvinylpyrrolidone (PVP) and to which the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) (50 mL L⁻¹) was added. Following addition of 1 mL of the isolation buffer, the samples were filtered through a 30-μm filter and incubated at room temperature for 45–60 min in the dark. The fluorescence of the nuclei was measured using a CyFlow Ploidy Analyser with CyView software (CyFlow PA, Partec, Görlitz, Germany) with UV-LED 365 nm. Samples with at least 2000 nuclei were measured. The level of ploidy expressed by the value of nuclear DNA fluorescence on the x-axis was read on the histograms. An external standard, tetraploid leaf sample taken from control shoots was used to establish the position of the fluorescence peak on the x-axis for the tetraploid; the position of the fluorescence peak for the octoploid should be twice the value on the x-axis compared to tetraploid.

Octoploidy of selected shoots was also confirmed by evaluation of relative nuclear DNA content using low cytometry with application of propidium iodide (PI) for DNA staining (FCM-PI). Similar to FCM-DAPI, samples (two leaves from the base of the shoot) were taken and were chopped together with 0.5 cm² leaf sample of internal standard in 0.5 mL nuclear isolation Partec buffer containing 1% PVP, PI (50 mL L⁻¹) and RNase (50 μg mL⁻¹) was added. As the internal standards, the young leaves of tomato (Solanum lycopersicum L.) ‘Stupicke’ (2C DNA = 1.96 pg) were used [23]. Following addition of 1 mL of the isolation buffer, the samples were filtered through a 30-μm filter and incubated at room temperature for 45–60 min in the dark. The fluorescence of the nuclei was measured using the CyFlow Ploidy Analyser with CyView software (CyFlow PA, Partec, Görlitz, Germany) with an Nd-YAG green laser at 532 nm. Data were analysed by means of software CyView (Sysmex Partec). The 2C DNA content of a sample was calculated as the sample peak mean divided by the mean of the standard plant peak and multiplied by the amount of DNA of the standard plant. Samples with at least 2000 nuclei were measured for five plants of each octoploid clone and their tetraploid counterparts, with two runs from each nuclei isolation extract.

Efficiency of polyploidization was calculated as the percentage of octoploids obtained from 16 primary shoot explants treated with a given antimitotic agents.

2.3. Confirmation of Octoploidy by Chromosome Counting

Slade preparation for somatic chromosome analysis was performed as described by Marasek-Ciolakowska et al. [24]. In brief, root tips were pre-treated with 2 mM 8-hydroxyquinoline for 4 h, fixed in 3:1 ethanol:glacial acetic acid solution for at least 12 h, and then digested in a mixture of enzymes comprised of 20% pectinase (Sigma), 1% cellulase (Calbiochem), and 1% cellulose ‘Onozuka R-10’ (Duchefa) at 37 °C for 1 h. Root meristems were squashed in a drop of 45% (v/v) acetic acid. After freezing in liquid nitrogen, cover slips were removed using a razor blade, and the preparations were dehydrated in absolute ethanol, air dried, and stained with 2.5 g mL⁻¹ 4′,6-diamidino-2-phenylindole (DAPI) (Serva). For each genotype, at least 10 instances of metaphase were photographed with a digital CCD camera PS-Fi1 (Nikon, Tokyo, Japan) attached to an epifluorescent microscope Optiphot-2 (Nikon Instruments Inc., Tokyo, Japan) using UV excitation for DAPI visualisation.

2.4. In Vitro Propagation and Rooting of Octoploids

Single shoots regenerated from each primary shoot explants with confirmed octoploid status by FCM-DAPI were cloned to 20–30 shoots and each octoploid clone was labelled as S8x-1 and S8x-2 for ‘Smoky’, and M8x-1 to M8x-4 for ‘Martin’. The octoploid clones and tetraploid counterparts were propagated on multiplication medium. After four weeks the total numbers of shoots per explant (primary shoot) were assessed (standard condition). Four-week-old shoot cultures maintained on the multiplication medium were cooled at 4 °C for three months. After this time shoot cultures were propagated on the multiplication medium and after four weeks the total numbers of shoots per explant were assessed (after cooling at 4 °C). The uncooled shoots were rooted. In vitro rooting was carried out using a two-step procedure: I—five days in darkness on the medium containing ½ MS salts and MS vitamins (Duchefa), 30 g L⁻¹ sucrose, 1.0 mg L⁻¹ indole-3-butyric acid (IBA) (Duchefa) and 6 g L⁻¹ plant agar (Duchefa), pH 5,6; II—30 days in standard 16 h photoperiod on medium ½ MS salts and MS vitamin, 30 g L⁻¹ sucrose, 4 g L⁻¹ active carbon (Duchefa) and 6 g L⁻¹ plant agar (Duchefa), pH 5.6. After 4 weeks, the percentage of rooted shoots and the number of roots per shootwere assessed. In greenhouse rooted shoots were acclimatized in a substrate (Klasmann-Deilmann TS1, Geeste, Germany) in plastic boxes, initially covered with polyethylene foil, shaded and kept in high humidity which was gradually reduced. The day after planting, the plants were sprayed with 10 mg L⁻¹ GA₃. From the 10th day, foliar fertilization 0.4% NPK 18-18-18 (Kristalon Yara, Oslo, Norway ) was carried out. Microplants grew in natural light at a temperature of 26 °C/20 °C (Table S1).

2.5. Ethylene Measurements

Due to slow growth, leaf yellowing and tendency to premature dormancy of octoploid plants, ethylene measurements were performed. The production of ethylene during the in vitro propagation of octoploid shoots was analysed in comparison to tetraploid shoots. The shoots derived from 4-week subculture were placed in 10 mL vials, tightly closed and kept for 2 h. Gas samples were taken from the headspace and injected into a gas chromatograph (Hewlett-Packard model 4890D) equipped with a flame ionization detector and a glass column packed with chromosorb 102. Ethylene production was expressed as nl g FW⁻¹ h⁻¹. All analyses were performed in seven replicates.

2.6. Stomatal Size and Density

Microscopic observations of the number and length of the stomata of octoploid and tetraploid plants were performed in plants grown for two months in greenhouse conditions. The samples of the abaxial epidermis isolated from the third leaf, at a 10-cm distance from their tips were mounted on slides for microscopic observations and stained with toluidine blue according to the procedure of Dyki and Habdas [25]. For each genotype, the frequency of stomata per 1 mm² and length of stomata (100 stomata/per genotype) were determined. The observations were performed using a Nikon Eclipse 80i microscope (Eclipse 80i, Nikon, Tokyo, Japan) with the program NIS-Elements BR ver. 2.30 (Nikon Instruments Inc., Tokyo, Japan), at 100 and 400× magnification, respectively.

2.7. Statistical Analyses

Data on growth parameters, nuclear DNA content and microscopic stomata measurements were analysed statistically using one-way analysis of variance (Statistica 13.1) separately for each cultivar and the means were compared by Tukey’s test at p = 0.05.

3. Results and Discussion

In mitotic polyploidization, chromosome duplication occurs in somatic cells. Colchicine is the antimitotic agent most commonly used to induce polyploidy [26]. However, due to its harmful effects, there had been an increase in the use of other antimitotic agents that were considered less toxic, such as oryzalin, trifluralin, and APM [27,28,29,30].

In our research, among the antimitotics used, trifluralin at the concentration of 50 and 100 mg L⁻¹ showed the greatest phytotoxic activity, causing the complete death of explants (Table 1). On the other site, this compound, at the same concentration was successfully used for polyploidization of Hemerocallis [31] and in concentration of 2 µM for Ranunculus asiaticus and 3 or 10 µM for Helleborus sp. [27,28]. Compared with the control, all of the antimitotics used in our research, especially those used at higher concentrations, decreased the total number of shoots per explants both in the first and second subculture.

In total, six octoploids were obtained for both tested cultivars. Four octoploids were selected for ‘Martin’ after treatment with 250 mg L⁻¹ of colchicine with polyploidization efficiency of 25% and two ones for ‘Smoky’ after treatment with 5 mg L⁻¹ of APM with polyploidization efficiency of 12.5% (Figure 1a,b). Previously, APM was successfully used for polyploidization of black currant (Ribes nigrum L.) and showed a slight phytotoxic effect [32]. APM induced also chromosome doubling in vitro in many other plant species, e.g., banana [33].

In our research, the octoploidy of the obtained clones was well documented by FCM-PI and chromosome count (Figure 1, Figures S1 and S2). The resulting nuclear DNA content of about 4.6 pg for octoploids is in line with the predicted value twice as high as that found in the tetraploids. Moreover, the cytological analyzes performed clearly showed that in all octoploids the number of chromosomes is 136 and in tetraploids 68 (Figure 1 and Figure S1). This is the first documented case of obtaining autoctoploids in the genus Amelanchier. The nuclear DNA content of the control tetraploid plants was 2.28 and 2.29 pg in ‘Smoky’ and ‘Martin’, respectively (Table 2). The obtained results of the nuclear DNA contents for the tetraploid of Amelanchier sp. are generally consistent with the values reported by other authors. Talent and Dickinson [34] showed that the nuclear DNA content of A. alnifolia cv. ‘Smoky’ was 2C DNA = 2.68 pg and according to Burgess et al. 2014 [35], the cultivar ‘Smoky’ was the tetraploid. A slightly higher value reported for ‘Smoky’ than that obtained in our research for this cultivar (2.28 pg) was probably due to the fact that we used as an internal standard Solanum lycopersicum with 2C DNA = 1.96 pg and Talent and Dickinson [34] used Pisum sativum with 2C DNA = 8.8 pg. We also used a different extraction buffers and equipment, which could affect the obtained results. The nuclear DNA content was also measured in other species and it was 2.59 pg in A. stolonifera [36] and 2.52 pg in A. lamarckii [37], indicating their tetraploidy, and 1.39 pg in diploid A. arborea [34].

Among the methods of vegetative propagation of the Amelanchier alnifolia, in vitro culture technology is the most effective and commonly used. In Canada, Pruski et al. [38] were the first to report a micropropagation method for this species. We proposed a certain modification of the medium, which consisted of the increased concentrations of CaCl₂ and MgSO₄ in the MS medium by 50%; the composition of growth regulators reported by Pruski et al. [38] was enriched with GA₃ and IAA, which ensured shoots of better quality [39].

The in vitro shoot multiplication rate of the newly obtained octoploids, compared with that of their tetraploid counterparts, differed between tested cultivars. For ‘Smoky’, the multiplication rate was similar for tetraploid and octoploid plants or even higher for the S8x-2 octoploid clone (Table 3). For ‘Martin’, the multiplication rate of octoploids was lower, but the differences were significant only for one of the octoploid clones (Figure 2a). A three-month cooling of the shoot cultures increased the multiplication rate in all octoploids, and for ‘Martin’ also in their tetraploid counterparts. Such enhancement of the multiplication ability following low-temperature treatment was observed in our previous studies on gooseberry (Ribes grossularia L.) micropropagation [40]. Compared with tetraploids, octoploids revealed poor rooting ability. Octoploid shoots had significantly fewer roots, and the percentage of rooted shoots was lower. A similar phenomenon was described by Podwyszyńska and Cieślińska [41] in newly formed apple autotetraploids.

During the stages of shoot multiplication, rooting and acclimatization of newly formed octoploids, a strong tendency toward premature dormancy and very slow growth of plants were observed (Figure 2). We suspected that this phenomenon might be caused by the more intense production of ethylene in octoploids. However, analysis of ethylene production during the in vitro propagation revealed that tetraploid plants released significantly more ethylene than octoploid ones. For ‘Smoky’, the differences were significant, while for ‘Martin’ the differences were smaller, but a similar tendency in ethylene production was observed (Table 4). The reason for the poor growth and premature dormancy of Saskatoon berry octoploids during the initial period of their growth and the presumably transitory nature of this phenomenon could be caused by the much higher rate of DNA methylation as recorded in newly obtained mitotic polyploids of other species such as apple and Brassica rapa [15,20,42,43]. It is presumed that gradual demethylation of the genome during its stabilization over the following years of cultivation of synthetic polyploid plants will restore their normal ability to actively growth, as it was observed in other newly formed tetraploids, where the high DNA methylation index decreased after low-temperature treatment [44].

One of the most frequently observed plant features of newly obtained polyploids, compared with their counterparts of lower ploidy level, are the larger stomata [13,14,20,45]. Also, in our studies octoploids of Saskatoon berry cultivars had significantly larger stomata and lower stomatal density (Table 5). A strong positive correlation between stomata size, and the level of ploidy was considered as a morphological marker of the level of ploidy for many plant species [46]. A smaller number of stomata per unit area might translate into better water management efficiency of plants and, consequently, greater their tolerance to drought.

4. Conclusions

The described method of chromosome doubling was effective and allowed to obtain Saskatoon berry octoploids of tested cvs. ‘Smoky’ and ‘Martin’;
Octoploids both cvs. ‘Smoky’ and ‘Martin’ were obtained after treatment with 250 mg L⁻¹ colchicine and 5 mg L⁻¹ APM;
Trifluralin revealed the highest phytotoxic activity among the antimitotics used in Saskatoon berry mitotic polyploidization;
Periodic cooling of in vitro shoot cultures increased the multiplication rate of octoploid plants of tested cultivars;
In octoploid Saskatoon berry plants, the length of the stomata was significantly greater and their number was smaller compared with their tetraploid counterparts;
The newly obtained octoploids Saskatoon berry showed slow growth and a tendency to premature dormancy;
It is assumed that the phenomenon of slow growth and premature dormancy of newly formed octoploids Saskatoon berry may be associated with a change in the level and pattern of DNA methylation. Further research is required to confirm this hypothesis as well as to find factors that stimulate the growth of synthetic octoploids.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12051215/s1, Figure S1: Chromosome counts of Amelanchier genotypes. a. ‘Smoky’ (2n = 4x = 68). b. ‘Smoky’ S 8-1 (2n = 8x = 136). c. ‘Smoky’ S 8-2 (2n = 8x = 136). d. ‘Martin’ (2n = 4x = 68). e. ‘Martin’ M 8x-1 (2n = 8x = 136). f. ‘Martin’ M 8x-2 (2n = 8x = 136). g. ‘Martin’ M 8x-3 (2n = 8x = 136). h. ‘Martin’ M 8x-4 (2n = 8x = 136). Bars represent 5 µm.; Figure S2: Histograms of nuclear DNA/ploidy level estimation using flow cytometry (FCM-PI) with internal standards: Solanum lycopersicum (2C DNA = 1.96 pg) for the Amelanchier alnifolia genotypes. a. ‘Smoky’ control plant S4x (2C DNA = 2.28 pg). b. ‘Smoky’ S 8-1 (2C DNA = 4.60 pg). c. ‘Smoky’ S 8-2 (2C DNA = 4.64 pg). d. ‘Martin’ control 4x (2C DNA = 2.29 pg). e. ‘Martin’ M 8x-1 (2C DNA = 4.55 pg). f. ‘Martin’ M 8x-2 (2C DNA = 4.63 pg). g. ‘Martin’ M 8x-3 (2C DNA = 4.57 pg). h. ‘Martin’ M 8x-4 (2C DNA = 4.56 pg). Table S1: Stages of obtaining, micropropagation, rooting and acclimatization of Saskatoon berry octoploids.

Author Contributions

Conceptualization, D.K. and M.P.; methodology, D.K., A.T., M.P., S.P., A.M.-C. and Ł.S.; formal analysis, D.K. and A.T.; investigation, D.K., A.T., M.P., S.P., A.M.-C. and Ł.S.; writing—original draft preparation, D.K. and M.P.; writing—review and editing, D.K., M.P. and S.P.; funding acquisition, D.K. and Ł.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Agriculture and Rural Development (grant number 3.11/21).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Induction and detection of octoploids. (a) In vitro shoot cultures of Saskatoon berry cv. ‘Smoky’ in the control medium and with the addition of 5 and 10 mg L⁻¹ APM. (b) Histograms of cytometric analysis FCM-DAPI; the position of the peak of nuclear DNA fluorescence on the X axis for tetraploid (left) and octoploid (right); on the Y axis, the number of nuclei with a specified level of fluorescence. (c) Metaphase chromosomes of tetraploid cv. ‘Martin’ (2n = 4x = 68). (d) Metaphase chromosomes of octoploid ‘Martin’ (2n = 8x = 136). Histograms of nuclear DNA estimation using flow cytometry (FCM-PI) with internal standard: Solanum lycopersicum (2C DNA = 1.96 pg) for the genotypes: (e) ‘Martin’ control 4x (2C DNA = 2.29 pg), (f) ‘Martin’ M 8x-3 (2C DNA = 4.57 pg).

Figure 2. In vitro propagation and rooting of octoploids (a) In vitro shoot multiplication of ‘Martin’. (b) In vitro shoot rooting of ‘Martin’. (c) In vitro rooted shoots (4x was about 3 cm, 8x was about 1.5 cm long). (d) Acclimatization of the Saskatoon berry ‘Martin’.

Table 1. The shoot multiplication and the primary shoot explant survival of Amelanchier alnifolia cvs. ‘Smoky’ and ‘Martin’ in vitro in the first and second subcultures following antimitotic treatments.

Antimitotics (mg L⁻¹)	1st Subculture		2nd Subculture
	Number of Shoots/Explant	Explant Survival (%)	Number of Shoots/Explant	Explant Survival (%)
	‘SMOKY’
Control	10.0 a *	100	8.5 a	100
Oryzalin 5	7.1 ab	96.4	4.0 ab	93.7
Oryzalin 10	4.9 b	87.5	3.1 b	87.5
Colchicine 125	7.1 ab	100	5.3 ab	87.5
Colchicine 250	5.8 b	93.7	4.6 ab	93.7
Trifluralin 50	-	0	-	0
Trifluralin 100	-	0	-	0
APM 5	7.6 ab	93.7	5.9 ab	93.7
APM 10	3.7 b	81.3	3.7 ab	56.2
p **	0.000		0.044
	‘MARTIN’
Control	11.8 a	100	6.6 ab	100
Oryzalin 5	5.1 b	100	10.1 a	100
Oryzalin 10	3.0 b	100	4.6 b	87.5
Colchicine 125	7.4 ab	93.7	7.8 ab	87.5
Colchicine 250	3.6 b	93.7	5.6 ab	87.5
Trifluralin 50	-	0	-	0
Trifluralin 100	-	0	-	0
APM 5	7.6 ab	93.7	5.4 ab	100
APM 10	5.4 b	100	3.5 b	100
p	0.000		0.003