Micropropagation and molecular characterization of Thymus sibthorpii Benth. (Lamiaceae), an aromatic-medicinal thyme with ornamental value and conservation concern

Thymus sibthorpii Benth. (Lamiaceae), with accession number 01,1796-22, is a biotype of native Greek thyme with ascending stems and potential use as a new medicinal-aromatic crop and ornamental plant. An efficient and reliable protocol for in vitro clonal propagation of T. sibthorpii from nodes and meristem tip explants was developed. Shoot proliferation succeeded on a new basal medium (BB) without plant growth regulators, as prior experiments with 6-benzyladenine generated hyperhydricity. Eight different basal media were compared; on two formulations using the new BB 5.9 and 5.6 shoots per explant were produced. Regenerated single shoots were rooted in the BB medium, supplemented with 5 μM of indole-3-butyric acid, and produced 3.1 roots along with 2.5 adventitious shoots. Three types of acclimatization were assessed: in vitro, using two different systems (no significant differences); ex vitro, using eight soil substrates under greenhouse and outdoor nursery conditions (in two of them, 100% of plantlets survived); and in field cultivations, established at eight geographically distant areas of Greece (100% survival rate at all locations). Molecular characterization of T. sibthorpii was evaluated with one nuclear ribosomal DNA and seven chloroplast DNA markers, followed by DNA sequence comparisons with a total of 30 different Thymus species, subspecies, and varieties. The trnH/psbA, trnL/trnF, and matK genes were the most efficient markers for molecular characterization of T. sibthorpii. The molecular markers rpoC1 and petB/petD did not match to any Thymus species and therefore, these DNA sequences provide new sequence information for entire Thymus taxa.

     

Regeneration of Elaeagnus angustifolia from leaf segments of in vitro-derived shoots

Shoot regeneration was achieved from in vitro-produced leaves of Elaeagnus angustifolia L. Half-leaf explants from the terminal part of the shoot produced more shoots than explants from the basal part of the in vitro-derived shoots on agar-solidified WPM medium supplemented with 1 M benzyladenine (BA). In liquid medium of the same formulation, compact shoots that did not elongate were formed on the explants. Leaf cross-section explants (1 mm thick) produced shoots both on solid and liquid medium with 1 M BA, whereas again compact shoots were formed with 10 M BA. Further shoot development on these explants was promoted by their transfer to fresh solid medium containing 1 M BA and 1 M gibberellic acid (GA₃).Abbreviations BA benzyladenine - GA₃ gibberellic acid - WPM woody plant medium     

GIS-facilitated ex situ conservation of the rare Greek endemic Campanula incurva Aucher: Seed germination requirements and effect of growth regulators on in vitro proliferation and rooting

In order to develop conservation protocols for Campanula incurva, the geographical information systems (GIS) were used to unveil its ecological requirements; this facilitated the selection of substrates and of appropriate temperatures for cultivation and guided propagation experiments and acclimatization. Seed germination was tested under (i) dark, (ii) 16-h photoperiod, (iii) immersion in 400 ppm gibberellic acid (GA₃) followed by incubation at dark, and (iv) immersion in 400 ppm GA₃ followed by incubation at 16-h photoperiod (all at 21 ± 1°C). Dormancy was not detected. Germination exceeded 85% in 10 days. Shoot tips were established in vitro in Murashige and Skoog (MS) medium with 1 μM 6-benzyladenine (BA) and 0.1 μM indole-3-butyric acid (IBA). The effect of 1–8 μM BA and 1–8 μM kinetin on shoot proliferation was studied. Moreover, 8 μM BA was combined with 0, 1, 5, and 10 μM IBA to investigate effects of cytokinin/auxin. The highest number of microshoots/explant (4.03) was obtained with 8 μM BA. Microshoots were transferred to half strength MS and full strength MS media with 0, 0.5, 1, 5, and 10 μM IBA to evaluate their root induction ability. Half strength MS medium with 5 μM IBA resulted in 100% rooting (16.80 average number of roots/microshoot). Plantlets produced were successfully acclimatized.     

In vitro propagation of medicinal and aromatic plants: the case of selected Greek species with conservation priority

Worldwide, many medicinal and aromatic plants (MAPs) are still collected from the wild and only a small fraction of them are exclusively sourced from cultivation. This practice when performed non-sustainably threatens species and populations. Micropropagation of MAPs is a powerful tool to conserve rare, threatened, and valuable MAPs, and to massively produce high-value plant material for cultivation without seasonal constraints. In this study, the in vitro propagation protocols of 22 Greek native MAPs assigned with conservation priority were assessed (herbaceous perennials, bulbous, subshrubs, and trees), including 17 range-restricted plants and 5 taxa of Orchidaceae. For the latter, current micropropagation efforts include seed germination, callus induction, and protocorm formation for successful plantlet development; however, these propagation protocols are still fragmentary. For the rest (n = 17), a five-stage detailed procedure is outlined (plant material, establishment, proliferation, rooting, and acclimatization), while materials, treatments, and data per stage are shown comparatively and discussed. Emphasis is given on the selection and preparation of plant material obtained from nature for research, sustainable use, and ex situ conservation actions, and on their effectiveness for conservation purposes or mass production needs. The protocol effectiveness was calculated using a specific equation to estimate the potential number of acclimatized plants raised from a single explant within a year. All protocols can facilitate conservation, and almost half of them could be used for commercialization with high cost (five cases), intermediate cost (eight), or low cost (four), which enables their possible sustainable use.

     

GIS-facilitated in vitro propagation and ex situ conservation of Achillea occulta

A Geographical Information Systems (GIS)-facilitated approach for the in vitro propagation and ex situ conservation of the conservation priority species Achillea occulta is presented. To realize the species?? ecological requirements, the coordinates of the original habitat were linked with thematic layers derived from digital databases in a GIS environment. From wild plants, shoot tips were established in vitro in a basal medium with 4???M 6-benzyladenine (BA) and 0.5???M indole-3-butyric acid (IBA). A modified basal MS medium (modMS, double amount of Fe) proved to be the most effective for in vitro adventitious shoot production. The effect of BA in combination with ??-naphthaleneacetic acid (NAA) or IBA on shoot proliferation was also tested. The highest number of new microshoots/explant (3.5), with 0.93?cm shoot height was obtained when the modMS was supplemented with 5???M BA and 2.5???M IBA. To evaluate the root induction ability, microshoots produced were transferred to modMS media supplemented with 0?C20???M IBA and 0?C20???M NAA. Rooting proved to be very difficult and only by adding 20???M IBA, a 12.5% rooting percentage was achieved. Acclimatization succeeded only during early spring. Young plants transplanted at the Balkan Botanic Garden of Kroussia produced flowers and seeds in the first year. This GIS-approach provided useful guidelines for A. occulta??s (a) effective propagation (selection of greenhouse temperatures, temperatures during in vitro culture, suitable period for cuttings and acclimatization of plantlets), and (b) ex situ cultivation (selection of watering regime, temperatures, locations and exposures for growing sites).     

Effect of temperature and light on seed germination of Erysimum naxense and Erysimum krendlii

Seed germination of two local Greek endemics was studied (Erysimum naxense, Erysimum krendlii). Seed viability was determined by using the tetrazolium method and germination was studied in synchronized cycles of five and four alternating temperatures [10/5 (for E. naxense only) and 15/10, 20/15, 25/20, and 30/25°C for both species, in cycles of 16 h day/8 h night], and in five light regimes (red, blue, green, white, and dark). Germination of E. naxense and E. krendlii seeds was determined daily for six and five weeks, respectively, with the data analyzed as viability adjusted accumulative seed germination at the end of each week. E. naxense’s seed viability was higher (90%) than that of E. krendlii (64%); seed germination (%) of both increased at low alternating temperatures (10/5°C, 15/10°C, 20/15°C). Germination of E. naxense seeds at low temperatures was light-independent, whereas at high temperatures it was increased with red light. Germination of E. krendlii seeds was inconsistently affected by light at the temperatures studied. Percentages of seed germination of both species were higher in experimental conditions similar to the ones of their natural habitats during autumn and/or spring (facilitated with Geographic Information Systems). These conclusions provide guidelines for species-specific propagation protocols and ex situ conservation.     

Conservation of important plants from the Ionian Islands at the Balkan Botanic Garden of Kroussia,N Greece: using GIS to link the in situ collection data with plant propagation and <Emphasis Type="Italic">ex situ</Emphasis> cultivation

The Balkan Botanic Garden of Kroussia (BBGK) is dedicated to the ex situ conservation of native plants of Greece and the Balkans. The BBGK has formulated a conservation strategy for the collection of wild plant material for propagation, prioritizing mainly the endemic, rare, endangered, threatened and vulnerable plants of Europe found in different regions of Greece. Its aim is to contribute to the implementation of Target 8 of the Global and European Strategies for Plant Conservation at local, regional and international scales. In order to (i) define the ecological profile of the in situ requirements preferred and/or tolerated by each selected species, (ii) develop rapid and effective species-specific propagation protocols, and (iii) improve the cultivation of species of conservation concern in BBGK’s nurseries and ex situ conservation sections, geographical coordinates and in situ collection data obtained for each taxon were imported into a Geographic Information System environment (GIS). This information was then linked with several digital GIS thematic layers, including topographic, geological, edaphic, climatic, precipitation and temperature data derived from digital databases. Based on this approach, sexual and asexual propagation of plants from the Ionian Islands were conducted and rapid and effective baseline protocols were developed for 29 taxa (species and subspecies); four are presented here in detail and species-specific ex situ propagation and cultivation guidelines are given. Most of the taxa originating from the Ionian Islands were propagated by cuttings (55.2%) or seeds (34.5%), while the rest were propagated by root division at a rate from 1.7 to 2. The first round of propagation achieved a success rate ranging from 15 to 50% for 3 taxa, from 60 to 80% for 8 taxa and from more than 80 to 100% for 16 taxa, while the ex situ cultivation of the wild and propagated plant material has, so far, been successful. The application of GIS exemplified here presents a sensible and invaluable tool with a broad-scale potential in enhancing the prospects of the ex situ conservation of priority species collected from diverse environmental conditions in man-made habitats such as botanic gardens.     

Micropropagation and salt tolerance of in vitro grown Crithmum maritimum L.

Crithmum maritimum (Apiaceae), a perennial halophyte native in Greece, could be used as an alternative culture at problematic soils. It presents significant economical potentials as its essential oils are in high demand from the medicinal and cosmetic industry. The response of the species on in vitro conditions was studied. MS proved to be the most effective of the basal media tested for in vitro adventitious shoot production, resulting in significantly increased number of new microshoots/explant and higher shoots. 6-Benzyladenine (BA) at 2.5 μM increased shoot proliferation. The combination of α-Naphthaleneacetic acid (NAA) (1–2.5 μM) with BA (2.5 μM) had a positive influence at simultaneous proliferation and rooting resulting in high rooting percentage (82.5–95%) and increased number of roots. Rooting percentage reached 100% and number of roots increased significantly when 0.5 μM and 1 μM IBA was combined with ½MS and full strength MS. The in vitro response to salinity stress (0–300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher concentrations of NaCl but shoot height was enhanced. Acclimatization procedure was successful.