Taxonomic Identification and Molecular DNA Barcoding of Collected Wild-Growing Orchids Used Traditionally for Salep Production.

Molecular DNA barcoding combined with botanical taxonomy can be used for the identification and conservation of collected Greek orchids used for salep production as well as in the regulation of fair salep trade. A modified CTAB protocol was used for DNA extraction, amplification of barcoding regions (ITS, matK, rbcL, trnH-psbA), and sequencing. Sequencing data were assembled using Bioedit software, and the BLAST algorithm was used on the NCBI database for species identification at the genus level. Molecular barcoding data based on genetic similarity identification was in full coherence with taxonomic classification based on morphological data. The combination of ITS and matK exhibited a greater capacity to identify a species among the Greek salep samples. Out of the 53 samples examined, 52.9% were classified as Dactylorhiza spp. and 33.3% as Anacamptis spp., whereas only 6 samples were identified as Orchis spp. (11.8%). Given that a superior-quality salep beverage comes from tubers of the latter, the number of samples classified as such in northwestern Greece is unexpectedly low. A database of 53 original reference sequences from wild-growing samples of Greek origin was generated, providing a valuable resource for the identification of other salep samples from different regions. The DNA barcoding results unveiled that salep samples from northwestern Greece are related to nine members of four different genera of Orchidaceae. All species are nationally protected and covered by the CITES convention, while many of these orchids are included in the EU Directive 92/43/EEC appendix as "Other Important Species". Thus, expedited coordinated management actions are needed to ensure their survival in the future.

Rosmarinic Acid Production from Origanum dictamnus 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.

Cretan Dittany (Origanum dictamnus 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.

Effect of explant type (leaf, stem) and 2,4-D concentration on callus induction: influence of elicitor type (biotic, abiotic), elicitor concentration and elicitation time on biomass growth rate and costunolide biosynthesis in gazania (Gazania rigens) cell suspension cultures.

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 of G. rigens.

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 77-90% 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.

Globular body production, their anatomy, DNase gel analysis and NDP kinase activity in root tips of Poncirus trifoliata L.

Green globular bodies were developed from Poncirus trifoliata L. root tip explants as a response to addition in the substrate of different growth regulators. From the globular bodies, shoots initiated and grew. Median section of the globular bodies reveals that they are composed of parenchyma cells and originate from the pericycle. The activity of DNases during shoot formation from globular bodies was influenced by the type and concentration of plant growth regulators that were added in the nutrient substrate. Peptide bands formation was also influenced by the increase of BA concentration. Consequently, BA, NAA and IAA combination influenced 5'-triphosphonucleosides (NTPs) appearance and activity in the presence of metal. Peptide bands resulted from the electrophoretic analysis of endogenous protein phosphorylation, proved to be catalytic subunits of NDP kinases, as they all phosphorylate diphosphonucleosides. The enzymes DNases and NDP kinases could be used as a scientific tool for the study of shoot formation from P. trifoliata L. green globular bodies.

Melatonin enhances root regeneration, photosynthetic pigments, biomass, total carbohydrates and proline content in the cherry rootstock PHL-C (Prunus avium × Prunus cerasus).

The present study, investigates the effects of melatonin (0, 0.05, 0.1, 0.5, 1, 5 and 10 µM) on the morphogenic and biochemical responses in the cherry rootstock PHL-C (Prunus avium L. × Prunus cerasus L.), from shoot tip explants. The incorporation of melatonin (0-10 µM) in the Murashige and Skoog (MS) medium, greatly influenced rooting either positively or negatively. Melatonin, irrespective of its concentration, had a negative effect concerning the number of roots. However, application of 0.5 µM melatonin significantly increased the root length; while 1 µM melatonin increased the root length by 2.5 times, and the fresh weight of the roots by 4 times, in comparison to the control. Although 0.05 µM melatonin increased rooting by 11.11%, 5 µM melatonin had a significant reduction on the number, the fresh weight of roots, and the rooting percentage. Melatonin concentration of 0.1 µM resulted in the greatest chlorophyll (a + b) content, and 5-10 µM reduced the chlorophyll concentration by 2 times, compared to the control. The high melatonin concentrations (5 and 10 µM), increased the levels of proline and carbohydrates in leaves by 3-4 times. In the roots, 0.5 µM of melatonin concentration increased the carbohydrate levels by 1.5 times, while 0.05, 0.1 and 1 µM melatonin concentration significantly reduced the proline content.

Melatonin promotes adventitious root regeneration in in vitro shoot tip explants of the commercial sweet cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and MxM 60 (P. avium × P. mahaleb).

The objectives of this study were to test the effects of melatonin (N-acetyl-5-methoxytryptamine), a natural compound of edible plants on the rooting of certain commercial sweet cherry rootstocks. Shoot tip explants from previous in vitro cultures of the cherry rootstocks CAB-6P (Prunus cerasus L.), Gisela 6 (P. cerasus × P. canescens), and M × M 60 (P. avium × P. mahaleb) were included in the experiment. The effect of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) alone or in combination with melatonin was tested concerning their rooting potential. Seven concentrations of melatonin (0, 0.05, 0.1, 0.5, 1, 5, and 10 µM) alone or in combination with 5.71 µM of IAA or 4.92 µM of IBA were tested. For each rootstock, 21 treatments were included. The explants were grown in glass tubes containing 10 mL of substrate. The parameters measured include rooting percentage, number of roots per rooted explant, root length, and callus formation. The data presented in this study show that melatonin has a rooting promoting effect at a low concentration but a growth inhibitory effect at high concentrations. In the absence of auxin, 1 µM melatonin had auxinic response concerning the number and length of roots, but 10 µM melatonin was inhibitory to rooting in all the tested rootstocks. The final conclusion of this experiment is that exogenously applied melatonin acted as a rooting promoter and its action was similar to that of IAA.