Slow growth in vitro conservation of coffee (Coffea spp.)

The effects of reduced sucrose concentrations and low temperature on a collection of coffee microcuttings have been examined. Sucrose concentrations of 0.5 g l^-1 and 20 g l^-1 and temperatures of 20°C and 27°C were compared in three accessions: the Arabusta (interspecific hybrid) and Coffea arabica L. cv. Caturra amarillo and cv. Mokka de Tahiti. After six months, low sucrose concentrations reduced microcutting growth, rooting and survival rate. At 20°C, microcutting growth was also reduced, but leaf loss and survival rate were promoted. The genotypic differences at six months were minor. After one year without subculture, survival rate was influenced by sucrose concentration and by genotype. These two species can be cold-stored six months at 20°C on a medium containing at least 20 g l^-1 sucrose.Abbreviations BA 6-benzylaminopurine - MS Murashige & Skoog     

Variability in storage response within a coffee (Coffea spp.) core collection under slow growth conditions

Anin vitro core collection of African coffee germplasm, structured in 32 diploid diversity groups, was established and conserved under slow growth for 3 years (6 subcultures). The initial objective was to store twenty accessions per group, with four replicates per accession. A statistical model was developed to analyse observations of survival rates within each diversity group. The goodness of fit of the model was shown. Survival analysis indicated a broad variability of the accessions in their response to the storage conditions and confirmed the importance of structuring the coffee complex down to the intraspecific level. Intra- and inter-group differences had consequences on the genetic representativity of thein vitro core collection. For practical purposes, conservation was carried on when the intra-group genetic drift was less than 50%.Abbreviations BA 6-benzyladenine - CAR Central African Republic - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - FAO Food and Agriculture Organization - IBPGR International Board for Plant Genetic Resources - IDEFOR-DCC Institut Des Fôrets - Département Café Cacao - ORSTOM Institut français de recherche scientifique pour le développement en coopération     

In vitro culture of common mullein (Verbascum thapsus L.)

Summary Verbascum thapsus L. is a medicinal herb and has been used to treat inflammatory disease, asthma, spasmodic coughs and migraine headaches. Studies were initiated to establish an in vitro culture protocol for V. thapsus. Explants (leaf dises, petioles and roots) were cultured on Murashing and Skoog minimal organics (MSMO) medium with benzyladenine (BA) or kinetin. Best shoot proliferation was obtained from leaf dise and petiole explants at 13.32 μM BA. Leaf dises were cultured on MSMO medium with 13.32 μM BA in combination with naphthalene acetic acid (NAA) or 2,4-dichlorphenoxyacetic acid (2,4-D). More shoot development was obtained with 13.32 μM BA and 5.37 μM NAA. Shoots were transferred to rooting media containing different levels of NAA and 2,4-D. Most of the shoots formed roots on media with 5.37 μM NAA. Plants were transferred to vermiculite and subsequently to potting media and maintained in the greenhouse.     

Effect of growth regulator and culture conditions on shoot multiplication and rhizome formation in ginger (Zingiber officinale Rosc.) in vitro

Summary Shoot multiplication of Zingiber officinale cv. V₃S₁₈ was achieved by meristem culture on a Murashige and Skoog (MS) basal medium supplemented with 26.6 μM 6-benzylaminopurine (BA), 8.57 μM indole-3-acetic acid (IAA), and 1111.1 μM adenine sulfate and 3% (w/v) sucrose. In vitro rhizome formation from in vitro-raised shoots was achieved on MS medium supplemented with 4.44 μM BA, 5.71 μM IAA, and 3–8% (w/v) sucrose after 8 wk of culture. Cultural variations such as photoperiod, carbohydrate, nutrient composition, and growth regulators were tested for the maximum yield of rhizomes. Among the different photoperiods used, a 24-h photoperiod helped in the formation of more rhizomes as compared with other photoperiods. Of the different carbohydrates used, sucrose helped to achieve rhizome formation as compared to other carbohydrates. The microrhizomes sprouted in a soil mixture within 2 wk of planting. The sprouted plantlets survived under field conditions with normal growth.     

Inoculation and colonization of coffee seedlings (Coffea arabica L.) with the fungal entomopathogen Beauveria bassiana (Ascomycota: Hypocreales)

The fungal entomopathogen Beauveria bassiana became established as an endophyte in coffee seedlings grown in vitro and inoculated with B. bassiana suspensions in the radicle. The fungus was recovered as an endophyte 30 and 60 days postinoculation, from stems, leaves, and roots, and at 60 days postinoculation one of the isolates was also recovered as an epiphyte. Fusarium sp., Rhodotorula sp., and four bacterial morpho-species were also detected, indicating these were present as endophytes in the seed.     

Factors affecting adventitious bud induction in Pinus elliottii (Engelm.) embryos cultured in vitro

Embryos of slash pine (Pinus elliottii Engelm.) were induced to form adventitious buds when placed in culture on nutrient media supplemented with cytokinin. Buds were induced on media containing Risser & White major salts. The high content in nitrogen of Murashige & Skoog formulation seems to be deleterious for this in vitro system, since morphogenic responses were only promoted when nitrogen concentration was drastically reduced in the macronutrient formulation. Factors such as concentration of cytokinin (6-benzyladenine) and time and method of exposure (liquid or solid induction medium) strongly influenced bud formation and development. The greatest number of buds and shoots were obtained from 22.0 M cytokinin, but these shoots showed less and slower development than those induced with low dosages of cytokinin. The presence of naphthaleneacetic acid in combination with cytokinin in the induction medium decreased the frequency of bud formation.Abbreviations (BA) 6-benzyladenine - (NAA) 1-naphthaleneacetic acid     

Promotion of petunia (Petunia hybrida L.) regeneration in vitro by ethylene

The influence of ethylene on shoot and root formation from petunia leaf explants was studied in cultures in test tubes placed in 51 glass jars. Reduction of the endogenously produced ethylene by inclusion of ethysorb (KMnO₄), an ethylene absorbent, caused a decrease of the number of shoots. On the other hand, supplementing the cultures with ethylene (0.01–10 ppm) caused a marked increase of the number of shoots without, however, any effect on the length and fresh weight. Ethylene treatments (1 ppm) were found to be most effective when they were applied in the second week of culturing of petunia explants. Addition of Co⁺⁺ to the medium resulted in a reduction of the endogenously produced ethylene and concomitantly reduced shoot formation. Similarly, inclusion of Ag⁺, an inhibitor of ethylene action, resulted in poor shoot formation. Ethylene also appeared to play a role on rooting of petunia microshoots in vitro in an auxin-free medium. Ethylene at a concentration of 10 ppm induced adventitious root formation considerably, whereas at low levels (0.01–1 ppm) it had no influence on rooting.     

Effects of N6-benzyladenine on shoots of five willow clones (Salix spp.) cultured in vitro

Growth and differentiation in shoot cultures of five willow clones on media of different BA concentrations were compared. The tendency of axillary shoots to develop on shoot cultures depended on the genotype, the type of shoot and the number of previous subcultures. The optimum concentration for shoot multiplication was either 5×10^-7 M or 10^-6 M. On BA concentrations of 10^-5 M or higher, browning and death of shoots occurred. Depending on the genotype, shoot elongation was best on media containing 0–5×10^-7 M BA. Rooting ability was also genotype dependent. Prolonged culture in vitro improved the rooting ability of the two most reluctant clones. BA concentrations of 5×10^-7 M or higher inhibited rooting almost completely, but this was not a permanent effect. All clones could be rooted on medium containing 10^-6 M NAA. Shoots were transferred to greenhouse conditions and rooted with varying degrees of success depending on shoot size and genotype.     

A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis sims. F. Flavicarpa Deg.)

Summary Mineral deficiency symptoms were observed in leaves of yellow passionfruit plantlets grown in MS medium (Murashige and Skoog, 1962) with 1.0 mg l⁻¹ (3.0 μM) gibberellic acid. Initially, leaves showed interveinal chlorosis, followed by bleaching of the leaves and retarded growth. Leaf mineral analysis was done and compared to mineral requirements suggested for passionfruit in the literature. Several modifications were made to the inorganic composition of MS medium, according to mineral deficiencies, mainly of Fe and Ca, and possible toxicity of Cl. The concentration of the elements in the new medium (MSM) was based on the mineral composition of leaves of healthy plants. The chemical equilibrium was checked using the software Geochem (Sposito and Mattigod, 1980) and final adjustments were made to ensure good availability of nutrients. To test the efficiency of the modified medium nodal segments were cultured in both MS and MSM supplemented with 3.0 mg l⁻¹ (13.3 μM) 6-benzyladenine. After three subcultures mineral analysis of the leaves was done. Severe mineral deficiency was observed on the leaves of plantlets cultured in MS, while plantlets cultivated in MSM had green leaves. A comparison of the mineral analysis of plantlets in both media showed a fairly large increase in Ca, Cu, Fe, Mg and S and decrease in levels of B and Cl in plantlets cultivated in MSM. A slight increase or decrease in other elements was also observed. Subculture of the chlorotic plantlets into MSM showed that the visual symptoms of mineral deficiency disappeared in 2–4 wk.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

In vitro conservation of enset under slow-growth conditions

Studies on in vitro storage of enset under slow-growth conditions were carried out to develop an efficient protocol for conservation of the genetic diversity of the crop. The response to different growth retardation treatments was examined using three enset clones collected from southwestern Ethiopia. In vitro cultures could be effectively maintained for 6 months at 15 °C and 18 °C on MS medium supplemented with 10 μM BAP, in the presence of mannitol at concentrations of 0, 1 or 2% as a growth retardant. Shoots were subsequently recovered and multiplied on MS medium supplemented with 10 and 20 μM BAP at 25 °C and rooted shoots were successfully transferred to the greenhouse. Incubation at the lower temperature (15 °C) and the presence of mannitol in the culture medium had a significantly positive effect on maintenance, measured by the number of recovered shoots after storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro propagation of banana (Musa spp.): initiation,proliferation and development of shoot-tip cultures on defined media

In vitro multiplication of banana (Musa spp.) from shoot-tip explants isolated from lateral suckers is described. Using explants with apical domes, a total of 22 banana cultivars were successfully cultured on a modified Murashige and Skoog's medium containing 6-benzylaminopurine (BA) and indolebutyric acid (IBA). Shoot-tip explants could be induced to produce multiple shoot initials in the presense or absence of apical domes, but the survival rates were higher if apical domes were retained. Cultivars varied widely in their multiplication rates in response to cytokinins, BA being consistently more effective than kinetin (Kn). Although Kn was less effective in this regard, it stimulated vigorous root growth. Rooted plantlets were successfully established in soil.     

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

Summary Shoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m⁻²·s⁻¹ photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.     

In vitro propagation of Typhonium flagelliforme (Lodd) blume

Summary An in vitro culture system was developed for Typhonium flagelliforme using buds from the rhizomes. The mineral salts of four media were tested. These were Murashige and Skoog (MS), Nitsch and Nitsch (NN), Gamborg B5 (GB5) and White (W) of which MS medium was found to be the best medium for in vitro culture of T. flagelliforme. The addition of as low as 0.1 mg l⁻¹ (0.54 μM) α-naphthalene acetic acid (NAA) with the presence or absence of N⁶-benzyladenine (BA) in the MS medium caused abnormal shoot formation. The best medium for maximizing shoot number combined with normal complete plantlets from each bud was MS medium supplemented with 0.3 mg l⁻¹ (1.33 μM) BA and 0.5 mg l⁻¹ (2.46 μM) indole-3-butyric acid (IBA). The best acclimatization process was to transfer the normal plantlets, with all the leaves removed, into sand plus coconut husks substrate (1∶1) and placed in intermittent water mists house or shaded plant house with 50% light exclusion. Ninety two percent of the plantlets survived using this acclimatization method.     

Effect of various growth regulators on growth in vitro of cherry shoot tips

An investigation was undertaken to examine the effects of nine different growth regulators on growth in vitro of shoot cultures of the semi-dwarfing cherry rootstock Colt (Prunus avium × P. pseudocerasus). The effects of each supplement on shoot extension and proliferation and also leaf and callus production were noted, and it was found that BAP has the ability to proliferate shoots, IBA nullifies this effect and that kinetin, ABA, GA₃ and ethylene inhibit the growth of colt cultures. Conditions were established which resulted in a) optimum shoot growth prior to subsequent rooting or grafting; b) maximum shoot proliferation for rapid clonal multiplication and c) minimum shoot growth. This study will form the basis of an investigation into germplasm conservation of temperate fruit trees by both cryogenic storage and minimal growth techniques.     

In vitro culture of Phyllanthus caroliniensis (Euphorbiaceae)

An efficient micropropagation protocol was developed for the medicinal plant Phyllanthus caroliniensis (Euphorbiaceae) using nodal segments for axillary shoot proliferation. Maximum multiplication (21–23 shoots per explant) was achieved on MS or AR media supplemented with either 5.0 μM BA, 1.25–5.0 μM kinetin or 2.5–5.0 μM 2iP. Rooting was achieved with 80–100% of the microshoots on MS medium without growth regulators, although 1.25 μM NAA and 1.25–5.0 μM IAA promoted significant increases in the number of roots per explant. Regenerated plants were successfully acclimatized and about 88% of plantlets survived under ex vitro conditions. Flowering was observed on in vitro grown plantlets and after 3–4 weeks of acclimatization. High frequency callus initiation and growth was achieved when nodal segment explants were inoculated in the vertical position on MS medium supplemented with 5.0 μM 2,4-D. Root cultures were successfully established on MS medium containing 1.1 μM NAA. The optimized micropropagation, callus and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolism studies. This revised version was published online in June 2006 with corrections to the Cover Date.     

Seasonal changes in photoprotective mechanisms of leaves from shaded and unshaded field-grown coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> L.) trees

Coffee is native to shady environments, but often grows and yields better without shade. Thus, it may be reasoned that coffee leaves should display enough plasticity to acclimate themselves to contrasting light environments. However, little is known about mechanisms associated with such plasticity in coffee. This work aimed, therefore, to explore differences in leaf photoprotective mechanisms. Plants were grown in the field and received either 48 or 100% natural light. Evaluations were made using outer leaves from the sun-facing sides of the coffee hedgerow in Viçosa (Brazil) in August and October, when growth and photosynthetic rates are expected to be minimal and maximal, respectively, and in December, when temporary depressions in those variables are common. Regardless of light treatments, coffee leaves showed: (1) very low photosynthetic rates (generally below 2.5 μmol m^?2 s^?1), (2) chronic photoinhibition in August (dry, cool season) that was accompanied by strong loss of pigment concentration, and (3) discrete, dynamic photoinhibition in October and December (rainy, warm season). Compared with shaded leaves, sunlit leaves generally exhibited lower pigment concentration, lower quantum yield of electron transport, steeper inclinations and similar electron transport rate. Total ascorbate pool tended to be larger in sunlit than in shaded leaves (but with similar redox state), whereas activities of key antioxidant enzymes, as well as malondialdehyde accumulation and electrolyte leakage, were similar between those leaf types. As a whole, the photosynthetic apparatus of the coffee tree showed a low phenotypic plasticity to varying irradiance.     

Biotechnology and the conservation of forest genetic resources: in vitro strategies and cryopreservation

In vitro techniques have a clear role within ex situ conservation strategies for trees and crop genetic resources, particularly where it is important to conserve specific genotypes or where normal propagules such as recalcitrant seed may not be suitable for long-term storage. These involve the use of conventional micropropagation, restricted growth techniques and cryopreservation. Although these techniques have been used primarily with herbaceous species, increasing attention is being given to woody species. Cryopreservation techniques for both woody and herbaceous species and new approaches which do not require freeze-induced cell dehydration, referred to as the encapsulation-dehydration and the vitrification techniques are described. Illustrative data are presented for the cryopreservation of willow using the encapsulation-dehydration technique.     

Growing of potato microplants in the presence of alginate-silverthiosulfate capsules reduces ethylene-induced culture abnormalities during minimal growth conservation in vitro

Alginate capsules containing anionic complex silverthiosulfate (STS) [Ag(S₂O₃)₂ ^3-] were placed in the culture tubes over minimal growth media for studying whether STS could be used at higher concentrations to sustain ethylene-inhibiting effect during conservation of microplants in six potato (Solanum tuberosum L.) genotypes in vitro. Different concentrations of STS (0.1, 0.5, 1.0, 2.0, 3.0 and 4.0 mM) were incorporated into the alginate capsules, and 12 alginate-STS capsules were tested in semisolid (7 g l^–1 agar) minimal growth medium containing 20 g l^–1 mannitol and 40 g l^–1 sucrose. This indirect supplementation of STS through alginate capsules rendered reduced total availability of STS in the minimal growth medium as compared to when it was directly supplemented in the medium at a given concentration. Growing of microplants in the presence of alginate-STS capsules improved the microplant growth and reduced the culture abnormalities over a period of 16 months under minimal growth conditions. Most significant improvement in microplant growth was in terms of green leaf production and leaf senescence. Vitrification, flaccidity and other growth abnormalities, viz., leaf loss, abnormal stem swelling and necrosis were not observed when the microplants were conserved in the presence of alginate-STS capsules. To foster optimum microplant growth and reduce culture abnormalities, potato microplants could favourably be maintained in the presence of 0.5–1.0 mM alginate-STS capsules during minimal growth conservation. Higher concentrations of alginate-STS capsules (>1.0 mM) were in general detrimental to potato microplant growth and survival during prolonged storage in vitro. Release kinetics of STS from the alginate-STS capsules, its distribution in the medium and accumulation of silver in potato microplants were studied using ^110mAg. The release rate of STS from the capsules was found to be directly proportional to the concentrations of alginate-STS capsules. A distinct concentration gradient of ^110mAg in the medium with increasing depth from top to bottom, and its accumulation in the potato microplants may be attributed to the improved anti-ethylene action of STS at higher concentrations through alginate capsules.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.