The effect of various pretreatments, culture conditions, and storage time on in vitro germination of seeds, as well as the effect of explant origin and plant growth regulators on in vitro propagation of Teucrium capitatum L. (Teucrium polium sp. capitatum Arcang., Lamiaceae) were examined. Seeds, collected from native plants and stored at room temperature for 3, 7, and 12 months, were cultured for germination in vitro in petri dishes with solid half-strength (½) Murashige and Skoog (1962) growth medium (MS) at 5, 10, 15, 20, 25, and 30 °C, and 16 hours light or continuous darkness. Pretreatments, such as cold stratification, scarification with sandpaper, dipping in concentrated sulfuric acid (H2SO4 > 95%), or dipping in boiling water were tested. Seeds without any pretreatment germinated at lower than 10%. Dipping in concentrated H2SO4 for 15 or 20 minutes was the most effective pretreatment, but still seed germination achieved was low (36%). Seeds preserved their germination capacity for at least 1 year, and germinated satisfactorily at a wide temperature range, from 15 to 25 °C (optimum), while photoperiod did not affect seed germination. Explants excised from in vitro-grown seedlings were established in vitro on MS medium with 1.0 mg·L−1 6-benzyladenine (BA) at much higher rates (≈90%) compared with those collected from plants grown from cuttings in a greenhouse (25%), while explants collected from adult wild plants failed to do so. Explants from seedlings showed strong variability in their response; those excised from branched seedlings formed shoots at significantly higher percentage (90%) at the establishment stage (cultured on MS medium with 0.5–2.0 mg·L−1 BA) than explants excised from unbranched seedlings (36% to 43%), while during subcultures on MS medium with 1.0 mg·L−1 BA, explants from branched seedlings also showed higher multiplication rates than those from unbranched ones. BA at 0.5–2.0 mg·L−1 induced shoot multiplication during both establishment and multiplication stages (7–8 and 14–15 shoots per explant, respectively), while kinetin and 6-γ-γ-(dimethylallylamino)-purine (2iP) were less effective than BA, and zeatin the least appropriate. Microshoot rooting was enhanced by 1-week culture on (½) MS medium with 1.0–4.0 mg·L−1 indole-3-butyric acid (IBA), followed by transfer to auxin-free, (½) MS medium (93% to 98%, 7–8 roots per microshoot), compared with culture on the same medium continuously for 5 weeks (69% to 80%, 5–6 roots per microshoot) or at lower IBA concentrations. Plantlets were acclimatized to ex vitro conditions at 98% on a peat–perlite (1:1, v/v) mixture.
Maria Papafotiou and Aekaterini N. Martini
Konstantinos F. Bertsouklis and Maria Papafotiou
Arbutus unedo L., A. andrachne L., and their natural hybrid, A. ×andrachnoides Link, are the three Arbutus species of the Eastern Mediterranean Macchia. A. unedo is used as an ornamental plant and as cut foliage, whereas the other two species have the potential to be introduced to the floricultural industry. This study was carried out to clarify whether the seeds of these Arbutus species possess dormancy to determine the temperature range for their germination and the effects of storage period on germination. Seeds of the three species, which were stored dry at 25 °C for either three or 11 months, germinated at very high percentages (82% to 99%) and in a short period of time (24 to 46 days) when incubated at 15 or 10 °C without any pretreatment proving that they do not possess dormancy; germination was faster at 15 °C. At 20 °C only seeds of A. unedo and A. andrachne stored for three months germinated (29% to 34%), whereas at 25 °C, there was practically no germination. Seeds stored for three months germinated (61% to 75%) when incubated at 5 °C, but the germination was delayed and the seedlings did not grow further than the appearance of the radicle unless transferred at a higher temperature (10 to 25 °C). After 27 months of dry storage at 25 °C, seeds did not germinate when incubated at the range of 10 to 25 °C even after pretreatment with cold stratification.
Georgia Vlachou, Maria Papafotiou and Konstantinos F. Bertsouklis
Seed ecophysiology and micropropagation of Clinopodium nepeta, an aromatic Mediterranean plant with pharmaceutical and horticultural uses was investigated. The optimum germination temperature of seeds stored at room temperature for 0, 6, or 12 months was 15 to 20 °C (100% germination completed in 10 to14 days) and cardinal temperatures were defined at 10 and 30 °C (80% to 82% and 62% to 76% germination, respectively). Six or 12 months of storage did not seem to affect germination, although 12-month-old seeds germinated at higher percentage and completed germination earlier at 15 °C than at 20 °C. Concerning micropropagation, shoot multiplication at subcultures of both adult plant- and seedling-origin nodal explants was tested on Murashige and Skoog (MS) medium supplemented with various cytokinin types, i.e., zeatin (ZEA), 6-benzyladenine (BA), kinetin (KIN), and 6-γ-γ-(dimethylallylamino)-purine (2IP), at various concentrations from 0.0 to 8.0 mg·L−1. Both explant types presented a rather similar response during in vitro culture. Increasing concentration of all cytokinin types resulted in an increase in shoot number per responding explant (1.1–5.3) and in most cases a decrease in shoot length (0.6–3.4 cm). Increasing cytokinin concentration induced hyperhydricity to a number of shoots (0.1–6.5) per explant, mostly when ZEA and BA were used. Supplementing the MS medium with 8.0 mg·L−1 BA combined with 0.1 mg·L−1 1-naphthaleneacetic acid (NAA) led to almost elimination of hyperhydricity and very satisfactory shoot production (80%/88% explant response and 6.5/7.5 shoot number per responding explant for seedling- / adult-origin explants, respectively). Alternatively, increasing the agar concentration to 12.0 g·L−1 and supplementing the medium with 8.0 mg·L−1 BA only, resulted in the same effect on eliminating hyperhydricity, such as the addition of NAA, and in the best shoot multiplication response achieved in this study (100% explant response, 9.4/9.9 shoots per explant for seedling-/adult-origin explants, respectively). Microshoots rooted abundantly (92% to 100%) on half-strength MS medium, either Hf or supplemented with 0.5 mg·L−1 to 4.0 mg·L−1 indole-3-butyric acid (IBA). The addition of IBA to the rooting medium, regardless of its concentration, affected only the root length by increasing it 2- to 3-fold. Microshoot clusters produced on multiplication media rooted at 96% when cultured on Hf half-strength MS medium. Rooted microshoots and shoot clusters survived at 80% to 100%, respectively, after ex vitro acclimatization in peat:perlite 1:1 (v/v).
Lamprini Tassoula, Maria Papafotiou, Georgios Liakopoulos and Georgios Kargas
The possibility of using Convolvulus cneorum L., a native Mediterranean xerophyte, with compact dome-like canopy and extended blooming period, on extensive green roofs in areas with semiarid Mediterranean climate was investigated in a 27-month experimental period, which included three summers (the dry season of the year). The aim was to preserve the local character and biodiversity, as well as to reduce water consumption and construction weight. Convolvulus cneorum rooted cuttings were planted in the beginning of July 2011 in experimental modules on a fully exposed flat roof at the Agricultural University of Athens, with a green roof infrastructure (substrate moisture retention and protection of the insulation, drainage element, and filter sheet). Two types of substrate with 10 cm depth were used, one with soil, i.e., grape marc compost:perlite:soil:pumice (3:3:2:2, v/v) and a lighter one without soil, i.e., grape marc compost:perlite:pumice (3:3:4, v/v). Two irrigation frequencies were applied during the dry periods, i.e., every 5 days (normal) and 7 days (sparse) in 2011 and 2012 and every 4 days (normal) and 6 days (sparse) in 2013. The chemical properties of the two substrates were similar, while their physical properties differ slightly as the substrate that contained soil was holding more water at saturation and it had lower saturated hydraulic conductivity and higher easily available water (EAW). The substrate type affected growth since plant height and diameter, shoot number, and aboveground dry weight were promoted by the soil substrate. Irrigation frequency did not affect plant growth. However, plants cultivated on soil substrate and irrigated normally had the highest growth, particularly compared with plants in soilless substrate under sparse irrigation. Flowering was abundant in April (spring) and in the first year flower number was promoted by the soil substrate. During the dry periods, sparse irrigation resulted in increased stomatal resistance one day before irrigation, indicating that water availability was marginal for the plants, while normal transpiration rate was restored the day after irrigation. According to photosystem II photochemical parameters measured one day before and the morning after an irrigation event, no evidence of damage to the photosynthetic apparatus was recorded in any of the treatments. In general, after 27 months of culture, plant size and roof coverage was appearing more or less similar in all the experimental treatments, therefore the combination of the lighter soilless substrate with sparse irrigation is highly suggested for C. cneorum cultivation on Mediterranean green roofs.
Aekaterini N. Martini, Maria Papafotiou and Stavros N. Vemmos
The aim of this study was to develop an efficient protocol for in vitro propagation of the rare and endangered ×Malosorbus florentina, not only enabling conservation of the species, but also its use as an ornamental. Explants excised from adult plants, shoot tip explants, and explants collected in March and April showed more browning and had higher content of total phenolics than explants excised from juvenile tissue, nodal explants, and those collected during any of the other months of the year. Shoot tip explants from adult plants were more difficult to establish in vitro (14%) compared with explants from micropropagated plantlets or sprouts of burned plants (29% to 36%). Nodal explants excised from seedlings were established at the highest percentage (83%), giving the most shoots per explant (5.2). Generally, in vitro cultures established from adult plants, with the exception of one culture, showed lower multiplication rates compared with cultures from juvenile plants. Nodal explants from the base of sprouts produced a higher percentage (60%) of shoots than explants from upper locations (20% to 31%), but any differences in proliferation rates of established cultures ceased after the third subculture. Microshoots from juvenile cultures were more capable of rooting (51% to 58%) than were those from adult plants (16% to 32%), whereas 83% of the plantlets were acclimatized ex vitro independently of their origin, but plantlets of juvenile origin, although developing the same height as those originating from adult plants, had shorter internodes and thus more compact shape.
Maria Papafotiou, Niki Pergialioti, Lamprini Tassoula, Ioannis Massas and Georgios Kargas
Green roofs could be a way to increase vegetation in the center of old Mediterranean cities. The need for conservation of local character and biodiversity requires the use of native plant species, whereas the deficiency of water, particularly in semiarid regions, requires the use of species with reduced irrigation needs. Moreover, the aged buildings lead to the use of lightweight green roof constructions. Therefore, research was undertaken to investigate the possibility of using three Mediterranean aromatic xerophytes, Artemisia absinthium L., Helichrysum italicum Roth., and H. orientale L., at an extensive green roof in Athens, Greece. Simultaneously, the possibility of using locally produced grape marc compost was investigated. Substrate type and depth and irrigation frequency effects on growth of these species were studied. Rooted cuttings were planted mid-May in plastic containers with a green roof infrastructure fitted (moisture retention and protection of the insulation mat, drainage layer, and filter sheet) and placed on a fully exposed third floor flat roof at the Agricultural University of Athens. Two types of substrates were used, grape marc compost:soil:perlite (2:3:5, v/v) and peat:soil:perlite (2:3:5, v/v, as a control), as well as two substrate depths, 7.5 (shallow) and 15 cm (deep), and two irrigation frequencies, sparse (5 or 7 days in shallow and deep substrate, respectively) and normal (3 or 5 days in shallow and deep substrate, respectively). Increased contents of macroelements, total phosphorus (P) and potassium (K) in particular, were recorded in the compost-amended substrate, whereas both substrates had similar physical properties. Plant growth was recorded from May to October. The deep compost-amended substrate, independent of irrigation frequency, resulted in taller plants with bigger diameter and aboveground dry weight in all species. However, a remarkable result was that shallow compost-amended substrate with sparse irrigation resulted in similar or even bigger plant growth of all plant species compared with deep peat-amended substrate with normal irrigation. Thus, all three species were found suitable for use in Mediterranean extensive or semi-intensive green roofs, whereas the use of grape marc compost in the substrate allowed for less water consumption and the reduction of substrate depth without restriction of plant growth at the establishment phase and the first period of drought.
Christos Lykas, Constantinos Kittas, Nikolaos Katsoulas and Maria Papafotiou
The use of chemical growth retardants is a standard practice for compact gardenia plant production. The aim of this study was to investigate the possibility of using a photoselective polyethylene greenhouse covering film as an alternative to chemical treatment for production of compact potted gardenia (Gardenia jasminoides Ellis) plants. Two types of experiments were carried out: 1) on gardenia cuttings rooted in rooting benches; and 2) on young potted plants grown under low tunnels. In both experiments, two types of cover materials were used: 1) a photoselective polyethylene (P-PE), filtering light within the wavelength range 600 to 750 nm; and 2) a common polyethylene film (C-PE) routinely used in greenhouse practice. Values of photosynthetically active radiation (in a wavelength of 400 to 700 nm), cover materials' spectral properties (in a wavelength range of 400 to 1100 nm), air temperature, and relative humidity were recorded inside the rooting benches and under the low tunnels. Plant growth parameters (main shoot length and leaf area and lateral shoot number, leaf area, and fresh and dry weight) were determined along the growth cycle. Cuttings rooted under the P-PE film received light with high ζn values (ratio of Rn: 655 to 665 nm to far red FRn: 725 to 735 nm) and high blue (B: 400 to 500 nm) to red (R: 600 to 700 nm) ratio (B:R) and were 68.7% shorter and had 21% lower leaf area compared with cuttings rooted under the C-PE film. Similarly, plants that were rooted and then grown under the low tunnels covered with the P-PE film, compared with plants rooted and grown under C-PE film, were 59% shorter, had 85% lower leaf area, 89% lower fresh weight, and 86% lower dry weight, whereas they did not produce lateral shoots. However, plants rooted under the C-PE film and then grown under the P-PE-covered low tunnels were 26% shorter and developed fewer laterals than plants rooted and grown under tunnels covered with C-PE film. Finally, plants rooted under the P-PE film and then grown under tunnels covered with C-PE film developed into compact, well-shaped plants, because they had a drastic reduction of height (56%) without an effect on leaf area, shoot and leaf fresh and dry weight, and the number of lateral shoots.
Maria Papafotiou, Barbara Avajianneli, Costas Michos and Iordanis Chatzipavlidis
Compost from cotton gin trash was evaluated as a peat substitute in Codiaeum variegatum L. production. Rooted cuttings were grown for 8 months in media containing cotton gin trash compost, sphagnum peatmoss, and perlite in 1:3:4, 1:1:2, and 3:1:4 ratios respectively, and their growth was compared with a control medium of 1 peat : 1 perlite (by volume). Even when 75% of peat in the control medium was replaced by cotton gin trash compost, plant height, leaf number, and leaf size were unaffected, whereas root dry weight was increased in the medium with 25% peat replacement. Although replacement of peat by cotton gin trash compost did not affect quantitative characteristics of croton foliage growth, it affected the foliage color, as plants in media with cotton gin trash compost had more areas colored red in their leaves. The increase of red coloration was proportional to the increase of cotton gin trash compost in the medium. The phenomenon was more intense in the lower leaves than the apical ones. Anthocyanin concentration measurements showed that the gradual increase of cotton gin trash compost level in the growth medium caused a gradual increase of the anthocyanin concentration in the leaves. This effect is discussed in relation to chemical properties of the media, as electrical conductivity, pH, and nutrient concentrations. The gradual increase of cotton gin trash compost level caused an analogous increase of N, P, and K concentrations in the medium. Also, media with cotton gin trash compost had high electrical conductivity at the beginning of the culture period, related to the cotton gin trash compost level, which was reduced to values similar to that in the control medium after 50 days of culture. Na concentration in the media ranged similarly to electrical conductivity. The pH was positively related to cotton gin trash compost level, and media with a high cotton gin trash compost level had increased pH during the culture period compared with the control.