Jorge Edgard Mayer

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology. However, the complexity of the patent landscape has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria–plant interactions to achieve plant transformation.

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.

Aluminum stress stimulates the accumulation of organic acids in root apices of Brachiaria species

Aluminum-resistant Brachiaria decumbens Stapf cv. Basilisk (signalgrass) and closely related, but less resistant Brachiaria ruziziensis Germain & Evrard cv. Common (ruzigrass) both accumulated high concentrations of aluminum (Al) in roots. Approximately two thirds of the total Al was complexed by soluble low-molecular-weight ligands, suggesting that it had been taken up into the symplasm. We therefore investigated whether these species might employ Al-chelating organic acids for internal detoxification of Al taken up by root apices, the primary site of Al injury. Unlike root apices of other Al-resistant plant genotypes, which secrete organic-acid anions to detoxify Al externally, apices of Brachiaria species accumulated organic acids within the tissue. A comparison with whole roots showed that this preference for accumulation (as opposed to secretion) was restricted to apices. Citric acid, and to a lesser extent trans-aconitic acid, accumulated in a uniform dose-dependent manner in root apices of both species as their Al content increased under Al-toxic growth conditions. Their accumulation was accompanied by a stimulation of malate synthesis in Al-resistant B. decumbens, while it occurred at the expense of malate in Al-sensitive B. ruziziensis. These data suggest a role of organic acids in the internal detoxification of Al in root apices of both Brachiaria species, presumably contributing to their comparatively high basal level of Al resistance. Yet internal detoxification of Al by organic acids does not appear to be the principal mechanism responsible for the superior resistance of B. decumbens. Aluminium-Stress stimuliert die Anreicherung von organischen Sauren in Wurzelspitzen von Brachiaria-Arten Sowohl das Aluminium-resistente Gras Brachiaria decumbens Stapf cv. Basilisk als auch das nah verwandte, aber weniger resistente Gras Brachiaria ruziziensis Germain & Evrard cv. Common akkumulierten hohe Aluminiumkonzentrationen in den Wurzeln. Etwa zwei Drittel des gesamten Aluminiums (Al) waren durch niedermolekulare Liganden komplexiert, was darauf hinweist, dass es im Symplasten lokalisiert war. Aus diesem Grund untersuchten wir, ob diese Pflanzenarten Al-komplexierende organische Sauren zur internen Entgiftung von Al in Wurzelspitzen (das gegenuber Al empfindlichste Organ) nutzen. Im Gegensatz zu Wurzelspitzen anderer Al-resistenter Pflanzengenotypen, die organische Saureanionen zur externen Al-Entgiftung sekretieren, reicherten beide Brachiaria-Arten organische Sauren im Wurzelspitzengewebe an. Ein Vergleich mit ganzen Wurzeln zeigte, dass diese Tendenz zur Akkumulation (im Gegensatz zur Sekretion) auf die Wurzelspitzen beschrankt war. Citrat, und in geringerem Ausmas trans-Akonitat, reicherten sich in Wurzelspitzen beider Arten mit steigendem Al-Gehalten bei Al-Angebot an. Ihre Akkumulation war in B. decumbens von einer Stimulation der Synthese von Malat begleitet, wahrend sie in B. ruziziensis auf Kosten von Malat erfolgte. Diese Ergebnisse deuten auf eine Beteiligung organischer Sauren an der internen Entgiftung von Al in Wurzelspitzen beider Brachiaria-Arten hin, was wahrscheinlich zu deren relativ hohem Resistenzniveau beitragt. Eine interne Entgiftung von Al durch organische Sauren scheint jedoch nicht der hauptsachliche Mechanismus zu sein, der fur die hohere Resistenz von B. decumbens im Vergleich zu B. ruziziensis verantwortlich ist.

Agrobacterium-mediated transformation of Stylosanthes guianensis and production of transgenic plants

Abstract Transgenic plants of the tropical forage legume Stylosanthes guianensis (Aubl.) Sw. CIAT 184 were regenerated from kanamycin- and phosphinotricin-resistant calli derived from Agrobacterium -inoculated leaf sections. Inoculations were carried out using the disarmed Agrobacterium tumefaciens strain EHA 101 harboring the binary vector pGV1040. pGV1040 T-DNA contains the bar and npt II genes as selectable markers, and the gus A gene as a screenable marker. Kanamycin was deleterious to plant regeneration, consequently phosphinotricin resistance was used for selection. Presence of the bar, npt II and gus A genes was detected during the regeneration process as well as in the transformed regenerated plants. Mendelian inheritance of the bar gene in the selfed progeny of the transformed plants was demonstrated.

Roots of nutrient-deprived Brachiaria species accumulate 1,3-di-O-trans-feruloylquinic acid

A novel di-hydroxycinnamoylquinic acid ester, 1,3-di-O-trans-feruloylquinic acid (DFQA), was isolated from roots of nutrient-deprived Brachiaria species--the most widely sown tropical forage grasses in South America. In contrast to other so far characterized quinic-acid esters, DFQA exists in a chair conformation with the carboxylic group in the axial orientation. It accumulates in older parts of the root system, but not in root apices or shoots. Higher levels were found in B. ruziziensis, which is poorly adapted to infertile acid soils, than in well adapted B. decumbens. DFQA was also found in the soil, most likely as a result of root decay, because it was not detected in root exudates of plants cultivated in solution culture. Nitrogen and phosphorus deficiency--but not aluminum toxicity or deprivation of other nutrients--stimulated its synthesis in roots. Its accumulation was correlated with a shift in biomass partitioning toward the root system.

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.

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

ABSTRACT 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.

Evaluation of a DNA probe for the quantitative detection of common bacterial blight in common bean and its application in a breeding program

SummaryBreeding of Phaseolus vulgaris L. for resistance to common bacterial blight (CBB) can be done with visual evaluations of symptoms to distinguish broad resistance classes, but a more quantitative measure was needed for genetic studies of resistance. A novel method of evaluation was developed by quantifying Xanthomonas campestris pv. phaseoli (XCP) in bean leaf tissue infected with CBB using a 32P-labeled probe and densitometric analysis of hybridization signals. Quantification of bacterial populations using the probe was highly correlated (r=0.98) with the number of colony forming units (CFU) from plate counts of the same leaf samples. The probe was used to follow XCP population dynamics on susceptible (BAT 41) and resistant (OAC 88-1) bean genotypes. OAC 88-1 supported a maximum XCP population which was approximately tenfold less than BAT 41. The probe was also used to study an F2/F3 population segregating for resistance. Narrow sense heritability estimates were less for resistance measured on the basis of bacterial populations (0.18–0.26) than on visual scores of symptoms (0.29–0.38). The anticipated response to selection for CBB resistance would be less based on bacterial numbers than based on symptom expression in this population. In breeding for resistance to CBB, selection based on visual symptoms combined with measurements of XCP populations using a DNA probe can be used to develop bean genotypes that are both resistant to symptom development and bacterial multiplication.