The Balkan Botanic Garden of Kroussia, Northern Greece :

in this paper the profile of the Balkan Botanic Garden of Kroussia (BBGK) is outlined and basic illustrations are provided. Τhe location, establishment, facilities, organization and staff of BBGK, as well as the topography, soils and climate of its grounds, are presented. the plant conservation targets and policies formulated by bbgk in order to fulfil its mission are also described. This includes eight different complementary policies. These are the Only Native Plants policy, Important Plant Species (IPS) policy, Explicit Plant Documentation policy, Propagation of the IPS First policy, DNA Barcoding policy, Combined ex situ and in situ Conservation policy, Evaluation for Sustainable Exploitation policy and the Environmental Awareness on Native Plants policy. The living native plant collections of the BBGK are presented quantitatively and the number of plant sections and displays designed in the BBGK and in the newly established Garden of Environmental Awareness are described. BBGK’s mission is to support research, maintenance, propagation, evaluation, conservation and sustainable use of the native plants of Greece and the Balkans, combined with raising public awareness of the environment

Incorporating Biogeographical Principles in Horticulture :

The design and creation of the Ionian Islands Unique Rock Garden (IIURG), an innovative plant display in Thessaloniki, Greece, is described. This includes seven naturalistic rock gardens hosting twenty-two plant taxa of conservation priority. The unique nature of the IIURG is based on two features: the emulation of the natural geographic characteristics of the Ionian Islands (Greece) and the integration of the endemic elements of the islands’ native flora, focusing on the biogeographical significance of each. In this way, the IIURG represents an innovative concept in the horticulture of botanic gardens which embodies the natural biogeography and its basic concepts such as ‘islands’, ‘native’ and ‘endemism’. In aesthetic terms the garden looks like a natural sculpture and functions as a focal point. It is a carefully constructed plant display using environmentally sustainable principles with low levels of maintenance and irrigation required. The aim is that the IIURG should be used as a useful tool for awareness-raising on the conservation of prioritised plants

Endophytic Bacteria From the Roots of the Medicinal Plant Alkanna tinctoria Tausch (Boraginaceae): Exploration of Plant Growth Promoting Properties and Potential Role in the Production of Plant Secondary Metabolites

Alkannin and shikonin (A/S) are enantiomeric naphthoquinones produced in the roots of certain plants from the Boraginaceae family such as Lithospermum spp. and Alkanna spp. They possess antimicrobial, anti-tumoral and wound healing properties. The production of secondary metabolites by Alkanna tinctoria might be influenced by its endomicrobiome. To study the interaction between this medicinal plant and its bacterial endophytes, we isolated bacteria from the roots of wild growing Alkanna tinctoria collected near to Athens and Thessaloniki in Greece. Representative strains selected by MALDI-TOF mass spectrometry were identified by partial 16S rRNA gene sequence analysis. In total, 197 distinct phylotypes of endophytic bacteria were detected. The most abundant genera recovered were Pseudomonas, Xanthomonas, Variovorax, Bacillus, Inquilinus, Pantoea, and Stenotrophomonas. Several bacteria were then tested in vitro for their plant growth promoting activity and the production of cell-wall degrading enzymes. Strains of Pseudomonas, Pantoea, Bacillus and Inquilinus showed positive plant growth properties whereas those of Bacteroidetes and Rhizobiaceae showed pectinase and cellulase activity in vitro. In addition, bacterial responses to alkannin and shikonin were investigated through resistance assays. Gram negative bacteria were found to be resistant to the antimicrobial properties of A/S, whereas the Gram positives were sensitive. A selection of bacteria was then tested for the ability to induce A/S production in hairy roots culture of A. tinctoria. Four strains belonging to Chitinophaga sp., Allorhizobium sp., Duganella sp., and Micromonospora sp., resulted in significantly more A/S in the hairy roots than the uninoculated control. As these bacteria can produce cell-wall degrading enzymes, we hypothesize that the A/S induction may be related with the plant-bacteria interaction during colonization

Cretan Dittany (<i>Origanum dictamnus</i> L.), a Valuable Local Endemic Plant: In Vitro Regeneration Potential of Different Type of Explants for Conservation and Sustainable Exploitation

Origanum dictamnus L. is a medicinal local endemic to the Island of Crete, Greece. Its propagation through biotechnological tissue culture techniques is essential due to its augmented multi-industrial sector demand. For direct organogenesis, among different culture media variants (MS, Gamborg B5), and cytokinins [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyl adenine (2-iP)], the MS + added with BA (2.2 μM) was the most effective treatment for shoots and roots formation. For indirect organogenesis, all explant types (leaves, petioles, roots) showed a 100% callusing rate after 2 months in all media variants tested; ODK1: 20 μM thidiazuron (TDZ) + 5 μM indole-3-butyric acid (IBA) or ODK2: 0.5 μM kinetin + 5 μM 2,4-dichlorophenoxy acetic acid (2,4-D). The leaves and petiole explants assured a low rate of shoot regeneration (20%) in ODK1. Afterwards, leaf-, petiole-and root-callus derived from both media were transferred to four new media plant growth regulators—free or with BA + IBA + gibberellic acid (GA3). After 10 months from callus transferring, the petiole callus gave rise to roots (20–75%) while the leaf callus exhibited 10–30% shoot or 30% root regeneration. In this study, indirect organogenesis of O. dictamnus was carried out for the first time, thus various organs can be used for plant regeneration, and the developed protocol may be applicable in the horticulture industry

In vitro propagation of Alkanna tinctoria Tausch.: a medicinal plant of the Boraginaceae family with high pharmaceutical value

Alkanna tinctoria Tausch. (Boraginaceae), commonly known as alkanet or dyers’ bugloss/alkanet, is a perennial plant rich in naphthoquinone enantiomers, such as Alkannin and Shikonin (A/S), which possess a wide range of pharmaceutical properties and are used as cosmetics, food additives, and natural dyes. This plant is mostly exploited from the wild, increasing the risk of its extinction as reported for other A/S producing plants extracted from their natural environment. Its cultivation under controlled conditions remains difficult and the need for alternative production systems both for preserving this endangered species and for increasing the production of A/S at a marketable level, has become a necessity. In the present study, a protocol for the in vitro production of A. tinctoria plants using shoot-tip explants was developed. Several culture media, concentrations of hormones, sugar, and gelling agents were tested to improve proliferation, rooting, and acclimatization of micropropagated shoot-tip explants from plants collected in the wild. Surface disinfection was optimal after immersion of shoot-tips and/or nodal explants in Signum® fungicide (30 min), ethanol 70% (1 min), and sodium hypochlorite 3% (10 min). Shoot proliferation was the highest on Murashige-Skoog basal medium enriched with 1.1 μM 6-benzyladenine, 0.15 μM α-naphthalene acetic acid, and 0.3 μM gibberellic acid with a proliferation rate of 3.9 every two weeks. For rooting, the Root Culture 1 (RC1) modified medium free of ammonium nitrate and enriched with 2.85 μΜ indole-3-acetic acid was the more adequate with 80% of roots formation after 30 days. Finally, acclimatization was optimal (100% survival rate) following transfer of the rooted explants in pots containing a peat moss:perlite (1:1, v/v) mixture, kept under a 90% relative humidity fog system for 10 days, followed by a decrease in relative humidity of 5% every day until 40% and a gradual increase in light intensity. The protocol developed allowed the production under in vitro culture conditions of a sufficient number of A. tinctoria plants with high levels of ex vitro survival, opening the door to industrial exploitation of its secondary metabolites and to the conservation of this important medicinal plants

GIS-Facilitated Effective Propagation Protocols of the Endangered Local Endemic of Crete Carlina diae (Rech. f.) Meusel and A. Kástner (Asteraceae): Serving Ex Situ Conservation Needs and Its Future Sustainable Utilization as an Ornamental

Conservation and sustainable exploitation of threatened endemic plants with medicinal and/or horticultural/ornamental value can be achieved through the development of effective propagation protocols. After unveiling the bioclimatic preferences of Carlina diae (Asteraceae) with geographic information systems (GIS), four propagation trials were conducted using seeds of this endangered local Cretan endemic for in vivo and in vitro germination, as well as seasonal vegetative propagation trials (softwood cuttings) and micropropagation (nodal explants). Seed germination was accomplished at a level of 77&ndash;90% in vivo (30 days) and 96% in vitro (10 days) using an MS medium with 2.9 &mu;M gibberellic acid (GA3). The optimum treatments for cuttings&rsquo; rooting were 1000 and 2000 ppm indole-3-butyric acid (IBA) (11&ndash;16 roots, 2&ndash;3 cm long, 100% rooting) within 40 days in mist. In vitro shoot propagation exhibited a 2.8 proliferation rate after six successive subcultures on an MS medium with 2.9 &mu;M GA3. Both ex vitro rooting and acclimatization were successful in 40 days, with 96% microshoot rooting and an equal survival rate. The GIS-facilitated effective species-specific propagation protocols developed in this study can consolidate the perspective of successful re-introduction of ex situ-raised material of C. diae into wild habitats and may serve its sustainable exploitation for high-added value ornamental products

Seed Germination Trials and Ex Situ Conservation of Local Prioritized Endemic Plants of Crete (Greece) with Commercial Interest

The in vivo germination course (15, 30, 45, and 60 days) of nine threatened local endemic plants of Crete (Greece) was studied due to conservation concerns and commercial interest in economic sectors. We used seeds directly collected from the wild sown in peat (Terrahum):perlite (1:1 v/v ratio)covered with coarse-grained vermiculite in a greenhouse mist bench with a substrate temperature of 19 &plusmn; 2 &deg;C. The onset of in vivo germination was defined by the visible radicle protrusion (6th&ndash;9th day from sowing). After 60 days, 100% germination was observed for seeds of Campanula cretica, Dianthus&nbsp;fruticosus subsp. creticus, and Petromarula pinnata, followed by Draba cretica (91%) and Sanguisorba cretica (86%). Average&ndash;low germination capacity was observed for seeds of Calamintha cretica (26%), Lomelosia minoana subsp. minoana (38%), and Origanum microphyllum (23%), and very low capacity was observed for Onopordum bracteatum subsp. creticum (1.5%). After keeping seeds at 4&ndash;5 &deg;C for three months, pre-treatments were performed (immersion in 50 or 250 ppm of gibberellic acid solution for 24 h) on three taxa with low germination capacity, thus resulting to the germination acceleration of Calamintha cretica seeds and increased germination capacity of Onopordum bracteatum subsp. creticum seeds. Apart from the facilitation of future species-specific conservation actions, the study showcases that the obtained results herein may permit an upgrade in the feasibility and readiness timescale assessments for the sustainable exploitation of studied taxa in different economic sectors

Rosmarinic Acid Production from <i>Origanum dictamnus</i> L. Root Liquid Cultures In Vitro

In the present work Origanum dictamnus L. was studied as a suitable in vitro adventitious root culture system for the production of important bioactive molecules, such as rosmarinic acid (RA). Callus culture was initiated from leaf, petiole and root explants on solid MS medium supplemented with either 5 μM NAA + 5 μM kinetin (ODK3) or 5 μM NAA + 0.5 μM kinetin (ODK4). New roots formed from leaf, petiole and root calluses were aseptically transferred into Erlenmeyer flasks containing 100 mL liquid medium and shaken at 120 rpm in the dark. The liquid medium used was the MS supplemented either with 35 μM IBA + 2.5 μM kinetin (ODY1) or 5 μM NAA + 0.5 μM kinetin (ODY2). Biomass production parameters, RA content (%) and yield index (YI) were recorded for each treatment explant type, medium composition and incubation period. Results showed, in every case, the production of RA in vitro. Between the two liquid media (ODY1, ODY2) and the different culture periods, the ODY1 medium and the longest 200-day-culture period were more effective for RA and biomass production, regardless of the initial explant type used. The combination of ODK4-ODY1 resulted in higher RA (5.1% and 4.7%), fresh biomass production (19.0 g and 11.6 g), mean YI (93.7 mg and 51.4 mg) and YI per explant (3.75 mg and 2.06 mg) for roots derived from leaf calluses and root calluses, respectively. However, the solid ODK3 (200 days)–liquid ODY1 (40 days) transition treatment was more beneficial for roots derived from petiole calluses leading to an 18.8-fold increase in fresh biomass growth rate. RA accumulation and YIs were also significantly influenced by explant type, with the highest value produced from root petiole calluses (6.6% RA dry weight, 115.3 mg mean YI and 4.61 mg YI per explant) after 240 days

Hydroponic Cultivation of Vine Leaves with Reduced Carbon Footprint in a Mediterranean Greenhouse

Vine leaves are considered a delicacy food however they are only produced as a byproduct for a short harvest period due to grape cultivation practices and numerous chemical applications. In this work, vine plants were cultivated hydroponically in a greenhouse, to extend the cultivation period and along with high plant density, maximize fresh leaves yield. Four different substrates were tested—Perlite, Perlite-Attapulgite, Perlite-Zeolite, 1.7Perlite-higher density planting—with soil treatment as a control, and the experimental cultivation lasted a total of about seven months in the year 2021. Quantitative and qualitative characteristics such as leaves number and weight, color of leaves, nitrates, photosynthetic parameters, total phenols, and plant nutrient concentrations were assessed, while the product’s environmental impact was calculated. The 1.7Perlite treatment produced a 1.6–2.0 times higher number of leaves per hectare than the other hydroponic treatments and 8.7 times higher than the soil treatment, while no statistically significant differences were found regarding qualitative characteristics. Consequently, the 1.7P treatment resulted in a 1.4 to 7.6 times lower product carbon footprint compared to the other treatments. In future research, substrates water and nutrient retention will be further studied along with year-round production in a heated greenhouse with full climate control so that plants are kept evergreen

Hydroponic Cultivation of Vine Leaves with Reduced Carbon Footprint in a Mediterranean Greenhouse

Vine leaves are considered a delicacy food however they are only produced as a byproduct for a short harvest period due to grape cultivation practices and numerous chemical applications. In this work, vine plants were cultivated hydroponically in a greenhouse, to extend the cultivation period and along with high plant density, maximize fresh leaves yield. Four different substrates were tested&mdash;Perlite, Perlite-Attapulgite, Perlite-Zeolite, 1.7Perlite-higher density planting&mdash;with soil treatment as a control, and the experimental cultivation lasted a total of about seven months in the year 2021. Quantitative and qualitative characteristics such as leaves number and weight, color of leaves, nitrates, photosynthetic parameters, total phenols, and plant nutrient concentrations were assessed, while the product&rsquo;s environmental impact was calculated. The 1.7Perlite treatment produced a 1.6&ndash;2.0 times higher number of leaves per hectare than the other hydroponic treatments and 8.7 times higher than the soil treatment, while no statistically significant differences were found regarding qualitative characteristics. Consequently, the 1.7P treatment resulted in a 1.4 to 7.6 times lower product carbon footprint compared to the other treatments. In future research, substrates water and nutrient retention will be further studied along with year-round production in a heated greenhouse with full climate control so that plants are kept evergreen