1. Introduction
V. dentatum, commonly called arrowwood viburnum, is a multi-stemmed deciduous shrub with glossy leaves bearing clusters of white flowers in spring which turn into bluish berries in autumn [
1].The plant, which is adapted to low and high temperatures, as well as drought and alkaline soil, is used in ornamental horticulture, particularly as a landscaping edging plant and also as a gardening container-grown plant [
2]. Propagating viburnum by seed is a long process as it takes more than ten months to germinate, thus it is usually propagated by softwood shoot cuttings [
3]. Yet, in vitro propagation protocols for
V. dentatum have been developed, which could be used for large-scale production of abundant propagation materials for commercial use [
4,
5].
The encapsulation technique for producing artificial or synthetic seeds (synseeds) is an important application of micropropagation and can play a crucial role as an alternative to other conventional methods for large-scale propagation and long-term germplasm storage of plants [
6,
7]. Artificial seeds are defined as artificially encapsulated somatic embryos, shoot tips, shoot buds, nodal segments, cell aggregates or any tissue that can be used for sowing as a seed and that possess the ability to convert into a plant under in vitro or in vivo conditions [
8,
9,
10]. Artificial seed technology mimics that of the conventional seed technology and combines the advantages of seed (sexual) propagation with those of vegetative (asexual) propagation [
11,
12,
13]. By this method, the genetic uniformity among regenerated plantlets, in vitro mother culture and in the field-grown or in the greenhouse-grown mother plant is maintained in most cases, while elite genotypes can be stored for a short to long term and can be regenerated later upon demand for plant propagation material [
14,
15]. In addition, the method offers the easy handling of artificial seeds during storage, unhindered transportation among countries from customs barriers and/or quarantines and cost efficiency in comparison with the traditional method of in vitro culture, which requires frequent and costly subcultures [
16,
17,
18]. Although many gel materials have been tried for artificial seed production, an alginate matrix was proved to be optimal for encapsulation towards artificial seed production, mainly due to the sensible thickness, weak spinability of solution, low expense and fast gelation [
19,
20]. Successful production of artificial seeds and retrieval of plants have been reported for a large range of plant species including ornamentals, fruit and forest trees, cereals, vegetables, and medicinal and aromatic plants [
21,
22,
23], whereas there is no reference for
V. dentatum.Plantlets derived from in vitro cultures may exhibit somaclonal variation due to physical and chemical factors, type of explant source, longevity of the in vitro culture and genotype [
24,
25]. Thus, assessment for the genetic stability of the produced plantlets from artificial seeds is necessary using Random Amplification of Polymorphic DNA (RAPD) and/or Inter Simple Sequence Repeat (ISSR) analysis as it has been referred in a number of reports [
14,
25,
26,
27].
The aim of this work was to develop an effective procedure for encapsulating shoot explants in alginate in order to produce artificial seeds, alternatively to traditional micropropagation methods, for a fast, efficient and mass propagation of genetically uniform regenerants of V. dentatum for landscape use and also as a container-grown plant. Therefore, the selection of the proper shoot explants, the encapsulation procedure and the short-term storage at 4 °C of the artificial seeds as well as their subsequent germination followed by a stability assessment of the regenerants using ISSR analysis were investigated.
2. Materials and Methods
2.1. Plant Material, Establishment of In Vitro Cultures
Terminal softwood shoots were cut from a viburnum (
V. dentatum L. “Lucidum Aiton”) plant, grown in the greenhouse, suitable for flowering container-grown plants, as well as for gardening plantation. Explants of shoot tips (1.0–1.5 cm in length) were excised from these terminal softwood shoots and dipped for 5 min in 0.1% (
w/
v) Mercuric chloride (HgCl
2) solution and then were surface-disinfested for 5 min in 2.0% (
v/
v) Sodium hypochlorite (NaOCl) solution under vigorous stirring plus 0.05% (
v/
v) Tween-20 (BDH Chemicals, Poole, England), followed by 3 rinses with sterilized distilled water under aseptic conditions. Afterwards, each explant was placed in a single glass culture tube (100 × 25 mm) containing 10 mL of Murashige and Skoog (MS) nutrient medium [
28], with the addition of 5 μM of 6-Benzylaminopurine (BAP) (Sigma-Aldrich, St. Louis, MI, USA), 2% (
w/
v) sucrose and 0.65% (
w/
v) agar (Technobiochem, Athens, Greece). The pH of the medium was adjusted to 5.8 using 0.5N NaOH or 0.1N HCl prior to agar addition. The glass culture tubes were covered with aluminum foil and autoclaved at a temperature of 121 °C and under pressure of 122 kPa for 20 min. Two weeks later, shoot tip explants without any signs of contamination were subcultured in fresh solid MS nutrient medium, of the same composition and sterilization, for multiple shoot formation. Microshoots developed in these subcultures, over a 4-week period, were the donors of various explants for encapsulation.
2.2. Source of Explants for Encapsulation
To determine the proper type of explants for encapsulation, based on the easiness of explant sprouting during germination, shoot tips (3–4 mm in length) or nodal segments of equal size from the 1st, 2nd 3rd and 4th node below the shoot tip, were aseptically cut from the microshoots of the above-described subcultures and transferred to solid MS nutrient medium containing 5 μM of BAP for shoot induction. At the end of the 4-week in vitro culture, the frequency of shoot formation (percentage of sprouted explants), and the number and length of produced shoots were recorded. Based on the response of the various explants in vitro, the shoot tips and 1st-node segments were selected, inequal numbers, for the encapsulation and production of artificial seeds, unless otherwise specified.
2.3. Encapsulation of Explants, Germination of Artificial Seeds
For determining the effect of the inclusion of nutrient medium and sucrose in the encapsulation matrix of viburnum explants, shoot tips and first-node segments were dipped into a liquid MS nutrient medium (lacking of Ca) supplemented with 2.5% sodium alginate, as a gelling agent, and with or without 1% sucrose. Apart from this, other explants were dipped into 2.5% sodium alginate solution deprived of liquid MS nutrient medium and sucrose. Then, explants from the three treatments were picked up by a forceps and placed separately into three different beakers containing 50 mM of calcium chloride (CaCl2.2H2O), as a complexing agent, with continuous stirring for 30 min in order to avoid bead sticking and complete the ion-exchange (Na+/Ca2+) reaction. The resultant alginate beads were collected and rinsed twice with sterilized distilled water to remove excess calcium chloride and then were placed in glass culture tubes (100 × 25 mm) containing solid MS nutrient medium supplemented with 5 μM of BAP. Thereafter, the bead cultures were kept in darkness or under light; to determine their germination success, after a 4-week period, the regeneration response (percentage of sprouted beads), and the number and length of produced shoots were recorded.
The optimal concentration of calcium chloride for bead hardening was identified, after encapsulation of shoot tips and first-node segments in 2.5% sodium alginate, by immersing the coated explants in solutions of calcium chloride at concentrations of 25, 50, 100 or 200 mM for 30 min. The resultant beads after rinsing with sterilized distilled water were placed in glass culture tubes with solid MS nutrient medium and maintained under light for germination, which was evaluated as previously described.
2.4. Germination after Storage of Artificial Seeds
Shoot tip explants encapsulated in 2.5% sodium alginate that was mixed with liquid MS nutrient medium plus 1% sucrose and hardened for 30 min in 100 mM of calcium chloride, as previously described, were maintained at 4 °C in darkness for 4, 8 or 12 weeks in order to evaluate the influence of cold storage duration on the conversion of artificial seeds into plantlets. The resultant beads were placed on moistened with sterilized distilled water filter paper in sealed Petri dishes covered by black polyethylene film. At the end of each cold storage period, the beads were transferred and cultured on fresh solid MS nutrient medium with the addition of 5 μM of BAP. After four weeks in vitro, the germination was evaluated and compared with non-encapsulated (naked) similar explants which were maintained under the light of the same cold conditions on hormone-free solid MS nutrient medium and after storage were subcultured on fresh MS nutrient medium supplemented with 5 μM of BAP.
2.5. Microshoot Rooting, Plantlet Acclimatization
V. dentatum microshoots, produced from the germinated beads after their storage at 4 °C for 12 weeks, were transferred aseptically for rooting to solid MS nutrient medium supplemented with 0.5 μM of Indole-3-acetic acid (IAA) (Sigma-Aldrich, St. Louis, MI, USA). After 4 weeks, rooted microshoots were taken out of the culture medium, washed thoroughly under running tap water to remove any agar medium remnants and transferred to the greenhouse where they were transplanted to a 1:1 (v/v) substrate of peat and perlite in pots. Afterwards, the young plantlets were placed for acclimatization under 75, 50 or 25% shading from a polyethylene net cover, equipped with a water-fogging system controlled electronically, for various periods of time. In the case of 75% shading, the plantlets remained for one week under these conditions and then were placed for one week under 50% shading followed for one more week under 25% shading and afterwards were moved to a greenhouse bench under full-light irradiance (approx. 320 μmol.m−2.s−1) for an additional week. In the treatment of 50% shading, the plantlets remained under these conditions for one week and then were placed under 25% shading for one more week and finally they were transferred to full-light irradiance. In the next two treatments, the plantlets were placed under 25% shading for one or two weeks and then were moved to full-light irradiance for three or two weeks, respectively. At the end of the 4-week acclimatization period the survival rate (percentage of healthy plantlets) of the viburnum plantlets was calculated. The ambient temperature during the acclimatization period was 24 ± 2 °C, while the natural photoperiod was 12–13 h. The relative humidity under the polyethylene net cover decreased gradually from 95% (under 75% shading) to 85% (under 50% shading) and finally to 75% (under 25% shading), while the light irradiance varied from 145 (under 75% shading) and 180 (under 50% shading) to 220 μmol.m−2.s−1 (under 25% shading). After the 4-week acclimatization period, the survived young viburnum plantlets were maintained for further growth in greenhouse conditions.
2.6. Culture Conditions
All in vitro cultures of viburnum for microshoot formation and those of encapsulated explants designed for bead germination were kept in a growth chamber with a 16-h photoperiod from cool-white fluorescent lamps of 50 μmol.m−2.s−1 photon flux density at the culture level. The relative humidity in the growth chamber was 60–70%, while the temperature was set at 23 ± 0.1 °C.
2.7. Genomic DNA Extraction, Polymerase Chain Reaction (PCR) Amplification
Genomic DNA (gDNA) was extracted from the leaves of seven randomly selected artificial seed-derived plantlets of viburnum, four weeks after acclimatization, and the mother control plant using NucleoSpin Plant ΙΙ (Macherey Nagel, Düren, Germany) following the instructions of the package. Purified gDNA was quantified and its quality was verified by nanodrop 2000 (Thermo Electron Corporation, Waltham, MA, USA), while each sample was diluted to 20 ng/μL in TE buffer (10mM Tris: 0.1mM EDTA; pH 8.0) and stored at −20 °C. Ten Inter Simple Sequence Repeat (ISSR) primers were used with numbers ISSR-UBC 808, 809, 810, 811, 812, 815, 816, 818, 821 and 834 (University of British Columbia, Vancouver, BC, Canada) as more appropriate, from a total number of 37 primers available in the laboratory. ISSR amplifications were performed in a volume of 20 μL containing 40 ng total DNA, Horse-Power™ Taq DNA Polymerase MasterMix (Canvax Biotech, Cordoba, Spain), which contained all PCR reaction components (dNTPs, PCR buffer, Mg2+ and Taq DNA Polymerase) and 1 μM from each primer.
PCR was performed at an initial denaturation of 94 °C for 4 min, followed by 35 cycles of 1-min denaturation at 94 °C, 1-min annealing at specific temperature for each primer and 2-min extension at 72 °C with a final extension of 72 °C for 10 min. The SimpliAmpTM Thermal Cycler (Life Technologies, Thermo Scientific, Waltham, MA, USA) was used for the PCR amplifications. DNA amplification fragments were separated in a 1.5% agarose gel with the application of electricity, using 1 × TAE buffer (Tris-acetate-EDTA), stained with ethidium bromide (0.001%) and analyzed using the gel documentation system Transilluminator UV light (Biostep, Burkhardtsdorf, Germany). The size of the amplification products was estimated by BrightMAX™ 1kb DNA Ladder (Canvax Biotech, Cordoba, Spain). Two independent PCR amplifications were performed for each sample.
For ISSR profiles, the clean-separated and consistently reproducible fragments ranging from 200 to 1400 bp were scored as present or absent. For detecting any genetic change, all the ISSR results were compared with each other for all the DNA samples. A similar procedure of genetic homogeneity confirmation among in vitro-derived plantlets has been published for other ornamental plant species such as gerbera [
29],
Lilium longiflorum [
30],
Platanus acerifolia [
31] and
Phoenix dactylifera [
32].
2.8. Data and Statistical Analysis
The experiments were conducted twice in completely randomized designs. In the various experiments, 45 replicates per treatment were used with the exception of the plantlet acclimatization where 20 replicates were employed. The statistical analysis of the data was based on analysis of variance (ANOVA). Data in percentages were subjected to an arcsin transformation prior to statistical analysis and were transformed back to percentages for presentation in tables. The separation among means was carried out with Duncan’s multiple range test and significance was determined at p ≤ 0.05. The statistical analysis was conducted using the SPSS 22 software (SPSS Inc. Statistical Package for the Social Sciences, Chicago, IL, USA).