Ex vitro Rooting, Acclimatization and Genetic Stability of Lonicera caerulea var. kamtschatica

Axillary shoot cultures of two L. caerulea var. kamtschatica cultivars ‘Wojtek’ and ‘Zojka’ were initiated from 2-year-old nursery plants. Shoots were established and continuously multiplied for 2 years on the Murashige and Skoog (1962) medium (MS) modified with a supplementation 85.5 mg·dm⁻³ NaH₂PO₄·H₂O, 100 mg·dm⁻³ myoinositol, nicotinic acid, pyridoxine and thiamine (1.0 mg·dm⁻³ each), 15 mg·dm⁻³ 2iP, 1.0 mg·dm⁻³meta-topolin, 30 g·dm⁻³ sucrose and solidified with a mixture of gelling agents, 3 g·dm⁻³ agar (Biocorp, Poland) and 1.2 g·dm⁻³ Gelrite (Duchefa, Netherlands) (Gabryszewska et al. 2016). The pH of the medium was adjusted to 5.6 before autoclaving. Axillary shoot cultures were subcultured on the fresh medium every 6–8 weeks.

The shoots were rooted in vitro on the MS medium containing indole-3-butyric acid (IBA) or 1-naphthaleneacetic acid (IAA) at different concentrations (0.0; 1.0; 2.5; and 4.0 mg·dm⁻³) (Wojtania et al. 2018). The aim of this experiment was to determine the post-effect of auxin type and concentrations on ex vitro acclimatization. The in vitro rooted microcuttings were planted at the end of March in multi-cell plug trays of 30 mm diameter, with peat substrate, in plastic mini-greenhouses (Garland Products, UK) with two adjustable “dial” ventilators to control the humidity and optimize growing conditions. The plants were maintained for 4 weeks in a growth room under fluorescent lamps (50 μmol·m⁻²·s⁻¹) at 23 ± 2 °C. Starting with day 7 after the transfer to ex vitro conditions, the humidity was stepwise reduced by opening the ventilators and the plantlets were fertilized with Peters Professional Plant Starter (0.25 g·dm⁻³). After 6 weeks, the following data were collected: survival rate, fresh mass and length of cuttings as well as fresh mass, the number and length of roots.

The aim of this experiment was to determine ex vitro rooting and acclimatization ability in the greenhouse of axillary shoots derived from a 6-week-old in vitro cultures incubated on a multiplication medium. For ex vitro direct rooting, two types of explants were used: single microshoots about 1.0 cm long and clumps of 4–5 shoots. The experiment started in mid-June. Both explant types were planted in multi cell plug trays of 30 mm diameter with three different soil substrates – peat, peat + sand (2 : 1 v/v), peat + perlite (2 : 1) – in plastic mini-greenhouses maintained in the greenhouse. Cuttings were maintained under natural photoperiod and irradiance with day/night of 24 ± 5 °C/18 ± 5 °C. The acclimatization procedure was the same as in Experiment 1. After 6 weeks of growing ex vitro in the greenhouse, the following data were collected: survival and rooting rate, fresh mass and length of cutting as well as fresh mass, the number and length of roots.

The genetic stability of acclimatized plantlets of two L. caerulea var. kamtschatica cultivars (‘Wojtek’ and ‘Zojka’) was evaluated in relation to the mother plants (standards) by using ISSR and AFLP analyses.

DNA was extracted from fresh leaves collected from 15 randomly selected acclimatized microplants of each cultivar. Genomic DNA was extracted using the Gene MATRIX Plant & Fungi DNA Purification Kit (EURx) in two replicates for each sample tested. The concentration and purity of the DNA were determined using an Epoch spectrophotometer (BioTek). Four ISSR and four AFLP primer pairs were finally used in the study after an initial screening of 23 ISSR and 23 AFLP primer pairs for the production of a high number of distinct and countable bands (Tab. 1). For ISSR analysis, the PCR reaction was carried out in 20 μL reaction volume containing 20 ng DNA, 1 × DreamTaq™ Green Buffer (Thermo Fisher Scientific), 0.65 μL dNTPs (10 mM) (Promega), 0.45 μL ISSR primer (10 μM) and 0.5 U DreamTaq™ Green Polymerase (Thermo Fisher Scientific). Amplification was carried out in the T100 Thermal Cycler (BioRad) programmed for 45 ISSR cycles (30 s at 94 °C, 40 s at 55 °C, 90 s at 72 °C). All reactions were repeated twice. The PCR products obtained were separated on 1.5% agarose gel through electrophoresis and photographed using Syngen Biotech camera. The size of the bands was assessed against size standards Gene-Ruler™ 100 bp DNA Ladder Plus (Thermo Fisher Scientific). For AFLP analysis, genomic DNA (50 ng) was digested with MseI and PstI endonucleases and ligated with appropriate adapters (Vos et al. 1995). Preamplification of obtained fragments was carried out using primers complementary to the adapter's sequence. The preselection PCR reaction mixture contains 20 μL DNA, 1 × Taq Polymerase Reaction Buffer (Sigma-Aldrich), 0.8 μL dNTPs (10 mM) (Promega), 1.2 μL of each primer (10 μM) and 0.75 U Taq DNA Polymerase (Sigma-Aldrich). Amplifications were carried out in the thermal cycler programmed for 30 cycles (30 s at 94 °C, 30 s at 60 °C, 60 s at 72 °C). The selective PCR with primer pairs was obtained by extending the primers used in preamplification with two additional nucleotides at the 3’ end (Tab. 1). PCR was carried out in the reaction mixture containing 3 μL DNA 1 : 30 (v : v), 1 × Taq Polymerase Reaction Buffer (Sigma-Aldrich), 0.4 μL dNTPs (10 mM) (Promega), 0.5 μL PstI-NN primer (10 μM), 0.65 μL MseI-NN primer (10 μM) and 0.75 U Taq DNA Polymerase (Sigma-Aldrich). Amplification was carried out in the thermal cycler programmed for 13 cycles (30 s at 94 °C, 30 s at 65–56 °C with annealing temperature decreased by 0.7 °C in each cycle, 60 s at 72 °C), followed by 26 cycles (30 s at 94 °C, 30 s at 56 °C, 60 s at 72 °C). Products of selective PCR were separated on 6% denaturing polyacrylamide gel through electrophoresis on Dual Dedicated Height Nucleic Acid Sequencer (C.B.S. Scientific). The separated AFLP products were stained in a silver nitrate solution and the gel was dried, described and photographed. The size of the bands was assessed against size standards 10 bp DNA Ladder (Invitrogen) and 50 bp DNA Ladder (Invitrogen). Bands generated in ISSR and AFLP analysis were scored manually as present (1) or absent (0) from the photographs. Only bright and reproducible products were scored. During the analysis of electrophoregrams, the number of ISSR and AFLP products, their size and diversity between mother plants and plants obtained from micropropagation were evaluated.

In all experiments on the rooting and acclimatization, 25 microcuttings for each treatment were taken. The experiments were carried out twice. The results were statistically analyzed with the use of the STATISTICA program (13.1 PL 2012, Statsoft, Poland). An analysis of variance was performed with ANOVA, R.A. Fischer for a two-factor system. Means were compared using Duncan's test at p = 0.05.

For both cultivars, the highest survival rate (max. 88%) was observed for microcuttings rooted on the medium containing the lowest auxin level (1.0 mg·dm⁻³). However, the differences between auxin type and concentration on survival rate in ex vitro conditions were not significant. It has been also found that the microcuttings rooted on low auxin medium after transfer to ex vitro conditions showed higher fresh mass and length of shoots as well as all root parameters (fresh mass, number and length) as compared to those rooted on the medium with high auxin (Tab. 2). During acclimatization, wilting and shoot tip necrosis were also observed, and these were most frequent among shoots rooted in vitro in the presence of high auxin concentration (data not shown).

The results showed successful ex vitro rooting without auxin in the greenhouse of both blue honeysuckle cultivars (Tab. 3). The ability to form roots depended on genotype. Higher ex vitro rooting and survival rate in the greenhouse were observed for ‘Wojtek’ (max. 96%) than ‘Zojka’ (max. 88%). There were no significant differences in rooting/survival rate of ‘Zojka’ either between microcutting types or soil substrates. However, the plantlets of ‘Wojtek’ showed a higher rooting/survival rate when using individual microcuttings than shoot clumps (Fig. 1). Shoot clumps had a higher tendency toward the wilting and the shoot tip necrosis, leading to the death of some plantlets. The plants of both cultivars that were acclimatized in peat alone or in a mixture of peat and perlite exhibited higher mass of shoots and roots as well as length of shoots as compared to those grown in a mixture of peat and sand (Tab. 3). The micro-propagated plantlets appeared similar to mother plants.

Figure 1

The results showed that the ISSR primers generated a total of 40 and 36 amplification products ranging in size from 300 to 1400 bp for ‘Wojtek’ and ‘Zojka’ plants, respectively (Tab. 4). Among these bands, 0.0% and 2.77% were polymorphic for ‘Wojtek’ and ‘Zojka’ cultivars in relation to the mother plants. The AFLP primer pairs generated a total of 102 and 105 amplification products ranging in size from 50 to 900 bp for ‘Wojtek’ and ‘Zojka’ plants, respectively (Tab. 4). Among these bands, 0.98% and 1.90% were polymorphic for both ‘Wojtek’ and ‘Zojka’, compared to the mother plants.