Cryopreservation of banana's cv Grand Naine in vitro rhizomes.

The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA) cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

Comparison of tissue deterioration of ripening banana fruit (Musa spp., AAA group, Cavendish subgroup) under chilling and non-chilling temperatures.

BACKGROUND: In fleshy fruits, induced programmed cell death (PCD) has been observed in heat-treated tomato, and in ethylene-treated and low-temperature exposure in immature cucumber. No other fleshy fruit has been evaluated for chilling-injury-induced PCD, especially mature fruit with full ripening capacity. The purpose of this research was to identify and evaluate the presence of PCD processes during the development of low-temperature-induced physiopathy of banana fruit. RESULTS: Exposure of fruit to 5 °C for 4 days induced degradative processes similar to those occurring during ripening and overripening of non-chilled fruit. Nuclease from banana peel showed activity in both DNA substrates and RNA substrates. No exclusive low-temperature-induced proteases and nucleases were observed. DNA of chilled peel showed earlier signs of degradation and higher levels of DNA tailing during overripening. CONCLUSION: This study shows that exposure to low temperatures did not induce a pattern of degradative processes that differed from that occurring during ripening and overripening of non-chilled fruit. DNA showed earlier signs of degradation and higher levels of DNA tailing. Nuclease activity analysis showed bifunctionality in both chilled and non-chilled tissue and no chilling-exclusive protease and nuclease. Fleshy fruit might use their available resources on degradative processes and adjust them depending on environmental conditions. © 2018 Society of Chemical Industry.

Cloning and functional characterization of MusaVND1 using transgenic banana plants.

Vascular related NAC (NAM, ATAF and CUC) domain-containing genes regulate secondary wall deposition and differentiation of xylem vessel elements. MusaVND1 is an ortholog of Arabidopsis VND1 and contains the highly conserved NAC domain. The expression of MusaVND1 is highest in developing corm and during lignification conditions, the increase in expression of MusaVND1 coincides with the expression of PAL, COMT and C4H genes. MusaVND1 encodes a nuclear localized protein as MusaVND1-GFP fusion protein gets localized to nucleus. Transient overexpression of MusaVND1 converts banana embryogenic cells to xylem vessel elements, with a final differentiation frequency of 33.54% at the end of tenth day. Transgenic banana plants overexpressing MusaVND1 showed stunted growth and were characterized by PCR and Southern blot analysis. Transgenic banana plants showed transdifferentiation of various types of cells into xylem vessel elements and ectopic deposition of lignin in cells of various plant organs such as leaf and corm. Tracheary element formation was seen in the cortical region of transgenic corm as well as in epidermal cells of leaves. Biochemical analysis indicates significantly higher levels of lignin and cellulose content in transgenic banana lines than control plants. MusaVND1 overexpressing transgenic banana plants showed elevated expression levels of genes involved in lignin and cellulose biosynthesis pathway. Further expression of different MYB transcription factors positively regulating secondary wall deposition was also up regulated in MusaVND1 transgenic lines.

Phenylpropanoid pathway is potentiated by silicon in the roots of banana plants during the infection process of Fusarium oxysporum f. sp. cubense.

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense, is a disease that causes large reductions in banana yield worldwide. Considering the importance of silicon (Si) to potentiate the resistance of several plant species to pathogen infection, this study aimed to investigate, at the histochemical level, whether this element could enhance the production of phenolics on the roots of banana plants in response to F. oxysporum f. sp. cubense infection. Plants of cultivar Maçã, which is susceptible to F. oxysporum f. sp. cubense, were grown in plastic pots amended with 0 (-Si) or 0.39 g of Si (+Si) per kilogram of soil and inoculated with race 1 of F. oxysporum f. sp. cubense. The root Si concentration was increased by 35.6% for +Si plants in comparison to the -Si plants, which contributed to a 27% reduction in the symptoms of Fusarium wilt on roots. There was an absence of fluorescence for the root sections of the -Si plants treated with the Neu and Wilson's reagents. By contrast, for the root sections obtained from the +Si plants treated with Neu's reagent, strong yellow-orange fluorescence was observed in the phloem, and lemon-yellow fluorescence was observed in the sclerenchyma and metaxylem vessels, indicating the presence of flavonoids. For the root sections of the +Si plants treated with Wilson's reagent, orange-yellowish autofluorescence was more pronounced around the phloem vessels, and yellow fluorescence was more pronounced around the metaxylem vessels, also indicating the presence of flavonoids. Lignin was more densely deposited in the cortex of the roots of the +Si plants than for the -Si plants. Dopamine was barely detected in the roots of the -Si plants after using the lactic and glyoxylic acid stain, but was strongly suspected to occur on the phloem and metaxylem vessels of the roots of the +Si plants as confirmed by the intense orange-yellow fluorescence. The present study provides new evidence of the pivotal role of the phenylpropanoid pathway in the resistance of banana plants to F. oxysporum f. sp. cubense infection when supplied with Si.

Anatomical and physical changes in leaves during the production of tamales.

PREMISE OF THE STUDY: Tamale preparation has a long tradition in Mexico. To understand which material properties have been considered important for this purpose throughout the years, a study was conducted of the anatomical, chemical, and mechanical properties of the leaves of four plant species used in tamale preparation in Veracruz, Mexico: Calathea misantlensis, Canna indica, Musa paradisiaca, and Oreopanax capitatus. METHODS: Four cooking treatments were considered: fresh (F), roasted (soasado, R), steamed (S), and roasted plus steamed (R/S). Chemical, anatomical, and mechanical analyses were conducted before and after each treatment. Leaf samples were tested for tensile strength at both parallel and perpendicular orientation relative to the fibers. KEY RESULTS: Musa paradisiaca had the highest proportion of cellulose, while the remaining species shared similar lower proportions. Leaves were stronger and stiffer in the longitudinal direction of the fibers. Musa paradisiaca leaves had higher values of mechanical strength than the other species. The cooking process that most affected the mechanical properties was steaming. CONCLUSIONS: The chemical constituents of the leaves are closely correlated with their physical properties. The treatment that caused the greatest decrease in leaf physical integrity was steaming, while the combination of roasting and steaming showed similar results to those of steaming alone. No evident anatomical changes are produced by any of the treatments. This is one of the few studies comparing physical, chemical, and anatomical characteristics of leaves used for human consumption, before and after cooking.

Separation and identification of Musa acuminate Colla (banana) leaf proteins by two-dimensional gel electrophoresis and mass spectrometry.

To establish a proteomic reference map of Musa acuminate Colla (banana) leaf, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 44 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Three spots that were not identified by MALDI-TOF MS analysis were identified by searching against the NCBInr, SwissProt, and expressed sequence tag (EST) databases. We identified 41 unique proteins. The majority of the identified leaf proteins were found to be involved in energy metabolism. The results indicate that 2D-PAGE is a sensitive and powerful technique for the separation and identification of Musa leaf proteins. A summary of the identified proteins and their putative functions is discussed.

Cytogenetic evidence of mixed disomic and polysomic inheritance in an allotetraploid (AABB) Musa genotype.

BACKGROUND AND AIMS: Edible bananas originated mainly from two wild species, Musa acuminata Colla (AA) and Musa balbisiana Colla (BB), and triploid cultivars with an AAA, AAB or ABB genome are the most widely used. In the present study, chromosome pairing affinities are investigated in a sterile AB Indian variety and in its fertile colchicine-induced allotetraploid (AABB) derivative to determine the inheritance pattern of the tetraploid genotype. The potential implications of interspecific recombination and chromosomal composition of diploid gametes for Musa improvement are presented. METHODS: The pairing of different chromosome sets at diploid and tetraploid levels was investigated through a combination of conventional cytogenetic and genomic in-situ hybridization (GISH) analyses of meiotic chromosomes, leading to a likelihood model of the pairing behaviour. GISH analysis of mitotic chromosomes was also conducted to reveal the chromosome constitution of hybrids derived from crosses involving the allotetraploid genotype. KEY RESULTS: Analysis of chromosome associations at both ploidy levels suggested that the newly formed allotetraploid behaves as a 'segmental allotetraploid' with three chromosome sets in a tetrasomic pattern, three sets in a likely disomic pattern and the five remaining sets in an intermediate pattern. Balanced and unbalanced diploid gametes were detected in progenies, with the chromosome constitution appearing to be more homogenous in pollen than in ovules. CONCLUSIONS: Colchicine-induced allotetraploids in Musa provide access to the genetic background of natural AB varieties. The segmental inheritance pattern exhibited by the AABB allotetraploid genotype implies chromosome exchanges between M. acuminata and M. balbisiana species and opens new horizons for reciprocal transfer of valuable alleles.

Fluorescent chlorophyll catabolites in bananas light up blue halos of cell death.

Breakdown of chlorophyll is a major contributor to the diagnostic color changes in fall leaves, and in ripening apples and pears, where it commonly provides colorless, nonfluorescent tetrapyrroles. In contrast, in ripening bananas (Musa acuminata) chlorophylls fade to give unique fluorescent catabolites (FCCs), causing yellow bananas to glow blue, when observed under UV light. Here, we demonstrate the capacity of the blue fluorescent chlorophyll catabolites to signal symptoms of programmed cell death in a plant. We report on studies of bright blue luminescent rings on the peel of very ripe bananas, which arise as halos around necrotic areas in 'senescence associated' dark spots. These dark spots appear naturally on the peel of ripe bananas and occur in the vicinity of stomata. Wavelength, space, and time resolved fluorescence measurements allowed the luminescent areas to be monitored on whole bananas. Our studies revealed an accumulation of FCCs in luminescent rings, within senescing cells undergoing the transition to dead tissue, as was observable by morphological textural cellular changes. FCCs typically are short lived intermediates of chlorophyll breakdown. In some plants, FCCs are uniquely persistent, as is seen in bananas, and can thus be used as luminescent in vivo markers in tissue undergoing senescence. While FCCs still remain to be tested for their own hypothetical physiological role in plants, they may help fill the demand for specific endogenous molecular reporters in noninvasive assays of plant senescence. Thus, they allow for in vivo studies, which provide insights into critical stages preceding cell death.

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

BACKGROUND: Fusarium 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. RESULTS: An 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. CONCLUSIONS: Collectively, 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.

Homoeologous chromosome pairing between the A and B genomes of Musa spp. revealed by genomic in situ hybridization.

BACKGROUND AND AIMS: Most cooking banana and several desert bananas are interspecific triploid hybrids between Musa acuminata (A genome) and Musa balbisiana (B genome). In addition, M. balbisiana has agronomical characteristics such as resistance to biotic and abiotic stresses that could be useful to improve monospecific acuminata cultivars. To develop efficient breeding strategies for improving Musa cultivars, it is therefore important to understand the possibility of chromosome exchange between these two species. METHODS: A protocol was developed to prepare chromosome at meiosis metaphase I suitable for genomic in situ hybridization. A series of technical challenges were encountered, the main ones being the hardness of the cell wall and the density of the microsporocyte's cytoplasm, which hampers accessibility of the probes to the chromosomes. Key parameters in solving these problems were addition of macerozyme in the enzyme mix, the duration of digestion and temperature during the spreading phase. RESULTS AND CONCLUSIONS: This method was applied to analyse chromosome pairing in metaphase from triploid interspecific cultivars, and it was clearly demonstrated that interspecific recombinations between M. acuminata and M. balbisiana chromosomes do occur and may be frequent in triploid hybrids. These results provide new insight into Musa cultivar evolution and have important implications for breeding.

Cloning and characterization of a novel stress-responsive WRKY transcription factor gene (MusaWRKY71) from Musa spp. cv. Karibale Monthan (ABB group) using transformed banana cells.

WRKY transcription factor proteins play significant roles in plant stress responses. Here, we report the cloning and characterization of a novel WRKY gene, MusaWRKY71 isolated from an edible banana cultivar Musa spp. Karibale Monthan (ABB group). MusaWRKY71, initially identified using in silico approaches from an abiotic stress-related EST library, was later extended towards the 3' end using rapid amplification of cDNA ends technique. The 1299-bp long cDNA of MusaWRKY71 encodes a protein with 280 amino acids and contains a characteristic WRKY domain in the C-terminal half. Although MusaWRKY71 shares good similarity with other monocot WRKY proteins the substantial size difference makes it a unique member of the WRKY family in higher plants. The 918-bp long 5' proximal region determined using thermal asymmetric interlaced-polymerase chain reaction has many putative cis-acting elements and transcription factor binding motifs. Subcellular localization assay of MusaWRKY71 performed using a GFP-fusion platform confirmed its nuclear targeting in transformed banana suspension cells. Importantly, MusaWRKY71 expression in banana plantlets was up-regulated manifold by cold, dehydration, salt, ABA, H2O2, ethylene, salicylic acid and methyl jasmonate treatment indicating its involvement in response to a variety of stress conditions in banana. Further, transient overexpression of MusaWRKY71 in transformed banana cells led to the induction of several genes, homologues of which have been proven to be involved in diverse stress responses in other important plants. The present study is the first report on characterization of a banana stress-related transcription factor using transformed banana cells.

Chlorophyll breakdown as seen in bananas: sign of aging and ripening--a mini-review.

The ripening of bananas is seen by a characteristic change of their color from deep green to bright yellow. Likewise, their over-ripening and eventual rotting are accompanied by the appearance of an unappetizing brown. Chlorophyll breakdown is a major contributor to the visual signs of these processes in bananas. Outlined here are the basic structures of chlorophyll catabolites in higher plants, with particular reference to ripening and aging bananas. In these fruits, unique fluorescent chlorophyll catabolites accumulate and give rise to their fascinating blue luminescence.

Cryopreservation of scalps of Malaysian bananas using a pre-growth method.

The effect of preculture with different sugars and mannitol on cryopreservation of scalps of the banana (Musa) cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak was investigated. Scalps (0.3 square cm) were precultured on semi-solid MS-based medium, containing 0.4 or 0.5 M sucrose, glucose, fructose, trehalose or mannitol, for 14 days under a 16 h light and 8 h dark photoperiod prior to rapid cooling and storage in liquid nitrogen. Explants were rewarmed rapidly in a water bath at 40 degree C for 1 min, followed by recovery on two layers of sterile filter paper overlaying 25 ml aliquots of semi-solid MS-based medium with 5 mg per liter benzylaminopurine, 0.2 mg per liter indole acetic acid and 10 mg per liter ascorbic acid (PM8 medium) for 2 days in the dark. Subsequently, scalps were transferred onto 25 ml aliquots of semi-solid PM8 medium and incubated in the dark for 1 week prior to incubation in the light. Shoot regeneration from 5 - 48 percent of cryopreserved scalps of all the banana cvs., was observed only following preculture with 0.4 or 0.5 M glucose or fructose, and with 0.4 M trehalose for the cvs. Pisang Berangan and Pisang Awak. Preculture with 0.4 M glucose resulted in maximum shoot regeneration of cryopreserved scalps of 10 percent, 13 percent, 42 percent and 48 percent for the cvs. Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak, respectively. Concentrations of 0.5 M trehalose, or 0.4 and 0.5 M sucrose or mannitol were extremely toxic to scalps of all the cvs. investigated.

A saturated SSR/DArT linkage map of Musa acuminata addressing genome rearrangements among bananas.

BACKGROUND: The genus Musa is a large species complex which includes cultivars at diploid and triploid levels. These sterile and vegetatively propagated cultivars are based on the A genome from Musa acuminata, exclusively for sweet bananas such as Cavendish, or associated with the B genome (Musa balbisiana) in cooking bananas such as Plantain varieties. In M. acuminata cultivars, structural heterozygosity is thought to be one of the main causes of sterility, which is essential for obtaining seedless fruits but hampers breeding. Only partial genetic maps are presently available due to chromosomal rearrangements within the parents of the mapping populations. This causes large segregation distortions inducing pseudo-linkages and difficulties in ordering markers in the linkage groups. The present study aims at producing a saturated linkage map of M. acuminata, taking into account hypotheses on the structural heterozygosity of the parents. RESULTS: An F1 progeny of 180 individuals was obtained from a cross between two genetically distant accessions of M. acuminata, 'Borneo' and 'Pisang Lilin' (P. Lilin). Based on the gametic recombination of each parent, two parental maps composed of SSR and DArT markers were established. A significant proportion of the markers (21.7%) deviated (p < 0.05) from the expected Mendelian ratios. These skewed markers were distributed in different linkage groups for each parent. To solve some complex ordering of the markers on linkage groups, we associated tools such as tree-like graphic representations, recombination frequency statistics and cytogenetical studies to identify structural rearrangements and build parsimonious linkage group order. An illustration of such an approach is given for the P. Lilin parent. CONCLUSIONS: We propose a synthetic map with 11 linkage groups containing 489 markers (167 SSRs and 322 DArTs) covering 1197 cM. This first saturated map is proposed as a "reference Musa map" for further analyses. We also propose two complete parental maps with interpretations of structural rearrangements localized on the linkage groups. The structural heterozygosity in P. Lilin is hypothesized to result from a duplication likely accompanied by an inversion on another chromosome. This paper also illustrates a methodological approach, transferable to other species, to investigate the mapping of structural rearrangements and determine their consequences on marker segregation.

Morpho-histological study of banana (Musa spp. cv. Grande Naine [AAA]) cell suspensions during cryopreservation and regeneration.

In this work, a morpho-histological study of banana (Musa spp. cv. Grande Naine [AAA]) embryogenic cell suspensions during cryopreservation and regeneration was performed. It was demonstrated that the regeneration process of somatic embryos originating from cryopreserved cell suspensions was different from that of control cell suspensions. Somatic embryos originating from cryopreserved cell suspensions had a unicellular origin. The regeneration process was modified not only by freezing in liquid nitrogen but also by the plasmolyzing effect of the 0.5 M sucrose solution employed during pretreatment. This result explained the high number of embryonic structures formed on M3 medium, compared with the control. Proembryos blocked at the globular stage could pursue their development when they were plated on new culture medium at a lower density after 30 days of culture on M3 medium. The unicellular origin of somatic embryos produced from cryopreserved cell suspensions offers the prospect of using cryopreservation to select non-chimeral transformed plants.

Wall porosity in isolated cells from food plants: Implications for nutritional functionality.

Macronutrients in whole plant foods are enclosed inside cells. The metabolic response from these entrapped nutrients may depend on cell-wall porosity, by controlling the passage of digestive enzymes. As non-interacting size mimics of digestive enzymes, we investigated the diffusion of fluorescently-labelled probes across the walls of isolated plant cells from potato tuber, red kidney bean and banana. Diffusion properties of permeable probes, dextran (20-kDa and 70-kDa) and albumin, were quantified, using fluorescence recovery after photobleaching. The consistent reduction of diffusion rate in the presence of cell walls (around 40%) compared to free-diffusion rate was attributed to the limiting porosity of the wall matrix. A combination of the physical barrier effects demonstrated here and non-catalytic binding of enzymes to cell walls limits the hydrolysis of intracellular macronutrients. This and further understanding of the structural basis for the physical barrier properties would help to design foods from plant materials with enhanced nutrition.

In Vitro Proliferation of Female Buds for Induction of Somatic Embryogenesis from False Horn Plantain (AAB, cv. Curraré).

Most cultivated bananas (Musa spp.) are polyploids, and their fruits are seedless and propagated exclusively vegetatively; however, they can also be cloned by micropropagation techniques, viz., direct organogenesis (DO) or somatic embryogenesis (SE). Banana indirect SE (ISE), with an embryogenic callus phase, is possible using young male or female flowers as direct explant depending on the genotype or shoot tips (scalps). For the False Horn Plantain, cv. Curraré (AAB, plantain subgroup), which has a degenerating male bud, female flowers are used to regenerate plants through ISE. Here, a protocol for increasing the number of initial explant material from a single mother plant and its embryogenic response is described. For those purposes, hands with young female buds are in vitro proliferated in the presence of 1 µM indole-3-acetic acid and 2.5 µM thidiazuron. Friable embryogenic cultures, here called ISE-2, obtained from the new proliferative secondary female bud clusters are initiated on medium containing auxins. Embryogenic suspensions are then established from the ISE-2 cultures. Regeneration of plants is achieved from embryogenic suspensions after plating on semisolid medium free of plant growth regulators; greenhouse acclimatized plantlets are ready for banana farming. This study demonstrates that proliferative female buds are a proper choice for ISE.

Inhibition of Agrobacterium-induced cell death by antiapoptotic gene expression leads to very high transformation efficiency of banana.

The death of plant cells in culture following exposure to Agrobacterium tumefaciens remains a major obstacle in developing Agrobacterium-mediated transformation into a highly efficient genotype-independent technology. Here, we present evidence that A. tumefaciens exposure induces cell death in banana cell suspensions. More than 90% of embryogenic banana cells died after exposure to A. tumefaciens and cell death was accompanied by a subset of features associated with apoptosis in mammalian cells, including DNA laddering, fragmentation, and formation of apoptotic-like bodies. Importantly, these cellular responses were inhibited in cells expressing the animal antiapoptosis genes Bcl-xL, Bcl-2 3' untranslated region, and CED-9. Inhibition of cell death resulted in up to 90% of cell clumps transformed with Bcl-xL, a 100-fold enhancement over vector controls, approaching the transformation and regeneration of every "transformable" cell. Similar results using sugarcane, a crop plant known for recalcitrance to Agrobacterium transformation, suggest that antiapoptosis genes may inhibit these phenomena and increase the transformation frequency of many recalcitrant plant species, including the major monocot cereal crop plants. Evidence of inhibition of plant cell death by cross-kingdom antiapoptotic genes also contributes to the growing evidence that genes for control of programmed cell death are conserved across wide evolutionary distances, even though these mechanisms are not well understood in plants.

Banana (Musa sp.).

Cultivated bananas are vegetatively propagating herbs, which are difficult to breed because of widespread male and female sterility. As a complementary gene transfer method in banana, the described Agrobacterium protocol relies on highly regenerable embryogenic cell cultures. Embryogenic cells are infected and co-cultivated in the presence of acetosyringone with Agrobacterium tumefaciens harboring a binary plasmid vector to obtain a mixed population of transformed and untransformed plant cells. Transformed plant cells are promoted to grow for 2 to 3 mo on a cell colony induction medium containing the antibiotics geneticin or hygromycin as selective agents, while agrobacteria are counterselected by timentin. The whole procedure, including plant regeneration, takes approx 6 mo and results in an average frequency of 25 to 50 independent transgenic plants per plate, which equals 50 mg of embryogenic cells. This method has been applied to a wide range of cultivars and to generate large populations of transgenic colonies and plants for tagging genes and promoters in banana.

Functional characterization of secondary wall deposition regulating transcription factors MusaVND2 and MusaVND3 in transgenic banana plants.

NAM, ATAF, and CUC (NAC) domain-containing proteins are plant-specific transcription factors involved in stress responses and developmental regulation. MusaVND2 and MusaVND3 are vascular-related NAC domain-containing genes encoding for nuclear-localized proteins. The transcript level of MusaVND2 and MusaVND3 are gradually induced after induction of lignification conditions in banana embryogenic cells. Banana embryogenic cells differentiated to tracheary element-like cells after overexpression of MusaVND2 and MusaVND3 with a differentiation frequency of 63.5 and 23.4 %, respectively, after ninth day. Transgenic banana plants overexpressing either of MusaVND2 or MusaVND3 showed ectopic secondary wall deposition as well as transdifferentiation of cells into tracheary elements. Transdifferentiation to tracheary element-like cells was observed in cortical cells of corm and in epidermal and mesophyll cells of leaves of transgenic plants. Elevated levels of lignin and crystalline cellulose were detected in the transgenic banana lines than control plants. The results obtained are useful for understanding the molecular regulation of secondary wall development in banana.