Gazania rigens(L.) Gaertn. (Asteraceae) is a medicinal plant with high ornamental potential and use in landscaping. The therapeutic potential of sesquiterpene lactones (SLs) as plant natural products for pharmaceutical development has gained extensive interest with costunolide (chemical name: 6E,10E,11aR-6,10-dimethyl-3-methylidene-3a,4,5,8,9,11a-hexahydrocyclodeca[b]furan-2-one) used as a popular herbal remedy due to its anti-cancer, antioxidant, anti-inflammatory, anti-microbial, anti-allergic, and anti-diabetic activities, among others. In the present study, two explant types (leaf, stem) and four 2,4-dichlorophenoxy acetic acid (2,4-D) concentrations (0, 0.5, 1 and 2 mg/L) were tested for callusing potential. The results showed that stem explants treated with 1.5 mg/L 2,4-D exhibited higher callus induction percentage (90%) followed by leaf explants (80%) with 1 mg/L 2,4-D, after a 4-week period. Cell suspension cultures were established from friable callus obtained from stem explants following a sigmoid pattern of growth curve with a maximum fresh weight at 20 days of subculture and a minimum one at 5 days of subculture. In the following stage, the effects of elicitation of cell suspension cultures with either yeast extract (YE) or methyl jasmonate (MeJA), each applied in five concentrations (0, 100, 150, 200 and 250 mg/L) on cell growth (fresh and dry biomass) and costunolide accumulation were tested. After 20 days of culture, YE or MeJA suppressed cell growth as compared to the non-elicited cells, while costunolide accumulation was better enhanced under the effect of 150 mg/L MeJA followed by 200 mg/L YE, respectively. In the subsequent experiment conducted, the optimal concentration of the two elicitors (200 mg/L YE, 150 mg/L MeJA) was selected to investigate further elicitation time (0, 5, 10, 15 and 20 days). The results revealed that YE biotic elicitation stimulated cell growth and costunolide production, being maximum on day 20 for fresh biomass, on day 5 for dry biomass and on day 15 for the bioactive compound. Accordingly, cell growth parameters were maximized under the effect of abiotic elicitation with MeJA for 15 days, while highest costunolide content was achieved after 10 days. Overall, MeJA served as a better elicitor type than YE for biomass and costunolide production. Irrespective of elicitor type, elicitor concentration and elicitation time, maximal response was obtained with 150 mg/L MeJA for 10 days regarding costunolide accumulation (18.47 ppm) and 15 days for cell growth (fresh weight: 954 mg and dry weight: 76.3 mg). The application of elicitors can lead the large quantity of costunolide to encounter extensive range demand through marketable production without endangering ofG. rigens.Graphical Abstract

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 77–90% in vivo (30 days) and 96% in vitro (10 days) using an MS medium with 2.9 μM gibberellic acid (GA₃). The optimum treatments for cuttings’ rooting were 1000 and 2000 ppm indole-3-butyric acid (IBA) (11–16 roots, 2–3 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 μM 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.

Plant growth and development are strongly influenced by light quality conditions in their environment. In this study, in vitro shoot proliferation, photosynthetic pigments, leaf anatomy and photosystem II photochemistry of Gisela 6 cherry rootstock (Prunus cerasus x Prunus canescens) were investigated. The culture medium used was the MS (Murashige and Skoog) containing 4.4 μΜ 6-benzyladenine (BA), 0.05 μΜ α-naphthalene acetic acid (NAA) and 0.3 μM gibberellic acid (GA₃) (30 days, 16 h photoperiod, 22 ± 2 °C). Initially, two different light wavelength absorption spectra: 400–700 nm provided by white fluorescent light lamps (mainly blue–green, BG at equal ratio 1:1; WFL-40) and, 430–690 nm provided by two Light Emitting Diodes (LED) types, differing in the spectral ratio of multiple light emitting wavelengths [LED2-40 (blue–red, BR) and LED3-40 (blue–green–yellow–orange–red, BGYOR)] were studied, under 40 μmol m⁻² s⁻¹ of light intensity. Following that, three LED intensities-20 (LED1-20), 40 (LED2-40), and 120 μmol m⁻² s⁻¹ (LED4-120) were investigated under BR spectrum. WFL-40 exhibited non-significant differences with LED2-40 (shoot number 4.25, proliferation rate 4.30, shoot height 0.91 cm). LED2-40 displayed higher fresh and dry shoot biomass, better PSII functionality, increased Chl a/b, Chl b and carotenoids content as well as decreased number of stressed explants. Leaf anatomy under LED2-40 and WFL-40 was similar. LED4-120 presented the highest rate of stressed explants, with their leaf anatomy being severely affected, as leaves had a reduced mesophyll cell density. In LED4-120, the proportion of the absorbed energy used in photochemistry (Φ_PSII) was decreased whereas the increased quantum yield of non-regulated energy loss in PSII (Φ_NO) could not be compensated by the increased photoprotective energy dissipation (Φ_NPQ). Therefore, LED2-40 is proposed as the most beneficial for growing cherry rootstock Gisela 6 plantlets in vitro.

Plants know no political borders and some of them are restricted to small geographical territories of different countries in which they are endemic. In this study, we prioritized plants (PPs) of the cross-border area of Greece and the Republic of North Macedonia that are already threatened or nearly so (PPA), those which occur exclusively on either side of borderline and/or nearby countries (PPB), or those which are uncommon and rare in this region (PPC) with the aim to document in-situ the species-specific risks-threats; offer ex-situ conservation for them as a back-up solution for future re-introductions and sustainable exploitation; and raise public awareness and alertness about the importance of local biodiversity. In the framework of the project Conse-pp, 20 botanical expeditions were performed in 75 selected areas to collect samples and suitable propagation material from 130 PPs (147 accession numbers), also recording all types of threats-identified in-situ for each of them. No ex-situ conservation was detected for 40 PPs worldwide while for another 12 PPs only limited attempts have been made to date. The fully documented PPs are currently maintained under ex- situ conservation and acclimatization in the Balkan Botanic Garden of Kroussia (BBGK). In total, 156 propagation trials (sexual or asexual) have been made for these PPs to develop species-specific propagation protocols. Consequently, the production of new plant stocks raised ex-situ was achieved (n = 3254 individuals; first-time ex-situ conservation for 40 taxa), and this has enabled the establishment of three awareness-raising sites with PPs: (i) 1000 plants of 70 PPs in the newly designed Kardia Botanical Park in Thermi (Greece); (ii) 850 plants of 104 PPs in the phytodiversity awareness spot in BBGK and 130 individual PPs in the Garden of Environmental Awareness; and (iii) 42 plants of 14 PP taxa delivered to the botanic garden of Ss. Cyril and Methodius University in Skopje. This project outlines the necessity of the development of common plant conservation strategies for threatened plants in cross-border areas of neighboring countries and presents an integrated approach allowing for sustainable development and future in-situ protection measures and actions in the studied cross-border area.