Cloning and sequence analysis of pep-carboxylase from cassava

Cassava (Manihot esculenta Crantz) is very drought tolerant and hows high levels of photosynthesis at high temperatures. This makes cassava an important source of carbohydrates for several arid regions, especially in Africa. Some varieties perform better than others. Physiological performance, which covers a wide range between C3 and C4 plants, could be increased through directed breeding. In order to do so, a better understanding of the underlying molecular mechanisms is needed, as to develop well defined breeding criteria and tests that could aid to that purpose. Several enzymes are involved in the fixation of CO2 in plants, phosphoenolpyruvate carboxylase playing a central role. We want to analyze the compartmentation of this and other photosynthetic enzymes between palisade and mesophyll cells by using in situ hybridization of the radiolabelled genes on histological sections. For this purpose the homologou gene must be isolated. We constructed a cassava genomic DNA library from the variety 996 CIAT in an EMBL 3 derived bacteriophage cloning vector. The library was screened with a maize ppc probe (obtained from T. Nelson, Yale), and after three rounds of purification, a putative cassava ppc clone of 10 kb was obtained. We are in the process of mapping and sequencing our clone. The next steps will include carrying out Northern analysis to assess levels of expression in different varieties, carrying out relatedness studies with other sequence characterized ppc genes, and the establishment of in situ hybridization techniques

Characterization of bacterial amylolytic activities during cassava solid state fermentation

Cassava sour starch is a naturally fermented product, which is used in the elaboration of several traditional, flour-based products, like pandeyuca and rosquillas, specially in Colombia and Brazil. Fermentation and drying under the sun confer to the product its specific baking properties (panification power, flavor and aroma). The main problem encountered in the production process, is the fluctuation in product quality. We have set out to study this very interesting solid state fermentation, whose only substrate is cassava starch. The fermentation process takes mainly place under anaerobic and low pH conditions. The production of lactic acid correlates well with the quality of the product. We are presently studying the enzymatic activities of the lactic amylolytic flora (LAB), homo-and heterofermentative. Several LAB strains were isolated on selective MRS starch medium with aniline blue. After Gram, catalase, sugar metabolism (API), and mobility analysis, the best 12 growing bacterial strains were selected for further analysis. They all seem to belong to the genus Lactobacillus. Amylolytic activities of these selected strains were chromatographically enriched from the medium, and their enzymatic parameters (Km. Vmax) determined, as well as their pH and temperature optima. The 12 strains show varying degrees of amylolytic activities. this has been clearly demonstrated on activity polyacrylamide gels, on which the different levels of activity as well as the isozyme patterns were observed. Protein fingerprinting using SDS PAGE was performed on these strains, again demonstrating the non-identity of the strains. Judging by the amylolytic activities and cell count numbers during the fermentation process, it seems likely that Lactobacillus spp are the dominating genus throughout the process

Dichloromethane as an economic alternative to chloroform in the extraction of DNA from plant tissues

Processing of large numbers smaples of plant tissue samples for molecular mapping and gene tagging requires methods that are quick, simple, and cheap, and that eventually can be automated. Organic solvents used for DNA extraction can represent a significant proportion of the overall cost. In this study we examined dichloromethane as a replacement for chloroform to be used in combination with phenol

Biofortified crops to alleviate micronutrient malnutrition

Micronutrient malnutrition affects more than half of the world population, particularly in developing countries. Concerted international and national fortification and supplementation efforts to curb the scourge of micronutrient malnutrition are showing a positive impact, alas without reaching the goals set by international organizations. Biofortification, the delivery of micronutrients via micronutrient-dense crops, offers a cost-effective and sustainable approach, complementing these efforts by reaching rural populations. Bioavailable micronutrients in the edible parts of staple crops at concentrations high enough to impact on human health can be obtained through breeding, provided that sufficient genetic variation for a given trait exists, or through transgenic approaches. Research and breeding programs are underway to enrich the major food staples in developing countries with the most important micronutrients: iron, provitamin A, zinc and folate

Aluminum stress inhibits accumulation of phosphorus in root apices of aluminum-sensitive but not aluminum-resistant brachiaria cultivar

The phosphorus (P) content of whole roots and root apices has been reported to increase under aluminum (Al) stress, particularly in Al-sensitive plant varieties. We investigated the impact of Al toxicity on the P content of root apices of two tropical forage cultivars of the Brachiaria genus, grown in Al-containing low-ionic-strength nutrient solutions. Exposure to Al did not affect apical P concentrations in Al-resistant B. decumbens cv. Basilisk. In contrast to previous results obtained with other plant species, however, it caused a 70% decline in the apical P content of Al-sensitive B. ruziziensis cv. Common, despite the simultaneously increasing P levels in older root portions. This effect was largely due to a reduced accumulation of organically bound P, because less than 7% of the P in root apices was in the inorganic form in either species or treatment. The P content of root apices might be a useful indicator for evaluating the resistance of Brachiaria cultivars to prolonged Al stress

Genetic potential and stability of carotene content in cassava roots

People in vast areas of the tropics suffer from vitamin A deficiency, resulting in progressive eye damage and eventually leading to blindness. Improving the content of vitamin A precursors in staple crops could alleviate or solve such a problem. The objective of this work was to study the range of variability for carotene content in a sub-set of the global cassava germplasm collection, and to determine the inheritance of carotenes, as well as their stability in response to different processing methodologies. Genotypes with more than 2 mg carotenes/100 g of fresh roots have been selected as parental material for population development. Although root colour is highly correlated with carotene content, a quantitative evaluation of genotypes selected by colour is required in order to increase the efficiency of selection. Relatively few major genes are involved in the determination of carotene accumulation in cassava roots. Stability of carotenes in response to different processing methods is genotypically dependant, representing a trait to be evaluated after selecting for high carotene concentration in fresh roots. The results from this work have provided the basis for defining future strategies for the improvement of the nutritional quality of cassava