Additional virulence genes in conjunction with efficient selection scheme, and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation

A critical step in the development of robust Agrobacterium tumefaciens-mediated transformation system in recalcitrant grain legume, cowpea is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. A dramatic increase in efficiency of T-DNA delivery was achieved by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404. A geneticin based selection system permitted rapid and efficient identification of transgenic shoots without interfering with their regeneration, and eliminated the bulk of escapes. Supplementation of 0.5 μM kinetin to medium containing 5.0 μM benzyl aminopurine after 1 week of culture followed by 3 weeks of culture were found critical for optimal multiplication and elongation of transformed shoots from cotyledonary node explants. Combining these three developments, we recovered fertile transgenic plants at a frequency of 1.64%, significantly higher than previous reports. The presence, integration, expression and inheritance of transgenes were confirmed by molecular analysis. The protocol developed for cultivar Pusa Komal will facilitate the transfer of desirable traits into cowpea.     

Callose formation as parameter for assessing genotypical plant tolerance of aluminium and manganese

Callose ((1,3)--glucan) formation in plant tissues is induced by excess Al and Mn. In the present study callose was spectrophotometrically quantified in order to evaluate whether it could be used as a parameter to identify genotypical differences in Al and Mn tolerance. Mn leaf-tissue tolerance of cowpea and linseed genotypes was assessed using the technique of isolated leaf tissue floating on Mn solution. Genotypical differences in the density of brown speckles on the leaf tissue (Mn toxicity symptoms) correlated closely with the concentrations of callose for both plant species. In cell suspension cultures Mn excess also induced callose formation. However, differences in tolerance of cowpea genotypes using callose formation as a parameter could only be found in cultured cowpea cells if controls cultured at optimum Mn supply showed low background callose. As soon as after 1 h, Al supply (50 M) induced callose formation predominantly in the 5-mm root tip of soybean seedlings. Callose concentration in the 0–30 mm root tips was inversely related to the root elongation rate when roots were subjected to an increasing Al supply above 10 M. Three soybean genotypes differed in inhibition of root-elongation rate and induction of callose formation when treated with 50 M Al for 8 h. Relative callose concentrations and relative root-elongation rates for these genotypes were significantly negatively correlated.     

Differences in calcium efficiency between cowpea (Vigna unguiculata (L.) Walp.) cultivars

The cowpea (Vigna unguiculata (L.) Walp.) cultivars TVu 354 and Solojo were grown in solution culture at 10 to 1000 M Ca supply. The Ca supply did not vary by more than 10% during the experiment. The pH value was kept constant within 0.1 units at 4.0 by automatic titration. The cultivar TVu 354 proved to be much more Ca-efficient than Solojo. At 10 M Ca supply Solojo died, whereas TVu 354 was hardly affected in dry matter production. The differences in Ca efficiency were independent of the P supply. They could not be explained by differences in Ca uptake or Ca concentrations in the plant tissue. Short-term studies using ⁴⁵Ca, both in the dark and in the light, indicated better transport of Ca from the roots to the shoots and within the shoots to the younger leaves in the Ca-efficient cultivar TVu 354. However, the main reason for the differences between the cultivars in sensitivity to low Ca supply were differences in the Ca requirement of the plant tissue to maintain tissue organization and function. Sequential fractionation of the freeze-dried leaf tissue with hot water, 0.5 M NaCl, 1 M CH₃COOH, and 2 M HCl did not reveal cultivar differences in Ca binding state. The results clearly show that considerable genetic potential in tolerance to low Ca supply exists in cowpea. However, a better understanding of the physiological/biochemical reasons for low internal Ca requirement is needed.     

Decomposition of cowpea and millet amendments to a sandy Alfisol in Niger

Current inputs of organic materials to cropped lands on sandy Alfisols and Entisols in Sahelian West Africa are insufficient to arrest soil organic matter (SOM) decline. Crop residues and green manures require proper management in order to maximize their contribution to nutrient supply and SOM maintenance. The objectives of this study were to quantify the rates of C and N mineralization from cowpea (Vigna unguiculata (L.) Walp.) green manure, cowpea residue, and millet (Pennisetum glaucum (L.) R.Br.) residue under field conditions in Niger and to determine the effect of these organic amendments on pearl millet yield. Millet was grown (1) as sole crop, (2) as intercrop with cowpea, (3) as intercrop with cowpea that was incorporated as green manure during the second half of the growing season, (4) with incorporated cowpea residue (2000 kg ha^–1), (5) with millet residue mulch (3000 kg ha^–1), and (6) with N fertilizer. Carbon loss as CO₂ from soil with and without organic amendment was measured three times per week during the growing season. Nitrogen fertilizer increased millet yield only in a year with a favorable rainfall distribution. Cowpea grown in intercrop with millet during the first part of the growing season and subsequently incorporated as green manure between millet rows increased millet grain yield in a year with sufficient early rainfall, which could be attributed to the rapid rate of decomposition and nutrient release during the first 3 weeks after incorporation. In a year with limited early rainfall, however, densely planted green manure cowpeas competed for water and nutrients with the growing millet crop. Incorporated cowpea residue and millet residue mulch increased millet yield. Surface applied millet residue had high rates of decomposition only during the first 3 days after a rainfall event, with 34% of the millet residue C lost as CO₂ in one rainy season. Recovery of undecomposed millet residue at the end of the rainy season was related to presence or absence of termites, but not to seasonal C loss. Millet residue mulch increased soil organic C content of this sandy Alfisol in Niger. Cowpea and millet residues had a greater effect on SOM and millet yield than cowpea green manure due to their greater rate of application and slower rate of decomposition.     

Uscana lariophaga, egg parasitoid of bruchid beetle storage pests of cowpea in West Africa: the effect of temperature and humidity

The stored-product bruchid pests,Callosobruchus maculatus (Fabricius) andBruchidius atrolineatus (Pic) cause considerable production losses in cowpea in West Africa.Uscana lariophage Steffan parasitizes the eggs of the bruchids both in the field and in storage. As chemical control of bruchids in traditional granaries is not appropriate for poor farmers, enhancement of the efficacy of the parasitoid by environmental manipulation has been investigated. The effect of temperature on the capacity ofU. lariophaga to parasitize eggs has been studied at eleven constant and three fluctuating temperatures within the range 10 to 45°C. Longevity of the female wasp decreased with increasing temperature. The rate of development increased linearly at temperatures from 17.5 to 35°C, but decreased from 35 to 40°C. Mortality of the developing wasp remained below 20% from 20 to 37.5°C, but outside this range, mortality reached 100% at 15 at 42.5°C. Most parasitization occurred at temperatures of 25 and 30°C. Sex ratio (percentage females) increased with temperature in the high temperature range. The intrinsic rate of increase (r_m) forU. lariophaga was highest in the temperature range from 30 to 37.5°C and was higher than that ofC. maculatus at all temperatures. While the r_m value ofC. maculatus did not vary much at temperatures from 25 to 35°C, the r_m value of the wasp doubled. Relative humidity did not effect longevity, egg-laying capacity, mortality, development time and sex ratio of the wasps withC. maculatus as host. However, withB. atrolineatus as the host, development time and mortality increased at lower relative humidity levels. The results indicate that temperature is the major regulating factor on the parasitoid. As the type of storage structure and its location (sun or shade) affects the temperature inside the store, ways are being investigated of manipulating the storage environment through temperature regulation to increase the impact of the parasitoid.     

The management of populations of vesicular-arbuscular mycorrhizal fungi in acid-infertile soils of a savanna ecosystem

A field trial was conducted at two sites in the savanna ecosystem of eastern Colombia to compare the effects of inoculation with vesicular-arbuscular mycorrhizal fungi (VAMF) ofBrachiaria dictyoneura (a tropical grass), cassava (Manihot esculenta), the tropical forage legume kudzu (Pueraria phaseoloides) andSorghum sp., and two phosphate sources. The second stage of the trial studied the effect of these pre-crop treatments on the subsequent growth, nutrition and VAM status of cowpea (Vigna unguiculata) andStylosanthes capitata in the following season, compared with both crops sown in native savanna. Inoculation significantly increased the levels of VAM and plant yields in the early growth stages of all crops during the first season, particularly with the rock phosphate (RP) source. The most significant increases were observed in the mycorrhiza-dependent cassava and kudzu crops up to 15 weeks after sowing, and were associated with increased foliar uptake of P and Mg. The effectiveness of the introduced inoculum was greater at the field site with a sandier soil. In the second season the levels of VAM in roots of cowpea andS. capitata were all increased significantly in pre-cropped plots compared with a savanna control. The increased presence of VAM was associated with significantly increased yields on plots previously sown to cassava, kudzu andSorghum sp. The data support the idea that increasing the VAMF inoculum potential of these acid-infertile soils by inoculation or pre-crops can greatly increase the rate of establishment of mycorrhiza-dependent host plants.     

Host selection by ovipositing cowpea weevils: patterning of input from separate sense organs

Ablations were performed to identify the sense organs used in host selection by ovipositing cowpea weevils, Callosobruchus maculatus. Antennae, foretarsi and palpi (maxillary + labial) were removed singly or in combination, and females were offered pairwise choices of four host species. Removal of the palpi consistently had the greatest effect on host choice, whereas the relative importance of other organs depended on the pair of hosts tested. Different organs may provide conflicting input as to the preferred host; certain ablations led to a complete reversal in preference (as opposed to a simple loss of discrimination). Input from separate organs appeared to be received in a dominance hierarchy.

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Sélection de l'hôte lors de la ponte de Callosobruchus maculatus: modélisation des influx provenant séparément des divers organes sensoriels

Résumé Les ablations ont servi d'étape initiale lors de la détermination des organes sensoriels impliqués dans la sélection du lieu de ponte par C. maculatus. Les antennes, les tarses antérieurs, les palpes (maxillaires et labiaux) ont été retirés seuls ou en combinaison. Les femelles ont eu des choix binaires entre les graines de 4 légumineuses: Cicer arietinum, Glycine max, Phaseolus vulgaris, Vigna unguiculata. Chaque combinaison d'ablations était accompagnée d'un témoin chez lequel les mêmes ablations étaient unilatérales. C'est l'ablation des palpes qui a le plus modifié le choix de l'hôte; l'influence relative des autres organes dépendait des paires d'hôtes offerts. Différents organes peuvent fournir des influx opposés quant à l'hôte préféré certaines ablations ont conduit à une inversion complète des préférences. Il semble que les influx sensoriels formaient une gamma avec hiérarchie dominante. Dans les choix binaires, les préférences initiales des femelles intactes ont été supprimées avec l'ablation des palpes seuls, cependant elles n'avaient pas été modifiées par l'ablation conjointe des palpes et des tarses. Dans ces expériences, l'influx antennaire ne devenait dominant qu'après ablation des deux autres organes sensoriels. Les préférences des femelles avec ablations unilatérales ne différaient pas de celles des femelles intactes.

     

Effects of soil applied herbicides on leaf nitrate reductase and crude protein in the leaf and seed of two cowpea cultivars

A glass-house study was conducted to determine the effects of four commonly used herbicides (pendimethalin, metobromuron, metolachlor and prometryne) applied pre-emergence at rates of 0, 0.125, 0.625 and 1.25 kg ha^–1, on leaf nitrate concentration (NO₃–C), nitrate reductase activity (NRA), leaf crude protein and seed protein in two cowpea cultivars, 60 day (60D) and Ife brown (IB).Control and treated plants of both cultivars showed separate peaks for NO₃–C and NRA, 49 days after planting (DAP) and 35 DAP for 60D and IB respectively. Herbicide treatment generally enhanced NO₃–C but tended to decrease NRA in both cultivars. Howver, metobromuron at 0.625 kg ha^–1 increased NRA throughout the growth period with an optimum increase of 52.5%, over the control, at 35 DAP. Pendimethalin increased NO₃–C NRA and leaf protein but did not influence seed protein appreciably. In contrast metobromuron increased NO₃–C, decreased NRA, but increased seed protein by 29.6% over the control at 0.125 kg ha^–1 in 60D. Metolachlor and prometryne were most inhibitory to seed protein development. In addition, metolachlor reversed the interdependence of NO₃–C and NRA.     

Ecology and genetics of tropical rhizobia species

Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation. So far most information on Rhizobium concerns fast-growing rhizobia and their host legume. Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils. The majority of the developing countries are in tropical and sub-tropical regions. Except for R. japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species. In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia. By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions. In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested. This involves close cooperation between molecular biologists and microbial ecologists.     

Effect of tissue phosphorus concentration on the mineralisation of nitrogen from stylo and cowpea residues

Laboratory studies were conducted to investigate the effect of phosphorus concentration in residues of cowpea (t Vigna unguiculata, L. Walp) and stylo (t Stylosanthes hamata, L., cv Verano) on their rate of nitrogen mineralisation when incubated in a soil whose P status was deficient for plant growth. Residues with a range of P concentrations were obtained by applying varying rates of P to soil in which the plants were grown in the field or the glasshouse. Variations in P concentration of field- or glasshouse-grown residues were not accompanied by variations in other chemical components (C:N ratio, lignin and polyphenol concentrations). Both lignin and polyphenol concentrations were higher in the field-grown than in the glasshouse-grown residues. Lignin concentration was greater in cowpea than in stylo, but polyphenols were higher in stylo. Cowpea residues mineralised N less rapidly than stylo. N mineralisation from residues with low P concentration was consistently less than from those of higher P concentration; reduced mineralisation was observed for P concentration in the residues below 1.6 g kg^–1. When inorganic P was added to the residue-soil systems, N mineralisation from the residues was increased, though no interaction between the effects of adding inorganic P and P concentration in the residues was observed.