The use of in vitro culture to improve the propagation of Rhododendron ponticum subsp. baeticum (Boiss. & Reuter)

Rhododendron ponticum subsp. baeticum is endemic in the southern region of the Iberian Peninsula. The relict populations of this species are vulnerable, due mainly to difficult conditions for the establishment of seedlings, resulting in a virtual lack of sexual recruitment. In order to preserve the surviving populations, in vitro culture methods have been applied for both the sexual and the agamic propagation of the species. The in vitro germination of seeds was high when conducted with Anderson’s medium without plant growth regulators. The self-rooted seedlings obtained were easily transplanted to outside conditions. The presence of growth regulators in the medium interfered with the development of the seedlings, causing heavy callus formation. The in vitro growth of explants took place readily in Anderson’s medium plus 0.072 mg L⁻¹ of BA and 0.036 mg L⁻¹ of NAA although the explants did not form roots. Rooting was achieved by the basal dipping of the explants in hydroalcoholic solutions of 500 mg L⁻¹ IAA during the outside transplanting process. Therefore, the combination of in vitro grown explants together with ex vitro rooting, results in a good method for the agamic propagation of Rhododendron ponticum subsp. baeticum.     

Use of sugarcane bagasse as an alternative low-cost support material during the rooting stage of apple micropropagation

Summary Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

In vitro culture of Cedrela fissilis Vellozo (Meliaceae)

An efficient micropropagation protocol was developed for Cedrela fissilis (Meliaceae) using nodal segments from juvenile origin for axillary shoot proliferation. Shoot proliferation was significantly affected by salt formulation, explant origin and 6-benzyladenine concentration. Maximum multiplication rates (6–7 new plants were produced in the second subculture cycle per single cotyledonary node cutting) were achieved on Murashige and Skoog media supplemented with 1.25–5.0 M 6-benzyladenine. Addition of -naphthaleneacetic acid to these media caused significant inhibition on shoot proliferation and growth and stimulated callus formation. High frequency callus initiation and synergistic effects on callus growth were achieved on Murashige and Skoog medium supplemented with 6-benzyladenine at either 1.25, 2.5 or 5.0 M combined, respectively, with 2.5, 1.25–5.0 or 5.0 M -naphthaleneacetic acid. Rooting was achieved, after 10–12 days, with 87–100% of the node cuttings on half strength Murashige and Skoog medium either without growth regulators or supplemented with 2.5 M indole-3-butyric acid. Regenerated plants were successfully acclimatized on sterilized sand, for 21 days, but for further plant development the sand:soil (1:1) mixture was the best substrate. The survival rate of plantlets under ex vitro conditions was 100% after 3 months. The optimized micropropagation and callus culture protocols offer the possibility to use the organ/cell culture techniques for vegetative propagation, cryopreservation and secondary metabolism studies.     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Root distribution of a Mediterranean shrubland in Portugal

The distribution of roots of an Erica (Erica scoparia and Erica lusitanica) dominated Mediterranean maquis was studied using three different approaches: root counts on trench walls (down to 120 cm), estimation of the maximum rooting depth using an allometric relationship and estimation of fine root biomass and fine root length using soil cores (down to 100 cm). Roots were classified according to diameter (fine, 1.0 mm; small, 1.1–5.0 mm; medium, 5.1–10.0 mm; coarse, >10.0 mm) and species (Erica sp., Pteridium aquilinum, Rubus ulmifolius and Ulex jussiaei). The depth corresponding to 50% of all roots (D ₅₀) was determined by fitting a new model to the cumulative root distribution. Fine roots represented 96% of root counts. Root counts of Erica represented 59%, Ulex 34%, Rubus 6% and Pteridium 1%. Overall root counts showed a D ₅₀ of 26 cm. D ₅₀ was higher for Ulex (40 cm) and Erica (22 cm), than for Pteridium (9 cm) and Rubus (3 cm). D ₅₀ for fine roots was 27 cm, for small roots 11 cm, for medium roots 6 cm and for coarse roots 4 cm. The estimated average maximum rooting depth of the 28 deepest Erica roots was 222 cm. The deepest Erica root was estimated to reach 329 cm. A total of 82% of roots growing deeper than 125 cm were not reaching more than 175 cm. The overall fine root length density ranged from 4.6 cm/cm³ at 10 cm to 0.8 cm/cm³ at 80 cm. The overall fine root biomass ranged from 7.7 mg/cm³ at 10 cm to 0.6 mg/cm³ at 40 cm. D ₅₀ for root biomass was 12 cm and D ₅₀ for root length was 14 cm. Fine root biomass was estimated as 1.6 kg/m² and the respective root length as 18.7 km/m².     

Plantlet regeneration from subculturable nodular callus of Pinus radiata

An efficient planlet regeneration system via nodular callus formation is described for Pinus radiata. Subculturable nodular callus was induced at its highest frequency (93%) on embryonic explants excised from seeds at an early stage of germination (radicle length 2–5 mm). The optimal medium for nodular callus tissue proliferation was LP basal medium that was modified by reducing the concentration of potassium nitrate to 500 mg l^–1 and supplemented with 22.2 M 6-benzyladenine (BAP) and 2.85 M indole-3-butyric acid (IBA). Bud differentiation from the nodules was achieved by reducing BAP and sucrose concentrations in the culture medium. The maximum frequency of adventitious bud formation occurred on LP basal medium containing 2% sucrose and 0.44 M BAP on which about 61% of the transferred nodules formed buds. During the next 6 weeks of culture on the same cytokinin-free medium multiple shoots elongated from the buds. These shoots were excised and transferred to root initiation medium (RIM2.1), consisting of full-stregth SH macro- and micro-salts, 1000 mg l^–1 myo-inositol, 0.4 mg l^–1 thiamine-HCl, 2% sucrose and a combination of naphthaleneacetic acid (NAA), IBA and BAP at concentrations of 2.69, 4.93 and 0.11 M, respectively. After 5–15 days, root meristems were initiated on the stem bases. The highest rooting frequency was achieved when shoots were treated for 10 days on RIM2.1 medium, before being transferred to half-strength Schenk and Hildebrandt medium with 1% sucrose and without growth regulators for root growth.     

Tissue culture and wetland establishment of the freshwater monocots <Emphasis Type="Italic">Carex,Juncus, Scirpus</Emphasis>, and <Emphasis Type="Italic">Typha</Emphasis>

Summary Cell cultures of freshwater wetland monocots were regenerated, plants were grown in the greenhouse, and then established and evaluated in wetlands. Typha (cattail), Juncus (rushes), Scirpus (bulrushes), and Carex (sedges) were studied because they are common, dominant, high biomass wetland-adapted plants, tolerant of chemically diverse ecosystems. The goal was to define micropropagation and wetland establishment protocols. Tissue culture systems defined for numerous monocot crop species can be readily applied to wetland plants, with a few modifications. Issues addressed were selection of explant material, shoot and root regeneration conditions, culture age verses regenerability, greenhouse acclimatization needs, plant uniformity and requirements for wetland establishment. In vitro-germinated seedlings were an excellent source of pathogen-free regenerable tissue. T. latifolia, T. angustifolia, and J. accuminatus were regenerated from callus induced in the dark with picloram, then transferred to medium with benzyladenine in the light to promote shoot organogenesis. J. effusus, S. polyphyllus, and C. lurida could not be regenerated from callus, which turned black. They could be regenerated directly by culturing intact seedlings directly on cytokinin media in the light. Shoots rooted with little or no auxin. J. effusus rooting was promoted by the addition of charcoal to the medium. Covering plants for the first 2 wk with plastic facilitated greenhouse establishment. There were high rates of greenhouse and wetland survival. No abnormal plants were observed. These regeneration systems could be utilized for the production of wetland plants for potential application in habitat restoration and wetland creation, and would provide an alternative to field collection.     

Significance of rooting depth in mire plants: Evidence from natural <Superscript>15</Superscript>N abundance

Variation in stable nitrogen isotope ratios (¹⁵N) was assessed for plants comprising two wetland communities, a bog-fen system and a flood plain, in central Japan. ¹⁵N of 12 species from the bog-fen system and six species from the flood plain were remarkably variable, ranging from –5.9 to +1.1 and from +3.1 to +8.7, respectively. Phragmites australis exhibited the highest ¹⁵N value at both sites. Rooting depth also differed greatly with plant species, ranging from 5cm to over 200cm in the bog-fen system. There was a tendency for plants having deeper root systems to exhibit higher ¹⁵N values; plant ¹⁵N was positively associated with rooting depth. Moreover, an increasing gradient of peat ¹⁵N was found along with depth. This evidence, together with the fact that inorganic nitrogen was depleted under a deep-rooted Phragmites australis stand, strongly suggests that deep-rooted plants actually absorb nitrogen from the deep peat layer. Thus, we successfully demonstrated the diverse traits of nitrogen nutrition among mire plants using stable isotope analysis. The ecological significance of deep rooting in mire plants is that it enables those plants to monopolize nutrients in deep substratum layers. This advantage should compensate for any consequential structural and/or physiological costs. Good evidence of the benefits of deep rooting is provided by the fact that Phragmites australis dominates as a tall mire grass.     

Direct Calculation of a Tree Length Using a Distance Matrix

Comparative studies of tree-building methods have shown minimum evolution to be in general an accurate criterion for selecting a true tree. To improve the use of this criterion, this paper proposes a method for rapidly and directly calculating a length of a dichotomous tree without having to resort to branch length calculations. This direct calculation (DC) method applies to the complete final topology, giving equal importance to each branch after a dichotomy. According to this method, the tree length S _DC is S _DC =∑ _i ∑ _j (D _ij /2 ^Bij ) = (∑ _i<j ∑D _ij 2 ^Bmax−Bij )/2 ^{Bmax −1} where D _ij is the observed distance between taxa i and j, B _ij is the number of branches connecting i and j, Bmax is the greatest B _ij in the tree, and the powers of two are due to the dichotomy of the tree. This tree length expression may be used as a rapid method for selecting the shortest tree from a set of hypothetical or subobtimal trees. Received: 2 March 2000 / Accepted: 24 March 2000