Does pre-culture in sugar-rich media affect carbohydrate content and post-thawing recovery rate of cryopreserved potato (Solanum phureja) shoot tips?

2.1 In vitro culture

In vitro potato plants (Solanum phureja) were obtained from the GERMICOPA SA company (France). Mono-nodal stem cuttings of 1 cm of length were excised from 6 to 8 week-old in vitro potato plants on fresh “cutting” medium. The culture medium contained macroelements of Tendille and Lecerf (1974) i.e. (808.8 mg/L KNO₃, 160.1 mg/L NH₄NO₃, 141.7 mg/L Ca(NO₃)₂.4 H₂O, 123.3 mg/L MgSO₄·7H₂O, 104.4 mg/L KCl, 81.7 mg/L KH₂PO₄, 7.0 mg/L K₂HPO₄), microelements of Murashige and Skoog (1962): (6. 2 mg/L H₃BO₃, 0.025 mg/L CoCl₂·6H₂O, 27.8 mg/L FeSO₄·7H₂O, MnSO₄·22.3 mg/L 4H₂O, 0.83 mg/L KI, 0.25 mg/L Na₂MoO₄·2H₂O, 8.6 mg/L ZnSO₄·7H₂O, 5.57 g/L FeSO₄·7H₂O, 7.45 g/L Na₂-EDTA, 0.025 mg/L CuSO₄·5H₂O), vitamins of Morel and Wetmore (1951): (100 mg/L inositol, 1 mg/L thiamine.HCl, 1 mg/L nicotinic acid, 1 mg/L pyridoxine.HCl, 1 mg/L Ca pantothenate, 0.01 mg/L biotin, Na-Fe-EDTA, 30 g/L sucrose, 8 g/L agar, pH 5.8), 30 g/L of sucrose, and 8 g/L of Difco® Bacto agar. The pH was adjusted to 5.85 with NaOH (1 N) and HCl (1 N). Agar was added at a temperature of 100 °C, followed by autoclaving at 110 °C for 20 min.

2.2 Shoot tip excision

For propagation of shoot tip donor plants, mono-nodal stem cuttings were sub-cultured for seven days in deep petri dishes (Ø = 10 cm) and incubated under the above described conditions, under room temperature (20 ± 1 °C), relative humidity = 70 ± 5%, photoperiod = 16 h, and light intensity = 50 μmoles/m²/s. Potato shoot tips of a length of 0.5–1.0 mm (with few leaf primordia) were excised under the stereoscope.

2.3 Encapsulation procedures

Excised potato shoot tips were placed in liquid Ca-free medium, containing macroelements (500 mg/L Ca(NO₃)₂·4H₂O, 125 mg/L MgSO₄ 7H₂O, 1000 mg/L KCl, 125 mg/L KH₂PO₄, 1000  mg/L (NH₄)₂SO₄) of Morel and Muller (1964), microelements of Heller (1953) i.e. (0.1 mg/L MnSO₄·4H₂O, 1 mg/L ZnSO₄·7H₂O, 1 mg/L H₃BO₃, 0.01 mg/L KI, 0.03 mg/L CuSO₄·5H₂O, 0.03 mg/L AlCl₃, 0.03 mg/L NiCl₂·6H₂O, 1 mg/L FeCl₃·6H₂O), vitamins of Morel and Wetmore (1951), with EDTA-FeNa₂, 5 mg/L of gibberellic acid (GA₃), 0.001 mg/L of 1-naphtaleneacetic acid (NAA), 0.01 mg/L 6-benzylaminopurine (BAP), 34.2 g/L of sucrose (0.1 M), supplemented with 30 g/L of sodium alginate (3%). For encapsulation, shoot tips and solution were absorbed with a micropipette, and dropped in an alginate-free culture medium (same composition), supplemented with 14.7 g/L of calcium chloride (CaCl₂, 2H₂O). Grown beads have a diameter of 3–4 mm and contained 1–2 shoot tips per bead. Beads were rinsed twice with liquid Ca-free shoot tip medium and pre-cultured on semi-solid shoot tip medium, supplemented with different types of cryoprotectants (sugars and polyols).

2.4 Cryoprotection (pre-culture)

A standard concentration of 0.1 M of sucrose was maintained for the encapsulation and pre-culture media. A final osmolarity of 0.75 M was obtained, adding the equivalent of 0.65 M of each cryoprotectant to the pre-culture media (Bouafia et al., 1995). Eleven different cryoprotective pre-culture treatments were assessed: 0.1 M of sucrose plus 0.65 M of different monosaccharides (glucose, fructose and galactose), disaccharides (trehalose, cellobiose, maltose and lactose), triholoside (raffinose), mannitol, sorbitol and sucrose. Coated shoot tips were pre-cultivated for two days under the previously described conditions (see subsection: 2.1. In vitro culture).

2.5 Dehydration on silica gel and freezing in liquid nitrogen (LN)

Pre-cultured shoot tips were dehydrated for 4.5 h on silica gel at a temperature of 20 °C. Shoot tips’ water content was reduced to 0.20–0.22 g of water/g DM. Shoot tips were transferred into cryobiological tubes, directly plunged in LN and maintained for minimum 1 h in LN. Water content of beads [in %] was calculated as (fresh weight−dry weight)/fresh weight, which represents weight loss.

2.6 Rewarming and plant recovery

Shoot tips were rewarmed at room temperature (∼20 °C), directly placed on shoot tip medium, and incubated at 20 ± 1 °C and a relative humidity of 70 ± 5%, with a photoperiod of 16 h and light intensity of 50 μmoles/m²/s, provided by True lite Durolux fluorescent tubes.

2.7 Determination of total and soluble carbohydrates in shoot tips

Total carbohydrate content was measured in both untreated (taken directly from the mono-nodal stem cuttings) and coated shoot tips. Shoot tips were pre-cultured for 2 days on shoot tip culture media, supplemented with 0.65 M of trehalose, glucose and sucrose (total osmolarity = 0.75 M). Pre-culture conditions were the same as previously described (see subsection: 2.1. In vitro culture). After pre-culture, shoot tips were extracted from the coating using a scalpel and rinsed with demineralized water. Shoot tips were fixed in 0.5 mL of ethanol (96% vol.) and stored at −25 °C. The experiment was repeated three times with a sample size of 20–25 shoot tips per treatment.

Shoot tips were crushed in the presence of 1 mL of ethanol (96% vol.) and centrifuged at 2000g for 10 min (at 4 °C). The supernatant was collected (ethanolic extract), the pellet was resuspended in 1 mL of water, crushed and centrifuged again to obtain the aqueous extract. Supernatants were stored at −25 °C after that the ethanolic and aqueous extracts were vacuum evaporated for 2–5 h. The dry extract was diluted in 200 µL of distilled water. Charged molecules were removed from the extract solution using an ion exchange resin mini-column. Samples of 20 µL of extract solution were analysed using high performance liquid chromatography (HPLC). Quantitative measurement was carried out with the integration method. Two reference solutions were used, the first contained sucrose, glucose and fructose, and the second enclosed the same carbohydrates plus trehalose.

2.8 Data analysis

Variation in fresh (FMW) and dry weight matter (DMW) of empty beads as well as FMW/DMW ratio and weight loss in % (FMW-DMW/FMW) was assessed among a set of 11 cryoprotectants using one-way analyses of variance (ANOVA) followed by Tukey’s post hoc tests. Normal distribution of data points was tested using the Shapiro test. The effect of the cryoprotectant molecular mass on weight loss and fresh and dry weights of encapsulated potato shoot tips (in beads) was analysed using a generalized linear model (GLM), including Gaussian error family and identity link. Raw data used in computations is available online at: https://dx.doi.org/10.6084/m9.figshare.4542454.

The effect of dehydration (-LN), liquid nitrogen exposure (+LN) and pre-culture with different cryoprotectants on the shoot tips’ recovery rate was analysed with a two-way ANOVA test. Separated ANOVA tests were performed for two cryoprotectant categories (disaccharides, trisaccharides and polyols). Variations of concentrations of total soluble carbohydrates and individual sugars in potato shoot tips, pre-cultured on three different cryoprotectants (sucrose, trehalose and glucose), were analysed with Tukey’s HSD test. The variation of different soluble carbohydrate concentrations following total carbohydrates of different pre-culture treatments was tested using GLM with Gaussian distribution error and identity link. Statistical analysis was carried out using R software (R Core Team, 2019).

3.1 Fresh and dry matter weights of pre-cultured beads

Pre-cultured empty beads (without shoot tips) were used to determine if the cryoprotectant treatment altered the water retention potential of the beads. After pre-culture in liquid monosaccharide-rich culture media (glucose, fructose and galactose), empty calcium alginate beads showed DMWs ranging from 145.5 to 158 mg. FWM and DMW of empty alginate beads increased significantly after pre-culture in disaccharide-rich culture media. Beads pre-cultured in sucrose- and trehalose-rich media had FMWs of 439.3 ± 3.3 mg and 266.6 ± 2.1 mg, respectively. Beads pre-cultured in maltose-, cellobiose- and lactose-rich culture media showed FMWs of 282.0 ± 2.4 mg, 336.9 ± 3.2 mg, and 335.2 ± 3.0 mg, respectively (Table 1). The highest DMWs were observed when beads were pre-cultured in culture media containing raffinose (318.6 ± 2.6 mg) or sucrose (345.6 ± 2.8 mg). Low DMWs were observed in beads pre-cultured in mannitol- (138.6 ± 2.1 mg) and sorbitol-rich (150.1 ± 0.2 mg) culture media.

The ANOVA test showed significant differences for DMWs, FMWs, FMW/DMW ratio and weight loss between the assessed cryoprotectants (P < 0.001). Following Tukey test, the lowest values of FMW/DMW ratio were recorded with sucrose (1.271) and raffinose (1.27) whereas the highest scores were observed with fructose (1.366) and lactose (1.353). Regarding the weight loss, it was the lowest in mannitol and trehalose with an average of 21%, and the highest in fructose, galactose and cellobiose with about 26% (Table 1).

The used General Linear Model showed a significant positive relationship between changes in FMWs and DMWs of empty alginate beads and the molecular mass of the respective cryoprotectant. The use of cryoprotectants with heavy molecular mass induced a significant increase in FMW (GLM: F = 88.4, P < 0.001) and DMW (GLM: F = 93.0, P < 0.001) of the pre-cultivated empty beads (Fig. 1). However, weight loss decreased significantly as the molecular mass of cryoprotectants increased (GLM: F = 13.4, P < 0.001).

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Fig. 1

Relationship between the molecular mass of cryoprotectants and weigh loss, weights of fresh (FM) and dry matter (DM) of empty beads of potato. The lines represent a linear regression with a GLM fit (generalized linear model) and 95% confidence region in light grey. F (F-statistics) and P (P-value) are results of GLMs (Gaussian distribution error and ‘Identity’ link).

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3.2 Tolerance to pre-culture, dehydration and freezing

Coated pre-cultured shoot tips showed an average recovery rates (rr) of 53% without liquid nitrogen exposure (-LN) and average 39% for in liquid nitrogen frozen samples (+LN). Dehydration with mannitol showed the highest recovery rate of 100% (-LN). No regrowth was observed when shoot tips were dehydrated in monosaccharide-rich culture medium (water content of beads: 21–27%). The pre-culture of shoot tips in disaccharide-rich culture media considerably increased its tolerance to dehydration (-LN) and freezing in liquid nitrogen (+LN). Highest post-thawing recovery rates were observed for shoot tips pre-cultured in culture media containing trehalose (rr = 91%), maltose (rr = 88%), and sucrose (rr = 78%). The ANOVA test showed significant variations for disaccharide cryoprotectants and treatments (Table 2). Shoot tips pre-cultured in culture media supplemented with polyols showed lower recovery rates after LN exposure. The ANOVA showed significant differences for the recovery rates of shoot tips pre-cultured in trisaccharide- and polyol-rich culture media (P < 0.001) (Fig. 2, Table 2).

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Fig. 2

Recovery rates of shoot tips pre-cultivated in various cryoprotectant-based culture medium after dehydration and freezing in liquid nitrogen. Italic letters indicate results of Tukey's HSD tests, where the same letter are not significantly different at P > 0.05.

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3.3 Composition of shoot tips with soluble carbohydrates

The ANOVA showed significant differences for the total carbohydrate content of potato shoot tips pre-cultured in culture media containing three different types of cryoprotectants (F_{(3, 32)} = 2556, P < 0.001). For the control treatment (without pre-culture), shoot tips showed a total carbohydrate content of 33 ± 1.2 mg/g FMW. The highest carbohydrate content was observed when shoot tips were pre-cultured in trehalose-rich medium (Fig. 3). Contents of total sugars (i.e. sum of the sugars that were analysed) in controls averaged 14.6 mg/g FMW of sucrose, 9.7 mg/g FMW of glucose and 8.7 mg/g FMW of fructose (Fig. 4). After pre-culture in 0.75 M sucrose-rich medium, the total soluble carbohydrate content was 48.4 ± 1.6 mg/g FMW (1.46 times higher than that for the control treatment). The total concentration of carbohydrates (78.3 ± 0.8 mg/g FMW) significantly increased after pre-culture in trehalose-rich culture medium. This increase was related to the high sucrose content (57.5 mg/g FMW), representing 73.4% of the total carbohydrates of shoot tips. The contents of glucose (14%) and fructose (12.6%) constituted only minor portions of the total carbohydrate content. For the glucose-rich pre-culture medium, shoot tips showed a total carbohydrate content of 58.4 ± 0.7 mg/g FMW. Glucose represented 33% (19.3 mg/g FMW), whereas sucrose was dominant with 50.5% of total sugars (29.5 mg/g FM) (Fig. 3, Fig. 4). Total soluble carbohydrate content was significantly different for pre-culture treatments (P < 0.004) and type of soluble carbohydrates (P < 0.001). The composition of the soluble carbohydrates (glucose, fructose and sucrose content) varied significantly following the cryoprotectant molecule used in pre-culture. The variation of soluble carbohydrate contents was higher in sucrose and glucose (P < 0.001) compared to fructose (P = 0.035) (Table 3).

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Fig. 3

Contents of total carbohydrates measured in potato shoot tips directly pre-cultivated in three cryoprotectants “sucrose, trehalose and glucose” (n = 3, with each replicate composed of 20–25 shoots). Error bars represent standard deviations. The control included shoot tips not pre-cultivated. Letters between brackets indicate results of Tukey's HSD tests, where the same letter are not significantly different at P > 0.05.

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Fig. 4

Variation of soluble carbohydrate contents in potato shoot tips pre-cultivated based on the application of various cryoprotectants “sucrose, trehalose and glucose” (n = 3, with each replicate composed of 20–25 shoots). The control included un-precultivated shoot tips. Vertical bars represent standard deviations. Italic lowercase letters in black colour indicate results of Tukey's HSD tests associated with two-way ANOVA (Table 3), where the same letter are not significantly different at P > 0.05. Capital and coloured letters (top of bars) are Tukey's HSD tests associated with one-way ANOVAs (comparisons of each soluble carbohydrate between pretreatments).

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3.4 Relationships between rich-media total carbohydrates and soluble carbohydrates in shoot tips

The GLM testing the variation of different soluble carbohydrate concentrations following total carbohydrates and pre-culture treatments showed that soluble carbohydrate contents increased when total carbohydrates increased in the media. However, this relationship was not significant (GLM: t = 0.71, P = 0.491) (Table 4). The model indicated also that the contents of soluble carbohydrates increased when the pre-cultivation of potato shoot-tips was carried out in sucrose compared to control and other sugars (Fig. 5). The GLM also revealed that regardless of total carbohydrate concentrations in pre-culture treatments, no differences (P > 0.05) were observed in concentrations of the soluble carbohydrates in shoot-tips (Table 4).

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Fig. 5

Relationship between total carbohydrate content and different soluble carbohydrate concentrations for various pre-culture treatments of cryopreserved potato shoot tips. Values are set to soluble carbohydrate concentrations and the shape and colour of points represent the type of soluble carbohydrate. The coloured lines represent linear regressions obtained by a Gaussian GLM fit (generalized linear model) with confidence regions in light grey.

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