Rony Swennen

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

SummaryWe report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×107 protoplasts.ml−1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (106 protoplasts.ml−1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.

Transient gene expression in electroporated banana (Musa spp., cv. ‘Bluggoe’, ABB group) protoplasts isolated from regenerable embryogenetic cell suspensions

SummaryElectroporation conditions were established for transient expression of introduced DNA in banana (Musa spp., cv. ‘Bluggoe’) protoplasts isolated from regenerable embryogenic cell suspensions. The following parameters were found to be highly influential: electroporation buffer, polyethylene glycol treatment and its duration before electroporation, use of a heat shock, and chimaeric gene constructs. The maximum frequency of DNA introduction as detected by an in situ assay for transient expression of the uidA gene, amounted to 1.8% of total protoplasts. Since plants have recently been regenerated from banana protoplasts at a high frequency, the present results may contribute to the production of transgenic banana.

Variability in storage potential of banana shoot cultures under medium term storage conditions

Shoot cultures of 401 banana clones were conserved under slow growth conditions (16±1°C, 25μmol m−2 s−1). Storage duration-defined as 60% survival time of 20 shoot cultures of a clone-averaged 334 days. However, large differences occurred among the different genomic (sub)groups and even within the same (sub)group. East-African highland bananas and non-plantain AAB bananas can be stored for significantly longer periods. Shoot tip cultures of another 41 banana clones conserved at higher ambient temperature (22±3°C) needed to be subcultured sooner (every 220 days on average).

Positive selectable marker genes for routine plant transformation

SummaryPlant genetic transformation technologies rely upon the selection and recovery of transformed cells. Selectable marker genes used so far have been either antibiotic resistance genes or herbicide tolerance genes. There is a need to apply alternative principles of selection, as more transgenic traits have to be incorporated into a transgenic crop and because of concern that the use of conventional marker genes may pose a threat to humans and the environment. New classes of marker genes are now available, conferring metabolic advantage of the transgenic cells over the non-transformed cells. The new selection systems, as described in this review, are being used with success and superior performance over the traditional marker systems.

Banana rhizodeposition : characterization of root border cell production and effects on chemotaxis and motility of the parasitic nematode Radopholus similis

Rhizodeposition was collected from root tips of banana (Musa acuminata). Two varieties, Grande naine and Yangambi km5, susceptible and resistant towards the burrowing nematode Radopholus similis, respectively, were examined for root border cell production under inxa0vitro and in soil growing conditions. Two types of cells were observed in rhizodeposition: spherical cells containing large amyloplasts, called statocytes (8% of total), and long ellipsoidal border cells (92%). Border cell production was high, related to root length and not different between the two cultivars. Rhizodeposition from plants grown in soil contained similar amounts of border cells, but viability was lower than in inxa0vitro grown plants. Chemotaxis and motility assays were performed to test the effects of banana roots, roots without rhizodeposition, rhizodeposition, exudates and border cells on the behaviour of R.xa0similis. Roots of both the susceptible and resistant variety attracted R.xa0similis, but only in the presence of rhizodeposition. Isolated rhizodeposition of Yangambi km5 also attracted nematodes. Border cells and exudates did not affect nematode chemotaxis. Roots of both varieties induced quiescence in R.xa0similis in the chemotaxis assay. Thirty to fifty percent of the nematodes became temporary quiescent in the motility assay with rhizodeposition and exudates. The effect lasted for 24xa0h for Grande naine and up to 3xa0days for Yangambi km5. Rhizodeposition collected from plants grown in soil affected neither R.xa0similis chemotaxis nor motility. Overall, there were no indications that rhizodeposition is involved in preformed resistance against R.xa0similis in Yangambi km5.

Organic banana production in Ecuador: Its implications on black Sigatoka development and plant–soil nutritional status

Black Sigatoka, caused by the leaf fungus Mycosphaerella fijiensis Morelet, is a major constraint to banana production around the world. In Ecuador, the biggest banana-exporting country in the world, this disease has become increasingly aggressive. This has resulted in more fungicide applications, which have significantly increased costs in production and for the environment. Consequently, many banana growers have shifted to organic production, which produces greater economic returns as a result of higher sale prices. In addition, production costs are lower as no fungicides are applied. These organic bananas receive substantial amounts of organic products. This study describes the black Sigatoka disease and nutrient status in an organic banana plantation and compares it with a conventionally fertilized and fungicide-treated plantation. Black Sigatoka symptoms were evaluated in the vegetative and flowering stages under both production conditions and in vitro conditions. Univariate and multivariate descriptive statistics were used to analyze the parameters. Disease symptoms were more severe in leaves from the organic field than in leaves from the inorganic field, but the nutrient status (soil and foliar) did not differ between the two farms. Banana plants from the organic farm had 12 functional leaves at flowering and eight functional leaves at harvest. Average banana yields were over 40% lower for organic versus inorganic management; however, the average price received for organic bananas was over two times higher. Profit–cost analysis has shown that the organic banana farm was substantially more profitable than the inorganic one during the time period analyzed. These results indicated that bananas can be grown commercially without fungicides, and the lower productivity levels are compensated by higher prices of organic fruits in international markets. In addition, organic production has beneficial impacts on social and environmental issues.

Transient gene expression in transformed banana (Musa cv. Bluggoe) protoplasts and embryogenic cell suspensions

In order to introduce currently-available genes with agronomical value into banana, two genetic transformation protocols have been optimized.

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

SummaryWe report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.

Host plant response of Eumusa and Australimusa bananas ( Musa spp.) to migratory endoparasitic and root-knot nematodes

Twenty-five banana varieties of section Eumusa (AA-group) and seven of the section Australimusa (Fei-group) from Papua New Guinea were evaluated for resistance to Radopholus similis , Pratylenchus coffeae and Meloidogyne spp. The host plant responses were compared with the susceptible reference cvs Grande Naine and Cavendish 901. In vitro propagated plants were transferred to the glasshouse in loamy sand and inoculated with approximately 1000 migratory endoparasitic nematodes at 4 weeks after planting. Reproduction of R. similis and P.coffeae in the roots was determined at 8 or 10 weeks, respectively, after inoculation. Reproduction of Meloidogyne spp. was determined 8 weeks after inoculation with 3300 to 5000 eggs. No resistance to R. similis was found in the diploid varieties. The Fei variety Rimina and possibly Menei were resistant to R. similis . All varieties tested were susceptible to P.coffeae and Meloidogyne spp. Tests de resistance de bananiers Eumusa et Australimusa (Musa spp.) envers les nematodes endoparasites migrateurs et galligenes - Vingt-cinq varietes de bananier de la section Eumusa (groupe AA) et sept de la section Australimusa (group Fei) provenant de Papouasie-Nouvelle Guinee ont ete testees pour leur resistance envers Radopholus similis , Pratylenchus coffeae et Meloidogyne spp. Les responses de ces varietes ont ete comparees a celles des cultivars sensibles de reference Grande Naine et Cavendish 901. Des vitroplants ont ete mis en place en serre sur un sol argilo-sableux et inocules 4 semaines apres plantation avec environ 1000 R. similis ou P.coffeae dont la reproduction a ete determinee 8 et 10 semaines, respectivement, apres inoculation. La reproduction de Meloidogyne spp. la ete 8 semaines apres inoculation avec 3300 a 5000 oeufs. Aucune resistance a R. similis na ete observee chez les varietes diploides. Les varietes du groupe Fei Rimina et Menei se sont montrees resistantes a R. similis , avec un certain doute dans le cas de la derniere. Toutes les varietes testees sont sensibles a P.coffeae et Meloidogyne spp.

Number and accuracy of T-DNA insertions in transgenic banana (Musa spp.) plants characterized by an improved anchored PCR technique

Nineteen transgenic banana plants, produced via Agrobacterium-mediated transformation, were analyzed for the integration of T-DNA border regions using an improved anchored PCR technique. The method described is a relatively fast, three-step procedure (restriction digestion of genomic DNA, ligation of ‘vectorette’-type adaptors, and a single round of suppression PCR) for the amplification of specific T-DNA border-containing genomic fragments. Most transgenic plants carried a low number of inserts and the method was suitable for a detailed characterization of the integration events, including T-DNA border integrity as well as the insertion of non-T-DNA vector sequences, which occurred in 26% of the plants. Furthermore, the particular band pattern generated by four enzyme/primer combinations for each individual plant served as a fingerprint, allowing the identification of plants representing identical transformation events. Genomic Southern hybridization and nucleotide sequence analysis of amplification products confirmed the data obtained by anchored PCR. Sequencing of seven right or left border junction regions revealed different T-DNA processing events for each plant, indicating a relatively low frequency of precisely nicked T-DNA integration among the plants studied.