Effective micropropagation of kale (Brassica oleracea convar. Acephala var. sabellica): one of the most important representatives of cruciferous crops

Kale (Brassica oleracea convar. acephala var. sabellica) is one of the oldest Brassica vegetable varieties with the highest nutritional value and stress resistance. This work aimed to establish an efficient kale micropropagation protocol. Shoot tips were used as explants during axillary shoots multiplication. The most effective cytokinin (6 and 10 shoots per explant after the 1st and 2nd passage, respectively) was 2.5 mg dm−3 6-benzylaminopurine. Rooting was the most effective on MS medium supplemented with 1.0 mg dm−3 indole-3-acetic acid (IAA; 95% rooted shoots). The highest survival rate during acclimatization to ex vitro conditions occurred when plantlets were planted in a soil and perlite mixture. No changes in DNA content were detected using flow cytometry. This paper additionally emphasizes problems associated with the abnormal development of some multiplied Brassicaceae shoots. Nevertheless, the results confirmed that kale micropropagation was successful without any phenotypic aberration. An effective and successful kale micropropagation procedure was developed. Kale is one of the oldest Brassica oleracea varieties and obtaining a stable culture is highly desirable for further research, especially for focused stress response and resistance studies.


Introduction
The Brassicaceae is a large angiosperm family comprising many species valuable for research, agronomy, and industry. However, some Brassica species are recalcitrant in micropropagation (Mollika et al. 2011). Moreover, in vitro plant material responses are highly genotype-specific. Thus, even within a single species, large variation exists in regeneration potential (Farooq et al. 2019). Brassica oleracea cultivation is important for global food systems, acting as a source of leaf and root vegetables, fodder, and forage (Mabry et al. 2021). Kale (Brassica oleracea convar. acephala var. sabellica) is one of the oldest cruciferous plant varieties, dating back to ancient Greek and Roman times (Arias et al. 2021). It has the highest nutritional 1 3 value, and the highest anticancer sulforaphane content, among all Brassica vegetables (Jurkow et al. 2019;Sasaki et al. 2012). Furthermore, kale has the greatest cold tolerance of the B. oleracea complex (Altinok and Karakaya 2003), and can also tolerate other abiotic stresses, such as high levels of salinity and drought (Bauer et al. 2022). Up to date, kale cultures have only been obtainable from isolated microspores (Wang et al. 2011;Zhang et al. 2008), anthers (Kieffer et al. 1993), cotyledons, and hypocotyl explants (Dai et al. 2009), whereas biochemical research has focused on hairy roots (Lee et al. 2016). However, using hypocotyls or cotyledons as explants for micropropagation can cause genome size instability for regenerated plantlets due to the presence of endopolyploid cells. To minimize the probability of occurrence of such somaclonal variation, the recommended micropropagation starting materials are shoot tips. Such a procedure has not yet been developed for kale until now. Stable in vitro shoots culture will allow large quantities of homogenous material to be obtained for biochemical and genetic experiments, which are related to specific kale properties.
In vitro culture efficiency depends on several factors, such as plant species, genotype, explant type, medium composition, and physicochemical conditions. The most critical step during the optimization of a medium composition is selecting a plant growth regulator (PGR) type and its concentration or proportion. The most important PGR groups are cytokinins and auxins. Among cytokinins (CKs) two groups generally exist: adenine-type and phenylurea-type (Abu-Romman et al. 2015). Adenine-type CKs are compounds with an isoprene or aromatic side chain attached to the N 6 amino group. The most commonly used isoprenoid CKs are natural N 6 -(2-Isopentenyl)adenine (2iP) and zeatin. The most frequently used aromatic CKs are natural kinetin (Kin) and synthetic 6-benzylaminopurine (BAP) (Haberer and Kieber 2002;Barciszewski et al. 1996). In addition to the above-mentioned, synthetic, phenylurea-type CKs also exist, such as diphenylurea and thidiazuron (TDZ) (Fathy et al. 2022;Nogué et al. 2003).
The aim of this study was to develop a simple and efficient system for kale micropropagation via shoot tips. Three different cytokinins were analyzed, namely N 6 -(2-isopentenyl) adenine (2iP; natural, isoprenoid), 6-benzylaminopurine (BAP; synthetic, aromatic), and kinetin (Kin; natural, aromatic). In the micropropagation rooting stage, two different auxins were selected, namely natural Indole-3-acetic acid (IAA) and synthetic 1-Naphthalene acetic acid (NAA). Flow cytometry (FCM) was used to confirm the genome size stability of plant material originating from in vitro culture. Successful kale propagation under in vitro conditions can provide a starting point for research on other varieties. It also has great potential for improving crop quality in an ever changing environment, by selecting individuals with the highest stress resistance or producing plant material for genetic transformation.

Plant material and shoot proliferation
Seeds of kale 'Halbhoher grüner Krauser' (Brassica oleracea convar. acephala var. sabellica) were sterilized with 70% EtOH for 30 s, followed by 20% sodium hypochlorite solution for 20 min. The seeds were then rinsed several times in sterile, distilled water. For germination seeds were placed in 540 mL glass jars containing 60 mL MS (Murashige and Skoog 1962) medium with pH 5.75, supplemented with 3% sucrose and solidified with 0.8% agar (basal medium). Culture was maintained at 22 ± 1 °C under 14/10 photoperiod with a quantum irradiation intensity of 100 µmol m −2 s −1 photosynthetically active radiation (PAR). After 14 days, single shoot tips isolated from seedlings were transferred onto proliferation MS medium supplemented with one of the three selected cytokinins, viz. N 6 -(2-isopentenyl)adenine (2iP), 6-benzylaminopurine (BAP) or kinetin (Kin) at three concentrations (1 mg dm −3 , 2.5 mg dm −3 , 4 mg dm −3 ) for shoots multiplication and placed at the same conditions. As a thermolabile compound, 2iP was added to the medium post-autoclaving via sterile filtration (0.22 μm Millipore filters). Isolation and transfer of the shoot tips onto fresh medium was repeated twice. After first 4 weeks (after the 1st subculture) and after the following 3 weeks (after the 2nd subculture) the multiplication rate (the mean number of shoots per explant), percentage of rooted shoots, length of the highest shoot, fresh (FW) and dry (DW) weight of the obtained shoots were established.

Rooting
Shoots obtained on MS medium with addition of 2.5 mg dm −3 BAP were individually transferred to the rooting MS medium supplemented with indole-3-acetic acid (IAA) or 1-naphthaleneacetic acid (NAA) at three concentrations (0.5 mg dm −3 , 1.0 mg dm −3 , 1.5 mg dm −3 ) and to the MS medium without any PGR (control). Thermolabile IAA was added post-autoclaving via sterile filtration (0.22 μm Millipore filters). The percentage of rooted shoots, the length of the shoot and the longest root, and callus weight were established after 3 weeks of culture under the same conditions as germination of the seeds and shoots proliferation.

Acclimatization
Plantlets were removed from the culture jars, roots were thoroughly and gently washed in sterile water to remove residual medium and placed into commercial propagators containing three different sterile substrates: soil, soil and vermiculite (1:1) or soil and perlite (1:1). Plantlets in the propagators were placed in the growth chamber (22 ± 1 °C, 14/10 photoperiod with quantum irradiation intensity of 100 µmol m −2 s −1 PAR, humidity between 55 and 60%) and covered with a transparent lid. Initial watering was performed using sterile half-strength liquid MS medium (1/2 MS) without sucrose for 5 days and then pure water was used. After 1 week plantlets were gradually hardened to low humidity and fully uncovered after 3 weeks. Plantlets survival (%) was established after 4 weeks.

Flow cytometry
Leaves of plantlets after 1st passage, grown on MS supplemented with different CKs at 2.5 mg dm −3 , and of plantlets after a 3-week acclimatization, proliferated with 2.5 mg dm −3 BAP and rooted using MS medium supplemented with 1.0 mg dm −3 IAA, were used for sample preparation. Leaves of plants obtained from seeds, cultivated in pots for 12 weeks, were used as a control. Samples were prepared as previously described (Sliwinska and Thiem 2007) using nuclei isolation buffer (0.1 mM TRIS-Cl, 2.5 mM MgCl 2 6H 2 O, 85 mM NaCl, 0.1% [v/v] Triton X-100; pH = 7.0) supplemented with propidium iodide (50 µg cm −3 ) and ribonuclease A (50 µg cm −3 ). Solanum lycopersicum (1.96 pg/2 C; Doležel et al. 1992) served as an internal standard. The nuclear DNA content was estimated using a CyFlow SL Green (Partec GmbH, Münster, Germany) flow cytometer. For each sample, the nuclear DNA content in 5000-6000 nuclei was measured using linear amplification. Histograms were evaluated using the FlowMax (Partec GmbH, Münster, Germany) program. The coefficient of variation of the G 0 / G 1 peak of the Brassica species ranged from 2.65 to 4.87%. The nuclear DNA content was calculated using the linear relationship between the ratio of the 2 C peak positions of Brassica/Solanum on a histogram of fluorescence intensities.

Statistical analysis
For all steps of micropropagation eight explants/plantlets were used for each treatment and the experiments were repeated three times. FCM analysis was performed in five biological replications. Data were evaluated by ANOVA followed by post-hoc Tukey's test (p = 0.05) in Statistica ver.14 (StatSoft, Inc).

Results and discussion
In this work, shoot multiplication occurred after the 1st MS medium subculture using any of the selected CKs, except for the medium supplemented with 1 mg dm −3 Kin ( Table 1). The highest proliferation rate, among all tested CKs, was obtained on MS medium supplemented with BAP, and the most effective concentration of this PGR was 2.5 mg dm −3 (6 shoots per explant; Table 1; Fig. 1a). Exposure to the other two cytokinins, 2iP and Kin, was less effective, even for the highest concentration (4 mg dm −3 ). Shoot elongation was the highest on the medium with 2.5 mg dm −3 2iP (about 30 mm). However, shoot lengths remained similar regardless of CK type and its respective concentration. More pronounced differences occurred for shoot fresh (FW) and dry weight (DW). The highest FW (from 231 mg at 4.0 mg dm −3 to 287 mg at 1.0 mg dm −3 ) and DW (from 17 mg at 4.0 mg dm −3 to 28 mg at 2.5 mg dm −3 ) was obtained for shoots grown on a medium supplemented with BAP, and when 2iP at 2.5 mg dm −3 was added (FW 268 mg and DW 30 mg). Clustered adventitious shoots, obtained on the medium supplemented with 2.5 mg dm −3 and 4 mg dm −3 BAP, did not produce roots. An increased 2iP and Kin concentration decreased root formation.
After the 2nd subculture, the shoot proliferation rate increased (Table 1). Nevertheless, BAP remained the most effective CK. Furthermore, BAP at 2.5 mg dm −3 demonstrated significant superiority compared to the other treatments (Fig. 1b), with an average of 10 shoots per explant. Shoot lengths and weights were also higher after the 2nd multiplication, especially in response to 2.5 mg dm −3 2iP in the medium. This treatment resulted in shoots with an average length of 42 mm, FW of 1046 mg, and DW of 87 mg. Shoots from the medium supplemented with BAP did not form roots. For 2iP, fewer rooted shoots were observed after the 2nd passage compared to the 1st, in contrast to Kin, for which further micropropagation stimulated root formation (Table 1). Altogether, a three-way ANOVA indicated that all analyzed parameters of the multiplied shoots were significantly affected by CK type (A) and concentration (B), as well as the number of subcultures (C). The interaction of these factors (A × B × C) was also significant (Table S1). Different organogenic responses of varied plant species are associated with differences in exogenous CK uptake and its subsequent metabolism (Auer et al. 1992). In this study, BAP produced the highest stimulating effect on shoot multiplication, and inhibiting effect on root formation of kale. Similar results have been obtained for four other B. oleracea varieties (red cabbage, cauliflower, broccoli, and Savoy cabbage), as well as for fourteen cabbage genotypes, where adventitious shoot regeneration was higher on a medium with 1 mg dm −3 BAP compared to Kin (Pavlović et al. 2010(Pavlović et al. , 2012. For some varieties, the highest shoot regeneration frequencies and multiplication rates were obtained when CKs were added in combination with auxins at the lowest concentration. For instance, the highest shoot propagation rate was reported for ornamental kale hypocotyl explants exposed to 3.0 mg dm −3 BAP and 0.1 mg dm −3 NAA (Dai et al. 2009). Similarly, the highest B. campestris shoot regeneration rate, from cotyledonary explants, was obtained on medium supplemented with 5 mg dm −3 BAP and 0.5 mg dm −3 NAA (Zhang et al. 1998), or 3.0 mg dm −3 BAP and 0.2 mg dm −3 NAA (Mollika et al. 2011). Furthermore, broccoli micropropagation was the most effective in the presence of 1.5 mg dm −3 BAP and 1.0 mg dm −3 NAA (Azis et al. 2015). For B. juncea, the best shoot regeneration results were obtained for MS with 2.0 mg dm −3 BAP, 0.2 mg dm −3 NAA, and 0.5 mg dm −3 Kin (Mollika et al. 2011). BAP, in combination with auxin, is optimal for Brassica shoot regeneration and multiplication (Gerszberg et al. 2015;Maheshwari et al. 2011). However, in contrast to this and regardless of the concentration, auxin addition in this study stimulated root formation with a simultaneous reduction in shoot growth. Thus, a cytokinin-auxin combination for shoot multiplication was not considered here.
A high CK concentration leads to hyperhydricity in Brassica species, and consequently to leaf curling and vitrification (Kumar and Srivastava 2015;Ravanfar et al. 2009Ravanfar et al. , 2014. In some studies, obtained shoots considered as normally formed were stunted and curled, indicating vitrification and/or fasciation, similar to cauliflower hypocotyl explants treated with TDZ (Siong et al. 2012). In the present work, single abnormal shoots occurred regardless of CK addition; however, this occurred only at the highest applied concentration (4 mg dm −3 , Fig. S1). Such shoots appeared only after the 1st subculture and were discarded from the culture. After the 2nd subculture and onward, no more abnormalities were reported (not even once in over a year of in vitro culture; Fig. S2). Furthermore, only shoots that regenerated from callus demonstrated abnormal morphology, which suggests that the high stimulation of callus propagation may induce variation in plantlet morphology. A similar observation was assigned to the high concentration of TDZ (Dewir et al. 2018), CK commonly used in Brassica micropropagation. This study confirmed that other CKs can also cause abnormalities during the first few weeks of in vitro conditions. Adding an auxin to the medium, regardless of the concentration, significantly stimulated root formation in the kale shoots in comparison to the control obtained on the MS medium without PGRs suplementation (Table 2; Fig. 2). The formed roots were very thin and clumped, which made it impossible to count them; however, they were numerous in all treatments, except for 1.5 mg dm −3 NAA. For this reason, the percentage of rooted shoots, the length of the longest root and shoot, and callus weight were all used instead (Table 2). Based on this data, the most effective treatment for rooting is 1.0 mg dm −3 IAA. In this treatment, 95% of all the shoots formed roots, and these roots and shoots were the longest (57 and 66 mm, respectively). The highest rooting efficiency coincided with the lowest callus biomass (16 mg) also in shoots treated with NAA, for which the most effective concentration was 0.5 mg dm −3 . Generally, shoots formed shorter roots on medium supplemented with NAA compared to IAA. Similar effects were observed for kale cotyledon explants and broccoli shoots: in response to NAA, explants formed numerous thick and white roots, whereas IAA application resulted in a few, but long roots (Adil and Abbasi Fig. 1 The effect of different cytokinins in concentration of 2.5 mg dm −3 on shoots multiplication after the 1st (a) and 2nd (b) subculture. Bar = 1 cm  Ravanfar et al. 2009). In this study, an increased NAA concentration decreased root formation from shoots, but stimulated callus proliferation. Similarly, for B. rapa effective rooting occurred at a low (0.1 mg dm −3 ) NAA concentration (Zhao et al. 2021), and the tendency to form callus in the presence of increasing NAA levels occurred at cabbage shoot bases (Munshi et al. 2007). Here, the type of auxin, its concentration, and a combination of these affected root and shoot length, as well as callus formation, but only auxin concentration significantly affected rooting frequency (Table S2). Regardless of the rooting process, microplantlets acclimatized to ex vitro conditions (Table S3; Fig. S3). Comparing the substrata used for acclimatization of in vitro-derived kale microplantlets, the highest survival rate result from a mixture of soil and perlite (1:1). Successful acclimatization in a mixture containing perlite was also reported for cabbage (soil:perlite, 3:1) and Chinese cabbage (perlite alone) (Gerszberg et al. 2015;Sivanandhan et al. 2019). Perlite is well-known for soil aeration and water absorption (Ilahi and Ahmad 2017), thus mixture with perlite is the easiest way to provide optimal conditions for microplantlets. In this experiment it also limited the occurrence of mildew due to the high humidity under the cover.
Control of genetic stability is crucial during in vitro culture (Dubrovna and Bavol 2011). PGRs used for micropropagation, especially CKs, integrate into cellular machinery that regulate the cell cycle, and induce undesirable genome size changes (Howell et al. 2003;Schaller et al. 2014). Genomic irregularities mainly concern cell and callus culture (Larkin and Scowcroft 1981;Phillips et al. 1994;Skirvin et al. 1994). The most common method currently applied to detect changes in DNA content is FCM. FCM has widely been used for genome size and ploidy estimation for many species under in vitro conditions (Sliwinska 2018), including different Brassica species and varieties. For example, FCM analysis revealed genome size stability for B. rapa regenerants obtained from cotyledon explants (Zhao et al. 2021), B. juncea plantlets obtained via somatic embryogenesis (Faisal et al. 2021), and B. oleracea var. gongylodes regenerated in vitro from different explants (Ċosiċ et al. 2015). Similarly, no changes in DNA content for in vitro produced kale plants were observed in this study (Table 3). Control plants obtained from soil grown

Conclusions
An efficient protocol was developed for producing stable adventitious shoots and development of kale plants. The highest shoot multiplication rate was obtained on the MS medium supplemented with 2.5 mg dm −3 BAP. The rooting of the shoots was the most effective in the presence of 1.0 mg dm −3 IAA. Microplantlets acclimatized well to ex vitro conditions in a soil and perlite mixture. After extensive growth in the presence of cytokinins and adaptation to ex vitro conditions, no changes in DNA content occurred compared to plants obtained from soil grown seeds.
Author contributions MK designed experiment, maintained in vitro cultures, analyzed the results and drafted the manuscript; ES carried out the flow cytometric analysis, evaluated the results and contributed to the writing the manuscript.

Funding
The study was financially supported by the Ministry of Science and Higher Education through the Faculty of Biology, University of Warsaw (Grant No. 501-D114-01-1140100).

Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

Conflict of interest The authors have no conflict of interest.
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