Silicon Isotopic Fractionation by Banana (Musa spp.) Grown in a Continuous Nutrient Flow Device

The determination of the plant-induced Si-isotopic fractionation is a promising tool to better quantify their role in the continental Si cycle. Si-isotopic signatures of the different banana plant parts and Si source were measured, providing the isotopic fractionation factor between plant and source. Banana plantlets (Musa acuminata Colla, cv Grande Naine) were grown in hydroponics at variable Si supplies (0.08, 0.42, 0.83 and 1.66 mM Si). Si-isotopic compositions were determined on a multicollector plasma source mass spectrometer (MC-ICP-MS) operating in dry plasma mode. Results are expressed as δ²⁹Si relative to the NBS28 standard, with an average precision of ± 0.08‰ (±2σ_D). The fractionation factor ²⁹ε between bulk banana plantlets and source solution is −0.40 ± 0.11‰. This confirms that plants fractionate Si isotopes by depleting the source solution in ²⁸Si. The intra-plant fractionation Δ²⁹Si between roots and shoots amounts to −0.21 ± 0.08‰. Si-isotopic compositions of the various plant parts indicate that heavy isotopes discrimination occurs at three levels in the plant (at the root epidermis, for xylem loading and for xylem unloading). At each step, preferential crossing of light isotopes leaves a heavier solution, and produces a lighter solution. Si-isotopic fractionation processes are further discussed in relation with Si uptake and transport in plants. These findings have important implications on the study of continental Si cycle.     

Chromosome number variations in micropropagated true-to-type and off-type banana plants (Musa AAA Grande Naine cv.)

Summary The objective of the present study on banana plants (Musa AAA Grande Naine cv.), obtained byin vitro shoot tip culture, was to determine whether modifications in chromosome number could account for the appearance of the off-types with mosaiclike leaf defects or dwarf stature, the most frequent off-types observed after micropropagation. Chromosome counts were conducted on shoot tip samples treated with 8-hydroxyquinoline, digested in pectinase and stained with Schiff's reagent. On average, 160 counts were made for each treatment. Four types of plant material were studied: phenotypically true-to-type plants, dwarf off types, mosaiclike off-types obtained by micropropagation, as well as true-to-type plants obtained by standard propagation techniques of suckers with no micropropagation history. Some cells from all four types of plant material were found to have an abnormal chromosome number (i.e., 2n = 3x = 33), characteristic of triploid banarias. The percentages of aneuploid cells were 14%, 22%, 35%, and 5%, respectively. Descending aneuploidy was noted in micropropagated plants derived from true-to-type and dwarf off-type suckers. The statistical analysis revealed that the two latter types of plant material had the same percentage of aneuploid cells. Thus, the dwarfism could not be correlated with a change in the chromosome number. Conversely, ascending aneuploidy was observed in the mosaiclike material, with 34 or 35 chromosomes in almost 28% of the cells. This percentage was significantly higher than in true-to-type plants and highlight the genetic origin of the mosaiclike variation.     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Beauveria bassiana (Balsamo) Vuillemin as an endophyte in tissue culture banana (Musa spp.)

Beauveria bassiana is considered a virulent pathogen against the banana weevil Cosmopolites sordidus. However, current field application techniques for effective control against this pest remain a limitation and an alternative method for effective field application needs to be investigated. Three screenhouse experiments were conducted to determine the ability of B. bassiana to form an endophytic relationship with tissue culture banana (Musa spp.) plants and to evaluate the plants for possible harmful effects resulting from this relationship. Three Ugandan strains of B. bassiana (G41, S204 and WA) were applied by dipping the roots and rhizome in a conidial suspension, by injecting a conidial suspension into the plant rhizome and by growing the plants in sterile soil mixed with B. bassiana-colonized rice substrate. Four weeks after inoculation, plant growth parameters were determined and plant tissue colonization assessed through re-isolation of B. bassiana. All B. bassiana strains were able to colonize banana plant roots, rhizomes and pseudostem bases. Dipping plants in a conidial suspension achieved the highest colonization with no negative effect on plant growth or survival. Beauveria bassiana strain G41 was the best colonizer (up to 68%, 79% and 41% in roots, rhizome and pseudostem base, respectively) when plants were dipped. This study demonstrated that, depending on strain and inoculation method, B. bassiana can form an endophytic relationship with tissue culture banana plants, causing no harmful effects and might provide an alternative method for biological control of C. sordidus.     

Plant regeneration from cultured protoplasts of the cooking banana cv. Bluggoe (Musa spp., ABB group)

Summary Suspensions of embryogenic cells of a triploid banana (Musa spp., cv. Bluggoe) were initiated from the uppermost part of meristematic buds, and used as protoplast source. After 20 weeks in culture, the suspension contained a mixture of globular structures or globules and embryogenic cell clusters, as well as single cells. Two types of protoplasts were obtained from embryogenic suspension culture: small (20–30 m) and larger (30–50 m) protoplasts with a dense cytoplasm and large starch grains respectively. The small protoplasts probably originated from embryogenic cell clusters, and also from pseudocambial cells of globules, while larger protoplasts were probably released from oval starchy cells and those of the globule peripheral area. In co-culture with a suitable feeder, consisting of suspensions of diploid banana cells, the protoplasts of triploid banana reformed the cell wall within 24 h and underwent sustained divisions leading to the formation of small clusters of 2–3 cells within 7 days. The latter developed directly into embryos without passing through an apparent callus phase. 10% of such embryos gave rise to plantlets when subcultured in 2.2 M 6-benzylaminopurine and 2 M 4 amino-3,5,6-trichloropicolinic acid for 1 week, before transfer to MS medium containing 10 M 6-benzylaminopurine. The rest of the embryos underwent intensive direct secondary embryogenesis which could lead to the formation of plantlets with a frequency of up to 50% upon further transfer to hormone-free medium.Abbreviations BAP 6-benzylaminopurine - MS Murashige and Skoog (1962) medium - 2,4-D dichlorophenoxyacetic acid - UV ultraviolet light - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - Picloram 4 amino-3,5,6-trichloropicolinic acid     

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, 25mol 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).Abbreviations BA 6-benzyladenine - CIRAD Centre de Coopération Internationale en Recherche Agronomique pour le Développement - IAA indole-3-acetic acid - IBPGR International Board for Plant Genetic Resources - INIBAP International Network for the Improvement of Banana and Plantain - PPF photosynthetic photon flux - QDPI Queensland Department of Primary Industries     

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.     

Relative mycorrhizal dependency and mycorrhiza-nematode interaction in banana cultivars (Musa spp.) differing in nematode susceptibility

Four Musa cultivars, differing in nematode susceptibility, were selected to study their relative mycorrhizal dependency and to study the interaction between the arbuscular mycorrhizal fungus (AMF), Glomus mosseae, and two migratory endoparasitic nematodes, Radopholus similis and Pratylenchus coffeae. Mycorrhization with G. mosseae resulted in significantly better plant growth, even in the presence of R. similis and P. coffeae. No differences in relative mycorrhizal dependency (RMD) were observed among the four cultivars. G. mosseae suppressed nematode population build-up in Grande Naine and Pisang Jari Buaya. Only in the case of R. similis (Indonesian population) in Pisang Jari Buaya, no significant suppression was observed. In the case of P. coffeae, the AMF reduced the damage in the roots, caused by the nematodes. For R. similis, no reduction of damage was observed. In all, except one experiment, the frequency of the mycorrhizal colonisation was negatively affected by the nematodes.     

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma₂ and PCM, significantly affected the number of microcalli produced, which was 90 × 10³ per Petri dish on Ma₂ with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 10³ per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma₂ and the frequency of embryogenic cell aggregates was higher among microcalli on Ma₂-medium. However, no cell division was observed in protoplasts cultured on Ma₂ in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.     

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.     

Evaluation of micropropagated plantain and banana (Musa spp.) for banana streak badnavirus incidence under field and screenhouse conditions in Nigeria

Between 1991 to 1996, more than 50 Musa hybrids and 10 landraces were evaluated under field and screenhouse conditions for virus symptoms resembling those caused by banana streak badnavirus (BSV). The symptoms included chlorotic streaks, leaf deformation, stunting, cigar leaf death, distortion of the peduncle, bunch or fruits, and internal pseudostem necrosis. Immunosorbent electron microscopy (ISEM) of randomly selected plants with one or more of these symptoms confirmed the presence of BSV particles in 15 tropical Musa plantain hybrids (TMPx) and five Musa landraces. Under both field and screenhouse conditions, the incidence of symptomatic plants in the hybrids was significantly higher than in the landraces. The hybrids also generally had a higher concentration of BSV antigens, as determined by enzyme-linked immunosorbent assay (ELISA). By contrast, most BSV-infected landraces were symptomless and had very low or undetectable amounts of BSV antigens. There was a significant variation in incidence of symptomatic plants between genotypes, experiments and year of observation. These results are discussed in relation to the higher natural BSV incidence observed on some Musa hybrids as compared with their parental genotypes.     

Influence of Liquid Pulse Treatment with Growth Regulators on in vitro Propagation of Banana (Musa spp. AAA)

The effect of liquid pulse treatment of growth regulators on in vitro propagation of banana (Musa spp. AAA) was studied. Optimal shoot proliferation rates were achieved due to the pulse treatment of 6-benzylaminopurine (BA) and kinetin combination (1:1) at the concentration of 50 mg l^–1 for 60 min. Similarly high frequency of root induction was obtained due to pulse treatment with a NAA and IBA combination (1:1) at the concentration of 100 mg l^–1 for 60 min.     

Diversity of Meloidogyne spp. on Musa in Martinique,Guadeloupe, and French Guiana

Ninety-six isolates of Meloidogyne species collected from banana fields from Martinique, Guadeloupe, and French Guiana, were examined using esterase (Est) and malate dehydrogenase (Mdh) phenotypes. Adult females identified as M. arenaria, M. incognita, M. javanica, M. cruciani, M. hispanica, and Meloidogyne sp. showed species-specific phenotypes only for the esterase enzymes. Intraspecific variability among isolates of M. arenaria, M. incognita, and M. javanica was detected using Est and Mdh. Perineal patterns were used as a complementary tool together with enzyme characterization and were essential for checking the morphological consistency of the identification. The major species of M. arenaria and M. incognita were detected at 61.9% and 34.3% of the total number of isolates, respectively, and the other minor species at 3.8%. The mixed Meloidogyne species were detected in 45.1% of the samples. Genetic analysis was conducted using RAPD markers, which alone or in combination provided reliable polymorphisms both between and within species. RAPD analysis of the data resulted in clustering of species and isolates congruent with esterase phenotype characterization. The intraspecific variability in M. incognita and in M. arenaria represented 14.9% and 61.6% of the amplified polymorphic fragments, respectively. This high level of variation in M. arenaria isolates may indicate multiple origins for populations classified as M. arenaria or more than one species inside the same group, but more detailed morphological and DNA studies will be necessary to test this hypothesis.     

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     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

Effects of carbon sources and auxins on in vitro propagation of banana

The effects of carbon sources (sucrose, glucose, fructose and mannitol) and auxins [indolebutyric acid (IBA) and α-naphthaleneacetic acid (NAA)] on in vitro propagation of banana (Musa spp. AAA) were studied. Over all carbon sources tested, sucrose induced highest frequency of shoot proliferation. Optimal shoot proliferation rates were achieved on the Murashige and Skoog (MS) medium supplemented with sucrose and glucose combination (1:1) at the concentration of 30 g dm⁻³. Similarly, higher frequency of root induction was obtained at IBA and NAA combination (1:1; concentration of 2 mg dm⁻³) than at other concentrations of IBA or NAA alone or their combinations.     

Water relations of crok-oak (Quercus suber L.) under natural conditions

Daily and annual courses of leaf transpiration, stomatal conductance and shoot water potential of four Quercus suber individuals were compared in a semi-natural stand in southwest Portugal, from spring 1989 to early summer 1990.The trees investigated showed annual patterns typical of evergreen sclerophyllous species but varied in their range of stomatal operation. This appeared to be related to differences in hydraulic conductivity in the root-to-leaf pathway.Maximum stomatal conductance and transpiration rates occurred from March to June.Water stress was found to be moderate and winter cold stress due to low air and soil temperatures appeared to have an influence on plant water balance through their effects on flow resistances.Abbreviations g_sw stomatal conductance - g_max maximum stomatal conductance - PAR photosynthetically active radiation - RH relative humidity of the air - T leaf transpiration - Ta air temperature - TL leaf temperature - T_max maximum leaf transpiration - W air-to-leaf vapor pressure difference - shoot water potential - PD predawn shoot water potential - MIN minimum shoot water potential     

Restriction fragment length polymorphism (RFLP)-based phylogenetic analysis of Musa

Summary Random genomic probes were used to detect RFLPs in 19 Musa species and subspecies. A total of 89 phylogenetically informative alleles were scored and analyzed cladistically and phenetically. Results were in general agreement with morphology-based phylogenetic analyses, with the following exceptions: our data unambiguously places M. boman in section Australimusa, and indicates M. beccarii is very closely related to M. acuminata. Additionally, no support was found for the separation of section Rhodochlamys from section Musa. A comparison of morphology-based and RFLP-based phylogenetic analyses is presented.