The main objective of the project is the genetic improvement of maize for a more successful incorporation of maize into cropping systems on acid soils of the tropics.

The better understanding of the physiological mechanism leading to acid soil tolerance in general and Al resistance and P efficiency as principal requirements for adaptation to such soils, specifically, will lead to the development of quick screening techniques and allow the better focus of molecular approaches thus facilitating and enhancing the progress made by breeding. Also, the prospects and limitation of such a genetic approach compared to an agronomic approach based on the amelioration of soil acidity using lime, fertiliser, and organic amendments are being evaluated.

The soils from three of the field experimental sites used in the breeding programmes and for the agronomic trials have been carefully characterised using appropriate methodologies by CIRAD. Major emphasis has been laid on the quantitative description of the Al exchange and dissolution characteristics a prerequisite for the modelling of the rhizosphere reactions in acid soils in relation to Al-induced inhibition of root elongation. The analysis revealed that the soils from Cameroon and Columbia have a very different Al dynamics related to their differences in contents of amorphous Al and their CEC.

The breeding programmes at EMBRAPA-CNPMS, Brasil, IRAD, Cameroun, and INRA, Guadeloupe identified germplasm with improved adaptation to soil acidity.

Breeding efforts at EMBRAPA-CNPMS, Brasil, have identified maize genotypes contrasting in both tolerance to Al stress and P acquisition/utilization efficiency under P stress. Tolerance to Al toxicity has been present in several new maize cultivar releases. The recent release BRS 3060, a triplecross maize hybrid, is both tolerant to Al toxicity and more efficient in P acquisition in P stress conditions. BRS 3060 is one of the best benchmark standards for P acquisition efficiency in P stress soils. These genetic standards have been distributed to project partners where they have been used to conduct whole plant and molecular physiology-based research to determine mechanisms for tolerance to Al toxicity and differences in P acquisition processes. Identification of these mechanisms is important to help direct crop improvement programs, particularly those based on plant biotechnology. Recombinant Inbred Lines (RILs) derived from a cross of contrasting maize lines for tolerance to Al toxicity have been used to identify QTLs associated with tolerance to Al toxicity. Six QTLs have been identified that explain 60% of variation of relative seminal root growth (RSRG) in nutrient solution with Al. Improved cultivars have been developed using technology and genetic resources developed at Embrapa Maize and Sorghum and released to private sector seed producers, and will continue to be developed and released independent of this project, but building on the technology generated from this project.

IRAD, CAMEROUN screened and evaluated inbred lines extracted from the acid soil-tolerant population ATP-SR. 180 test-crosses were made and were evaluated for their adaptation to acid Al-toxic soil. 37 maize cultivars from different origin, among them the cultivars agreed upon in the common breeding approach, were evaluated more intensively. Generally, classification for acid soil-tolerance/Al resistance was confirmed with, however, some exceptions. Some of the cultivars of Brasilian origin and advanced selections showed high yielding capacity independent of soil acidity correction.

In GUADELOUPE INRA conducted experiments at two soil correction levels. The objective was to measure the performance of experimental varieties, double cross hybrids, single crosses and synthetics selected for their adaptation to acid soil environments in Brasil, Colombia, Cameroon and Guadeloupe. In addition, plant characteristics correlated to responses on acid soil were assessed by determining their genetic variance, their heritability, and their genotype by environment interaction in order to predict genetic progress for yield and acid soil tolerance. ASI (anthesis to silking interval), number of ears per plant, plant height, ear rot, and number of kernels per unit surface exhibited interesting variation under acid soil as to be used as indirect selection criteria for acid soil-tolerance breeding. Genotypes from CIMMYT and Brasil with high yielding capacity on acid soil could be identified. Some selections from Guadeloupe proved to be especially adapted to the Guadeloupe environment.

In a joint breeding effort for which INRA, Guadeloupe, had taken the main co-ordinating responsibility, eleven tropical maize populations from different genetic origins and covering a wide range of adaptation to acid-soils abilities were crossed. The diallel was evaluated in Guadeloupe and Cameroon in acid and non-acid soil environments. Results assessed genetic progress for adaptation to soil acidity and permitted to identify traits of interest within the parental populations. Heterosis appeared higher than expected. Both additive and non-additive gene actions with predominance of non-additive effects were observed, suggesting to develop heterotic pools. Therefore, new crosses were made in Cameroon, source germplasms from Brazil and Guadeloupe were added, and a new set of trials was prepared in Guadeloupe for multi-location evaluation. The diallel trials were sown in Guadeloupe (INRA), Cameroon (IRAD) and Columbia (CIMMYT and CORPOICA).

In CAMEROUN a main activity was the evaluation of the diallel mentioned above. The evaluation of the results clearly indicate that selected genotypes from the own programme but also from CIMMYT, Cali, and Brasil can contribute greatly to enhance progress in breeding for acid soils in Cameroon. In addition, testcross and hybrid development was advanced and evaluated with the objective to identify inbred lines with high combining ability for yield on acid Al- and/or Mn-toxic soils and to identify hybrids with high yielding capacity and acid soil tolerance for release to farmers.

The diallel analysis by INRA revealed that both additive and non-additive gene effects for yield and secondary traits such as anthesis-silking interval and early vigor and cob set and high heterosis values will make a reciprocal recurrent selection process very promising for the development of open-pollinated cultivars and hybrids a conclusion which is also supported by the results in Cameroon. The results clearly showed complementary traits of interest for plant adaptation to soil acidity within the parental populations, such as plant vigor at young stage, earlyness, prolificacy, size and rate of growth. Although also in Guadeloupe the value of the CIMMYT germplasm and some of the material from Brasil for the development of acid soil-tolerant germplasm could be confirmed, there were quite distinct differences in the germplasm performance between Guadeloupe and Cameroon, clearly showing the need of multilocational testing and combined analysis of the data across locations. Across locations evaluation of this diallel and combined analysis provide valuable information on the genetics of adaptation and will allow the breeders to identify stable superior germplasm for the development of cultivars for acid soils of the tropics.

Different maize cultivars used in field experiments in Brasil, Guadeloupe, and Cameroon were characterised for Al resistance using different quick screening methods, medium-term root elongation in low ionic nutrient solution (CIRAD), hematoxilyn staining (UAB, EMBRAPA), and Al-induced callose formation (UH) with fairly comparable classifications. It could be confirmed that the selection of cultivars for the agronomic experiments conducted in Cameroun and in Columbia have been made on correct assumptions concerning adaptation to soil acidity based on Al resistance. A major effort was made by UH in co-operation with CIMMYT, Cali, with the objective to evaluate the prospects of using Al-induced callose formation as an indicator not only for Al resistance as previously demonstrated but also for yielding capacity in an acid, Al-toxic soil. A non-destructive test was developed allowing the testing of single seedlings for callose formation and subsequently yield formation after transplanting to the field.

The physiological studies by EMBRAPA-CNPMS continued to be focused on the better understanding of the physiological, morphological mechanisms of P efficiency and the effect of P deficiency on N uptake and N assimilation. A series of controlled experiments were designed and conducted using contrasting genetic standards to identify mechanisms controlling tolerance to Al toxicity and improved phosphorus efficiency and to study mode of inheritance of these traits. We now understand that the inheritance for tolerance to Al toxicity is simpler and more straightforward. However, it appears that more than one mechanism is involved in Al resistance in maize, and different tolerant genotypes may actually have different mechanisms or multiple mechanisms. The results demonstrated that one of the traits associated with P acquisition efficiency is greater root growth of more P-efficient genotypes at low P supply.

Rainfall distribution during the growing period in the Cerrados may be sporadic and irregular, leading to a scenario of repeated wetting and drying of the soil profile. As the soil dries, phosphate concentration in the soil solution approaches zero, NO3-N concentration is reduced and the NH4-N/NO3-N ratio increases. Cultivars unable to cope in this scenario will produce poorly, but those that are more resilient and can temporarily endure repeated cycles of low P levels and utilize the NH4+-N more efficiently should have greater and more stable production. Several studies have been initiated to help better understand what occurs in this type of scenario.

Research is currently underway at EMBRAPA to evaluate organic acid exudates of maize roots under both P and Al stress, principally citrate and malate, with the objective of understanding the effect of these molecules on tolerance to toxic Al and the efficiency of P absorption by the root. Currently, new procedures are being evaluated to improve the efficiency of qualifying and quantifying these organic acids in complete nutrient solution. High affinity phosphate transporter probes have been obtained to study the induction of maize high affinity phosphate transporter genes under P stress in the genetic maize standards for P uptake efficiency.

The understanding of mechanisms that regulate the tolerance to nutrient stress or improved plant nutrition in this acid soil complex, at the plant, cell and molecular levels; and the development of genetic markers is expected to accelerate the breeding program of developing high yielding cultivars better adapted to the acid soil complex of the tropical acid savannas of the world.

The research activities at the Autónoma University of Barcelona mainly focussed on the following aspects: (i) Time-dependent changes in apoplastic and vacuolar Al in Al-resistant and Al-sensitive maize cultivars in relation to Al-induced injury, (ii) Exudation of organic acids as a possible mechanism for detoxification of phytotoxic Al species in the rhizosphere, and (iii) The possible role of different, operationally defined polyamine fractions as stress markers or as internal protective substances in maize.

A detailed analysis of root elongation under Al-stress revealed clear differences in the time-dependence of the responses of Al-sensitive and Al-resistant maize cultivars. These differences in time dependence of Al-induced injury between Al-sensitive and Al-resistant cultivars were accompanied by clear differences in the accumulation and compartmentation of Al in the root tips as revealed by different Al staining techniques. Energy dispersive X-ray microanalysis (EDXMA) confirmed the time-dependent change in Al distribution in root tips of Al-resistant maize cultivars. The failure to detect Al in cell walls of the Al-resistant cultivar after 24 h exposure, while Al was found after shorter exposure times indicate that in resistant cultivars resistant mechanisms are activated that lead to a change in the speciation of apoplastic Al.

The possibility that in resistant cultivars internal detoxification mechanisms may play an additional role is indicated by the finding that Al was rapidly detected in root tip vacuoles after 4 h exposure to Al as electron-dense vacuolar deposits resembling the mineral composition of phytate.

The quantification of the organic acid-anion exudation under sterile conditions revealed that Al-resistant cultivars exuded organic acids anions in nutrient solution, particularly oxalate, from root tips, both under control conditions and when exposed to Al.

A possible role of polyamines in the protection of plants against Al toxicity is indicted: Al-resistant cultivars showed significantly higher concentrations of soluble spermine (spm) and spermidine (spd) levels than Al-sensitive cultivars. The exposure to 50 ÁM Al for 24 h caused a much higher increase of the soluble polyamine levels in sensitive than in resistant cultivars. This change in the concentrations of free polyamines is in line with the role of this polyamine fraction as stress indicator. On the other hand, in Al-resistant cultivars the bound polyamine fraction was approximately ten times higher than in the sensitive cultivars. The higher concentration of bound polyamines in Al-resistant cultivars is in line with the idea of a protective role of polyamines against Al.

Physiological research activities at the Univiversity of Hannover concentrated on four aspects during the project: (i) Validation of the screening for adaptation of maize cultivars to acid soils with high Al supply using homogenous Al supply to the roots in nutrient solution (Al avoidance), (ii) Comparative physiological characterisation of the P and Al stress responses of cultivars differing in Al resistance and P efficiency, (iii) Development of a technique to assess differences between maize cultivars in the mobilisation capacity of maize cultivars for soil and fertiliser P, and iv) Verification of the importance of root cell-wall properties for Al resistance.

When Al was applied to only one half of the root system of maize plants of both the Al-resistant cultivar and Al-sensitive cultivars, neither inhibition of root elongation of Al-treated roots nor growth of the roots not treated with Al were enhanced compared to roots uniformly treated with Al or not treated with Al at all, respectively. This result was independent of the P concentration of the nutrient solution. We therefore conclude that in maize an Al avoidance mechanism does not interfere with the assessment of genotypic Al resistance by application of Al homogeneously to the entire root system.

Differences between cultivars in P uptake at P-limiting e were closely related to the P content of the seeds. Therefore, we studied physiological responses to P deficiency in solution culture-grown plants which meight contribute to P-acquisition efficiency. P deficiciency was expressed in an increase of the root/shoot ratio of all cultivars. Although the cultivars differed in their root there was no overall significant decrease of the root diameter of P-deficient plants as was expected. All cultivars showed a net proton excretion per unit of rootsurface. However, only 4 cultivars responded to P deficiency by a significantly enhanced net proton excretion-rate. Exudation of organic acid anions significantly differed between P, Al treatments and cultivars. Al supply rather than P deficiency clearly enhanced the release of organic acid anions particularly and specifically of citrate. Cultivars classified as Al-resistant had a higher citrate exudation-rate than Al-sensitive cultivars. The main characteristic of Al-resistant ATP-Y was the capability to maintain a high citrate exudation rate over 24h whereas in all other cultivars the rate declined with Al-treatment duration.

For the characterisation of the P mobilisation capacity of maize seedlings a sandwich technique was developed.. The central compartment contained a sand/perlite mixture and was supplied regularly with all nutrients required for plant growth except P which had to be mobilised from the side compartments. The side compartments were separated from the central compartment (with the roots) by a 40 Ám nylon membrane, which allowed only root hairs and root exudates to penetrate into the soil compartments. The 10 cultivars differed widely in the depletion of readily extractable P. Unfortunately, in spite of promising initial results the NaOH-P fraction reflecting P mobilisation through root exudates did not reveal clear depletion of rhizosphere P in this fraction. The depletion curves allowed the calculation of the P depletion from the 2 fraction and it's comparison to P uptake. No relationship was obvious between the reported adaptation of the cultivars to acid, Al-toxic soils with low P availability.

Zea mays L suspension cells were modified in cell wall properties by long-term treatment to grow on 200 mM NaCl or DCB or short-term treatment with pectolyase or pectin methylesterase. Cells were then incubated for 2 h with different AlCl3 concentrations or 20 ÁM digitonin. After 2h Al treatment of the cell suspension-cultures, there was a close negative relationship between the degree of cell-wall pectin methylation (DM) and relative callose formation or Al contents.

Due to the lack of suitable transgenics in maize, the role of the DM for Al sensitivity was further supported using potato plants modified to differentially express pectin methyl-esterase, controlling the negative charge of the cell-wall pectins. Based on root elongation, Al-induced callose formation and Al contents of root tips as parameters transgenic potato mutants over-expressing PME proved to be more Al-sensitive than the wild type, the Al-resistant and even the Al-sensitive cultivar.

In the area of agronomy CORPOICA, Colombia, and IRAD, Cameroun, conducted complex long-term field experiments with the objective to evaluate the long-term implications of strategies for improving maize production on acid soils: introduction of improved, soil acidity-tolerant germplasm, amelioration of soil acidity using lime or organic amendments, or both. At both locations the maize cultivars improved for soil-acidity tolerance out-yielded the non-adapted and local cultivars at zero level of correction. The latter cultivars responded more to lime application. All cultivars responded to organic matter application. Highest maize grain yields were achieved with chicken manure application, but also application of cowpea (COLOMBIA) and particularly of Cassia septabilis (CAMEROUN) green manure produced yield increases. The results cleary show that at both locations organic amendments can at least partly substitute for lime application in soil acidity-sensitive maize cultivars. However, at the Cameroon site, lime application was necessary and highest yield were only achieved when both, lime and chicken manure were applied. While in Colombia P application was very beneficial, P application did not give a significant grain yield increase for the two cultivars on the acid soil of Cameroon. Statistical analysis by CORPOICA of the 3-years field experimental results confirmed the superiority under control conditions of the adapted, acid soil-tolerant maize cultivar. Chicken manure but not cowpea shoots strongly improved the yield of the non-adapted cultivars particularly be reducing soil acidity and improving base saturation of the soil. There is no indication by soil and plant analysis so far that soils are deteriorated by the use of acid soil-tolerant germplasm, but high productivity can only be achieved by both genetic and agronomic strategies.

The evaluation of the root growth studies conducted in close co-operation with CIRAD using the software package “RACINE” developed by CIRAD revealed that root-length densities in the topsoil were mainly affected by application of organic matter (cowpea and chicken manure). Organic matter application enhanced roots length by soil volume, especially in the chicken-manure application and cultivar Sikuani. There were, however, no significant consistent differences between maize cultivars. Software “RACINE” has been improved and has proved to calculate root-length densities from root counts on a vertical transversal plane using a locally improved model. The methodological studies (software, models) conducted within the project allowed to show that it is possible to describe root-length density (RLD) distribution from root counts in the profile (grid method) and to infer availability to the root system of soil nutrients and water. Soil management practices (manure, lime) increased RLD in the upper layers of the of studied acid soil, but it was not possible to improve the physical characteristics of deep soil layers for root exploitation (below 30 cm). Therefore, the studied agronomic practices did not clearly improve the availability of soil moisture and nutrients from the subsoil.

In the area of plant-soil relationships CIRAD in co-operation with INRA-UFR, Monpellier, developed a soil ionic exchange model using a simplified system and calibrated it using soil from the field experimental sites in Cameroon and Colombia. The fitting of the intrinsic dissociation constants of the ions was carried out using a wide range of pH and Al/Ca ratios in solution. The model was validated using a culture device allowing the study of Al dynamic between soil and roots. The results show that the mechanistic model is capable to describe the dynamics of the ions, mainly Al3+, H+ and Ca2+ in the rhizosphere of maize cultivated on acid soils but requires to take into account permanent and variable exchange sites as well as the kinetics of solubilisation of soil constituents containing Al. The model shows that the rate of Al adsorption on the root, i.e Al-tolerance or Al-sensitivity, is limited by a kinetic factor relative to the rate of Al dissolution which associate the dissolution and the transport of Al of mineral surface with solution. Moreover, it is shown that the equilibrium of Al in solution can himself refer with various type of Al material more or less soluble. The slower rate of Al dissolution in the case of the soil of Cameroon authorises rhizospheric pH lower than in the case of the soil of Colombia. For this last soil, the Al concentration in solution is equal to that defined by the solubility of the gibbsite. In the case of the Cameroon the Al concentration near the root is lower than that defined by the product of solubility of the gibbsite. Others compounds of the Al silicates could then define the Al concentration in solution. The plant decreases the concentration of the ions in the soil solution that can be saturated with regard to Al hydroxides. The model allows to describe the the dynamics of the ions in the rhizosphere. These results highlight the importance of the processes of dissolution and diffusion of Al in the rhizosphere and the necessity to take into consideration Al-complexing compounds such as organic acids.

In conclusion, the better understanding of the inheritance and of the physiological mechanism leading to acid soil tolerance in general and Al resistance and P efficiency as principal requirements for adaptation to such soils, specifically, lead to the development of quick screening techniques and allowed the better focus of molecular approaches thus facilitating and enhancing the progress made by breeding. Consequently, new varieties with improved yields on acid soils have been released in Brasil and Cameroon.

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