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Influence of Genotype and Slaughter Age on the Content of Selected Minerals and Fatty Acids in the Longissimus Thoracis Muscle of Crossbred Bulls

Simple Summary In some European countries, including Poland, beef production is based mostly on dairy cattle herds. Beef quality can be improved by the commercial crossbreeding of dairy cows with beef bulls. The offspring are characterized by higher fattening performance and more desirable carcass characteristics. The experiment was performed on 72 bulls produced by crossing Polish Holstein-Friesian (PHF) cows with bulls of three most popular beef breeds: Hereford (HH), Limousin (LM), and Charolais (CH), to determine the effects of genotype and age at slaughter on the content of selected minerals (K, Na, Mg, Zn, and Fe) and fatty acid profile in beef. The meat of PHF × LM crosses had a lower content of K and Mg, compared with the remaining crossbred bulls. The Fe content per kg of meat was higher in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The best sire breed for crossing with dairy cows cannot be clearly indicated based on the present findings. However, the results of this study suggest that bulls should be slaughtered at 21 months of age to achieve the optimal values of most analyzed traits and parameters, in particular the fatty acid profile. Abstract The aim of this study was to determine the effects of genotype and slaughter age on the mineral content and fatty acid profile of beef. The experiment was performed on 72 crossbred bulls produced by crossing Polish Holstein-Friesian (PHF) cows with bulls of three beef breeds: Hereford (HH), Limousin (LM), and Charolais (CH), slaughtered at 15, 18, and 21 months of age. Samples of the longissimus thoracis muscle were collected to determine their mineral (potassium—K, sodium—Na, magnesium—Mg, zinc—Zn, and iron—Fe) and fatty acid composition. The meat of PHF × LM crosses had a lower (p ≤ 0.01) content of K and Mg, compared with the remaining crossbred bulls. The Fe content per kg of meat was 4 mg higher (p ≤ 0.01) in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The content of monounsaturated fatty acids (MUFAs) in intramuscular fat was 2.77% higher in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The n-6/n-3 polyunsaturated fatty acids (PUFA) ratio did not exceed 4.0, and it was most desirable in PHF × LM crosses (2.84) and in the oldest bulls (2.92).

Introduction
Red meat, including beef, is an important source of highly biologically active proteins, fat, vitamins, and minerals (in particular, iron-Fe and zinc-Zn). Fat is the richest source of dietary energy, and it supplies essential nutrients such as fat-soluble vitamins and fatty acids [1]. Bovine fat contains fatty acids that deliver health benefits to humans. n-3 and n-6 polyunsaturated fatty acids (PUFAs) must be derived from the diet because they cannot be synthesized de novo by the human body or other monogastric mammals. n-3 and n-6 PUFAs act as carriers of fat-soluble vitamins (A, D, E, and K) and play a key role in the immune response. Linoleic acid, a member of the n-6 fatty acid family, contributes to lowering serum cholesterol levels and reducing the risk of cardiovascular disease. It is an important structural constituent of cell membranes, a function thought to be related to dermatitis in infants [2]. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), members of the n-3 fatty acid family, help prevent cardiovascular disease and arrhythmia, lower plasma triacylglycerol levels, decrease blood pressure, reduce platelet aggregation, and exert anti-inflammatory effects [3,4]. Conjugated linoleic acid (CLA) is a group of isomers of linoleic acid found mostly in the meat and dairy products derived from ruminants. The major CLA isomers are cis-9 trans-11, and trans-10 cis-12. Research has shown that CLA has immunomodulatory, antimutagenic, anticarcinogenic, antidiabetic (type II diabetes), and antihypertensive properties. It can also play a role in preventing obesity, atherosclerosis, lifestyle diseases, and the metabolic syndrome [5][6][7]. Vaccenic acid is a trans fatty acid, but it is believed to provide health benefits to humans. It is the predominant trans fatty acid found in red meat, and it does not affect the blood levels of total or low-density lipoprotein (LDL) cholesterol [8]. Zn is important for the immune system; it participates in the regulation of cell growth and proliferation, is a component of numerous metalloenzymes, and contributes to antioxidant defense. The major sources of Zn in the human diet are red meat, seafood, poultry, cereals, dairy products, legumes, and vegetables [9]. Red meat is a rich source of readily bioavailable Zn, whereas cereals have a high content of phytic acid, a strong inhibitor of Zn absorption [10]. Fe plays an important role as an oxygen carrier in hemoglobin (in red blood cells) and myoglobin (in muscle cells), and it is involved in numerous metabolic processes [1]. Heme Fe found in meat is more bioavailable than non-heme Fe found in plant-based foods [9]. Magnesium (Mg) has a strong relation with the immune system, it is involved in both non-specific and specific immune responses, also referred to as innate and acquired immune responses, respectively [11].
Mineral concentrations in meat change depending on the diet [12], genotype and age of an animal [13], muscle type [14][15][16], and geographic conditions [17,18]. The fatty acid composition of beef is mostly affected by the breed, age, and sex category of an animal [19,20], as well as dietary factors [21,22]. The differences in the fatty acid profile between cattle breeds result from the differences in gene expression and the activity of enzymes that participate in fatty acid synthesis [23]. As animals grow older, the content of subcutaneous tissue and muscular fat increases, whereas the ratio of PUFAs to saturated fatty acids (SFAs) decreases [24]. Furthermore, pre-slaughter stressors can impact on ruminant carcass and meat quality. Loudon et al. [25] observed that plasma creatine kinase was positively correlated with ultimate pH, indicating that cattle with higher muscle trauma had a higher rate of glycogen depletion pre-slaughter.
In Poland, beef production is based mostly on dairy cattle herds. Beef quality can be improved by the commercial crossbreeding of dairy cows with beef bulls. The offspring are characterized by higher fattening performance and more desirable carcass characteristics [26]. The most popular beef cattle breeds in Poland are Hereford (HH), Limousin (LM), and Charolais (CH), and bulls of these breeds are usually used as the paternal component for crossing with dairy cows. The aim of this study was to determine the effects of genotype and age at slaughter on the content of selected minerals and fatty acid profile in meat from crossbred bulls produced by crossing Polish Holstein-Friesian (PHF) cows with HH, LM, and CH bulls.

Animals
The study was conducted upon the approval of the Ethics Committee of the University of Warmia and Mazury (decision no. 121/2010). The experimental material comprised 72 crossbred bulls produced by crossing PHF cows with HH, LM and CH bulls. Crossbred calves were purchased at 2-3 weeks of age. The animals underwent a seven-day quarantine period and were raised until six months of age. They were fed milk replacer supplemented with hay and concentrate, followed by grass silage. The fattening period began when the calves were 181 days old. The animals were fattened in a semi-intensive system, and they were fed ad libitum a total mixed ration (TMR) composed of grass silage and concentrate (Tables 1 and 2). The concentrate to silage ratio, on a dry matter (DM) basis, was 25:75 (Table 2). When the bulls reached a body weight (BW) of 300 kg, concentrate composition was modified to account for age-related changes in the dietary protein to energy ratio. Bulls with BW below 300 kg were fed a TMR containing concentrate I (triticale, 71%; rapeseed meal, 25%; premix (Cargill Poland Ltd., Warsaw, Poland), 4%), and bulls with BW above 300 kg were fed a TMR containing concentrate II (triticale, 77%; rapeseed meal, 19%; premix, 4%). The animals were fattened in a free-stall barn until 15, 18, and 21 months of age.

Carcass Quality
At the end of the fattening period, the bulls were transported to a meat processing plant, where they were fasted for 24 h and slaughtered. The animals were weighed before slaughter, stunned, slaughtered, bled, dressed, and split into two sides that were then chilled for 96 h at +4 • C. Electrical stimulation was not applied to the carcasses. Half-carcasses were weighed within an accuracy of 0.5 kg, using an automatic in-line scale. Carcass conformation and fatness were evaluated by a trained grader, based on the EUROP system criteria [27]. Dressing percentage (percentage ratio of carcass weight to slaughter weight) was calculated. After 96 h of carcass chilling, samples weighing approximately 200 g were collected from the longissimus thoracis (LT) muscle between the 11th and 13th thoracic vertebrae. Vacuum-packaged samples were transported (+4 • C) to the laboratory. Slaughter and post-slaughter processing were carried out in accordance with Council Regulation (EC) No 1099/2009 of 24 September 2009 on the protection of animals at the time of slaughter [28]. During the experiment, the animals remained under veterinary care and were regularly visited by an animal care specialist.

Mineral Content of Meat
In order to determine the content of selected minerals, samples of the LT muscle were homogenized, and approximately 0.5 g of the muscle tissue was wet mineralized with 7 mL of 65% Suprapur nitric acid (Merck, Darmstadt, Germany) in the MARS Xpress 5 microwave digestion system (CEM Corporation, Matthews, NC, USA). Each mineralization procedure involved two blank samples and two samples of Standard Reference Material ® 1577c Bovine Liver (NIST, Gaithersburg, MD, USA). All samples were mineralized and analyzed in duplicate. The mineralized samples were cooled and quantitatively transferred to 25 cm 3 volumetric flasks. The content of selected minerals (potassium-K, sodium-Na, magnesium-Mg, zinc-Zn, and iron-Fe) in beef samples was determined by flame atomic absorption spectrometry (flame AAS) with the use of the AA240FS Fast Sequential Atomic Absorption Spectrometer (Varian, Palo Alto, CA, USA) [29,30].

Fatty Acid Profile
Intramuscular fat (IMF) was extracted from ground meat samples by the Soxhlet method [31] using the B-811 extraction system (Büchi, Flawil, Switzerland), with n-hexane as the solvent (POCH, Gliwice, Poland). Fatty acid methyl esters (FAMEs) were obtained by methylation in a methanol-chloroform-H 2 SO 4 mixture (POCH, Gliwice, Poland; STANLAB, Lublin, Poland; STANLAB, Lublin, Poland, respectively) according to the modified Peisker method [32] and Standard PN-EN ISO 5509 [33]. The content of 33 fatty acids in IMF was determined with the use of the CP-3800 gas chromatograph (Varian, Palo Alto, CA, USA) equipped with a split/splitless injector and a flame-ionization detector (FID). Samples (1 µL) of FAMEs were placed on the Varian Capillary Column Select™ FAME (length: 100 m; inner diameter: 0.25 mm) using a Hamilton syringe and the CP-8410 autosampler (Varian, Palo Alto, CA, USA). Helium was used as the carrier gas. Injector temperature was 260 • C. The total time of a single analysis was 68 min. Fatty acids were identified by comparing their retention times with the retention times of individual reference standards and their mixtures, purchased from Supelco (Sigma Aldrich, Bellefonte, PA, USA). Analyses of the samples and reference standards were performed under identical separation conditions. The instrument was controlled, and the data were acquired and processed using the GALAXIE Chromatography Data System. Fatty acids were divided into the following categories; saturated fatty acids (SFAs), unsaturated fatty acids (UFAs), including monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs), as well as n-6 and n-3 PUFAs. The following ratios were calculated; MUFA/SFA, PUFA/SFA, and n-6/n-3 PUFA.

Statistical Analysis
The data were analyzed statistically using Statistica version 13.3 software [34]. The effects of genotype (PHF × HH, PHF × LM and PHF × CH) and slaughter age (15,18, and 21 months) on the fatty acid profile and mineral content of beef were determined by the least squares method using the formula where Y ijk is the value of the analyzed parameter, µ is population mean, A i is the effect of genotype (PHF × HH, PHF × LM, PHF × CH), B j is the effect of slaughter age (15,18,21), (AB) ij is the genotype × slaughter age interaction, and e ijk is random error. The differences between means were estimated by Tukey's test.

Results and Discussion
As expected, older animals were characterized by higher BW at slaughter (p ≤ 0.01) ( Table 3). The final BW of crossbred bulls was not affected by sire breed although the beef breeds used for crossing (HH, LM, and CH) represent different maturity types. Genotype had a significant effect on carcass dressing percentage and fat content. The dressing percentage of PHF × LM crosses was 1.79% higher (p ≤ 0.01) than that of PHF × CH crosses. Dressing percentage is a key indicator of carcass quality in cattle. The LM breed is known for its high carcass quality, which was confirmed by Chambaz et al. [35] in whose study, dressing percentage was 4.4% higher in LM steers than in CH steers. In the present experiment, PHF × HH crosses received the highest fatness score, and the difference relative to PHF × LM crosses was statistically significant (p ≤ 0.05). Carcass dressing percentage, conformation, and fatness were significantly affected by age at slaughter. Dressing percentage was higher in heavier animals, which could result from their higher carcass fatness. In a previous study investigating young Holstein-Friesian bulls [36], higher BW at slaughter was associated with higher carcass fat content. The mineral composition of the LT muscle of crossbred bulls is presented in Table 4. Genotype had a significant influence on the concentrations of K, Mg, and Fe. Meat from PHF × CH and PHF × HH crosses had a higher (p ≤ 0.01) content of K and Mg than meat from PHF × LM crosses. Fe content was higher (p ≤ 0.05) in meat from PHF × CH crosses than in meat from PHF × LM crosses. The content of Zn and Fe in the LT muscle was affected by age at slaughter. The meat of animals slaughtered at 18 months of age had the highest (p ≤ 0.05) Zn content (63.9 mg/kg of fresh meat), whereas the meat of the oldest bulls had the highest Fe content (19 mg/kg of fresh meat). The effect of cattle breed on the K and Mg content of beef was also noted by Pilarczyk [37] and Domaradzki et al. [38]. In the study by Pilarczyk [37], the Fe content of meat was also affected by the genotype. Pilarczyk [37] found that meat from CH bulls was more abundant in K than meat from HH and Simmental bulls. K concentrations in meat samples collected from LM, Red Angus, Salers, CH, HH, and Simmental bulls [37,39] were considerably lower (2377.0-2506.1 mg/kg of fresh meat) than those noted in the current experiment (4750.2-4979.8 mg/kg of fresh meat). The K content of meat from Angus bulls, reported by Mateescu et al. [40], was also lower than the values determined in the present study. One-hundred grams of meat supplied 5.7-6% of the adequate intake for Na for adults. In the case of K, consumption of 100 g of cooked beef supplied 19.8-20.8% of the daily standard for men and 25.9-27.1% for women [41]. In contrast to our findings, Doornenbal and Murray [42], and Kotula and Lusby [43] demonstrated that age at slaughter affected K levels in beef. However, Doornenbal and Murray [42] observed changes in K concentrations in cattle aged 3 to 12 years. Kotula and Lusby [43] reported that the K content of meat was influenced by age at slaughter in steers aged 1 to 6 years, and it decreased from 392 mg/100 g of wet tissue in 24-month-old animals to 332 mg/100 g of wet tissue in 72-month-old animals. However, the cited authors [43] noted no significant changes in the concentrations of K, Na, Mg, or Zn in Angus steers aged 12 and 24 months. In the current study, neither breed nor age at slaughter exerted significant effects on the Na content of beef. The lowest Mg content of meat was noted in meat from PHF × LM crosses and bulls slaughtered at 18 months of age (183.0 mg/kg of fresh meat and 199.3 mg/kg of fresh meat, respectively). In the remaining groups, Mg concentrations exceeded 200 mg/kg of fresh meat. Consumption of 100 g of cooked beef satisfied 8.1-9.6% and 6.2-7.3% of the recommended daily allowance (RDA) for Mg for women and men, respectively [41]. Pilarczyk [39] found that meat from HH bulls had lower Mg content than meat from CH bulls, which is consistent with the present results. Regardless of cattle breed, Pilarczyk [37,39] noted higher average Mg concentration in beef (263.4 mg/kg of fresh meat), compared with our experiment. In the present study, the Zn content of the LT muscle ranged from 38.5 to 63.9 mg/kg of fresh meat. The present study shows that cooked beef is an important source of Zn, as an edible portion of 100 g met satisfies 58.3-96.8% of their daily requirement for males and 80.2-133.1% for females [41]. Meat from bulls slaughtered at 18 months of age and PHF × HH crosses was the richest source of Mg. Beef was a rich source of Fe and the consumption of 100 g of cooked meat satisfies 13.2-16.7% and 29.7-37.6% of the RDA for females and males, respectively [41]. Littledike et al. [44] compared mineral concentrations in nine beef cattle breeds and found that Fe content was lowest in LM and Red Poll cows. In the current study, Fe content ranged from 15.0 to 19.0 mg/kg of fresh meat, and it was lowest in PHF × LM crosses. In the experiments conducted by Cabrera et al. [14] and Ramos et al. [45], Fe content ranged from 30 to 45 mg/kg of wet tissue and from 35 to 40 mg/kg of wet tissue in HH and Braford steers, respectively. In the present study, age at slaughter had a significant effect on Fe concentration in beef, which increased with age. Giuffrida-Mendoza et al. [13] demonstrated that Fe levels remained unchanged in samples collected from water buffalo and Zebu-influenced cattle aged 17 and 19 months, but decreased significantly at 24 months of age. According to Duckett et al. [46], differences in the mineral composition of beef can be attributed to differences between genetic groups, feed types and finishing systems. According to García-Vaquero et al. [47], in animals characterized by an adequate mineral status, essential trace element concentrations in muscle tissue depend on individual muscle metabolism.
The concentrations of fatty acids, major fatty acid groups, and ratios in the IMF of crossbred bulls are presented in Tables 5-7. The IMF content of the LT muscle was affected by age at slaughter and, as expected, it increased with age up to 1.80%. A significant difference in IMF content was noted between bulls slaughtered at 15 and 18 months of age. According to many authors [48,49], beef from LM cattle is lean. In the present study, IMF content was lowest in PHF × LM crosses, and highest in PHF × HH crosses, which resulted from physiological differences between early-and late-maturing breeds. Such a low IMF content of meat was affected by diets based on grass silage supplemented with concentrate. In the current experiment, age at slaughter had a significant effect on the concentrations of SFAs, UFAs and the MUFA/SFA ratio (p ≤ 0.01), and on the concentrations of MUFAs (p ≤ 0.05) in the LT muscle of bulls. The IMF of animals slaughtered at 21 months of age had a significantly higher content of UFAs and MUFAs, compared with those slaughtered at 15 months of age. Age at slaughter had a significant influence on the percentage share of the following fatty acids in IMF: oleic, C15:0 anteiso (p ≤ 0.01), capric, lauric, pentadecanoic, margaric, stearic, and gadoleic (p ≤ 0.05). The IMF of bulls slaughtered at 15 months of age had the highest concentrations of capric acid and lauric acid, whereas the IMF of the oldest bulls had the lowest content of the following fatty acids: capric, lauric, C15:0 anteiso, pentadecanoic, margaric and stearic. The IMF of the oldest bulls had the highest proportion of UFAs, including oleic acid (a significant difference was noted between animals slaughtered at 21 and 15 months of age) and gadoleic acid. The n-6/n-3 PUFA ratio was affected by genotype and age at slaughter. A significant (p ≤ 0.01) difference was observed between PHF × LM crosses (2.84) and PHF × CH crosses (3.3). The most desirable n-6/n-3 PUFA ratio was noted in bulls slaughtered at 21 months of age (2.92), with a significant (p ≤ 0.05) difference relative to those slaughtered at 15 months of age.
In the present study, UFAs were the predominant fatty acids in the IMF of bulls, and their proportion was lower than that of SFAs only in bulls slaughtered at 15 months of age. The concentrations of UFAs and MUFAs increased, whereas the proportion of SFAs decreased with age. In the fatty acid profile of IMF, PUFAs accounted for 6.73-7.47%, and their proportion was highest in PHF × CH crosses and in bulls slaughtered at 18 months of age. The most abundant fatty acids were oleic acid (34.17-36.82%), palmitic acid (25.17-26.24%), and stearic acid (17.35-19.38%). The predominant SFAs in beef are palmitic acid, stearic acid, and myristic acid. Lauric acid, myristic acid, and palmitic acid are associated with elevated plasma total and LDL cholesterol concentrations, which are not increased by stearic acid [4]. Elaidic acid, the major industrially produced trans fat, can exert adverse effects on human health by negatively affecting cholesterol metabolism [8]. In the current experiment, its concentration ranged from 0.470% to 0.523%. The minimum recommended PUFA/SFA ratio in the human diet is 0.45 [50]. Its values noted in this study were considerably lower (0.137-0.156), due to too low PUFAs concentrations in the analyzed samples. According to the British Department of Health [50], the n-6/n-3 PUFA ratio should not exceed 4.0, and it was determined at 2.84-3.30 in this study. A high dietary n-6/n-3 PUFA ratio is considered a risk factor for selected lifestyle diseases. The n-6/n-3 PUFA ratio in the modern Western diet is much too high, reaching 10-15:1, whereas its optimal range is 2-4:1 [3].
In comparison with the present study, De Freitas et al. [51] noted a lower proportion of SFAs (46.42%), a similar proportion of MUFAs (43.38%), and a considerably higher proportion of PUFAs (10.05%) and the n-6/n-3 PUFA ratio (4.87) in the meat of HH steers. Contrary to our findings, Ugarković et al. [52] found lower SFAs concentrations and higher MUFAs concentrations in beef from HH cattle, compared with the CH breed. The following fatty acids analyzed in this study deliver health benefits: vaccenic acid, oleic acid, linoleic acid, CLA, α-linolenic acid, arachidonic acid, EPA, docosapentaenoic acid (DPA), and DHA. The oleic acid content of the LT muscle increased with age. The concentrations of linoleic acid, CLA, α-linolenic acid, arachidonic acid, EPA, and DPA were highest in PHF × CH crosses, whereas PHF × LM crosses were characterized by the highest levels of vaccenic acid and DHA. Cis-9 trans-11 is the most prevalent CLA isomer, accounting for 80-90% of the total CLA in ruminant tissues [5]. In the current experiment, its content ranged from 0.278% to 0.302%.

Conclusions
Different beef breed sires used for commercial crossing affected the content of K, Mg, and Fe in beef. The LT muscle of PHF × LM crosses had the lowest content of K, Mg, and Fe. Bulls slaughtered at a later age were characterized by higher Fe content and higher concentrations of MUFAs, in particular, oleic acid and gadoleic acid. The n-6/n-3 PUFA ratio did not exceed the nutritionally desirable value of 4.0, and it was lowest in PHF × LM crosses and in bulls slaughtered at 21 months of age. The best sire breed for crossing with dairy cows cannot be clearly indicated based on the present findings. However, the results of this study suggest that bulls should be slaughtered at 21 months of age to achieve the optimal values of most analyzed traits and parameters, in particular the fatty acid profile.

Conflicts of Interest:
The authors declare no conflict of interest.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

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Abscisic acid in preservation of Taraxacum pieninicum in the form of synthetic seeds in slow growth conditions

Taraxacum pieninicum Pawł. is listed as critically endangered species, for which currently applied protection methods are insufficient. The aim of this study was to investigate the possibility of T. pieninicum storage in the form of synthetic seeds under slow-growth conditions in combination with ABA treatment, as one of the ex situ protection methods of this species. The obtained results indicated that darkness was much more favorable condition for synseed storage and did not generate additional stress during cold exposure in contrast to the light conditions. The preculture of shoot tips on the medium supplemented with ABA led to a decrease in the shoots proliferation rate and inhibition of their growth. ABA clearly inhibited growth of the encapsulated shoot tips also during cold storage. Biochemical parameters showed that ABA effectively reduced the negative effect of the cold stress, what was found on the basis of analyzes of H2O2 and TBARS levels in the stored material. Moreover, synseeds stored under light conditions and treated with ABA exhibited decreased level of endogenous jasmonic acid what indicated interaction between those two phytohormones at a low temperature. The study also demonstrated that in vitro culture, cold storage and ABA treatment had no effect on the flowering process of this species after acclimatization to ex vitro conditions. The aim of this research was to assess ABA effect on post-storage regrowth and the stress factors level in plants obtained after long-term storage of Taraxacum pieninicum synthetic seeds.

Introduction
Taraxacum pieninicum belongs to the Asteraceae family and is a stenochoric species, occurring in only one site in the Central Pieniny in the Western Carpathians (Poland) (Piękoś-Mirkowa and Mirek 2009). This species is considered to be probably the oldest endemic species in Pieniny Mts. from the Paleogene (Puchalski et al. 2014). Creating collections in botanical garden as a method of its protection is not very effective due to the difficulty of keeping it in these areas. As the only species of the Taraxacum genus, it does not produce sesquiterpene lactones, such as taraxic acid and triterpenes, commonly referred to as taraxacin in older literature, which gives the leaf a bitter Communicated by Maurizio Lambardi.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1124 0-020-01924 -0) contains supplementary material, which is available to authorized users. taste (Schütz et al. 2006). Lack of biochemical protection means that it is eagerly eaten by various herbivore species. In addition, seeds lose their viability (Honek et al. 2005), therefore the in vitro method was considered appropriate for protection of this species. An efficient regeneration system for T. pieninicum has been developed (Trejgell et al. 2013), nevertheless maintaining cultivars in in vitro conditions is costly and carries the risk of contamination (Pence 2010;Cruz-Cruz et al. 2013). For these reasons optimization of the slow growth conditions is highly desirable to maximize time between passages. Lowering the temperature of T. pieninicum shoot tips culture allowed its storage for several months (Kamińska et al. 2018a), although increased survival of the explants was noted after encapsulation (Kamińska et al. 2018b). The possibility of extending the storage time of encapsulated explants by using abscisic acid as a growth retardant was evaluated in this paper.
Growth retardants are synthetic or natural chemical compounds that can cause structural changes or affect plant life processes by direct effect on their hormonal balance. Synthetic retardants that inhibit plant elongation growth are usually gibberellin antagonists that affect their metabolism (Espindula et al. 2009). Natural growth retardants include phytohormones, as abscisic acid (ABA) and jasmonic acid (JA), which show diverse physiological activity (Heinrich et al. 2012).
Endogenous and exogenous ABA is considered as auxins and gibberellins antagonist, leading to inhibition of cell division and elongation growth (Humplík et al. 2017;Lorrai et al. 2018). In conditions of drought, salinity or low temperature stress, this compound promotes closing stomata to reduce water loss by transpiration (Kim et al. 2010), affects the change in the morphology of the root system (Sah et al. 2016) and activates many genes encoding enzymes responsible for the biosynthesis of osmoregulatory substances and LEA-type proteins (Fujita et al. 2011).
The greatest importance in the reaction of plants to abiotic stress is attributed to abscisic acid. The role of ABA in acclimatization to cold is confirmed by increased freezing tolerance in plants growing at room temperature treated with ABA (Palva et al. 2002). In addition, the involvement of ABA in the activation of genes expression related to cold acclimatization has been documented many times. In our previous studies, we have shown the possibilities of storing T. pieninicum synseeds and the usage of jasmonic acid as a growth retardant (Kamińska et al. 2018a, b). The aim of this research was to assess the effect of abscisic acid on the condition of plants obtained after long-term storage of Taraxacum pieninicum synthetic seeds. Verification of the ABA influence on T. pieninicum stored explants was based on the alterations in post-storage regrowth, biochemical stress factors, endogenous ABA and JA content.

Plant material and culture conditions
Seeds of T. pieninicum were obtained from the collection of the Polish Academy of Sciences Botanical Garden-Center for Biological Diversity Conservation in Powsin. The seeds were sterilized with 70% ethanol for 30 s, followed by 20% sodium hypochlorite solution for 20 min. After this time, the seeds were washed four times with sterile distilled water and were transferred onto MS medium (Murashige and Skoog 1962) without plant growth regulators. MS medium was supplemented with 3% sucrose, solidified with 0.8% agar and adjusted to pH 5.75-5.8 prior to autoclaving for 20 min at 121 °C.
Shoot tips of 12-day-old seedlings were isolated and transferred onto proliferation medium to induce the process of development of axillary buds and their proliferation. MS medium (see above) supplemented with 1.1 μM benzylaminopurine (BAP) and 0.14 μM naphthyl-1-acetic acid (NAA) was used for shoots multiplication (MS BAP ). Cultures were kept in a growth room at 26 ± 1 °C, under continuous light with a quantum irradiation intensity of 80 μmol m −2 s −1 (optimal growth conditions). After 4 weeks of culture, obtained axillary buds were isolated and transferred on fresh growth medium with the same composition.

Synthetic seeds production
Individual shoot tips after isolation from the culture of T. pieninicum axillary shoots were placed on a Petri dish containing 3% sodium alginate dissolved in liquid MS medium. A single explant covered with alginate solution was added dropwise to a 100 mM calcium chloride solution. Polymerization of alginate was carried out for 25 min with gentle stirring of the calcium chloride solution. Using sterile strainer, the calcium chloride solution was removed and the obtained synthetic seeds were rinsed with sterile distilled water.

Abscisic acid treatment
Two methods of ABA exposure were used: a four-week shoots preculture in the optimal growth conditions on MS BAP medium supplemented with ABA preceding encapsulation (further indication in the paper-preculture) and addition of ABA to the alginate solution during shoot tips encapsulation (inside). For both experiments four concentrations of ABA were used: 19 μM, 38 μM, 57 μM and 76 μM. After preculture proliferation rate (number 1 3 of shoots per explant) and visual evaluation of the shoots growth were additionally assessed.

Cold storage conditions
Synseeds were placed on 50 mL MS medium without plant growth regulators in polycarbonate culture boxes (Magenta™ vessel, Sigma-Aldrich). Storage was carried out for 3, 6 or 9 months at 4 ± 1 °C in conditions of continuous light with a reduced quantum irradiation intensity to 40 μmol m −2 s −1 or in the dark. Synseeds non-treated with ABA were used as a control.

Post-storage regrowth
Directly after cold-storage a visual evaluation of the shoots and roots length was performed, then alginate coat was gently removed, leaves were collected for biochemical analyzes and isolated shoot tips were transferred onto MS BAP medium to the optimal growth conditions. The proliferation rate was assessed after 4 weeks of 2 subcultures. For rooting shoots after 2nd subculture were transferred onto MS medium without plant growth regulators for 4 weeks. Rooting ability was investigated in terms of percent shoots able to form roots, number of roots per shoot and root length. Roots were gently washed with sterile distilled water and then plantlets were transferred to pots containing sterile vermiculite and sand (1:1 v/v). The pots were covered with a transparent lid to maintain humidity (60-70%). Plantlets grown at 23 ± 1 °C under white fluorescent light (80 μmol m −2 s −1 ) for 4 weeks after which they were transferred to the pots containing sterile soil and then were transferred to the field conditions. Survival rate and ability to flower of the plantlets were noted in the next year after acclimatization (April/May).

Biochemical analyzes
Directly after cold-storage leaves of stored explants were analyzed in terms of chlorophyll, H 2 O 2 , TBARS, soluble sugars, free proline and endogenous phytohormone (ABA, JA) content. Leaves from stored explants non-treated with ABA were used as a control. Additional non-stored plant tissue from optimal growth conditions was used for phytohormone analyzes. Chlorophyll, H 2 O 2 , soluble sugars and proline content was also determined in leaves after 4-weeks preculture on medium supplemented with ABA under optimal growth conditions. Chlorophyll was extracted from 20 mg of plant tissue in 96% ethanol and determined by the method of Nair and Chung (2015). To determine the total chlorophyll content (a and b) the formula developed by Lichtenthaler (1987) was used: Chl a+b μg mg −1 = 5, 24 ⋅ A 664,2 + 22, 24 ⋅ A 648,6 μg mL −1 ⋅ 10 mL∕m sample (mg) Hydrogen peroxide was determined according to Velikova et al. (2000). For extraction 100-200 mg of leaves was used and determination of H 2 O 2 was based on the iodide-reduction method with 1 M KI solution. TBARS level was estimated following the protocol described by Song et al. (2011). Sample for this analysis contained approximately 50 mg of leaves. Free proline was determined spectrophotometrically using method based on a reaction with ninhydrin (Bates et al. 1973). Extraction and determination of total soluble sugars was carried out by the phenol-sulfuric acid method (PSA) described by Dubois et al. (1956). Glucose was used to prepare standard curve. Endogenous level of abscisic acid and jasmonic acid was determined by using mass spectrometry combined with liquid chromatography (LC-MS). Analysis was carried out for explants stored for 9 months on MS medium without ABA and treated with this phytohormone at concentrations of 38-76 μM. Sample contained 50-100 mg of leaves homogenized in liquid nitrogen (LN). For the extraction of phytohormones 80% methanol (v/v) was added. In this step also small amount of antioxidant butylhydroxytoluol (BHT), 5 ng of deuterated ABA (d 6 ABA) and 10 ng deuterated JA (d 5 JA) as internal standards were added. The mixture was shaken overnight (at least 18 h). After incubation, the samples were centrifuged and methanol was evaporated. To remove chlorophyll, the samples were acidified to pH 2 by the addition of hydrochloric acid. After centrifuged the extracts, the supernatant was subjected to solid phase extraction (SPE) using silica packed columns (Discov-ery® DSC-18 SPE Tube). The columns were activated with 100% methanol and conditioned with formic acid. The applied samples were purified with 1 M formic acid and a solution of 1 M formic acid in 20% methanol (v/v). Elution was performed using 80% methanol (v/v). Before analysis samples were concentrated 7.5 times and centrifuged. For phytohormones determination the LCMS-8045 tandem mass spectrometry (Shimadzu Corp.) was used. Chromatographic separation was carried out on a Kinetex® 2.6 μm XB-C18 100 Å reverse phase column (150 × 2.1 mm). Water with 0.1% formic acid (v/v) (A) and methanol with 0.1% formic acid (v/v) (B) were used as the mobile phase. The separation was carried out in a linear gradient of 40-90% (v/v) methanol for 7 min at a flow rate of 0.3 mL/min at 30 °C. In mass spectrometry, the samples were subjected to negative electrospray ionization (ESI) and ions were fragmented by collision-induced dissociation (CID). The ionization voltage was − 3 kV. Analysis of individual phytohormones was based on multiple reactions monitoring (MRM).

Data collection and statistical analysis
The results presented in this paper were expressed as the mean ± standard error. Parameters from preculture (proliferation rate), storage (survival) and post-storage regrowth (proliferation rate and rooting ability of the shoots) were evaluated for 16 explants in each variant in three replicates. The leaves of survived explants from each vessel were grounded in LN together and biochemical analyzes were evaluated for 3 samples (1 representative sample from all of the explants stored in 1 vessel). Each biochemical test was performed in 3 biological and 3 technical replicates. The normality of the distribution of obtained results was tested by the Shapiro-Wilk test. In the case of data with non-normal distribution, the significance of differences was determined using non-parametric Mann-Whitney U test. Data with normal distribution and obtained in biochemical analyzes were analyzed using Tukey's test. In addition, the significance of the impact of the studied factors on the stored plant material and their interactions on the accumulation of selected chemical compounds was analyzed using two-and three-way ANOVA.

Preculture with ABA in optimal conditions
The obtained results indicated that ABA, at all tested concentrations, significantly decreases shoot proliferation rate in comparison to the control (18.9 ± 1.6 shoots per explant). Proliferation rate of ABA-treated shoots ranged from 10.1 ± 1.4 (19 μM ABA) to 3.3 ± 0.4 (76 μM ABA) shoots per explants (Fig. 1a). In addition, it was observed that increasing ABA concentration slightly decreased shoots length (Fig. 1b).

ABA treatment during cold-storage
Preliminary observation indicated that exposure to ABA reduced growth of the shoots obtained during synseed storage in comparison to the control, both after 3 and 6 months of storage (Figs. S1-S2) and after 9 months, where a particularly significant reduction of shoot length occurred in the presence of 76 μM ABA inside artificial endosperm in both light and dark conditions (Fig. 2). In addition, shoots stored in the dark in all tested ABA variants did not form roots, regardless of the ABA concentration. However, when storing synseeds under light conditions ABA-treated shoots during preculture were able to form roots during cold storage for 6 and 9 months, but their elongation was inhibited (Figs. 2, S1-S2).

Regrowth of the stored explants under optimal conditions
After cold-storage the survival and shoots proliferation ability under the optimal growth conditions were analyzed. After 9 months storage treatment with ABA did not affect explants survival (Fig. 3). Over 75% of synseeds remained viable, Fig. 1 The effect of ABA on T. pieninicum shoot proliferation in the optimal growth conditions: proliferation rate (a) and phenotype of shoots cluster (b) on MS medium supplemented with 1.11 μM BAP and 0.14 μM NAA and different ABA concentration after 4 weeks of the culture. Bar = 1 cm. Means with different letters are significantly different followed by Kruskal-Wallis test at p ≤ 0.05 1 3

Fig. 2
Taraxacum pieninicum shoots obtained from synthetic seeds during 9 months storage at 4 °C under light conditions (a) and in the dark (b) after different treatments with ABA. Bar = 1 cm regardless of the ABA treatment. Light conditions during storage decreased synseeds viability, although differences to the dark were not statistically significant in ABA-treated explants. Similar results were obtained after 6 months storage. Reduced survival of the synseeds treated with ABA was observed only after 3 months storage particularly under light conditions and when ABA was added inside synseed structure (Fig. S3).
The light conditions during cold-storage had the most significant impact on the post-storage proliferation ability of the explants under optimal growth conditions. Higher proliferation rate was indicated after storage in the dark in comparison to the light conditions, significant differences were noted for most of the used variants. After 9 months of storage ABA-treated synseeds remained comparable proliferation ability to the control stored in the light (7.6 ± 1.2 shoots per explant) and dark (13.7 ± 1.4) conditions (Fig. 4a). The method of ABA exposure was significant mostly after shorter storage time (Fig. S4). Similar relationships were observed after the second post-storage subculture (Fig. 4b).
In comparison to the control, prior ABA treatment did not inhibit shoots proliferation ability. The highest proliferation rate in 2nd subculture was noted for shoots obtained from synseeds containing 38 μM ABA inside and stored in the dark for 9 months (23.7 ± 1.8; Fig. 4b) and also for 6 months (21.3 ± 1.7; Fig. S5).
The last stage of T. pieninicum post-storage regeneration was rooting shoots on the MS medium. Regardless of the storage variant, the shoots were able to root (Fig. 5), except for the few ABA variants stored for 3 and 6 months (Fig.  S6). The highest percentage of rooted shoots was obtained after storage for 9 months in the dark with 76 μM ABA inside synseed (Fig. 5a). ABA treatment did not reduce the number of roots developing from the shoots obtained after storage, regardless of the storage time (Figs. 5b, S7), but inhibited roots elongation, regardless of its exposure method and concentration (Figs. 5c, S8). However, ABA did not affect the ability of the regenerants to acclimatize. All plantlets transferred to ex vitro conditions were able to acclimatize. The morphological appearance of the regenerants treated even with highest ABA concentration did not differ to the control plants and plantlets were able to flower after transfer to the field conditions (Fig. 6).

Biochemical analyzes
After 4-weeks preculture on medium supplemented with ABA in the optimal growth conditions it was indicated that ABA significantly decreased chlorophyll content in the multiplicated microshoots (Fig. 7a). The usage of the highest ABA concentration (76 μM) significantly increased accumulation of H 2 O 2 ( Fig. 7b) but in none of the used concentration ABA affected soluble sugars accumulation (Fig. 7c). Treatment of the explants with ABA in the two highest concentrations (57 μM and 76 μM) significantly increased proline accumulation in the leaves (Fig. 7d).

Fig. 3
Survival of the encapsulated shoot tips of T. pieninicum after 9 months storage 4 °C. Statistically significant differences assessed using Kruskal-Wallis test at p ≤ 0.05: to the control plants in the same light conditions are denoted *(p < 0.05), **(p < 0.01), ***(p < 0.001); between light conditions are denoted # (p < 0.05), ## (p < 0.01), ### (p < 0.001); between ABA treatment in the same light conditions are denoted ◊ (p < 0.05), ◊◊ (p < 0.01), ◊◊◊ (p < 0.001) Fig. 4 The effect of light conditions and ABA-treatment on T. pieninicum proliferation rate of shoots during regrowth on MS medium with 1.11 μM BAP and 0.14 μM NAA in the 1st (a) and 2nd (b) subculture after 9 months storage at 4 °C. Statistically significant differences assessed using Kruskal-Wallis test at p ≤ 0.05: to the control plants in the same light conditions are denoted *(p < 0.05), **(p < 0.01), ***(p < 0.001); between light conditions are denoted # (p < 0.05), ## (p < 0.01), ### (p < 0.001); between ABA treatment in the same light conditions are denoted ◊ (p < 0.05), ◊◊ (p < 0.01), ◊◊◊ (p < 0.001) Fig. 5 The effect of light conditions and ABA-treatment on T. pieninicum rooting ability of shoots on MS medium after 9 months storage at 4 °C assessed by % of rooted shoots (a), number of roots per rooted shoots (b) and length of roots (c). Statistically significant differences assessed using Kruskal-Wallis test at p ≤ 0.05: to the control plants in the same light conditions are denoted *(p < 0.05), **(p < 0.01), ***(p < 0.001); between light conditions are denoted # (p < 0.05), ## (p < 0.01), ### (p < 0.001); between ABA treatment in the same light conditions are denoted ◊ (p < 0.05), ◊◊ (p < 0.01), ◊◊◊ (p < 0.001) Fig. 6 Flowering T. pieninicum shoots in the field conditions, regenerated and acclimated after storage in the form of synthetic seeds with 96 μM ABA inside for 9 months at 4 °C under the light conditions (a) and in the dark (b) After 9 months of storage the highest chlorophyll content was determined in control plants (Fig. 8a). The same results were obtained after 6 months of cooling, while after 3 months the highest chlorophyll accumulation was reported in leaves of shoots obtained from synseeds containing ABA in artificial endosperm (Fig. S9). Two-way ANOVA indicated that ABA application method had a significant effect on chlorophyll content after 3 and 6 months of storage, while ABA concentration had no significant effect regardless of the storage time (Table 1).
Increasing ABA concentration in the preculture treatment led to an increase in the accumulation of the hydrogen peroxide in the plant tissue after 9 months of storage, what was particularly evident in the material stored under light conditions (Fig. 8b). A similar effect was noted for explants stored in capsules containing ABA under light conditions. In the dark ABA inside synseed had no effect on H 2 O 2 levels. Furthermore, after storage in the dark in each ABA-treated variant there were no significant differences compared to the controls. A three-way ANOVA showed a significant individual effect after 9 months of storage of ABA treatment method, ABA concentration and light conditions on H 2 O 2 accumulation (Table 2). However, the interaction of these factors did not significantly affect analyzed parameter. Preculture with ABA also increased H 2 O 2 accumulation after 3 months storage (Fig. S10a). However statistically significant differences to the control was noted only for tissue stored in the dark. ABA addition inside synseed had no effect on H 2 O 2 accumulation after 3 months storage. After 6 months of storage preculture with ABA and addition of ABA to the synseeds stored in the dark reduced H 2 O 2 accumulation (Fig. S10b).
Directly after storage, TBARS content was also determined as a marker for lipid peroxidation. After 9 months of storage ABA inhibited TBARS accumulation in comparison to the control, especially when synseeds were stored under light conditions (Fig. 8c), although increasing ABA concentration significantly increased accumulation of determined compound. Three-way ANOVA showed not only significant effect of the light conditions on TBARS content, but also ABA concentration and method of its exposure (Table 3). After shorter storage time ABA did not indicated significant effect on TBARS content in plant tissue (Fig. S11).
The accumulation of soluble sugars depended on all analyzed variation factors ( Fig. 8d; Table 4). After 9 months of storage under light conditions a significantly higher than in control plants level of soluble sugars was determined in explants treated with 38 μM ABA and 57 μM ABA during preculture. Increased sugar accumulation was also shown in leaves from variant where ABA  (e) in T. pieninicum shoots directly after 9 months storage at 4 °C (a). Statistically significant differences assessed using Tukey test at p ≤ 0.05: to the control plants in the same light conditions are denoted *(p < 0.05), **(p < 0.01), ***(p < 0.001); between light conditions are denoted # (p < 0.05), ## (p < 0.01), ### (p < 0.001); between ABA treatment in the same light conditions are denoted ◊ (p < 0.05), ◊◊ (p < 0.01), ◊◊◊ (p < 0.001) was inside synseeds structure at a concentration of 19 μM and 38 μM and synseed were stored also under light conditions. After 6 months of storage ABA inhibited accumulation of soluble sugars in tissue cooled in both light conditions, while after 3 months ABA did not show any significant effect on analyzed parameter (Fig. S12).
The level of accumulated proline depended on the method of ABA treatment, its concentration and light conditions during storage ( Fig. 8e; Table 5). After 9 months of storage proline level increased with an increase of ABA concentration inside synseeds structure, especially in light-stored explants. In contrast, the material treated with 19-57 μM ABA during preculture accumulated significantly less proline under light conditions than control plants. Similar results were obtained after 6 months of storage, but ABA did not affect proline accumulation after the shortest storage time (Fig. S13).
In response to the cold stress, endogenous ABA level increased significantly in shoots obtained from the synseeds non-treated with ABA stored under light conditions (38.0 ± 4.0 ng/g FW; Fig. 9a), while in the dark ABA accumulation after cooling (5.8 ± 2.6 ng/g FW) was comparable to the non-stored control (4.9 ± 0.6 ng/g FW; data not shown in the figure). The explants stored under light conditions and exposed to ABA at a concentration of 38 μM and 76 μM (both ABA-treatment) accumulated less endogenous ABA compared to the cold-stored control, while in the dark 38 μM ABA stimulated accumulation of endogenous ABA in the explants. Treatment with higher ABA concentration during storage in the dark did not affect the endogenous level of the phytohormone compared to the stored control and in addition determined level was comparable to non-stored control.
Three-way ANOVA showed that the endogenous ABA level depended on ABA treatment method, its concentration and the light conditions (Table 6). A significant correlation between the examined factors was noted only for the ABA concentration and light conditions. Like ABA, the level of JA increased significantly in control shoots after cold-storage under the light conditions (187.4 ± 25.0 ng/g FW) in comparison to the non-stored control (31.2 ± 3.2 ng/g FW) (Fig. 9b). It was showed that ABA treatment, irrespective of its concentration and method of application, led to a statistically significant decrease in the level of JA in explants stored under light conditions. In shoots from all analyzed variants of synseed storage in the dark, the JA level was comparable to the control. Three-way ANOVA showed that light conditions had a significant effect on JA levels individually, as well as in correlation with ABA treatment method or ABA concentration (Table 6).

Discussion
Explant encapsulation used during storage of T. pieninicum is designed to protect plant tissue from low temperature damages and to physically reduce initial growth rate through  . 9 The effect of the light conditions and ABA treatment on the content of endogenous ABA (a) and JA (b) in shoots of T. pieninicum obtained from artificial seeds after storage for 9 months at 4 °C; the significance of differences was assessed by the Tukey test at α = 0.05 and determined: to the control plants in the same light conditions are denoted *(p < 0.05), **(p < 0.01), ***(p < 0.001); between light conditions are denoted # (p < 0.05), ## (p < 0.01), ### (p < 0.001); between ABA treatment in the same light conditions are denoted ◊ (p < 0.05), ◊◊ an alginate coating (Kamińska et al. 2018a). Slowing the explants growth during storage is highly desirable due to the longer time without passage what reduce costs and the risk of contamination. The simplest method to slow down the growth of the plant material is to lower the culture temperature (Divakaran et al. 2006). Storage of synseeds at a reduced temperature was used to protect endangered species such as Celastrus paniculatus (Fonseka et al. 2019), Ceropegia barnesii (Ananthan et al. 2018), Cymbidium aloifolium (Pradhan et al. 2016), Eclipta alba (Salma et al. 2019), Mondia whitei (Baskaran et al. 2015), Spilanthes acmella (Sharma et al. 2009) and Withania coagulans (Rathore and Kheni 2017). Morphological observations combined with biochemical analyzes have shown that light is a stress factor during storage of T. pieninicum synseeds, what was with agreement with our previous results also those obtained for non-encapsulated shoot tips of this species (Kamińska et al. 2016). Many studies indicated numerous relationships between light stress and low temperature conditions. Light-induced stress may result from an imbalance between the absorption and usage of a light energy (Ensminger et al. 2006;Beck et al. 2007). Absorption of a large amount of light in low metabolic activity conditions resulted from a lowered environmental temperature may led to damage of D1 protein, the site of attachment of plastoquinone QB. The inability to transfer energy to the PS reaction center may lead in turn to ROS production (Roach and Krieger-Liszkay 2014). Furthermore, under cold conditions electron transport may be inhibited by changing the structure of the cell membrane (Ensminger et al. 2006) and by reducing the activity of photosystem II (Borawska-Jarmułowicz et al. 2014) what may increase stress on which plant tissue is exposed.
Storage of synseeds in the dark seems to be a better method due to a more effective growth inhibition in comparison to the light conditions. This was confirmed for Hibiscus moscheutos synseeds which might be stored in the dark at 5 °C for almost 20 months with 80% viability (West et al. 2006) and for Splachnum ampullaceum synseeds stored under the same conditions for 30 months with maintaining viability of 50% explants (Mallón et al. 2007). Better results after storing the plant material in the dark compared to a light conditions were also obtained for Vitis berlandieri x V. riparia synthetic seeds after 9 months of cooling (Benelli 2016).
In order to further growth reduction of shoots during T. pieninicum synseeds storage in this study natural growth retardant abscisic acid was examined. Results showed that ABA limited shoots growth during the preculture preceding storage, as well as during synseeds storage (inside treatment) in both light variants. This is a potential indicator of the possibility of extending storage due to slower nutrient compounds consumption. Numerous studies indicated the inhibitory effect of ABA in plant in vitro culture. ABA inhibited the elongation growth of Citrus lemon (Kotsias and Roussos 2001), Oryza sativa (Pattanagul 2011) and Teucrium polium shoots (Rabba'a et al. 2012) during in vitro culture.
Numerous reports suggested that the addition of ABA to the medium allow extension of the storage time of viable explants e.g., Malus domestica (Kovalchuk et al. 2009), Tetrastigma hemsleyanum (Peng et al. 2015) or Lilium davidii and L. longiflorum (Yun-peng et al. 2012). However, medium supplementation with ABA reduced the survival of adventitious shoots of Vitis heyneana (Pan et al. 2014). Also in Physalis peruviana the addition of ABA to the synseeds reduced the survival of nodal cuttings and inhibited their elongation growth (Yücesan et al. 2015).
An important step in the in vitro preservation of plant material is the possibility of their micropropagation after storage. Regardless of the ABA treatment and duration of synseeds cold-storage, the shoot tips of T. pieninicum have been propagated under optimal growth conditions on the medium supplemented with BAP, but this process was more effective after storage in the dark, what was also noted after storage of non-encapsulated shoots of T. pieninicum (Kamińska et al. 2016). Similar results were published by Hausman et al. (1994) storing shoot tips of Populus tremula x P. tremuloides for 3 months at 4 °C in the dark without influence on shoot proliferation rate in comparison to nonstored control. Piccioni et al. (1996) reported regrowth of encapsulated explants of M.26 apple rootstock after 2 and 4 months at 4 °C. Effective propagation after synseeds storage was also noted for Olea europea and Quercus cerris after 6 weeks at 4 °C (Micheli et al. 1998;Tsvetkov and Hausman 2005), Corymbia torelliana × C. citriodora and Khana senegalensis after 12 months at 14˚C in the dark (Hung and Trueman 2012). ABA treatment did not affect T. pieninicum shoots ability to proliferate during post-storage regrowth. Similarly, addition of ABA to the medium did not affect the proliferation rate of Cedrus libani explants after 6 months of cold-storage (Renau-Morata et al. 2006). Also, the preculture of Castanea sativa shoots on the medium supplemented with 5 -10 μM ABA did not affect their survival, but highest ABA concentration reduced shoots ability to proliferate after storage at 4 °C (Capuana and Di Leonardo 2013). In turn, in the study of da Silva and Scherwinski-Pereira (2011) the addition of ABA into MS medium reduced survival and proliferation rate of Piper aduncum and P. hispidinervum after 6 months of storage. Taraxacum pieninicum shoots were effectively rooted on MS medium without auxin. Only shoots obtained after 3and 6-months storage in capsule containing highest ABA concentration did not root. However, root organogenesis occurred after transfer of shoots to the vermiculite-sand mixture in ex vitro conditions what was also reported for other species from the Asteraceae family, e.g. Carlina acaulis (Trejgell et al. 2009).
Storage of the plant material using in vitro cultures, due to the presence of cold stresses and growth regulators (especially cytokinins) during regrowth stage may lead to genetic variability. However, during shoots multiplication from shoot tips usually the DNA level remained stable (Clarindo et al. 2008). In our previous research we verified the genetic stability of the plants obtained after long-term storage of non-encapsulated shoot tips on medium supplemented with ABA. No differences were observed using flow cytometry, RAPD, ISSR and SCoT markers between regrown plantlets after storage and plants cultivated from seeds in a soil (Kamińska et al. 2016(Kamińska et al. , 2020. The metabolic content changes under the influence of a low temperature may be associated with (a) catalytic activity or stability of enzymes involved in biosynthesis or degradation of specific compounds; (b) biosynthesis of compounds resulting from cellular damage; (c) regulating the concentration of given compounds to maintain homeostasis or (d) biosynthesis and accumulation of compounds involved in developing tolerance (Janmohammadi 2012). Therefore, the levels of chlorophyll, H 2 O 2 , TBARS / MDA, soluble sugars, proline as well as ABA and JA were analyzed.
Hordeum vulgare and Zea mays explants stored under high light intensity and low temperature were found to increase chlorophyll and xanthophyll content in response to ABA additions, what would indicated the role of ABA in protection of photosystem II (Jia and Lu 2003). The results presented in this paper did not indicate the participation of ABA in this process in T. pieninicum during storage in conditions of reduced light intensity because a decrease in Chl content in explants was noted. Furthermore, decreased level of Chl was also observed after preculture with ABA in optimal growth conditions without exposure to the low temperature. The cold conditions during storage of plant material also reduced the Chl content in Avena nuda seedlings (Liu et al. 2013). However, short-term storage of synseeds did not affect its level in T. pieninicum shoots obtained under optimal growth conditions. Similar results were reported for Rauvolfia tetraphylla synseeds stored for 4 weeks at reduced temperature (Faisal et al. 2013).
Numerous scientific reports indicated that under reduced temperature conditions there is an accumulation of ROS that leads to oxidative stress in plant tissues. Hydrogen peroxide is the most stable form of ROS, and its accumulation is considered as a direct response of plant cells to a low temperature (Carvajal et al. 2015). An increase in H 2 O 2 content was noted after few hours of storage at 4 °C in Vitis vinifera (Rooy et al. 2017). In A. thaliana callus cells, the maximum increase in H 2 O 2 accumulation was observed after 2 days at 4 °C, but from the 5th day of cooling, the level of H 2 O 2 decreased (O'Kane et al. 1996). It was assumed that after the cessation of cold stress, the level of ROS decreases to the level occurring in optimal conditions (Einset et al. 2007). Excessive ROS accumulation can lead to chlorophyll degradation (Kavi Kishor and Sreenivasulu 2014), lipid peroxidation (Bhattacharjee 2005), and even to induction of programmed plant cell death (Desikan et al. 1998). Under stress conditions, level of endogenous ABA increase in plant tissue, what is one of the elements of the signal transductions pathway leading to an increase in chlorophyll photooxidation resistance (Pospíšilová et al. 2009) and activation of antioxidant enzymes (Jiang and Zhang 2001). Application of exogenous ABA may also reduce the effects of stress on plant tissue. However, in the case of T. pieninicum no such effect was found. A significant increase in H 2 O 2 accumulation has been shown in the light, while ABA may not be involved in the production or neutralization of ROS in this species. However, high concentration of ABA increased H 2 O 2 level in T. pieninicum leaves grown under optimal growth conditions.
The consequence of ROS accumulation may be a peroxidation of membrane lipids which is denoted by the accumulation of malonodialdehyde (Liu et al. 2013;Rooy et al. 2017). T. pieninicum explants stored in the light accumulated significantly more TBARS / MDA than in the dark. In addition, its level in plant tissue increased with prolonging storage time. The ABA application did not affect the level of the tested parameter after 3 and 6 months of storage. However, after 9 months significantly less TBARS/MDA was determined in plant tissue treated with ABA than in the control, especially after storage under light conditions, what may indicated the involvement of ABA in lipid peroxidation inhibition mechanisms. Similar results were obtained for Saccharum officinarum (Huang et al. 2015) and Vitis vinifera (Karimia et al. 2016), where foliar application of 50-200 μM ABA led to a decrease in TBARS content under reduced temperature conditions. An increased content of soluble sugars during acclimation to cold has been observed for many species, including A. thaliana (Klotke et al. 2004), Triticum aestivum (Kamata and Uemura 2004), Hordeum vulgare (Tabaei-Aghdaei et al. 2003) and Solanum tuberosum (Folgado et al. 2013). Tognetti et al. (1990) proposed that the increased accumulation of sugars in plant tissue enhanced resistance of the species to a low temperature. After 3 months of storage of T. pieninicum no effect of ABA on the accumulation of soluble sugars in tissue was noted. Additionally, treatment of explants with ABA during preculture and prolonged storage for up to 9 months in the dark also did not affect the level of accumulated sugars compared to controls. In contrast, literature reports point to a relationship between ABA accumulation and soluble sugars under cold stress (Rooy et al. 2017).
Accumulation of proline in the tissues of cooled plant material has proven to be of key importance in the process of cold acclimation, what has been shown for many species, including Saccharum officinarum (Rasheed et al. 2010), Vitis vinifera (Rooy et al. 2017) and Avena nuda (Liu et al. 2013). However, the increased proline accumulation does not necessarily indicate a high tolerance to coldness or freezing of a given species, but it is necessary to adapt the tissue to a condition that may lead to a disruption of its growth (Rooy et al. 2017). In Quercus rober proline abolished the inhibitory effect of cold on the growth of embryogenic tissue (Gleeson et al. 2004), while in Cicer arietinum proline stimulated the accumulation of sucrose and chlorophyll in plants exposed to low temperature (Kaur et al. 2011). Accumulation of proline did not depend on storage time in T. pieninicum. Higher proline content was determined in light-stored tissue than in the dark, what confirmed that light combine with the low temperature causes severe stress in this species. Treatment of ABA explants did not significantly affect proline accumulation during storage in the dark. However, the stimulating effect of ABA on proline accumulation was determined in shoots obtained from synseeds containing 76 μM ABA in artificial endosperm and stored for 9 months under light conditions. Also, high ABA concentration resulted in increased proline accumulation after preculture under optimal growth conditions. Numerous studies indicated that ABA led to increased proline accumulation (Kavi Kishor and Sreenivasulu 2014), such a relationship was noted for example for Triticum aestivum (Hou et al. 2010).
Exposure of T. pieninicum tissue to the low temperature led to a significant increase in endogenous ABA and JA only after storage under light conditions. It is well-known that the level of endogenous ABA in plant material can be controlled by light (Xu et al. 2014;Seo et al. 2009). In our study reduced shoot proliferation after storage under light conditions may probably resulted from the high levels of endogenous ABA in tissue. However, no significant differences between light conditions during storage and ABA accumulation were noted. It seems that the response of this species to cold-storage under continuous light is more complex and does not depend only on the accumulation of ABA in tissue. Treatment of explants with ABA did not lead to an increase in endogenous ABA accumulation in tissue compared to the cooled control. Under low temperature conditions, an increase in endogenous ABA levels was noted in Oryza sativa (Oliver et al. 2007) and Vitis vinifera (Rooy et al. 2017). In Actinidia deliciosa endogenous ABA levels increased until the second month of cooling at 0 ± 0.5 °C. However, longer storage led to a decrease in ABA accumulated levels (Yang et al. 2013).
In Arabidopsis exposure to JA led to a decrease in ABA accumulation, while exogenous ABA under abiotic stress stimulated JA biosynthesis (Adie et al. 2007;Fan et al. 2009;Wang et al. 2018). However, treatment of T. pieninicum tissue with ABA led to significantly lower JA content after light storage.
Summing up the results, it can be stated that treatment with ABA reduces the effects of cold stress in T. pieninicum tissues stored in the light, what was manifested by reduced H 2 O 2 levels in shoots subjected to the preculture on medium supplemented with ABA (19-57 μM). In addition, significant reduction of membrane lipid peroxidation was noted after tissue treatment with ABA before and during cold-storage. No stimulating effect of treatment with ABA on the level of chlorophyll and the proliferation rate of cooled shoots was observed. Signal transduction pathways of ABA and JA are interrelated because treatment of ABA tissue significantly reduced JA content. ABA and JA are involved in the protective mechanisms of plant tissues against low temperature stress, however, processes associated with acclimatization to the cold are multifactorial, and the studied phytohormones are not the only factors affecting the expression of genes associated with the plant's response to cold. Potential participation in this process might be also attributed to ethylene (Zhao et al. 2014), gibberellic acid (Richter et al. 2013), brassinosteroids (Singh et al. 2012), salicylic acid (Dong et al. 2014), ROS and cytosolic Ca 2+ (Chinnusamy et al. 2007).

Conclusions
In conclusion, light combined with low temperature is a stress factor during storage of T. pieninicum, regardless of the ABA exposure. Treatment with abscisic acid of T. pieninicum explants limits their growth, which contributes to the extension of their storage time at reduced temperature at least to 9 months and alleviates the effects of cold stress. Shoots of T. pieninicum after cold storage and ABA treatment are able to regrowth effectively under optimal growth conditions, while field acclimatized regenerants after storage and propagation in vitro are able to flower.