The present study demonstrates the potential of the alginate encapsulation of shoot tips and nodal segments of Gardenia jasminoides Ellis, the short-term cold storage of artificial seeds and subsequent successful conversion to desirable, uniform and genetically stable plantlets. Shoot tips and first-node segments below them, derived from shoots of in vitro cultures, responded better than second-to-fourth-node segments on agar-solidified Murashige and Skoog (MS) nutrient medium and thus, they were used as explants for alginate encapsulation. Explant encapsulation in 2.5% sodium alginate in combination with 50 mM of calcium chloride resulted in the production of soft beads, while hardening in 100 mM of calcium chloride formed firm beads of uniform globular shape, suitable for handling. The addition of liquid MS nutrient medium in the sodium alginate solution doubled the subsequent germination response of the beads. The maintenance of alginate beads under light favored their germination response compared to maintenance in darkness. Encapsulated shoot tip explants of gardenia, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in their regeneration response (73.3, 68.9, 53.3%, respectively), whereas, non-encapsulated explants (naked), stored under the same time durations of cold conditions, exhibited a sharp decline in regeneration response up to entirely zeroing (48.9, 11.1, 0.0%, respectively). Shoots, derived from 12-week cold-stored encapsulated explants, were easily rooted in solid MS nutrient medium with the addition of 0.5 μM of Indole-3-acetic acid (IAA) and after transplantation of the rooted plantlets individually to pots containing a peat–perlite (3:1, v/v) substrate, they were successfully acclimatized in the greenhouse under the gradual reduction of 75 or 50% shading with survival rates of 95–100%. The genetic stability of the acclimatized plantlets was assessed and compared with the mother plant using inter simple sequence repeat (ISSR) markers. ISSR analysis confirmed that all regenerated plantlets were genetically identical to the mother plant. This procedure of artificial seed production could be useful for the short-term storage of germplasm and the production of genetically identical and stable plants as an alternative method of micropropagation in Gardenia jasminoides.

Pistacia lentiscus L. is a dioecious shrub or small tree with high drought resistance, native in the entire Mediterranean basin. The variety ‘Chia’ (mastic tree) is cultivated for the production of mastic gum (a resin produced after wounding of the trunks and thick branches) in the Island of Chios (Greece), but it also has a significant ornamental value. In the present study, ten male and ten female genotypes of P. lentiscus var. Chia from the natural habitats of Chios were selected and examined with respect to the rooting efficiency of their shoot cuttings. The germination ability of the seeds was also tested. The 20 plants were morphologically described, mainly with respect to traits related to their ornamental value. Furthermore, leaves were collected from the 20 genotypes, from which genomic DNA (gDNA) was isolated, followed by amplification of gDNA fragments using the polymerase chain reaction (PCR) and inter simple sequence repeat (ISSR) primers. This was done aiming both at the determination of their genetic distance and the establishment of possible correlations between the amplified bands and certain morphological traits. The results of the study showed that there were differences among the genotypes for both methods of propagation. Regarding the rooting of the shoot cuttings, the best genotype was 8M (cv. ‘Psilophyllos’), achieving a rooting rate of 62.5% in winter with the application of indolebutyric acid (potassium salt of IBA), while regarding the seed germination capacity the genotype 2F exhibited the highest germination rate (57%). Genetic analysis using ISSRs separated the plants into four groups, one group consisting of male genotypes, one of the female genotypes, one consisting of members from both genders and a fourth containing a single male genotype. The genetic analysis of the male genotypes only produced a dendrogram showing the cultivars clustering in three different groups. Regardless of the genetic analysis, it seems that there were correlations between the ISSR markers and the leaf traits and also the gender and the asexual propagation. These correlations can assist future breeding programs of P. lentiscus var. Chia.

The market demand for interesting varieties and cultivars of Ilex aquifolium necessitates the exploration and sustainable exploitation of plant individuals thriving in nature without human care. In this work, an effort was made to develop a simple and reliable protocol for vegetative propagation of I. aquifolium plants, with desirable-for-market characteristics, grown in a mountain area of Halkidiki, Greece, and at the same time to proceed with their genetic identification using molecular markers. From these plants, new plants were vegetatively produced which afterward were used as stock plants for providing the needed shoot cuttings for the experiments of rooting and leaves for their genetic analysis. Factors studied in formulating a propagation protocol included the season of cutting collection and the application of 0.2% 1-naphthaleneacetic acid (NAA), as well as the type of shoot cuttings (terminal, subterminal) and the application of auxin. It was found that application of NAA was crucial for rooting response and number of roots formed, whereas the season effect was not significant on rooting. Terminal cuttings treated with 0.2% NAA exhibited the highest rooting percentage (100%) and formed abundant roots (25.7) compared to subterminal ones. All rooted cuttings, after being potted and transferred to acclimatization greenhouse, were successfully hardened. In the spring of the next year, the produced plants blossomed abundantly and formed fruits (bright red berries) presenting their characteristic ornamental appearance that was maintained until Christmas. For the identification procedure, the genotypic profile of the stock plants was also investigated by inter-simple sequence repeat (ISSR) genetic analysis, revealing that they were genetically the same both among themselves and when compared with a certified I. aquifolium ‘Agrifoglio Commune’ individual, but they differed genetically from I. aquifolium ‘Argentea Marginata’ and I. aquifolium ‘Hellas’.

The present study demonstrates an efficient protocol for alginate encapsulation, interim cold storing of artificial seeds and conversion to genetically stable plants of Viburnum dentatum L. “Lucidum Aiton”. Explants of shoot tips and first-node segments, excised from in vitro-derived viburnum microshoots, were encapsulated in 2.5% sodium alginate mixed with liquid MS nutrient medium and hardened in 50 mM of calcium chloride producing solid, soft and uniform beads. These artificial seeds achieved 28.9% germination under light, forming 4.3 microshoots per bead. However, with 100 mM of calcium chloride for hardening, the beads were firm and of a uniform globular shape and suitable for handling and exhibited a germination response of 48.9%. Encapsulated shoot tip explants of viburnum, which were stored at 4 °C for 4, 8 or 12 weeks, showed a gradual decline in regeneration response (73.3, 62.2, 51.1%, respectively), while non-encapsulated explants, stored under same conditions, did not survive after the fourth week of cold storage. Microshoots from cold-stored encapsulated explants, which were rooted in solid MS nutrient medium with 0.5 μΜ of Indole-3-acetic acid (IAA) and transplanted to a substrate of peat-perlite (3:1, v/v), acclimatized successfully after application of 75 or 50% shading, which was gradually reduced, and were established with minimum losses in a greenhouse. For the genetic stability of the artificial seed-derived plantlets and compared with the mother plant, an assessment was conducted using Inter Simple Sequence Repeats (ISSRs) analysis. The ISSR profiles proved the genetic uniformity and clonal stability of the regenerated plantlets and their genetic resemblance to the mother plant. The present regeneration procedure could be used as an alternative method for the micropropagation of V. dentatum.

Rosmarinus officinalis L. belongs to the Lamiaceae family and is cultivated worldwide due to its diverse uses as an ornamental evergreen shrub in gardening, as well as a food seasoning and a natural medicine. The present research was conducted to study the morphological traits of seven wild-grown rosemary populations in Greece, as well as the propagation of two selected populations with the most desirable ornamental characteristics, by the use of shoot cuttings and in vitro cultures. From the study of the morphological traits of the seven populations, those with interesting features were grown in the areas of Amaliada and Piperia and, thus, were chosen for studying their propagation. Rooting of the shoot cuttings was carried out during the four seasons of the year, using potassium salt Indole-3-butyric acid (K-IBA) at concentrations of 0.5 and 1 g·L⁻¹, in various substrates under the intermittent mist or fog system. It was found that the shoot cuttings rooted easily in all four seasons, but there were better results for the population of ‘Piperia’ in autumn, with 1 g·L⁻¹ K-IBA (80%), and ‘Amaliada’ in spring, with 0.5 g·L⁻¹ K-IBA (82.5%), while higher rooting percentages were achieved in the fog system, on a substrate consisting of perlite and peat, in a ratio of 2:1 (85%). For the in vitro cultures, shoot tips excised from the two selected populations were successfully disinfested by pre-soaking in an antioxidant solution and then, by sterilizing them in 0.6% (w/v) NaOCl, followed by transferring them onto a Murashige and Skoog (MS) nutrient medium. ‘Amaliada’ cultures proved to be the most productive population (2.1 shoots per explant), with the highest shoot formation frequency (91.6%), when cultivated on the MS nutrient medium without plant growth regulators. For ‘Piperia’ cultures, the highest shoot formation frequency (66.6%) was achieved on the MS nutrient medium supplemented with 0.25 or 0.5 mg·L⁻¹ 6-Benzyloaminopurine (BAP) and 0.1 mg·L⁻¹ Indole-3-acetic acid (IAA). Spontaneous root formation frequency was noticed on the MS nutrient medium, containing 0.5 mg·L⁻¹ BAP and 0.1 mg·L⁻¹ IAA, for both ‘Amaliada’ and ‘Piperia’ cultures (50% and 41.6%, respectively) in a single stage, with root lengths of 7.1 and 5.3 cm, respectively. Rosemary plantlets, with roots formed in vitro after transplanting them in soilless substrate, were acclimatized adequately in the greenhouse environment (~70%).