Cavendish Banana Cultivars Research Articles

Early detection of bacterial wilt in bananas caused by Ralstonia solanacearum using reflectance spectroscopy

Bacterial wilt is one of the most important vascular diseases that generate high worldwide losses. The main strategy to control it involves early detection of infected plants that mitigate its spread in the field. This research characterized the spectral response of healthy plants, infected with Ralstonia solanacearum E.F. Smith race 2 and subjected to water stress, in two banana cultivars by spectroscopy reflectance. The spectral data were used to detect vascular wilt during the incubation period of the disease in Gros Michel and Cavendish banana cultivars. The reflectance data were collected from the leaves using a portable spectrometer. Both varieties of healthy plants showed a typical low reflectance in the visible range, with a peak of green of around 12% in Williams and 5% in Gros Michel. In the measured infrared range, the two varieties presented values between 60 and 70% in the latest days post-infection measurements, decreasing to ~ 50% after 12 dpi. The results obtained indicate that plants infected with R. solanacearum have no initial increase in reflectance in the visible (Vis) range, whereas decrease rapidly after 6 dpi in the 700–1000 nm range. This methodology identifies three wavelengths (710, 770, and 965 nm) that allow differentiation between ill and healthy plants after 6 days post-inoculation, with a percentage of correct classification that ranges from 96 to 100%. The spectral response characterization in healthy plants and those subjected to various types of stress is a fundamental input for the development of early disease detection systems based on spectroscopy techniques.

Bunch Weight Determination in Relation to the Source-Sink Balance in 12 Cavendish Banana Cultivars

This study was carried out to compare bunch weight determination in relation to the source-sink balance in a selection of 12 Cavendish banana cultivars. Fruit number and individual fruit weight are the main bunch weight components. The fruit number depended on the growth potential during floral induction, which we estimated according to the active leaf area at floral induction (ALAfi). We studied fruit weight determination by measuring the source-sink balance during bunch growth, carbohydrate reserves and bunch-sucker biomass partitioning. Throughout the experiment, we monitored the global radiation (Rg), which exhibited seasonal variations. We noted a source variation from the first to the second cycle during bunch growth because of a decrease in the Rg and active leaf area. Our results showed significant differences between Cavendish cultivars regarding bunch weight, fruit number and susceptibility to source variations. There was an overall positive linear relation between ALAfi and fruit number, but the ALAfi/fruit number ratio varied between cultivars. We also found that the bunch weight was mainly determined by the fruit number rather than the fruit weight when the source was not limited. Finally, we assessed the cultivar susceptibility to source decreases, thereby revealing some cultivars of interest for agroecological cropping systems.

Effective biocontrol of banana fusarium wilt tropical race 4 by a bacillus rhizobacteria strain with antagonistic secondary metabolites

Fusarium oxysporum f.sp. cubense Tropical Race 4 (FOC TR4), is one of the newly emerged dreaded pathogens of banana with an established global presence. Fungicides have not been effective in controlling the disease. There are very few effective antagonistic microbes reported for the biological management of this pathogen.In the present study, suppressive soil was selected for isolation of potential biocontrol plant growth-promoting bacteria. The bacterial strain CSR-D4 Bacillus licheniformis was identified with significant antagonistic attributes and plant growth-promoting properties. This strain exhibited a high inhibition percentage (77.59%) against the FOC TR4 pathogen in the dual plate assay. Further, the bioactive metabolite of the strain conferring biocontrol of FOC-TR4 was also identified through the Liquid Chromatography-Mass Spectrophotometry technique. Our analysis revealed the presence of many antifungal metabolites in the isolate CSR-D4, the majority of which were identified as iturin, fengycin, surfactin, and bacillomycin. These results indicated the involvement of metabolites in the antifungal activity of the strain CSR-D4 against FOC TR4. During sick-pots studies taken with susceptible Cavendish banana cultivar Grand Naine, application of B. licheniformis CSR-D4 showed a significant reduction in the disease incidence percentage (10%). B. licheniformis CSR-D4 treated plants also showed enhanced activity of defense-related enzymes such as β-1,3 glucanase, peroxidase, chitinase, polyphenol oxidase, and phenyl-alanine ammonium lyase compared to FOC TR4 alone treated plants that suggest the occurrence of induced host tolerance in Bacillus licheniformis CSR-D4 treated plants. Our results suggest the antagonistic potential of rhizobacterium Bacillus licheniformis CSR-D4 strain through secreted metabolites mediated the effective biocontrol of Fusarium oxysporum f.sp. cubense Tropical Race 4.

Canopy cover evolution, diurnal patterns and leaf area index relationships in a Mchare and Cavendish banana cultivar under different soil moisture regimes

The biggest abiotic threat to banana (Musa spp.) production is water deficit, but physiological indicators in plantations are lacking. Canopy Cover (CC) seems to be a relevant parameter, but so far not used in banana fields. Field experiments were conducted to determine the effect of optimal irrigation (FI) versus rainfed (RF) on CC and Leaf Area Indices (LAI) in two experiments with different cultivars (Mchare ‘Huti Green’ [HG, AA] and Cavendish ‘Grand Naine’[GN, AAA]) (n = 3 for HG, n = 4 for GN) until harvest of cycle 1 (C1), studying C1 and C2 plants. Soil moisture was followed using Time Domain reflectometry. CC and LAI were reduced 8–9 weeks after the start of soil moisture divergence between RF and FI treatments in both experiments (p < 0.05), leading to a reduction in CC growth rate (r) and maximum CC (CCmax) in RF plots (p < 0.05). On a daily timescale, CC varied diurnally (i.e. was reduced) under high evaporative demands, whereby soil moisture depletion increased the CC reduction. Cultivar specific CC-LAI curves were created following the Lambert-Beer equation, whereby HG had a lower extinction coefficient than GN (0.52 vs. 0.67). CC growth over time seems a promising indicator for water deficit in the field. Diurnally, CC is more affected by evaporative demand than soil moisture depletion, although soil moisture depletion increases CC diurnal drops under high ET0.

Agronomic evaluation of new Cavendish banana cultivars for growing in different protected cultivation areas in Turkey

Protected cultivation (in greenhouses) is an effective cropping system to produce bananas in Turkey and much more profitable than open-field conditions. 'Grand Nain' is the most popular cultivar grown in greenhouses in Turkey, but it may be possible to increase yield and fruit quality, and subsequently grower incomes, by testing new cultivars. The objective of this study was to evaluate in subtropical conditions the performance of five new Cavendish cultivars ('CV 902', 'Jobo, MA13', 'Williams' and 'MA13') previously selected in open-field tropical conditions, compared to ‘Grand Nain’ (control). All new Cavendish cultivars were introduced in Turkey from VITROPIC SA, France. All cultivars were produced by in vitro micropropagation of meristems, and then deflasked and grown in nurseries to 25 cm for planting. The trials were undertaken in three different locations (Alanya, Anamur and Bozyazi, Turkey) in greenhouses. Various parameters were recorded over two crop cycles including: stem circumference, stem height, total leaf number, bunch stem circumference, number of hands and finger per bunch, finger length and circumference at harvest and bunch weight. The Bozyazi location showed better performance in many aspects of plant growth compared to the other two sites. 'Williams' had on average the highest pseudostem circumference and height but also the highest bunch weight (50.27 kg). The bunch weight of 'MA13' and ‘CV 902’ was 47.65 and 42.66 kg, respectively, whereas the control (‘Grand Nain’) and 'Jobo' had a lower bunch weight. 'Williams', 'MA13' and 'CV 902' were more productive than 'Grand Nain' in greenhouse conditions. In addition, no somaclonal variations were observed in any of the three experimental locations. These results suggest that the new selections have potential to provide higher yields in Turkey in protected cultivation, providing higher returns to growers, compared to local selections.

Genome ancestry mosaics reveal multiple and cryptic contributors to cultivated banana.

SummaryHybridizations between closely related species commonly occur in the domestication process of many crops. Banana cultivars are derived from such hybridizations between species and subspecies of the Musa genus that have diverged in various tropical Southeast Asian regions and archipelagos. Among the diploid and triploid hybrids generated, those with seedless parthenocarpic fruits were selected by humans and thereafter dispersed through vegetative propagation. Musa acuminata subspecies contribute to most of these cultivars. We analyzed sequence data from 14 M. acuminata wild accessions and 10 M. acuminata‐based cultivars, including diploids and one triploid, to characterize the ancestral origins along their chromosomes. We used multivariate analysis and single nucleotide polymorphism clustering and identified five ancestral groups as contributors to these cultivars. Four of these corresponded to known M. acuminata subspecies. A fifth group, found only in cultivars, was defined based on the ‘Pisang Madu’ cultivar and represented two uncharacterized genetic pools. Diverse ancestral contributions along cultivar chromosomes were found, resulting in mosaics with at least three and up to five ancestries. The commercially important triploid Cavendish banana cultivar had contributions from at least one of the uncharacterized genetic pools and three known M. acuminata subspecies. Our results highlighted that cultivated banana origins are more complex than expected – involving multiple hybridization steps – and also that major wild banana ancestors have yet to be identified. This study revealed the extent to which admixture has framed the evolution and domestication of a crop plant.

CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit

Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.

Assessing Variations in Host Resistance to Fusarium oxysporum f sp. cubense Race 4 in Musa Species, With a Focus on the Subtropical Race 4.

Fusarium oxysporum f. sp. cubense (Foc) has severely curtailed banana production in the tropical regions of the world. The tropical race 4 (TR4) of Foc was detected in Australia in the 1990s and it is virulent to all Cavendish type banana cultivars, which represents the majority of banana production in Australia. Genetic resistance to Foc race 4 is urgently needed. To characterize sources of resistance, we have assessed the Foc resistance response of 34 Musa cultivars with plants grown under controlled settings. Amongst diploid banana cultivars carrying the AA genome, resistance is found in Musa acuminata sub-species including malaccensis ‘Pahang’ and burmannica ‘Calcutta4.’ In the polyploid group, the hybrids such as ‘FHIA-18’ and ‘FHIA-25’ are highly resistant against both Foc-TR4 and subtropical race 4 (Foc-STR4). Interestingly, ‘FHIA-2’ and ‘CAM020’ appear to be resistant to Foc-TR4 but susceptible to Foc-STR4, suggesting potential differences in the resistance mechanisms against the different race 4 strains. Using a GFP tagged Foc-STR4 strain challenged onto both resistant and susceptible M. a. malaccensis lines, a high inoculum dosage rapidly induced vascular wilt in the susceptible M. a. malaccensis lines at 2.5 weeks. This was associated with an accumulation of micro-conidia in the rhizome and the movement of the fungus through the xylem vessels. In contrast, the fungal movement was restrained in the rhizome of the resistant M. a. malaccensis lines and no sporulation was observed. Overall, this research suggests that the resistance response is dependent to an extent on inoculum dosage and that the plant host’s response, in the rhizome, plays an important role in inhibiting the fungus from spreading to the rest of the plant. Identifying race 4 resistant accessions can help to understand mechanisms of resistance and provide banana breeders with the genetic resources to integrate resistance genes into commercial varieties.

Quantitative proteomics analysis reveals resistance differences of banana cultivar ‘Brazilian’ to Fusarium oxysporum f. sp. cubense races 1 and 4

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases in banana production. Foc is classified into three physiological races. However, the resistance mechanisms of banana against different Foc races are poorly understood. In this study, we performed a comparative proteomics analysis to investigate the resistance mechanisms of ‘Brazilian’ against Foc1 and Foc4. The proteomes of ‘Brazilian’ roots inoculated with Foc1 and Foc4 and mock inoculated control at 48 h were analyzed using TMT based quantitative analysis technique. A total of 7325 unique protein species were identified, of which 689, 744, and 1222 protein species were differentially accumulated in Foc1 vs. CK, Foc4 vs. CK, and Foc1 vs. Foc4, respectively. The differential accumulations of candidate protein species were further confirmed by RT-qPCR, PRM, and physiological and biochemical assays. Bioinformatics analysis revealed that the differentially abundance protein species (DAPS) related to pattern recognition receptors, plant cell wall modification, redox homeostasis, and defense responses were differentially accumulated after Foc1 and Foc4 infection, suggesting that ‘Brazilian’ differed in resistance to the two Foc races. Our study lay the foundation for an in-depth understanding of the interaction between bananas and Foc at the proteome level. SignificanceThe banana fusarium wilt disease is one of the most destructive disease of banana and is caused by Fusarium oxysporum f. sp. cubense (Foc). Foc is classified into three physiological races, namely, Foc1, Foc2, and Foc4. Among these races, Foc1 and Foc4 are widely distributed in south China and significantly lose yield. Although both physiological races (Foc1 and Foc4) can invade the Cavendish banana cultivar ‘Brazilian’, they have significant pathogenicity differences. Unfortunately, how the resistance differences are produced between two races is still largely unclear to date. In this study, we addressed this issue by performing TMT-based comparative quantitative proteomics analysis of ‘Brazilian’ roots after inoculation with Foc1 and Foc4 as well as sterile water as the control. We revealed that the series of protein species associated with pattern recognition receptors, plant cell wall modification, redox homeostasis, pathogenesis, phytohormones and signal transduction, plant secondary metabolites and programmed cell death etc. were involved in the response to Foc infection. Notably, the potential role of lipid signaling in banana defense against Foc are not reported previously but rather unveiled for the first time in this study. The current study represents the most extensive analysis of the protein profile of ‘Brazilian’ in response to Foc inoculation and includes for the first time the results from comparison quantitative proteomics analysis between plants inoculated with a pathogenic strain Foc4 and a nonpathogenic strain Foc1 of ‘Brazilian’, which will lay the foundation for an in-depth understanding of the interaction between bananas and Foc at the proteome level.

Histological and Gene Expression Analyses in Banana Reveals the Pathogenic Differences between Races 1 and 4 of Banana Fusarium Wilt Pathogen.

Fusarium oxysporum f. sp. cubense, the causative agent of Panama disease, is classified into three races: Foc1, Foc2, and Foc4. However, the histological characteristics, the accumulation of fusaric acid (FA), and resistant gene expression in banana infected with different races remain unclear. In this study, we compared the infection processes, FA contents, and gene expression levels in a Cavendish banana cultivar (Musa AAA Brazilian) inoculated with Foc1 and Foc4. Results showed that Foc4 can rapidly extend from the roots to the leaves, whereas Foc1 expands slowly from the roots to the rhizomes but cannot expand further upward. In addition, the colonization of plants by Foc4 was significantly higher compared with Foc1, as was the content of FA in those infected plant tissues. We observed that a large amount of starch granules was produced in the rhizomes and the number of starch granules was significantly higher after infection with Foc1 than after infection with Foc4. We further found that starch has an important inhibitory effect on the phytotoxicity induced by FA, thus leading to more resistance to the pathogens in the plants with high amounts of starch accumulation than in those with a low amount of starch accumulation. Moreover, the expression levels of 10 defense-related genes were analyzed and the results showed that the induction levels of those genes were higher after infection with Foc1 than after infection with Foc4. These results suggest that the observed differences in the invasion of host tissues and FA accumulation, and the number of starch granules and expression of defense-related genes, may contribute to a difference in virulence between the two races and the resulting difference in host resistance response, respectively.

Prediction of Flower Emergence and Evaluation of Cropping Potential in Selected Banana Cultivars (Musa sp.) Cultivated in Subtropical Conditions of Coastal Alabama

The development of more cold-tolerant short-cycle banana cultivars has made subtropical production possible, but fruiting may be unreliable in colder margins, such as the coastal region of Alabama, as a result of cold winter temperatures and other suboptimal growing conditions. Thus, the objectives of this study were to determine plant growth parameters that predict flowering, and to evaluate vegetative and reproductive growth of Cavendish and non-Cavendish banana cultivars. Pseudostem circumference and the height-to-circumference ratio (HCR) for tall cultivars and HCR for medium cultivars exhibited linear or quadratic relationships when regressed to the number of days from planting to inflorescence emergence (DPE), and hence were the best predictors of inflorescence emergence. The banana cultivars Double, Grand Nain, Cardaba, Ice Cream, and Goldfinger demonstrated cropping potential by producing mature bunches in the cooler environment of the subtropics and currently offer the best possibilities for banana production in Alabama.

Differential gene expression in banana roots in response to Fusarium wilt

Banana wilt disease, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a devastating disease in the banana industry. The molecular mechanism underlying the wilt resistance of AAA-type banana cultivars has not been fully characterized. Our objective was to analyse the genes differentially expressed in roots during the time of flower bud differentiation in plants grown in fields infested with Foc TR4. We compared the Foc TR4-tolerant ‘Formosana’ banana and the susceptible ‘Brazil’ banana. cDNA libraries were constructed from RNA isolated from the roots of both cultivars, and generated approximately 40 million and 35 million high-quality reads, respectively. We mapped 28 353 810 ‘Formosana’ reads and 26 917 421 ‘Brazil’ reads to the banana genome. A search of the NR (non-redundant) database resulted in the annotation of 34 408 genes. Additionally, 107 genes were differentially expressed between the two cultivars. Among the 48 differentially expressed genes with known functions, 41 were expressed more highly in ‘Formosana’ than in ‘Brazil’. These genes were divided into several categories, including specific resistance mechanism-related enzyme genes and phytoalexin synthesis-related genes. The expression levels of eight genes were validated by a quantitative real-time polymerase chain reaction. Our results indicate that ‘Formosana’ may enhance its tolerance to Foc TR4 by increasing the expression of defence-related genes (possibly constitutively). This cultivar also exhibits up-regulated expression of non-specific stress-related genes, which may enhance its overall disease tolerance. This study provides important transcript-level details potentially useful for clarifying the molecular mechanism underlying the disease resistance of Cavendish banana cultivars.

English

A study was undertaken to determine the thermo-tolerance of 4 month old dwarf Cavendish banana plants of cultivar &quot;Malindi&quot; planted using suckers. The electrolyte leakage procedure was used to generate sigmoidal curves from which the thermo-tolerance was determined at 50% electrolyte leakage. A completely randomized design (CRD) with three replicates was used. Leaflets (1.2 cm width &times; 5 cm length) from third fully opened leaves of plants fertilized by composted dairy cow manure and mineral fertilizers were exposed to heat regimes ranging from 30 to 80&deg;C (at 5&deg;C increments) for 30 min and 23&deg;C (room temperature) as control. The results revealed that there was a positive strong correlation between heat regimes and electrolyte leakage percentage (r2 = 0.73, p &le; 0.01). Lethal temperature for Malindi banana plants was 58&deg;C. The present investigation provided an accurate determination for heat tolerance of dwarf Cavendish banana cultivar &quot;Malindi&quot; and thus helps banana farmers to well manage their plants during such harsh weather conditions. &nbsp; Key words: Dwarf Cavendish banana, electrolyte leakage, heat stress, thermo-tolerance.

Comparison of volatile aroma compounds in Dwarf Cavendish banana (Musa spp. AAA) grown under organic or traditional cultivation

SummaryThe aroma profiles of ripened fruit of a Dwarf Cavendish banana cultivar grown under traditional (Tr) or organic (Or) cultivation were studied to determine differences in their volatile organic compounds (VOCs). VOCs were extracted using 100% (v/v) dichloromethane, and analysed using gas chromatography (GC) with flame ionisation detection, or by GC–mass spectrometry. According to a sensory panel analysis, extracts of both types of banana were representative of the typical banana odour. A total of 66 VOCs were identified and quantified in Tr bananas, and 76 in Or bananas. Organic bananas contained qualitatively and quantitatively more VOCs. Of all the aroma compounds detected and measured, esters were present in the highest concentrations, followed by aldehydes. The main esters were: isoamyl acetate, 2-pentanol acetate, isoamyl butanoate, 3-methylbutyl-3-methylbutanoate, and n-butyl acetate. The total concentration of esters in Tr bananas was 28.27 mg kg–1, and in Or bananas was 36.34 mg kg–1. Based on the sensory panel analysis, Or bananas were preferred to Tr bananas produced by traditional cultivation because of their improved aroma, texture, and sweetness. A similar conclusion was obtained by chemical and biochemical analysis. Organic cultivation increased soluble solid contents (SSC), fruit firmness, total sugar (Ts), D-fructose, and sucrose concentrations, showing statistically significant differences (P ≤ 0.05).

Comparison of aroma compounds in Dwarf Cavendish banana (Musa spp. AAA) grown from open-field and protected cultivation area

Aroma profile from ripened fruits of Dwarf Cavendish banana cultivars grown under open-field (OF) and protected area (PA) cultivation was studied in order to determine aroma differences. Aroma compounds were extracted with dichloromethane, and analysed by gas chromatography (GC)-flame ionisation detection and GC–mass spectrometry. According to sensory analysis, the aromatic extract of banana was representative of banana odour. A total of 80 components were identified and quantified in OF-banana and 76 in PA-banana. Open-field banana contained more aroma compounds both qualitatively and quantitatively. Of all aroma compounds measured, esters were present in the highest amounts, followed by aldehydes. The main esters were isoamyl acetate, 2-pentanol acetate and isoamyl butanoate. The total amount of esters in OF-banana was 23.8mg/kg and in PA-banana 20.5mg/kg. Based on the sensory analysis, OF-banana was preferred over bananas produced in protected areas, because of their aroma quality.

Isolation and characterization of an exopolygalacturonase from Fusarium oxysporum f.sp. cubense race 1 and race 4.

BackgroundFusarium wilt is an economically devastating disease that affects banana production. Although Cavendish banana cultivars are resistant to Fusarium oxysporum f.sp. cubense race 1 (FOC1) and maitain banana production after Gros Michel was destructed by race 1, a new race race 4 (FOC4) was found to infect Cavendish.ResultsAn exopolygalacturonase (PGC2) was isolated and purified from the supernatant of the plant pathogen Fusarium oxysporum f.sp. cubense race 4 (FOC4). PGC2 had an apparent Mr of 63 kDa by SDS-PAGE and 51.7 kDa by mass spectrometry. The enzyme was N-glycosylated. PGC2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. To obtain adequate amounts of protein for functional studies between the PGC2 proteins of two races of the pathogen, pgc2 genes encoding PGC2 from race 4 (FOC4) and race 1 (FOC1), both 1395 bp in length and encoding 465 amino acids with a predicted amino-terminal signal sequence of 18 residues, were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC2 products, r-FOC1-PGC2 and r-FOC4-PGC2, were expressed and purified as active extracellular proteins. Optimal PGC2 activity was observed at 50°C and pH 5. The Km and Vmax values of purified r-FOC1-PGC2 were 0.43 mg.mL-1 and 94.34 units mg protein-1 min-1, respectively. The Km and Vmax values of purified r-FOC4-PGC2 were 0.48 mg.mL-1 and 95.24 units mg protein-1 min-1, respectively. Both recombinant PGC2 proteins could induce tissue maceration and necrosis in banana plants.ConclusionsCollectively, these results suggest that PGC2 is the first exoPG reported from the pathogen FOC, and we have shown that fully functional PGC2 can be produced in the P. pastoris expression system.

Influence of genotype and age of explant source on the capacity for somatic embryogenesis of two Cavendish banana cultivars (Musa acuminata Colla, AAA)

The embryogenic capacities of flower explants from one- and two-week-old male inflorescence buds from Musa acuminata Cavendish, AAA, genotypes ‘Williams’ and ‘Grand Naine’ were investigated. Explants of hands with immature flowers were excised and induced for embryogenesis. Highly significant differences were found in the embryogenic response, depending on the genotype and the developmental stage of the buds from which the explants were excised. After four months of induction, the total percentage of callus formed ranged from 97.81% in explants of two-week-old ‘Williams’ buds to 52.11% in explants of two-week-old ‘Grand Naine’ buds. Embryogenic callus formation was, on average, higher in the two-week-old ‘Williams’ explants (10.01%) than in the one-week-old explants (0.78%). The opposite was true for ‘Grand Naine’, with 7.51% embryogenic callus produced in explants of one-week-old buds compared to 2.49% in two-week-old buds. Selected embryogenic calluses that were successfully established on proliferation medium led to embryogenic cell suspensions with good regeneration capacities. Fifteen to thirty-five percent of the embryos germinated, demonstrated high plant-conversion capacity (99%). The effect of the interaction between the developmental age of explant and the genotype on the embryogenesis response is discussed.

A molecular diagnostic for tropical race 4 of the banana fusarium wilt pathogen

This study analysed genomic variation of the translation elongation factor 1α (TEF‐1α) and the intergenic spacer region (IGS) of the nuclear ribosomal operon of Fusarium oxysporum f. sp. cubense (Foc) isolates, from different banana production areas, representing strains within the known races, comprising 20 vegetative compatibility groups (VCG). Based on two single nucleotide polymorphisms present in the IGS region, a PCR‐based diagnostic tool was developed to specifically detect isolates from VCG 01213, also called tropical race 4 (TR4), which is currently a major concern in global banana production. Validation involved TR4 isolates, as well as Foc isolates from 19 other VCGs, other fungal plant pathogens and DNA samples from infected tissues of the Cavendish banana cultivar Grand Naine (AAA). Subsequently, a multiplex PCR was developed for fungal or plant samples that also discriminated Musa acuminata and M. balbisiana genotypes. It was concluded that this diagnostic procedure is currently the best option for the rapid and reliable detection and monitoring of TR4 to support eradication and quarantine strategies.

RECENT OCCURRENCE OF FUSARIUM OXYSPORUM F. SP. CUBENSE TROPICAL RACE 4 IN ASIA

ISHS III International Symposium on Banana: ISHS-ProMusa Symposium on Recent Advances in Banana Crop Protection for Sustainable Production and Improved Livelihoods RECENT OCCURRENCE OF FUSARIUM OXYSPORUM F. SP. CUBENSE TROPICAL RACE 4 IN ASIA

Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in an important Cavendish banana cv. Robusta (AAA)

Establishment of an efficient protocol for regeneration and genetic transformation is required in banana for the incorporation of useful traits. Therefore an efficient method has been developed for somatic embryogenesis, plant regeneration and transformation of Cavendish banana cultivar Robusta (AAA). Embryogenic cell suspension culture (ECS) was established using immature male flowers. Percentage appearance of embryogenic callus and distinct globular embryos was 10.3 and 11.1, respectively. ECS obtained was cocultivated under different cocultivation conditions with Agrobacterium tumefaciens strain EHA105 harboring pCAMBIA 1301 plant expression vector. Up to 30 transgenic plants/50 mg settled cell volume (SCV) was obtained with cocultivation in semisolid medium whereas no transgenics could be obtained with parallel experiments carried out in liquid medium. Histochemical GUS assay in different tissues of putatively transformed plants demonstrated expression of uidA gene. Among the putatively transformed plants obtained, a set of 4 were confirmed by PCR analysis and stable integration of the transgene by Southern analysis. GUS specific activity measured by a MUG (4-methylumbelliferyl-β-d-glucuronide) based flourometric assay revealed increase in transient GUS expression in semisolid as well as liquid cocultivation with centrifugation. This is the first report showing somatic embryogenesis and Agrobacterium tumefaciens mediated transformation using embryogenic cell suspension cultures in an important Cavendish banana cultivar Robusta. The present protocol will make possible agronomic improvement of this important commercially grown cultivar by introduction of disease resistance characteristics and antisense-mediated delayed fruit ripening strategies. Further, it will also assist in functional characterization of new gene or promoter elements isolated from this or other cultivars of banana.