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 describes a method for obtaining in vitro germination of zygotic embryos from open-pollinated wild seedy bananas and controlled hybridizations. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. This part describes the required laboratory materials, medium preparation, embryo extraction and culture. Results. Embryos typically begin germination in the dark about 15 days after inoculation on germinating medium. After 30––40 days of culture, plantlets are subcultured on a plant growth medium and placed in the light to achieve plant growth and rooting. The ratios of zygotic embryos obtained vary with the types of crosses achieved between Musa sp. genotypes.

Introduction. This protocol aims at measuring the mechanical characteristics of bananas, especially peel and fruit hardness, and pulp firmness; it can also allow the mechanical characterisation of green or ripening fruit of different pedo-climatic origins and/or varieties. Materials and methods. This part describes the required laboratory materials and the three steps necessary for the measurement of the mechanical characteristics of bananas. Results. The data allow the drawing of a curve characterising the firmness of the fruit (slope), hardness of the peel (peak) and hardness of the pulp (plateau).

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.

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. The technique presented provides molecular markers for DNA methylation studies in the Musa sp. genome. This technique can be used for studying DNA methylation in natural populations, in mapping populations, and also in plant organs or during plant development. 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 producing MSAP autoradiography with HpaII / MspI (CCGG methylation) and for EcoRII / PspGI (CCWGG methylation). It mentions the main problems which can occur. Results. The protocol makes it possible to detect CCWGG and CCGG methylated sites.

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 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 protocol aims at measuring the storage life potential of banana fruit, and at determining the physiological age of fruit. 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 five steps necessary for calculating the banana green life duration, which corresponds to the number of days between the fruit harvest and climacteric crisis. Results. The measurement of O2 and CO2 concentrations allows one to detect the climacteric peak which marks the end of the banana green life.

Introduction. This protocol aims at detecting and evaluating sensitivity shifts in Pseudocercospora musae (teleomorph: Mycosphaerella musicola) populations towards fungicides that are currently sprayed to control Sigatoka disease of banana. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. Necessary laboratory materials, and details of the seven steps of the protocol achieved during four days of experiments are described. Results. The protocol results in the observation of conidial germination of P. musae (pattern or germ tube elongation) according to the sensitivity to fungicides.

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 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 (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 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. 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. The techniques presented allow a long-term conservation of Cylindrocladium strains to preserve them from major morphological and physiological changes. The principle of the methods applied, key advantages, starting plant material, time required and expected results are presented. Materials and methods. The necessary laboratory materials, and details of the nine steps achieved for the monospore purification of Cylindrocladium strains, their long-term maintenance, and the production of conidia and microsclerotia are described. Results. When revived, purified strains generate actively growing colonies within 48–72 h. Such colonies can then be readily used for conidia or microsclerotia production.

Introduction. This protocol aims at detecting Cylindrocladium spp. in root and soil samples from banana fields and at quantifying the inoculum. The principle of the two methods applied [Root Slice Plating Method (RSPM) and Soil Isolation Method (SIM)], the key advantages of these methods, starting plant and soil materials, the time required and the expected results are presented. Materials and methods. Necessary laboratory materials, and details of the six steps achieved for the use of the two methods are described. Results. When using the RSPM, typical Cylindrocladium colonies are isolated in petri dishes. When using the SIM, typical Cylindrocladium colonies originate from microsclerotia embedded or not in banana root tissue. The inoculum density can be calculated.

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 aims at detecting and quantifying quiescent infections of Colletotrichum musae on bananas. The principle, key advantages, starting plant material, time required and expected results are presented. Materials and methods. The materials required and details of the three steps of the protocol (fruit sampling, fruit ripening and anthracnose lesion quantification) are described. Possible troubleshooting is discussed. Results. The protocol results in the quantification of anthracnose lesions on the fruits, which makes it possible to predict postharvest losses due to anthracnose (peel rot), and also to propose a better management of postharvest fungicide applications.