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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.

Base Sequence↗

Purification and characterization of pectate lyase from banana (Musa acuminata) fruits.

Pectate lyase (PEL) has been purified by hydrophobic, cation exchange and size exclusion column chromatographies from ripe banana fruit. The purified enzyme has specific activity of 680 +/- 50 pkat mg protein(-1). The molecular mass of the enzyme is 43 kDa by SDS-PAGE. The pI of the enzyme is 8 with optimum activity at pH 8.5. Analysis of the reaction products by paper and anion exchange chromatographies reveal that the enzyme releases several oligomers of unsaturated galacturonane from polygalacturonate. The K(m) values of the enzyme for polygalacturonate and citrus pectin (7.2% methylation) are 0.40 +/- 0.04 and 0.77 +/- 0.08 g l(-1), respectively. PEL is sensitive to inhibition by different phenolic compounds, thiols, reducing agents, iodoacetate and N-bromosuccinimide. The enzyme has a requirement for Ca(2+) ions. However, Mg(2+) and Mn(2+) can substitute equally well. Additive effect on the enzyme activity was observed when any two metal ions (out of Mg(2+), Ca(2+) and Mn(2+)) are present together. The banana PEL is a enzyme requiring Mg(2+), in addition to Ca(2+), for exhibiting maximum activity.

Catalysis↗

The acyclic nucleoside phosphonate analogues, adefovir, tenofovir and PMEDAP, efficiently eliminate banana streak virus from banana (Musa spp.).

We report that the anti-retroviral and anti-hepadnavirus molecules, adefovir, tenofovir and 9-(2-phosphonomethoxyethyl)-2,6-diaminopurine (PMEDAP), efficiently eradicate the episomal form of Banana streak virus (BSV) from banana plants. Up to 90% of plants regenerated from BSV-infected highly proliferating meristems were virus free following a 6-month treatment period with 10 microg/ml (a non-phytotoxic concentration) of either compounds.

Adenine↗

Expression of a ripening-related cytochrome P450 cDNA in Cavendish banana (Musa acuminata cv. Williams).

As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

Amino Acid Sequence↗

Inhibition of acetate ester biosynthesis in banana (Musa sapientum L.) fruit pulp under anaerobic conditions.

The effect of anaerobic conditions on acetate ester biosynthesis in ripened banana pulp was investigated. Incubation of the pulp in less than 1% O(2) resulted in a significant reduction in the formation of ethyl acetate. Regardless of the presence of a large amount of endogenous ethanol and the remaining exogenous isobutyl alcohol after complete anaerobic incubation with the pulp, the production of acetate ester decreased. The effect of addition of pyruvate, isobutyl alcohol, acetate, and methyl hexanoate on acetate ester formation in 100% N(2) was also investigated. The addition of pyruvate and isobutyl alcohol to the pulp gave lower acetate esters in N(2) than in air, whereas the pulp incubated with acetate and isobutyl alcohol produced more acetate ester in both conditions. Therefore, the lack of acetyl CoA, or more precisely acetate, in the tissue is the main reason for the inhibition of acetate ester formation under anaerobic conditions. The activity of beta-oxidation measured by incubation with methyl hexanoate was detected only in the samples incubated in air. The formation of acetyl CoA, derived from pyruvate through mitochondria and through beta-oxidation, was inhibited by anaerobic conditions, which suggests that mitochondrial activity and/or beta-oxidation are essential for ester biosynthesis.

Acetates↗

Functional proteome analysis of the banana plant (Musa spp.) using de novo sequence analysis of derivatized peptides.

We report the use of chemical derivatization with MALDI-MS/MS analysis for de novo sequence analysis. Using three frequently used homology-based search algorithms, we were able to identify more than 40 proteins from banana, a nonmodel plant with unsequenced genome. Furthermore, this approach allowed the identification of different isoforms. We also observed that the identification score obtained varied according to the position of the peptide sequences in the query using the MS-Blast algorithm.

Algorithms↗

Number, position, diameter and initial direction of growth of primary roots in Musa.

To understand soil colonization by a root system, information is needed on the architecture of the root system. In monocotyledons, soil exploration is mainly due to the growth of adventitious primary roots. Primary root emergence in banana was quantified in relation to shoot and corm development. Root emergence kinetics were closely related to the development of aerial organs. Root position at emergence on the corm followed an asymptotic function of corm dry weight, so that the age of each root at a given time could be deduced from its position. Root diameter at emergence was related to the position of the roots on the corm, with younger roots being thicker than older ones. However, root diameters were not constant along a given root, but instead decreased with the distance to the base; roots appear to be conical in their basal and apical parts. Root growth directions at emergence were variable, but a high proportion of the primary roots emerged with a low angle to the horizontal. Further research is needed to evaluate whether these initial trajectories are conserved during root development. Results presented in this study are in good agreement with those reported for other monocotyledons such as maize and rice. They give quantitative information that will facilitate the development of models of root system architecture in banana.

Musa↗

Malate synthase gene expression during fruit ripening of Cavendish banana (Musa acuminata cv. Williams).

Malate synthase (MS) is a key enzyme responsible for malic acid synthesis in the glyoxylate cycle, which functions to convert stored lipids to carbohydrates, by catalysing the glyoxylate condensation reaction with acetyl-CoA in the peroxisome. In this study, the cloning of an MS cDNA, designated MaMS-1, from the banana fruit is reported. MaMS-1 was 1801 bp in length encoding a single polypeptide of 556 amino acid residues. Sequence analysis revealed that MaMS-1 possessed the conserved catalytic domain and a putative peroxisomal targeting signal SK(I/L) at the carboxyl terminal. MaMS-1 also shared an extensive sequence homology (79-81.3%) with other plant MS homologues. Southern analysis indicated that MS might be present as multiple members in the banana genome. In Northern analysis, MaMS-1 was expressed specifically in ripening fruit tissue and transcripts were not detected in other organs such as roots, pseudostem, leaves, ovary, male flower, and in fruit at different stages of development. However, the transcript abundance in fruit was affected by stage of ripening, during which transcript was barely detectable at the early stage of ripening (FG and TY), but the level increased markedly in MG and in other fruits at advanced ripening stages. Furthermore, MaMS-1 expression in FG fruit could be stimulated by treatment with 1 microl l(-1) exogenous ethylene, but the stimulatory effect was abolished by the application of an ethylene inhibitor, norbornadiene. Results of this study clearly show that MS expression in banana fruit is temporally regulated during ripening and is ethylene-inducible.

Amino Acid Sequence↗

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.

Agrobacterium tumefaciens↗

Vomiting, abdominal distention and early feeding of banana (Musa paradisiaca) in neonates.

The objective of this cohort study was to assess the relationship between banana given as early solid food with the symptoms of intestinal obstruction (SIO) among neonates, in a rural community in West Lombok District, West Nusa Tenggara Province, Indonesia. Mothers having newborn infants were interviewed and 3,420 neonates were followed for 28 days. Compared with infants who were not given solid food, the relative risk (RR) for infants given food other than banana as early solid food was 1.87, 95% CI 0.48-8.24, p=0.4, while for infants given banana only as early solid food the RR was 9.15, 95% CI 1.96-42.58, p 0.0005. After adjustment for birthweight, colostrum, and breastfeeding, the odds ratio for infants given banana and the appearance of SIO was 2.99, 95% CI 2.65-5.14; p=0.0012. These data indicate that banana given as early solid food is an important risk factor for the appearance of SIO in neonates.

Gastric Dilatation↗

Plant defense induced in in vitro propagated banana (Musa paradisiaca) plantlets by Fusarium derived elicitors.

Perception of microbial signal molecules is part of the strategy evolved by plants to survive attacks by potential pathogens. To gain a more complete understanding of the early signaling events involved in these responses, we used fungal components of Fusarium under in vitro condition and checked the rise in signal molecule, salicylic acid (SA), and marker enzymes in defense reactions against the pathogen. SA level increased by 21 folds in elicitor treated plantlets as compared to that of control plantlets and there was marked increase in phenylalanine ammonia-lyase(PAL), peroxidase(POX), polyphenol oxidase(PPO) along with higher total phenolic content. Present results indicated that use of fungal components had successfully induced systemic resistance in in vitro cultured banana plantlets.

Catechol Oxidase↗

Cloning and developmental and tissue-specific expression of banana (Musa acuminate AAA) lectin gene.

A cDNA encoding banana lectin (BanLec) was obtained by using RT-PCR from banana fruit and sequenced. Comparison of this cDNA sequence with the sequence of BanLec gene registered in GenBank (Accession No. AF001527) showed that both sequences shared high similarity with only nine nucleotieds and four amino acids difference. RT-PCR analysis of the expression patterns of BanLec gene in different tissues and different fruit ripening stages of banana suggested that this cDNA not only expressed specifically in fruit but also expressed developmentally during fruit ripening. Relative quantification of expression level of BanLec gene in different tissues and different ripening stages were carried out by using computer software.

Base Sequence↗

Selection of assessment methods for evaluating banana weevil Cosmopolites sordidus (Coleoptera: Curculionidae) damage on highland cooking banana (Musa spp., genome group AAA-EA).

Cosmopolites sordidus (Germar) is an important pest on bananas and plantains. Population build-up is slow and damage becomes increasingly important in successive crop cycles (ratoons). Yield loss results from plant loss, mat disappearance and reduced bunch size. Damage assessment requires destructive sampling and is most often done on corms of recently harvested plants. A wide range of damage assessment methods exist and there are no agreed protocols. It is critical to know what types of damage best reflect C. sordidus pest status through their relationships with yield loss. Multiple damage assessment parameters (i.e. for the corm periphery, cortex and central cylinder) were employed in two yield loss trials and a cultivar-screening trial in Uganda. Damage to the central cylinder had a greater effect on plant size and yield loss than damage to the cortex or corm periphery. In some cases, a combined assessment of damage to the central cylinder and cortex showed a better relationship with yield loss than an assessment of the central cylinder alone. Correlation, logistic and linear regression analyses showed weak to modest correlations between damage to the corm periphery and damage to the central cylinder. Thus, damage to the corm periphery is not a strong predictor of the more important damage to the central cylinder. Therefore, C. sordidus damage assessment should target the central cylinder and cortex.

Animals↗