Genomic changes associated with somaclonal variation in banana (Musa spp.)

The molecular basis of somaclonal variation is not precisely known, but both genetic and epigenetic mechanisms have been proposed. The available evidence points toward the existence of labile portions of the genome that can be modulated when the cells undergo the stress of tissue culture. Therefore, the hypothesis that there are identifiable and predictable DNA markers for the early diagnosis of somaclonal variation has been tested. Representational difference analysis was used to isolate unique fragments of DNA (difference products) between visible culture-induced off-type and normal banana plants. Markers generated from six difference products differentiated between some of the off-type and normal pairs. The genomic region around one of these difference products has been extensively characterized and has a high degree of polymorphism, with variation in up to 10% of the nucleotides sequenced in the region. This same region has been shown to vary in other pairs of off-type and normal banana plants derived from tissue culture as well as in plants propagated commercially in vitro. The data are consistent with the hypothesis that there is at least one particularly labile portion of the genome that is especially susceptible to the stress imposed during tissue culture and that is associated with higher rearrangement and mutation rates than other portions of the genome. Consequently, the regions that are reported here have the potential to be used as early detection tools for identifying somaclonal variants.     

Production of selectable marker gene-free Cavendish banana (Musa spp.) using a steroid-inducible recombinase platform

Transgenic Research - Genetic improvement of commercially accepted banana cultivars is strongly reliant on the ability to introduce genes that encode important agro-traits such as disease...     

Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Musa spp.)

The utilisation of cryopreservation for the eradication of cucumber mosaic virus (CMV) or banana streak virus (BSV) from Musa spp. was investigated. Banana plants, cv. Williams (AAA, Cavendish subgroup), were mechanically infected with CMV or naturally infected with BSV, and proliferating meristems were produced from the infected plants. Excised meristematic clumps were cryopreserved through vitrification using PVS-2 solution. The health status of regenerated in vitro plants was first checked by means of ELISA. The putative virus-free material was subsequently tested a second time following greenhouse acclimatisation. The frequency of virus eradication for CMV and BSV was 30% and 90%, respectively, following cryopreservation. In comparison, the frequency of virus-free plants regenerated directly from highly proliferating meristems, corresponding to a spontaneous eradication rate, reached 0% and 52% for CMV and BSV, respectively. The conventional meristem culture resulted in 0% CMV-free plants and 76% BSV-free plants, while the cryoprotective treatment resulted in 2% CMV-free plants and 87% BSV-free plants. To understand the mode of action of cryopreservation for the eradication of viral particles, we examined the structure of the meristem tips by light microscopy. The cryopreservation method used only allowed survival of small areas of cells located in the meristematic dome and at the base of the primordia.     

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

Summary We 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.     

Iron and zinc supplies mitigate cadmium toxicity in micropropagated banana (Musa spp.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Cadmium (Cd) is a toxic heavy metal that negatively affects plant growth and physiology. Plants display multiple changes in physiological and...     

A statistical approach to study the dynamics of micropropagation rates, using banana (Musa spp.) as an example

The use of micropropagation to obtain large numbers of high-quality planting material has increased in recent years. Behavior in culture, mainly in terms of multiplication rate, varies among genotypes, directly affecting plant production planning. To study multiplication rates over time, suckers of banana, Musa spp., cv. Maçã, were collected in the field and the shoot apex introduced in vitro for micropropagation. The number of new shoots produced in each of the six multiplication cycles was recorded and the data analyzed statistically. Variability in total shoot production and differences in multiplication rates was considerable among families, which consisted of the initial explant and its progeny. Moreover, the adjusted Poisson regression models for the number of shoots showed that the multiplication rate in this cultivar tends to decrease with time: after the seventh subculture, new shoots may form at a very low rate. Interpretation of the first and second derivatives of the regression model allowed determination of the maximum speed of multiplication and the time at which the multiplication rate begins to decline.     

Intron-mediated enhancement of the banana bunchy top virus DNA-6 promoter in banana (Musa spp.) embryogenic cells and plants

 Intron-containing fragments derived from the 5′ untranslated regions of the maize ubi1, maize adh1, rice act1 and sugarcane rbcS genes were tested for their enhancing effects on the banana bunchy top virus DNA-6 promoter (BT6.1) in banana (Musa spp. cv. Bluggoe) embryogenic cells. The rice act1 and maize ubi1 introns provided the highest levels of intron-mediated enhancement of GUS expression, increasing native BT6.1 promoter activity by about 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about tenfold, whereas the adh1 intron had no significant effect. In regenerated transgenic banana plants, the ubi1 intron significantly enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35 S promoter and did not appear to affect the tissue specificity of the promoter. Received: 28 July 2000 / Revision received: 21 August 2000 / Accepted: 4 October 2000     

Effects, distribution and uptake of silicon in banana (Musa spp.) under controlled conditions

Three contrasted genotypes of Musa spp. (M. acuminata cv Grande Naine, M. acuminata spp. Banksii and M. balbisiana spp. Tani) were grown for 6 weeks under optimal conditions in hydroponics and were submitted to a wide range of Si supply (0–1.66 mM Si) to quantify the Si uptake and distribution in banana, as well as the effect of Si on banana growth. The level of Si supply did not affect plant growth, nor the rate of water and nutrient uptake. The rate of Si uptake and the Si concentration in plant tissues increased markedly with the Si supply. At the highest Si concentrations (1.66 mM), silicon absorption was essentially driven by mass flow of water (passive transport). However, at lower Si concentrations (0.02–0.83 mM), it was higher than its uptake by mass flow and caused the depletion of silicon in the nutrient solution, suggesting the existence of active processes in silicon transport. The distribution of silicon among shoot organs (pseudostem < petiole and midrib < young lamina < old leaf) confirmed the major role of transpiration in silicon accumulation and was not dependent on silicon supply. However, other mechanisms of transport might be operating in the roots and in the petiole and midrib of young leaves, whose silicon concentration was unexpectedly high at low Si supply (0.02 mM) compared to higher levels of Si. The three genotypes did not exhibit consistent differences in their responses to silicon supply.     

Amplified somatic embryogenesis from male flowers of triploid banana and plantain cultivars (Musa spp.)

Summary Somatic embryogenesis and plant regeneration of banana and plantain cultivars (Musa spp.) were obtained by culturing young male flowers. Multiplication and maintenance of embryogenic cultures were achieved by culturing somatic embryos in a temporary immersion system (SIT). A multiplication rate of 40 allowed us to obtain more than 6000 somatic embryos after 6 mo. of subculture. Plant recovery frequencies were 60 to 70%. This method was expanded to different banana and plantain genomic groups.     

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.     

Generation of transgenic plantain (Musa spp.) with resistance to plant pathogenic nematodes

Plant parasitic nematodes impose a severe constraint on plantain and banana productivity; however, the sterile nature of many cultivars precludes conventional breeding for resistance. Transgenic plantain cv. Gonja manjaya (Musa AAB) plants, expressing a maize cystatin that inhibits nematode digestive cysteine proteinases and a synthetic peptide that disrupts nematode chemoreception, were assessed for their ability to resist nematode infection. Lines were generated that expressed each gene singly or both together in a stacked defence. Nematode challenge with a single species or a mixed population identified 10 lines with significant resistance. The best level of resistance achieved against the major pest species Radopholus similis was 84% ± 8% for the cystatin, 66% ± 14% for the peptide and 70% ± 6% for the dual defence. In the mixed population, trial resistance was also demonstrated to Helicotylenchus multicinctus. A fluorescently labelled form of the chemodisruptive peptide underwent retrograde transport along certain sensory dendrites of R. similis as required to disrupt chemoreception. The peptide was degraded after 30 min in simulated intestinal fluid or boiling water and after 1 h in nonsterile soil. In silico sequence analysis suggests that the peptide is not a mammalian antigen. This work establishes the mode of action of a novel nematode defence, develops the evidence for its safe and effective deployment against multiple nematode species and identifies transgenic plantain lines with a high level of resistance for a proposed field trial.     

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.     

Banana (Musa spp.) as a model to study the meristem proteome: acclimation to osmotic stress

Banana (Musa spp.) multiple shoot meristems are an excellent model to study the meristem proteome. Using a 2-DE protocol developed for small amounts of tissue and MS-based cross species polypeptide identification, we have revealed the meristem proteome and investigated the influence of sucrose-mediated osmotic stress in a dehydration-tolerant variety. Proteins that were significantly up- or down-regulated due to the high-sucrose treatment were classified using non-parametric univariate statistics. Our results suggest that the maintenance of an osmoprotective intracellular sucrose concentration, the enhanced expression of particular genes of the energy-conserving glycolysis and the conservation of the cell wall integrity are essential to maintain homeostasis, to acclimate and to survive dehydration. By comparing the dehydration-tolerant variety with a dehydration-sensitive variety, we were able to distinguish several genotype-specific proteins (isoforms), and could associate the dehydration-tolerant variety with proteins involved in energy metabolism (e.g., phosphoglycerate kinase, phosphoglucomutase, UDP-glucose pyrophosphorylase) and proteins that are associated with stress adaptation (e.g., OSR40-like protein, abscisic stress ripening protein-like protein). This work shows that proteome analysis can be used successfully to perform quantitative difference analysis and to characterize genetic variations in a recalcitrant crop.     

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.     

Leaf silicon content in banana (Musa spp.) reveals the weathering stage of volcanic ash soils in Guadeloupe

Several plant species accumulate silicon, which is taken up by roots in soil solution. The Si concentration in soil solution can be governed by silicate dissolution and formation, and thus soil constitution. Here, we study the Si leaf content of mature banana plants (Musa acuminata cv Grande Naine) cropped on soils derived from andesitic ash in Guadeloupe through standard foliar analysis. The soils strongly differ in weathering stage and total Si content. The most desilicated soils (Andosol–Nitisol–Ferralsol) occur in the wettest areas, on the Eastern slopes (Es) of the volcano exposed to rain bearing winds. Least weathered soils (Andosol–Cambisol) occur on Western slopes (Ws). The average leaf Si concentration ranges from 2.7 to 3.9 g kg^?1 for bananas cropped in Es soils, and from 7.7 to 9.6 g kg^?1 in Ws soils. The leaf Si concentrations are lowest for the Es gibbsite-rich Andosols and Ferralsols. The leaf Si concentration is positively correlated with soil CaCl₂-extractable Si content, soil Si content and total reserve in weatherable minerals. The silicon content of banana leaves thus reveals the weathering stage of volcanic ash soils in Guadeloupe.     

16S ribosomal DNA characterization of nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril)

Nitrogen-fixing bacteria isolated from banana (Musa spp.) and pineapple (Ananas comosus (L.) Merril) were characterized by amplified 16S ribosomal DNA restriction analysis and 16S rRNA sequence analysis. Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans, Burkholderia brasilensis, and Burkholderia tropicalis were identified. Eight other types were placed in close proximity to these genera and other alpha and beta Proteobacteria.     

Potential implications of biopriming in banana (Musa spp) plantlets against banana bunchy top virus (BBTV)

Abstract

Transplant media as a means for the introduction of biological agents is currently being investigated in a variety of crops. This study aimed to investigate the impact of microbial inoculation in micropropagated banana plantlets to enhance their resistance against Banana bunchy top virus (BBTV). Virus indexed micropropagated plantlets of banana were subjected to root colonization followed by foliar spraying with bacterial strains Pseudomonas fluorescens Pf1, CHA0 and Bacillus subtilis EPB22 during primary and secondary hardening stage in the nursery, at the time of repotting and 3 months after planting in the pot. Microbe inoculated plantlets showed enhanced PR proteins and defense enzymes besides reducing banana bunchy top disease incidence under glasshouse condition. The results indicated the effective use of beneficial microbes in reducing the disease incidence of BBTV in tissue culture banana plantlets. In addition, the molecular characterization of endophytes isolated from banana plantlets, using SDS-PAGE and RAPD-PCR revealed that endophytes were categorized into two distinct groups. These results emphasize the significance of microorganisms in protection of young plantlets from transplanting stresses in field. Further, the use of beneficial microorganisms instead of chemicals sustains an ecological niche in the agricultural ecosystem.     

Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.