Isolation and partial characterization of a root-specific promoter for stacking multiple traits into cassava (Manihot esculenta CRANTZ).

Cassava can be cultivated on impoverished soils with minimum inputs, and its storage roots are a staple food for millions in Africa. However, these roots are low in bioavailable nutrients and in protein content, contain cyanogenic glycosides, and suffer from a very short post-harvest shelf-life, and the plant is susceptible to viral and bacterial diseases prevalent in Africa. The demand for improvement of cassava with respect to these traits comes from both farmers and national agricultural institutions. Genetic improvement of cassava cultivars by molecular biology techniques requires the availability of appropriate genes, a system to introduce these genes into cassava, and the use of suitable gene promoters. Cassava root-specific promoter for auxin-repressed protein was isolated using the gene walking approach, starting with a cDNA sequence. In silico analysis of promoter sequences revealed putative cis-acting regulatory elements, including root-specific elements, which may be required for gene expression in vascular tissues. Research on the activities of this promoter is continuing, with the development of plant expression cassettes for transformation into major African elite lines and farmers' preferred cassava cultivars to enable testing of tissue-specific expression patterns in the field.

Enhancer/Suppressor mutator (En/Spm)-like transposable elements of cassava (Manihot esculenta) are transcriptionally inactive.

Transposable elements contribute to the size, structure, variation, and diversity of the genome and have major effects on gene function. Sequencing projects have revealed the diversity of transposable elements in many organisms and have shown that they constitute a high percentage of the genome. PCR-based techniques using degenerate primers designed from conserved enzyme domains of transposable elements can provide quick and extensive surveys, making study of diversity and abundance and their applications possible in species where full genome sequence data are not yet available. We studied cassava (Manihot esculenta) En/Spm-like transposons (Meens) with regard to genomic distribution, sequence diversity and methylation status. Cassava transposase fragments characteristic of En/Spm-like transposons were isolated, cloned and characterized. Sequence analysis showed that cassava En/Spm-like elements are highly conserved, with overall identity in the range of 68-98%. Southern hybridization supports the presence of multiple copies of En/Spm-like transposons integrated in the genome of all cassava cultivars that we tested. Hybridization patterns of HpaII- and MspI-digested cassava genomic DNA revealed highly methylated sequences. There were no clear differences in hybridization pattern between the cultivars. We did not detect RNA transcripts of Meens by Northern procedures. We examined the possibility of recent transposition activities of the cassava En/Spm-like elements.

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava.

Agrobacterium-mediated transformation of friable embryogenic calli (FEC) is the most widely used method to generate transgenic cassava plants. However, this approach has proven to be time-consuming and can lead to changes in the morphology and quality of FEC, influencing regeneration capacity and plant health. Here we present a comprehensive, reliable and improved protocol, taking approximately 6 months, that optimizes Agrobacterium-mediated transformation of FEC from cassava model cultivar TMS60444. We cocultivate the FEC with Agrobacterium directly on the propagation medium and adopt the extensive use of plastic mesh for easy and frequent transfer of material to new media. This minimizes stress to the FEC cultures and permits a finely balanced control of nutrients, hormones and antibiotics. A stepwise increase in antibiotic concentration for selection is also used after cocultivation with Agrobacterium to mature the transformed FEC before regeneration. The detailed information given here for each step should enable successful implementation of this technology in other laboratories, including those being established in developing countries where cassava is a staple crop.

Isolation and characterisation of a cassava catalase expressed during post-harvest physiological deterioration.

Storage roots of cassava undergo a rapid, endogenous, post-harvest deterioration response that is thought to involve oxidative processes. A cassava catalase (MecCAT1) was isolated from a root cDNA library. The transcript is expressed predominantly in roots with little expression in leaves. Catalase enzyme activity and MecCAT1 transcript expression during the post-harvest period were compared in highly susceptible and less susceptible cultivars and suggest that high levels of catalase activity may play a role in delaying the deterioration response.

Hydrogen peroxide and flavan-3-ols in storage roots of cassava (Manihot esculenta crantz) during postharvest deterioration.

Cassava storage roots are an important staple food throughout the lowland humid tropics. However, cassava suffers from a poorly understood storage disorder, known as postharvest physiological deterioration (PPD), which constrains its exploitation. In an attempt to broaden the understanding of PPD, nine different cassava cultivars were analyzed for specific compounds accumulating during the process. The production of hydrogen peroxide (H(2)O(2)) is involved in the early stages of PPD in cassava roots. H(2)O(2) was quantified and localized histochemically at the tissue and cell level in deteriorating roots. This reactive oxygen species accumulated during the first 24 h after harvest, especially in the inner parenchymatic tissue. Three flavan-3-ols, (+)-catechin, (+)-catechin gallate, and (+)-gallocatechin, accumulated during the storage of cassava roots. However, these potential antioxidants cannot be related to early storage disorders or wound responses because they start to accumulate only after 4-6 days.

Phylogenetic grouping and identification of Rhizobium isolates on the basis of random amplified polymorphic DNA profiles.

Through the use of a single, random 15mer as a primer, between 1 and 12 DNA amplification products were obtained per strain from a selection of 84 Rhizobium and Bradyrhizobium isolates. A principal-coordinate analysis was used to analyse the resulting amplified DNA profiles and it was possible to assign isolates to specific groupings. Within the species Rhizobium leguminosarum, the biovar phaseoli formed a distinct group from the other biovars of the species, viciae and trifolii, which grouped together. Isolates of Rhizobium meliloti and Bradyrhizobium species formed their own clear, specific groups. Although it was possible to identify individual isolates on the basis of differences in their amplified DNA profiles, there was evidence that some amplified segments were conserved among individuals at the biovar and species levels.

Differentiation of Acanthamoeba strains from infected corneas and the environment by using restriction endonuclease digestion of whole-cell DNA.

Restriction endonuclease digestion of Acanthamoeba whole-cell DNA was used to study the relationship between 33 morphologically identical strains from keratitis cases (30 strains), contact lens storage containers (2 strains), and soil (1 strain). Samples digested with BglII, EcoRI, or HindIII and separated by agarose gel electrophoresis contained detectable mitochondrial DNA restriction fragment length polymorphisms (RFLPs). By comparing RFLPs, the strains could be assigned to seven multiple-strain and three single-strain groups. The largest of these contained nine strains, eight of which were isolated in keratitis cases in various locations worldwide and may indicate a group particularly associated with keratitis. Restriction endonuclease analysis of whole-cell DNA is proposed as a valuable technique for detecting mitochondrial DNA RFLPs in the differentiation of morphologically identical Acanthamoeba strains and may therefore be useful in resolving the complex taxonomy of the genus, which has hitherto been founded on subjective morphological criteria.

Laboratory investigation of Acanthamoeba keratitis.

Following the diagnosis of Acanthamoeba keratitis in a contact lens wearer, the antimicrobial susceptibility of the clinical isolate and the environmental source of the infection were investigated. Contrary to previous reports, in vitro antimicrobial testing showed that the infecting strain was inherently resistant to propamidine isethionate. Restriction endonuclease digestion analysis of Acanthamoeba whole-cell DNA of strains isolated from the patient's cornea, contact lens storage container, saline rinsing solution, and kitchen cold-water tap showed that the isolates were identical. This implicates, for the first time, domestic tap water as the source of Acanthamoeba sp. in this infection. It is therefore recommended that the use of homemade saline solutions and the rinsing of contact lenses in tap water be strongly discouraged.

High sequence conservation between isocitrate lyase from Escherichia coli and Ricinus communis.

The deduced amino acid sequences of isocitrate lyase (EC 4.1.3.1) from Escherichia coli and Ricinus communis (castor bean) were compared and regions of high homology between the two enzymes were identified. The castor-bean enzyme had a 14 amino acid amino-terminal, and a 25 amino acid carboxy-terminal extension and a 102 amino acid central insertion compared to the E. coli enzyme. Enzymatic data were used to attempt to identify specific amino acids in the active site. Comparisons with putative peroxisomal/gloxysomal targeting sequences were made and a region including part of the central insertion of the castor bean enzyme was tentatively identified.

Nucleic acid (cDNA) and amino acid sequences of isocitrate lyase from castor bean.

A cDNA library was constructed to mRNA enriched for isocitrate-lyase mRNA from castor-bean (Ricinus communis var. zanzibarensis) endosperms. Nine clones for isocitrate lyase (EC 4.1.3.1) were identified. The insert of 2.2 kb from clone ICL4 was sequenced and proved to contain the entire coding region, 1731 bp, for isocitrate lyase. The amino acid sequence of isocitrate lyase was deduced from the nucleic acid sequence. By analogy with muscle aldolase a lysine residue that possibly takes part in the binding of the substrate was identified. The 3' untranslated region contained three putative polyadenylation addition signals and two direct repeats.

Changes in levels of transcripts in endosperms of castor beans treated with exogenous gibberellic acid.

A complementary-DNA library to mRNA from castor-bean endosperm has been prepared. Three clones have been examined in detail. One of these is complementary to isocitrate-lyase mRNA. The other two clones code for proteins with M r , 42000 and 38000. All three clones have been used to measure levels of transcripts during seed germination. The three transcripts all increased during germination and the rate of their appearance is stimulated by exogenous GA3. The data strongly support the view that the action of GA3 in these seeds is to stimulate non-specifically the rate of transcription and, in turn, protein synthesis. Possible mechanisms for the action of the growth regulator are discussed.