Introduction. This protocol describes a method for obtaining well-established banana plants in the field, both for open-air and greenhouse cultivation, from hardened tissue culture nursery plants. The principle, starting material and time required are presented. Materials and methods. This part describes the process of land preparation, both in the case of planting and replanting, including preplant fertilizer requirements and irrigation recommendations, and the main horticultural practices appropriate both for planting out in the field and for the first months after planting. Special attention is given to correct irrigation, a key issue in the establishment process. Possible problem areas for troubleshooting are listed. Results. At the end of the process, vigorous plants with uniform growth and high yield potential are planted out and become optimally established in the commercial field.

Introduction. This protocol can be used as a guide for people who wish to construct their own nursery. It describes the methods for receiving, transplanting, managing and hardening young banana tissue culture plants after the weaning stage. The principle, key advantages, starting plant material and time required are presented. Materials and methods. This part describes the nursery construction, the nursery media, fertiliser and bags, the handling and transplanting of weaned plants into bags (3 steps), and the management schedule for hardening nursery plants (8 steps). Possible troubleshooting is listed. Results. All the characteristics of nursery-hardened plants in bags, ready for transplanting to the field, are formulated.

Introduction. The protocol describes a method for promoting the acclimatization and protection of rooted in vitro banana plants between rooting in the laboratory and their establishment in a nursery. The principle, key advantages, starting plant material and time required are presented. Materials and methods. This part describes the weaning house installation, the weaning medium used to transplant the vitroplants, the two steps for acclimatizing the plantlets: receipt of rooted laboratory plants in flasks and planting, and factors affecting the growth of the plantlets in weaning conditions (light, misting system, temperature, humidity, wind, pest control and nutrition). Possible problems for troubleshooting are listed. Results. Under normal conditions, new roots appear 4–5 days after the start of weaning, and new leaves start to grow within 8–10 days. At the end of the weaning phase, young plants are healthy, well developed and they can be established in a nursery.

Introduction. This protocol describes a method for obtaining protoplasts from banana leaves, calli and cell suspensions, and their sustainable development via somatic embryogenesis from embryogenic cell suspensions. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, and media preparation for protoplast production and culture. Results. The first protoplasts may be seen after 30 min of incubation in enzyme maceration. With protoplasts from embryogenic cell suspension, complete development into a whole plant, through somatic embryogenesis, is observed in 12 weeks. The first cell divisions occur on feeder layers 3–8 days after protoplast plating. Proembryo formation is observed 14–21 days after initiation of protoplast culture. The transfer of derived embryo plantlets, at 8–10 weeks after protoplast plating, onto growth regulator-free medium, leads to plant rooting and elongation.

Introduction. RNA isolation is a prerequisite to studying gene expression in banana and to understanding changes occurring in response to the environment. Standard extraction methods do not efficiently extract RNA from plants such as banana, with high levels of phenolics, carbohydrates, or other compounds that bind to and/or coprecipitate with RNA. Materials and methods. Five to seven RNA extraction methods were compared. Four crown-tissue storage methods were also compared. cDNA-AFLP was used to ensure that the obtained RNA was of sufficient quality for molecular applications and that RNA expression was unaltered by in situ storage. Results and discussion. The modified hot-borate method proved to be the best RNA extraction method, allowing high yields of good quality, undegraded RNA from the crown, fruit peel and pulp at all stages of ripening. The RNA obtained by this method was of sufficient quality for molecular applications such as cDNA-AFLP that give highly reproducible results. Freeze-drying of fresh tissues and tissue conservation in hot-borate buffer, two original storage methods, appear appropriate for preserving RNA in situ without ultra-low temperature. The RNA obtained was of high quality, undegraded, and useful for all downstream applications. The genome expression profile obtained by cDNA-AFLP analysis was unaltered by these methods for storing collected tissues. Conclusion. By applying all the suggested procedures in this work, it is possible to store and study gene expression in any banana fruit tissue, whatever the maturity stage, without affecting the RNA expression level.

Introduction. This protocol applies to the characterization of genome structure and evolution of M. acuminata and M. balbisiana genomes; construction of physical maps around markers of interest and QTLs; comparative genomic studies with other monocot species; and study of banana streak virus (BSV) integration patterns in banana nuclear genomes. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required materials, and the protocols for isolation and lysis of banana nuclei; high-molecular-weight (HMW) DNA Hind III digestion and pulsed-field gel electrophoresis (PFGE) analysis; large-scale HMW DNA Hind III digestion and ligation to the pINDIGOBAC–5 vector; and analysis of recombinant BAC clones. It mentions the main problems which can occur. Results. The described protocols will enable the efficient construction of BAC libraries from Musa species. These libraries can be used to construct physical maps and select specific genomic regions to be sequenced.

Introduction. This method is used to forecast the harvest date of banana bunches from as early as the plant shooting stage. It facilitates the harvest of bunches with the same physiological age. The principle, key advantages, time required and expected results are presented. Materials and methods. Details of the four steps of the method (installation of the temperature sensor, tagging bunches at the flowering stage, temperature sum calculation and estimation of bunch harvest date) are described. Possible problems are discussed. Results. The application of the method allows drawing a curve of the temperature sum accumulated by the bunches which have to be harvested at exactly 900 degree-days physiological age.

Introduction. This protocol aims to diagnose under laboratory conditions the main Mycosphaerella spp. pathogens of bananas and plantains. The three pathogens Mycosphaerella fijiensis (anamorph Paracercospora fijiensis), M. musicola (anamorph Pseudocercospora musae) and M. eumusae (anamorph Pseudocercospora eumusae) are, respectively, the causal agents of Black Leaf Streak disease, Sigatoka disease and Eumusae Leaf Spot disease. The principle, key advantages, starting plant material and time required for the method are presented. Materials and methods. The laboratory materials required and details of the thirteen steps of the protocols (tissue clearing and in situ microscopic observations, isolation on artificial medium and cloning of single-spore isolate, in vitro sporulation and microscopic observations of conidia, and long-term storage of isolates) are described. Results. Diagnosis is based on the observations of anamorphs (conidiophores and conidia) which can be observed directly from banana leaves or after sporulation of cultivated isolates if sporulating lesions are not present on banana samples.

Introduction. This protocol aims at monitoring the development of Sigatoka disease (Mycosphaerella musicola / Pseudocercospora musae) of banana plants in order to control this disease with a minimum of fungicide applications. A good timing of fungicide application will result in lower cost of control, lower risk of fungicide resistance and lower negative environmental impact. The principle, key advantages, starting plant material and expected results are presented. Materials and methods. Details of the seven steps of the protocol allowing banana plot preparation, data collection, stage of evolution of the disease (SED) calculation, and timing of fungicide applications are described. Possible troubleshooting is mentioned. Results. Weekly observation of selected banana plants allows filling in a data sheet used for the calculation of the SED. A graphic representation of the weekly evolution of this SED can then be used for timing of fungicide spraying decisions.

Introduction. This protocol aims at evaluating (a) the efficacy of new fungicides for the control of post-harvest diseases, (b) the efficacy of various application methods for the chemical control of post-harvest diseases, and (c) the quality of the fungicide solution during the same packing day where this solution is recycled. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. Materials required and details of the eighteen steps of the protocol (fruit sampling and inoculum preparation, wound anthracnose study, quiescent anthracnose study, and crown-rot study) are described. Results. Comparison between untreated control bananas and bananas treated with fungicide allows the calculation of the fungicide treatment efficacy.

Introduction. This protocol aims at monitoring the development of Black Leaf Streak (Mycosphaerella fijiensis Morelet) disease of banana plants in order to control this disease with a minimum of fungicide applications. A good timing of fungicide application will result in lower cost of control, lower risk of fungicide resistance and lower environmental negative impact. The principle, key advantages, starting plant material and expected results are presented. Materials and methods. Details of the six steps of the protocol allowing banana plot preparation, data collection, stage of evolution of the disease (SED) calculation, and timing of fungicide applications are described. Possible troubleshooting is mentioned. Results. Weekly observation of selected banana plants allows filling in a data sheet used for the calculation of the SED. A graphic representation of the weekly evolution of this SED can then be used for timing of fungicide spraying decisions.

Introduction. This rearing technique is applicable to migratory nematodes (e.g., Pratylenchus spp. and Radopholus similis) for mass production of nematodes for experimental purposes, conservation of nematodes, and direct studies of nematodes’ multiplication or reproduction mechanisms. The principle of the method applied, key advantages, starting plant material and time required are presented. Materials and methods. Necessary laboratory materials, and details of the nine steps required for the preparation of carrot discs, preparation of nematodes, inoculation of nematodes and collection of nematodes out of petri dishes are described. Possible troubleshooting is explained. Results. Several thousands of nematodes can be extracted out of one carrot disc after 6–8 weeks of culture, depending on nematode species and geographical population.

Introduction. This protocol aims at (i) analysing the nature of host-parasite interactions (resistance or susceptibility), and (ii) studying the variability of the pathogenicity of different pathogen populations (intra/inter-specific) with regard to both virulence and aggressiveness using artificial inoculation with Mycosphaerella fijiensis or Mycosphaerella musicola on plants and banana leaf pieces. The principle, key advantages, starting plant material and time required for the method are presented. Materials and methods. Laboratory materials required and details of the sixteen steps of the protocol (conidial inoculum preparation, artificial inoculation on banana plants and artificial inoculation on banana leaf pieces) are described. Possible troubleshooting is discussed. Results. Typical symptoms of the different diseases are obtained after artificial inoculation.

Introduction. This protocol aims at measuring fruit ethylene production during ripening. It can be used to compare ethylene production between different banana varieties or to compare ethylene production between fruit produced in different pedo-climatic conditions. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials and the three steps necessary for calculating the amount of ethylene produced during banana postharvest ripening. Possible troubleshooting is considered.

Introduction. This protocol aims at preparing total RNA for gene expression analysis by Northern blots, RT-PCR and real-time quantitative PCR; cDNA isolation by RT-PCR; and cDNA library construction. The principle, key advantages, starting plant material, time required for obtaining total RNA and expected results are presented. Materials and methods. This part describes the required materials and the 27 steps necessary for preparing RNA from peel and pulp fruit tissue: preparation of plant tissue powder, preparation of the complete RNA extraction buffer and isolation of RNA from ground banana fruit tissue. Results. Extraction of total RNA by the method described makes it possible to achieve electrophoresis under denatured conditions and in vitro reverse transcription. An example for Northern blot analysis is illustrated.

Introduction. The centrifugal-flotation technique allows all nematode species and life stages to be separated from root debris and residual soil particles to facilitate their observation. The principle of the method applied, key advantages, starting plant material and time required are presented. Materials and methods. Necessary laboratory materials, and details of the 19 steps required for extracting nematodes from plant tissue for counting are described. Possible troubleshooting is explained.

Introduction. This protocol aims at detecting and evaluating sensitivity shifts in Colletotrichum musae populations towards fungicides that are currently used for the control of postharvest diseases of bananas. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. The necessary laboratory materials and details of the twelve steps of the protocol for banana sampling and anthracnose lesion development, the agar plate preparation, single-spore isolation, germination test, and data analysis are described. Possible troubleshooting is mentioned. We obtain (a) for antimitotic products, the percentage of resistant strains in the population analysed; (b) for Sterol Biosynthesis Inhibitor (SBI) fungicides, the variation and distribution of the isolates' sensitivity within the population analysed according to their level of radial growth reduction by the fungicide.

Introduction. This protocol makes it possible to determine the chromosome number of banana clones in root tip squashes. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, and the four steps of the protocol used for the chromosome counts (root sampling and treatment, slide preparation, observation of useful cells and chromosome counts on useful cells). Possible troubleshooting is discussed. Results. The protocol usually results in condensed and intensely stained chromosomes.

Introduction. We present some protocols aiming at partially characterizing banana fruit quality through measurement of some key biochemical parameters. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials and the steps necessary for achieving four protocols making it possible to measure sugar, organic acids and free ACC contents, and in vitro ACC oxidase activity. Results. Standard results obtained by using the protocols described are presented in the figures.

Introduction. This protocol aims at (a) evaluating the resistance to post-harvest diseases within different genotypes of bananas, and (b) comparing different origins of bananas (geographic origin, physiological stage, etc.) for their susceptibility to post-harvest diseases. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. Materials required and details of the twelve steps of the protocol (fruit sampling and inoculum preparation, wound anthracnose resistance study, quiescent anthracnose resistance study and crown-rot resistance study) are described. Results. Typical symptoms of the different diseases are obtained after artificial inoculation.