In vitro plant regeneration via organogenesis of cowpea [Vigna unguiculata (L.) Walp.]

Shoot regeneration via organogenesis was achieved from axenic cowpea [Vigna unguiculata subsp. unguiculata L. (Walp.) Verde.] hypocotyls and cotyledons of advanced breeding lines and varieties. Cotyledons and embryos were excised from green immature pods. The apical parts of the embryos were removed and the hypocotyls were transferred to regeneration media. Cotyledons and hypocotyls were tested on media with gradients of several hormonal and putrescine combinations. Cowpea cotyledons and hypocotyls exhibited a pattern of shoot formation that occurred in three distinct phases. Multiple shoots developed within 45 days from the wounded region of the primary hypocotyl and cotyledons in different media containing a high cytokinin concentration. The induced plant explants were then grown for 20 days in low-intensity light (10 μmol m^–2 s^–1) on the same medium and numerous shoot buds emerged de novo from the upper part of the hypocotyl and the wounded part of the cotyledons. These buds had no apparent vascular connection with the parent tissues. The plant regeneration capability of this procedure was tested with several cowpea genotypes, five of which (83D-442, 86D-1010, 93K-624, Vita 3 and Ife Brown) responded positively with shoot development and were able to form roots and whole plants. Some somaclonal variation was observed. Received: 14 June 1996 / Revision received: 14 December 1996 / Accepted: 25 January 1997     

Mapping QTL for drought stress-induced premature senescence and maturity in cowpea [Vigna unguiculata (L.) Walp.]

Cowpea is an important crop for subsistence farmers in arid regions of Africa, Asia, and South America. Efforts to develop cultivars with improved productivity under drought conditions are constrained by lack of molecular markers associated with drought tolerance. Here, we report the mapping of 12 quantitative trait loci (QTL) associated with seedling drought tolerance and maturity in a cowpea recombinant inbred (RIL) population. One hundred and twenty-seven F₈ RILs developed from a cross between IT93K503-1 and CB46 were screened with 62 EcoR1 and Mse1 primer combinations to generate 306 amplified fragment length polymorphisms for use in genetic linkage mapping. The same population was phenotyped for maintenance of stem greenness (stg) and recovery dry weight (rdw) after drought stress in six greenhouse experiments. In field experiments conducted over 3 years, visual ratings and dry weights were used to phenotype drought stress-induced premature senescence in the RIL population. Kruskall–Wallis and multiple-QTL model mapping analysis were used to identify QTL associated with drought response phenotypes. Observed QTL were highly reproducible between stg and rdw under greenhouse conditions. Field studies confirmed all ten drought-response QTL observed under greenhouse conditions. Regions harboring drought-related QTL were observed on linkage groups 1, 2, 3, 5, 6, 7, 9, and 10 accounting for between 4.7 and 24.2% of the phenotypic variance (R ²). Further, two QTL for maturity (R ² = 14.4–28.9% and R ² = 11.7–25.2%) mapped on linkage groups 7 and 8 separately from drought-related QTL. These results provide a platform for identification of genetic determinants of seedling drought tolerance in cowpea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Potassium and magnesium relationship in cowpea (Vigna unguiculata (L.) Walp.)

Summary The effects of K and Mg application on dry matter yield and uptake of K, Mg and Ca in cowpea were studied in greenhouse at Haryana Agricultural University, Hissar (india). Dry matter yields of leaves, stems and roots increased by 17, 30 and 27 per cent over control due to application of 150 ppm K and 17, 16 and 26 per cent by 40 ppm Mg respectively. Potassium application has antagonistic effect on Ca concentration of leaves, stems and roots and synergistic on root Mg concentrations upto 25 ppm K. However, Mg had a synergistic effect on concentration of K upto 20 ppm Mg and antagonistic at 40 ppm Mg in all plant parts. Uptake of K, Mg and Ca increased by Mg application, but K increased only K uptake.     

Interactive effects of high temperature and elevated carbon dioxide concentration on cowpea [Vigna unguiculata (L.) Walp.]

Limitations in carbohydrate supplies have been implicated as a factor responsible for reproductive failure under heat stress. Heat stress affects two stages of reproductive development in cowpea [Vigna unguiculata (L.) Walp.], and genotypes are available with tolerance and sensitivity to heat during these different stages. The objectives of this study were to determine the responses of these cowpea lines to ambient and elevated [CO₂], under heat stress and optimal temperature, and test whether differences in carbohydrate supplies due to genotypes, CO₂ enrichment and heat stress are associated with differences in sensitivity to heat during reproductive development. Plants were grown in reach-in growth chambers and subjected to day/night temperatures of either 33/20 or 33/30°C, and [CO₂] levels of either 350 or 700 μmol mol^-1. Under intermediate night temperature (33/20°C), all lines set substantial numbers of pods. Under high night temperature (33/30°C) with either ambient or elevated [CO₂], one heat-sensitive line produced no flowers and the other set no pods, whereas the heat-tolerant line abundantly set pods. High night temperature reduced the overall carbohydrate content of the plants, especially peduncle sugars, and caused decreases in photosynthetic rates. The high pod set of the heat-tolerant line, under high night temperature, was associated with higher levels of sugars in peduncles compared with the heat-sensitive lines. The heat-tolerant line accumulated substantial shoot biomass, exhibited less accumulation of starch in leaves, and possibly had less down-regulation of photosynthesis in response to CO₂ enrichment and heat stress than the heat-sensitive lines. Elevated [CO₂] resulted in higher overall carbohydrate levels in heat-sensitive lines (starch in leaves, stems and peduncles), but it did not increase their heat tolerance with respect to flower production or pod set. Heat-induced damage to floral buds and anthers in the sensitive lines was associated with low sugars levels in peduncles, indicating that heat had greater effects on assimilate demand than on leaf assimilate supply. The heat-tolerant line was the most responsive genotype to elevated [CO₂] with respect to pod production under either high or intermediate temperatures.     

Allozyme diversity of cultivated cowpea Vigna unguiculata (L.) Walp.

A survey of allozyme variation in cultivar-groups of cowpea [Vigna unguiculata (L.) Walp.] was undertaken by examining 21 enzyme systems encoded by 36 loci in 271 accessions representing the five cultivar-groups. Very low levels of variation were found within accessions, which is typical of self-pollinating species. Little variation was also found among accessions. Compared with other legume crops, V. unguiculata is depauperate in allozyme variation. We found an average of 1.61 alleles per locus with 42% of the loci polymorphic and a total heterozygosity of 0.061. Of the variation present, 90% was found within cultivar-groups, while 10% was among cultivar-groups. Data analyses revealed continuous variation among cultivar-groups and geographic regions with the accessions failing to segregate into discrete morphophysiological or geographic clusters. However, evolved cultivar-groups (cv.-gr. Melanophthalmus and cv.-gr. Sesquipedalis) appear to be less diverse than their putative primitive cultivar-group progenitors. Due to the lack of availability of critical material, no clear center of origin can be established. However, the data presented suggest that Northeast Africa could be a possible center of domestication. Received: 18 February 1999 / Accepted: 4 November 1999     

Callose deposition in leaves of cowpea (Vigna unguiculata [L.] Walp.) as a sensitive response to high Mn supply

First macroscopic visible symptoms of Mn toxicity in cowpea (Vegna unguiculata [L.] Walp.) plants grown in solution culture were dark brown spots on the older leaves. Close to these spots, large quantities of substances which fluorescence with aniline blue were deposited, indicating formation of (1,3)-β-glucan (callose). Callose formation in the leaf epidermis was a more sensitive indicator of Mn toxicity than the appearance of macroscopic symptoms, or the Mn concentration in the leaf.     

Restriction site polymorphism-based candidate gene mapping for seedling drought tolerance in cowpea [Vigna unguiculata (L.) Walp.]

Quantitative trait loci (QTL) studies provide insight into the complexity of drought tolerance mechanisms. Molecular markers used in these studies also allow for marker-assisted selection (MAS) in breeding programs, enabling transfer of genetic factors between breeding lines without complete knowledge of their exact nature. However, potential for recombination between markers and target genes limit the utility of MAS-based strategies. Candidate gene mapping offers an alternative solution to identify trait determinants underlying QTL of interest. Here, we used restriction site polymorphisms to investigate co-location of candidate genes with QTL for seedling drought stress-induced premature senescence identified previously in cowpea. Genomic DNA isolated from 113 F_2:8 RILs of drought-tolerant IT93K503-1 and drought susceptible CB46 genotypes was digested with combinations of EcoR1 and HpaII, Mse1, or Msp1 restriction enzymes and amplified with primers designed from 13 drought-responsive cDNAs. JoinMap 3.0 and MapQTL 4.0 software were used to incorporate polymorphic markers onto the AFLP map and to analyze their association with the drought response QTL. Seven markers co-located with peaks of previously identified QTL. Isolation, sequencing, and blast analysis of these markers confirmed their significant homology with drought or other abiotic stress-induced expressed sequence tags (EST) from cowpea and other plant systems. Further, homology with coding sequences for a multidrug resistance protein 3 and a photosystem I assembly protein ycf3 was revealed in two of these candidates. These results provide a platform for the identification and characterization of genetic trait determinants underlying seedling drought tolerance in cowpea.     

Fast-growing bacteria from nodules of cowpea (Vigna unguiculata (L.) Walp.)

D akora , F.D. & V incent , J.M. 1984. Fast-growing bacteria from nodules of cowpea ( Vigna unguiculata (L.) Walp.) Journal of Applied Bacteriology 56 , 327–330.
First plating from nodules of cowpea frequently yielded fast-growing large colonies, either apparently uniform or associated with small colony forms typical of the expected slow-grower ( Bradyrhizobium ). Most cultures from single large colonies nodulated both cowpea and siratro ( Macroptilium atropurpureum ), but all such nodules revealed Bradyrhizobium alone or associated with a fast-growing form. Six of nine plants inoculated with a mixed inoculum of slow and fast forms had nodules occupied by both although in no case was the fast-grower able to secure solo invasion. Most of the fast-growing forms shared some internal antigens with Rhizo-biurn meliloti and/or R. trifolii ; none reacted with antiserum to Bradyrhizobium CB 756.     

Rhizobial-induced increase in internode length and identification of endogenous GAs of cowpea (Vigna unguiculata [L.] Walp) stems and nodules

Inoculation with Bradyrhizobium sp. strain 127E14 has been shown to cause a dramatic increase in the internode length of lima bean (Phaseolus lunatus L.), when compared to control plants inoculated with strain 127E15. This rhizobial-induced growth also occurs in cowpea (Vigna unguiculata [L.] Walp), an alternate host for the symbiont. Cowpea plants inoculated with strain 127E14 were 23% taller than those inoculated with strain 127E14 after 6 weeks of growth. Petiole length was found to be significantly greater in plants inoculated with strain 127E14. Cowpea plants treated at the apex with exogenous GA₃ or GA_4/7 responded by increasing internode length when compared to controls. As in lima beans, the rhizobial-induced growth response observed in cowpeas may be in response to an imbalance in the levels of GA-like substances within the plants. Gibberellins A₁, A₃, A₈, A₁₉, A₂₀, A₂₉, and A₄₄have been identified by GC-MS analysis in stems of cowpea, whereas the gibberellins A₁, A₁₉, A₂₀, A₂₉, and A₄₄ were found to be present in nodule tissue formed by strain 127E14. The presence of these GAs indicates that the early 13-hydroxylation biosynthetic pathway is operative in cowpea. GAs identified in cowpea nodules are similar to those found in lima bean nodules formed by the same rhizobia. The finding that rhizobial strain 127E14 induces GA-dependent growth responses in two host legumes further supports the hypothesis that the presence of this bacteria alters the GA balance within the plant.     

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.     

Nodulation and symbiotic nitrogen fixation of cowpea (Vigna unguiculata (L.) Walp)

Summary Rhizobium strains CIAT 301, CIAT 79 and SLM 602 were tested and found effective in the nodulation and nitrogen fixation of cowpea cv. MI-35 (Vigna unguiculata (L.) Walp) plants in growth chamber experiments. Fresh weight of nodules increased with plant age initially and stabilized in 20–30 days from planting, followed by a secondary flush of nodule growth after 30 days. Apparent nitrogen fixation per gram nodule fresh weight reached a maximum in 20–30 days after planting and then decreased, even though a flush of new nodules was produced.     

Mineralization of labeled N from cowpea [Vigna unguiculata (L.) Walp.] plant parts at two growth stages in sandy soil

Cowpea [Vigna unguiculata (L). Walp.] has great potential as green manure due to its rapid N accumulation and efficient N₂ fixation. The objective of this study was to measure the rate of N mineralization from cowpea plant parts harvested at onset of flowering (5 weeks) and mid pod-fill (7 weeks) under near optimum conditions. Cowpeas were grown in a greenhouse and supplied with ¹⁵NH₄ ¹⁵NO₃ to isotopically label tissue. Cowpea leaves, stems, and roots were incorporated into a sandy soil (Psammentic Paleustalf) and net N mineralized was measured several times during a 10 week incubation. The amount of N accumulated in 7-week old cowpeas was more than double that in 5-week old cowpeas. The portion of N mineralized after 10 weeks was 24% for 5-week old cowpeas and 27% for 7-week old cowpeas. The rate of N mineralization from leaves and stems increased with plant age, but decreased for roots. The amount of N mineralized from 7-week old cowpeas was more than double (235%) that from 5-week old cowpeas due to greater N accumulation and a more rapid rate of N mineralization of the more mature cowpeas. The greatest amount of N was released from leaves, which amounted to 74 and 65% of total N mineralization from 5- and 7-week old cowpeas, respectively. The percentage of N mineralized by 10 weeks was linearly related to the tissue N concentration of the plant parts and to their C/N ratio. These relationships allow a quick estimation of the amount of N that would mineralize from cowpea residues incorporated into soil based on their N concentration or C/N ratio.     

De Novo Purine Synthesis in Nitrogen-Fixing Nodules of Cowpea (Vigna unguiculata [L.] Walp.) and Soybean (Glycine max [L.] Merr.)

Partially purified, cell-free extracts from nodules of cowpea (Vigna unguiculata L. Walp. cv. Caloona) and soybean (Glycine max L. Merr. cv. Bragg) showed high rates of de novo purine nucleotide and purine base synthesis. Activity increased with rates of nitrogen fixation and ureide export during development of cowpea plants; maximum rates (equivalent to 1.2 micromoles N₂ per hour per gram fresh nodule) being similar to those of maximum nitrogen fixation (1-2 micromoles N₂ per hour per gram fresh nodule). Extracts from actively fixing nodules of a symbiosis not producing ureides, Lupinus albus L. cv. Ultra, showed rates of de novo purine synthesis 0.1% to 0.5% those of cowpea and soybean. Most (70-90%) of the activity was associated with the particulate components of the nodule, but up to 50% was released from this fraction by osmotic shock. The accumulated end products with particulate fractions were inosine monophosphate and aminoimidazole carboxamide ribonucleotide. Further metabolism to purine bases and ureides was restricted to the soluble fraction of the nodule extract. High rates of inosine monophosphate synthesis were supported by glutamine as amide donor, lower rates (10-20%) by ammonia, and negligible rates with asparagine as substrate.     

Maternal inheritance of plant variegation in cowpea, Vigna unguiculata (L.) Walp.

A chimeric plant was observed in the F₂ generation of a cross between a mutant cultivar, Ife BPC, and a germplasm line, TVu 2, in cowpea, Vigna unguiculata (L.) Walp. The chimeric plant had four lateral branches, one of which was intensely variegated, while the others were mostly green with few white sectors. F₃ progeny from the intensely variegated branch of this plant were all variegated, while seed derived from the mostly green branches produced only green progeny. In subsequent generations, the descendants of the variegated branch bred true for the variegated trait, while those of the mostly green branches were also true-breeding for green colour. No pure-green or pure-white shoots were observed in any of the variegated plants examined in this study. Consequently, no pure-green or pure-white seedlings were produced from seeds harvested from the variegated plants. The results of reciprocal crosses between variegated and normal green plants indicate that variegation is inherited in a strictly uniparental maternal fashion. This is the first report of a cytoplasmically inherited mutation affecting foliage colour in cowpea. Received: 10 March 2000 / Accepted: 16 May 2000     

Interaction between Cu toxicity and P deficiency in soil-grown cowpea (Vigna unguiculata (L.) Walp.)

Anthropogenic contamination with Cu is an important issue and it is necessary to understand how Cu toxicity influences the uptake/acquisition of nutrients by plants. An experiment was conducted with soil-grown cowpea (Vigna unguiculata (L.) Walp.) to investigate the interaction between Cu toxicity and P deficiency. Plant performance was related to the activity of Cu²⁺ at the outer surface of the root plasma membrane, {Cu²⁺} ₀ ^o , which was calculated from properties of the soil solution. The addition of Cu to the soil was found to reduce growth of plant shoots by inducing Cu toxicity, which was associated with a reduction in the shoot tissue Fe concentration. The critical value (50% reduction in shoot growth) determined for {Cu²⁺} ₀ ^o in this soil-based experiment (3.8 μM) corresponds well to values determined previously. Importantly, regression analyses indicated that although the alleviation of P deficiency improved overall growth, the P-status of the plant did not influence the apparent toxicity of the Cu. This result was unexpected, given that Cu inhibits the growth of roots hairs; these being important for the uptake of immobile nutrients such as P. This study advances our understanding of Cu toxicity and its impact upon nutrient uptake.     

Cellular compartmentation of ureide biogenesis in root nodules of cowpea (Vigna unguiculata (L.) Walp.)

Cowpea (Vigna unguiculata (L.) Walp.) nodules have been investigated by means of cytochemical and immunocytochemical procedures at the ultrastructural level in order to assess the role of the uninfected cells in ureide biogenesis. Uricase activity in the nodules was shown by cytochemical methods to be localized exclusively in the numberous large peroxisomes confined to the uninfected cells; the small peroxisomes in the infected cells did not stain for uricase. Uricase was also localized in the peroxisomes of uninfected cells by immunogold techniques employing polyclonal antibodies against nodule-specific uricase of soybean. There was no labeling above background of any structures in the infected cells. The results indicate that the uninfected cells are essential for ureide biogenesis in cowpea. Although tubular endoplasmic reticulum, the presumptive site of allantoinase, increases greatly in the uninfected cells during nodule development, it virtually disappears as the nodules mature. The inconsistency between the disappearance of the tubular endoplasmic reticulum from older nodules and the high allantoinase activity reported for older plants remains to be explained.Abbreviations DAB 3,3-diaminobenzidine - ER endoplasmic reticulum - GARG goat anti-rabbit immunoglobulin G - IgG immunoglobulin G - kDa knodalton - Mr apparent molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

In vitro somatic embryogenesis from cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

We report, an efficient protocol for plantlet regeneration from the cell suspension cultures of cowpea through somatic embryogenesis. Primary leaf-derived, embryogenic calli initiated in MMS [MS salts (Murashige and Skoog 1962) with B5 (Gamborg et al. 1968) vitamins] medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D), casein hydrolysate (CH), and l-Glutamic acid-5-amide (Gln). Fast-growing embryogenic cell suspensions were established in 0.5 mg l^–1 2,4-D, which resulted in the highest recovery of early stages of somatic embryos in liquid MMS medium. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. Mature monocotyledonary-stage somatic embryos were induced in liquid B5 medium containing 0.1 mg l^–1 2,4-D, 20 mg l^–1 l-Proline (Pro), 5 M Abscisic acid (ABA), and 2% mannitol. B5 medium was found superior for the maturation of somatic embryos compared to MS and MMS media. The importance of duration (5 d) for effective maturation of somatic embryos is demonstrated. A reduction in the 2,4-D level in suspensions increased the somatic embryo induction and maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction while mannitol for embryo maturation and maltose for embryo germination. Extension of hypocotyls and complete development of plantlet was achieved in half-strength B5 medium supplemented with 3% maltose, 2500 mg l^–1 potassium nitrate, and 0.05 mg l^–1 thidiazuron (TDZ) with 32% regeneration frequency. Field-established plants were morphologically normal and fertile. This regeneration protocol assures a high frequency of embryo induction, maturation, and plantlet conversion.     

Effects of Decapitation and Benzyladenine on Growth and Yield of Cowpea [Vigna unguiculata (L.) Walp.]

A study was undertaken to define more clearly the role of theearly loss of apical dominance on yield of cowpea [Vigna unguiculata(L.) Walp. cv. Vita-5]. Decapitation at the fifth leaf stageresulted in an increase in branching components, yields andharvest indices, while vegetative d. wt accumulation was reduced.Foliar-applied sprays of 6-benzyladenine had no effect on branchingunless combined with decapitation and no significant effectson yield over that of controls were observed. However, harvestindices were increased by 50 per cent. Vigna unguiculata (L.) Walp., cowpea, apical dominance, decapitation, 6-benzyladenine