Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition

Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m⁻² s⁻¹) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h⁻¹ for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity, emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included 40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation method in photoautotrophic micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Effect of In Vitro Culture Conditions on the Pattern of Photoinhibition during Acclimation of Gardenia Plantlets to Ex Vitro Conditions

We tested the effect of growing conditions during micropropagation on the fast kinetics of chlorophyll (Chl) fluorescence of Gardenia jasminoides Ellis plantlets during a 4-week acclimation to ex vitro. We studied whether photoautotrophic growing in vitro produced plantlets with less photoinhibition impairment during acclimation. Of the growing conditions stimulating photoautotrophy in vitro, only loose tube caps had a positive effect, whereas low sucrose or sucrose-free content in the medium and high PPFD showed a negative effect. Thus, plantlets cultured with 3 % (m/v) of sucrose were subsequently less photoinhibited throughout acclimation than those cultured with low sucrose (0.5 %) or sucrose-free media. Moreover, at the end of acclimation the former plantlets showed F_v/F_m and F_v/F₀ ratios typical of unstressed ex vitro plants as well as a higher Chl content and ratio of Chls to carotenoids. Plantlets cultured at a photosynthetic photon fluence density (PPFD) of 50 µmol m^–2 s^–1 also showed a better performance at the end of acclimation than those cultured at a higher (110 µmol m^–2 s^–1) PPFD. Thus except in the case of loose-tube closure, gardenia plantlets cultured in vitro under conventional sucrose concentration and PPFD are the least photoinhibited during acclimation. Nevertheless, significant interactions between the in vitro growing factors were observed at the end of acclimation.     

Growth of micropropagated bananas colonized by root-organ culture produced arbuscular mycorrhizal fungi entrapped in Ca- alginate beads

The effect of root-organ culture (ROC) produced arbuscular mycorrhizal fungi (AMF), i.e. Glomus proliferum, Glomus versiforme and Glomus intraradices, entrapped in Ca-alginate beads on the first stages development of micropropagated bananas (Musa spp. cv. Grande Naine) was investigated. The experimental design consisted of banana plants inoculated with one of the three AMF and two controls, i.e. Control-AL (with empty alginate beads), and Control (no beads). Forty plants were considered per treatment and cultured under greenhouse conditions in a completely randomized design. Eight plants per treatment were harvested 40, 80, 120, 160 and 200 days after inoculation and analysed for root colonization, growth parameters and nutrient concentration. In addition, spores were enumerated in the substrate at the same intervals. Ca-alginate entrapped ROC-produced AMF spores were able (1) to colonize the root system of a micropropagated banana cultivar under nursery conditions, (2) to increase plant P nutrition and biomass, and (3) to proliferate in the commercial nursery substrate, therefore increasing the fungal inoculum biomass. The entrapment of ROC-propagated spores, adaptable to a wide range of Glomeromycetes, represents thus a forthcoming alternative pathogen-free inoculum.     

Adventitious shoot regeneration from leaf explants and stem nodes of Lilium

A method for the regeneration of lily plantlets (Lilium spp.) through different morphogenic pathways is described. Plant regeneration was obtained from in vitro cultured leaves of four lily hybrids, cultured on Murashige and Skoog's basal medium supplemented with cytokinins (TDZ and BA) and auxins (NAA and IBA) at different concentrations. Direct shoot regeneration occurred with all tested media for the Asiatic lilies `Elite' and `Pollyanna' and also for the Oriental hybrid `Star Gazer'. Callus developed on TDZ-enriched medium from leaf segments of L. longiflorum cv. `Snow Queen' regenerated by direct organogenesis. This occurred on a medium with auxin/ cytokinin balance which was lower than other genotypes. There were fewer problems of sterilization with leaves from sprouted bulbs than in vitro scale culture. This suggests that the leaf-segments obtained in this way could be an alternative to the scales as a source of material for propagation. A protocol for micropropagation based on bulblets from in vitro shoot-tip-derived stem nodes was also used. The development of pseudo-bulbets is particularly advantageous since it allows for structures characterised by absent or low dormancy. Regenerated shoots have been rooted and successfully acclimatized to greenhouse conditions where they flowered after the second year giving plants with true-to-type shape and colour.     

Extracellular Matrix in Early Stages of Direct Somatic Embryogenesis in Leaves of Drosera spathulata

The growth and water relations of Paulownia fortunei in photoautotrophic cultures (nutrient medium lacking sucrose and growth regulator) with CO₂ enrichment (PWAH) or without CO₂ enrichment (PWAL) were compared with those in photomixotrophic shoot (PWC; 30 g dm⁻³ sucrose and 0.3 mg dm⁻³ N⁶-benzyladenine) and root cultures (PWR; 0.3 mg dm⁻³ indole-3-butyric acid). The photoautotrophic and photomixotrophic cultures were incubated under photosynthetic photon flux 125 and 60 μmol m⁻² s⁻¹, respectively. 100 % sprouting and significantly higher number of shoots (1.6) were obtained with PWAH as compared to PWAL and PWC. PWAH and PWAL stimulated spontaneous rooting from the cut end of axillary shoots. In PWAH, 84 % of shoots rooted with an average of 5.9 roots per shoot and 4.0 cm of root length in 21 d. Rooting of photomixotrophic shoot cultures were stimulated by an auxin treatment. In this case, 98.3 % of shoots were rooted with an average of 4.6 roots per shoot and 1.9 cm length. A microscopic observation on leaf abaxial surface prints from photomixotrophic shoot and root cultures showed widely open (6 – 8 μm) spherical stomata (12 – 14 μm) and from photoautotrophic cultures elliptical stomata (10 – 12 μm) with narrow openings (3 – 4 μm). Leaves from photomixo-trophic cultures had higher stomatal index as compared to photoautotrophic cultures. The rate of moisture loss from detached leaves was not varying significantly in different cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Growth and photosynthetic activity of micropropagated strawberry plants inoculated with endomycorrhizal fungi (AMF) and growing under drought stress

The plants produced by in vitro methods are free of any microflora contrary to natural systems where plants are colonized by symbiotic fungi. The present paper reports the experiments carried out to evaluate the role of arbuscular endomycorrhizal fungi in development of micropropagated strawberries and their photosynthetic activity (measured by chlorophyll fluorescence) under drought conditions. Mycorrhization strongly affected growth and tolerance to water deficiency of the plants cultivated in greenhouse. Wilting of not-mycorrhized plants was accompanied by drastic increase of Fo and Tfm and decrease of Fm. At the same time, the value of these parameters for mycorrhized plants did not change. Drastic decrease in the value of parameters Fv/Fm, Fv/Fo and Fo/Fm for plants without AMF appeared at the end of dry period. Rise of Fs and decrease Rfd was noted only for not-mycorrhized plants. The plants colonized by fungi, fully recovered their photosynthetic activity when watering was restored.     

Variation in potato microplant morphology in vitro and DNA methylation

Heterotrophic and autotrophic culture in agar and in polyurethane foam, the latter used as an alternative tissue support to agar, resulted in potato microplants with different in vitro morphologies. The microplants were visually characterised in terms of their relative developmental maturity, by comparing the respective leaf shapes in vitro with ontogenetic differences in leaf shape in glasshouse-grown potato plants. Cytosine methylation in the DNA of microplants of the different morphologies was determined using a method based on the AFLP technique but employing methylation-sensitive restriction enzymes (MSAP analysis) to test the hypothesis that DNA methylation could be used to characterise differences in microplant development in vitro. In three of the four treatments there was a good correlation between the visual assessment of relative morphological maturity and DNA base methylation levels. In these microplants there was increased DNA methylation in the leaves with mature leaf morphology represented by a decreased number of restriction fragments. The fourth in vitro morphology had the most juvenile leaf shape but did not have the predicted level of DNA methylation, having a relatively low number of restriction fragments. Subtraction analysis was used to discriminate the fragments that were unique to the juvenile and mature in vivo leaf morphologies. Comparison of the fragment patterns from the microplants with the latter reference profiles, confirmed the relationship with the total DNA methylation as detected by MSAP analysis, that is, the number of common fragments with the juvenile or mature in vivo leaf profiles, respectively. However, none of the fragment profiles, while sharing some common bands at random, was identical to any other; or to that of either the juvenile or mature in vivo leaf. The anomalous relationship of the microplants with most juvenile leaf shape and highest DNA methylation was confirmed. The measurement of DNA methylation in in vitro plants is discussed in the context of the development of a method to assess the quality of microplants produced by different in vitro protocols.     

Multiple shoot induction and leaf and flower bud abscission of Annonacultures as affected by types of ventilation

Nodal explants of Annona squamosa L. and Annona muricata L. were cultured in vitro under various types of ventilation: airtight vessel (sealed condition; number of air exchange 0.1 h^–1), natural ventilation (via a polypropylene membrane; number of air exchange 1.5 h^–1), and forced ventilation (5.0 cm³ min^–1 in a 60 cm³ vessel; number of air exchange 5.0 h^–1). In both species, numbers of leaves, leaf areas and numbers of nodes per shoot increased with improving standards of ventilation, while leaf abscissions were substantially reduced; all the leaves had abscised in the airtight vessels after 12–15 days, but none had done so with forced ventilation. Flower-bud abscission in A. muricatashowed a similar trend after 21 days. These effects were associated with reductions in the accumulation of ethylene within the culture vessels, produced by increasing the efficiency of ventilation; ethylene was not detected in those fitted with a forced ventilation system. CO₂ concentrations in culture headspaces and the net photosynthetic rates of the plantlets were also evaluated. CO₂ concentrations decreased well below the ambient in the natural and airtight vessels; however, under forced ventilation, CO₂ concentrations were significantly higher during the photoperiod, compared to those of the natural ventilation and airtight vessel treatments. In general, net photosynthetic rates per unit leaf area increased with increasing photosynthetic photon flux (PPF) and rates were highest in plantlets grown under forced ventilation, intermediate under natural ventilation and lowest in the airtight vessels.Eighteen different media were investigated for their effects on multiple shoot induction in both species. The best medium for multiple shoot induction and growth in A. squamosa was Murashige and Skoog medium (MS) + 6-benzylaminopurine (BA; 1.5 mg l^–1) + casein hydrolysate (1.0 g l^–1) and for A. muricata MS + BA (1.0 mg l^–1) + naphthaleneacetic acid (NAA; 0.1 mg l^–1).     

In vitro propagation of Spilanthes mauritiana DC., an endangered medicinal herb, through axillary bud cultures

Summary Spilanthes mauritiana DC., (Compositae), a East African medicinal herb containing pharmaceutically promising secondary metabolites, has successfully been raised in vitro. We have developed a clonal propagation protocol that uses juvenile plants as starting material. The addition of benzylaminopurine (BA) (1.0 μM) and naphthaleneacetic acid (NAA) (0.1 μM) to the culture medium resulted in maximum shooting response with minimal callusing. Shoots rooted best in vitro in MS medium supplemented with indole-3-acetic acid (IAA; 0.2 μM), and plants that had already developed roots showed better growth, with maximum survival rate, in the greenhouse after an initial hardening.     

in vitro propagation of Ochreinauclea missionis (Wall. EX G. Don), an ethnomedicinal endemic and threatened tree

Summary Biotechnology has offered a nonconventional method of plant propagation and has been intensively applied as a conservation strategy for sustaining biodiversity for rare plants. In vitro conservation through micropropagation of Ochreinauclea missionis, a rare, endemic and medicinal tree species of Western Ghats in Karnataka region of India is reported. Multiple shoots were initiated from nodal explants on Murashige and Skoog (MS) medium supplemented with 8.8 μM 6-benzylaminopurine (BA) and 0.3% (w/v) activated charcoal. Shoots were elongated in MS medium with a combination of 2.2 μM BA and 5.3 μM α-naphthaleneacetic acid (NAA) or growth regulator-free medium. Individual shoots with a minimum of one node were excised and rooted in vitro on MS medium with 0.3% activated charcoal or ex vitro rooted by treatment with 49 μM indole-3-butyric acid (IBA) for 30 min. Regenerants acclimated in Soil-rite exhibited 65% survival in the greenhouse.