Grain quality in chickpea (<i>Cicer arietinum</i> L.) is a major factor affecting its consumption for human nutrition and health benefits. Some of the major factors affecting chickpea grain quality are: seed weight, size, colour, protein, starch and amylose concentration, and amylopectin structure. The objectives of this study were to: 1) determine variation, repeatability and genotype by environment interaction on thousand seed weight, starch, amylose and protein concentration of chickpea cultivars adapted to western Canada; 2) assess variations in global chickpea germplasm for thousand seed weight, seed size, protein, starch and amylose concentrations; and 3) characterize the desi and kabuli type chickpea for starch concentration, composition, and amylopectin structure to study their effect on starch enzymatic hydrolysis. Limited variation was observed in seed composition of chickpea cultivars adapted to the western Canadian prairies. Significant genotype by environment interaction occurred for starch, amylose, and protein (except for kabuli) concentrations, seed yield and thousand seed weight indicating that testing over a wide range of environments is needed to identify genotypes for grain quality improvement. Repeatability of starch, amylose, and protein concentrations was low and inconsistent across chickpea market classes. Broad sense heritability was higher than repeatability across all traits for all market classes implying that repeatability estimates do not set upper limits to heritability if significant genotype by environment interaction is present. The negative relationship between seed constituents and yield indicates that selection for chickpea cultivars with desired seed composition may require compromise with yield and indirect selection. All the mini core accessions that had above average seed diameter score in both desi and kabuli also had above average score for thousand seed weight. Selecting mini core with promising intrinsic and extrinsic quality characteristics may reduce yield. Slowly digestible starch was negatively correlated with hydrolysis index in both pure starch and meal starch of desi and kabuli. Amylose had a strong relationship with resistant starch but not with rate of starch hydrolysis. Genotypes with a significantly higher rate of starch hydrolysis had significantly lower 60-80 µm starch granule size volume. Amylopectin B2 chains were related to slowly digestible starch of meal (except kabuli) and extracted starch. Resistant starch positively correlated with B1 fraction of amylopectin chain length in both desi and kabuli meal starch. Our results suggest that there is no major difference between starch composition in the two chickpea market classes, although only three genotypes of each class were tested. The meal components affect the starch hydrolytic properties and the effect is genotype specific. The results also show that amylopectin structure influences starch hydrolytic properties. These observations emphasize that complete characterization of seed components is needed to obtain meaningful results regarding the desired nutritional and health benefits attributed to any grain.

In this study, it was aimed to identify chickpea (Cicer arietinum) genes or gene fragments expressed upon Ascochyta rabiei infection using a tolerant chickpea cultivar ILC195 and fungal isolates with varying level of pathogenicity. PCR amplification of resistance gene analogs (RGA) and disease related genes, and mRNA differential display reverse transcription (DDRT) were used to get these expressed gene fragments in chickpea. The constitutively or differentially expressed PCR product fragments were cloned and sequenced. Out of nearly 300 clones, 160 sequences (expressed sequence tags, ESTs) could be analyzed and these sequences were disclosed in this study. About 100 of these ESTs were classified according to predicted &ldquo / molecular function&rdquo / , &ldquo / biological process&rdquo / and &ldquo / cellular component&rdquo / . The most common ppredicted functions of the products coded by these ESTs were &ldquo / Protein Fate&rdquo / , &ldquo / Metabolism&rdquo / , &ldquo / Cell Rescue, Defense and Virulence&rdquo / , &ldquo / Transcription&rdquo / , &ldquo / Transport&rdquo / , &ldquo / Energy&rdquo / , and &ldquo / Cell Fate&rdquo / . Six ESTs were subjected to Real-Time quantitative RT-PCR analysis to compare the response of ILC195 infected by one A.rabiei isolate with another resistant chickpea genotype (FLIP84-92C)/A.rabiei pathotype system. Some of these genes were differentially expressed among different chickpea/A.rabiei isolate combinations. Highly upregulated ESTs in all these combinations were a formate dehydrogenase (metabolism and detoxification), a serine carboxypeptidase (protein fate and communication) and a hypothetical protein probably similar to acyl-CoA synthetases. A genetic mapping study was carried out with EST specific primers and two EST markers were assigned in the current chickpea genetic map. However, no genetic linkage of them was detected with known chickpea quantitative trait loci for A.rabiei resistance.

The effect of micronization (at 130 and 150 °C) as a potential heat treatment to reduce ‘beany’ aroma and flavor of cooked chickpea (Cicer arietinum) and green lentil (Lens culinaris) flours was investigated. A simultaneous distillation solvent extraction method was developed to extract key volatile compounds with potential contribution to ‘beany’ aroma and flavor notes in micronized pulse flours and analyzed by gas chromatography-mass spectrometry. Concentrations of volatile compounds such as pentanol, hexanal, 2-hexenal, hexanol, heptanal, furan-2-pentyl, 2-octenal, nonanal, 2,4 decadienal, and 2,4- undecadienal were significantly (P<0.05) decreased with micronization. Low fat burgers fortified with 6% micronized chickpea and green lentil flours showed significantly higher acceptability for aroma, flavor, texture, color and overall acceptability (p<0.05) compared to non-micronized samples in a consumer acceptability test with 101 consumers. In addition, fatty acid analysis of burgers showed burgers containing micronized pulses had higher level of linoleic and linolenic acid content.

The effect of micronization (at 130 and 150 °C) as a potential heat treatment to reduce ‘beany’ aroma and flavor of cooked chickpea (Cicer arietinum) and green lentil (Lens culinaris) flours was investigated. A simultaneous distillation solvent extraction method was developed to extract key volatile compounds with potential contribution to ‘beany’ aroma and flavor notes in micronized pulse flours and analyzed by gas chromatography-mass spectrometry. Concentrations of volatile compounds such as pentanol, hexanal, 2-hexenal, hexanol, heptanal, furan-2-pentyl, 2-octenal, nonanal, 2,4 decadienal, and 2,4- undecadienal were significantly (P<0.05) decreased with micronization. Low fat burgers fortified with 6% micronized chickpea and green lentil flours showed significantly higher acceptability for aroma, flavor, texture, color and overall acceptability (p<0.05) compared to non-micronized samples in a consumer acceptability test with 101 consumers. In addition, fatty acid analysis of burgers showed burgers containing micronized pulses had higher level of linoleic and linolenic acid content.

The objective of this study was to optimize an efficient selection system and Agrobacterium mediated transformation of chickpea (Cicer arietinum L.). Cotyledonary node explants of Turkish chickpea cultivar G&ouml / k&ccedil / e were used to determine the effects of selective agents, two antibiotics (Kanamycin, Hygromycin) and two herbicides (PPT, Glyphosate) as well as four antibiotics (Augmentin, Carbenicillin, Cefotaxime, Timentin) for eliminating Agrobacterium on multiple shoot and root induction. Selective agents and antibiotics were applied to explants at different concentrations for one month and numbers of regenerated shoots and roots were recorded. Kanamycin at 100 mg/L, Hygromycin at 20 mg/L, PPT at 3 mg/L and Glyphosate at 5 mg/L were found to be appropriate to select chickpea transformants. Lowest concentrations of all selective agents (50 mg/L Kanamycin, 10 mg/L Hygromycin, 3 mg/L PPT, 1 mg/L Glyphosate) totally inhibited rooting of the regenerated shoots. Among the Agrobacterium-eliminating antibiotics, Cefotaxime and Augmentin each up to 600 mg/L had no adverse effect on shoot induction, whereas Timentin (300 mg/L) significantly increased and Carbenicillin (300 mg/L) significantly decreased shoot induction after four weeks of culture. Augmentin was determined to have no effect on rooting capacities of chickpea shoots. However Cefotaxime at all concentrations significantly decreased root induction. On the other hand only high concentrations of Carbenicillin (300 mg/L) and Timentin (200 mg/L) significantly decreased rooting. Sulbactam in combination with Carbenicillin and Cefotaxime displayed effective inhibition of bacterial growth. Furthermore, Agrobacterium mediated transformation procedure for cotyledonary node explants of G&ouml / k&ccedil / e, was also optimized by monitoring transient uidA expression on 4th, 9th, and 16th days after transformation. Transformation procedure was improved via mechanical injury of axillary region of explants and application of vacuum infiltration at 200 mmHg for 40 minutes.

Abstract Salinity has considerable adverse effects on agriculture through reduction in plant growth and water use. Sodium chloride salinity has both an osmotic effect on plant water relations, and a toxic effect on cellular processes. The relative contribution of these two effects to plant growth depends on a range of factors including plant specific tolerance mechanisms, such as Na and Cl exclusion, compartmentation of ions at a whole plant and cellular level, and synthesis of organic osmotic compounds for plant osmotic adjustment. Plants growing in saline soil would also experience reduced plant available water, due to the additional osmotic effect on soil water potential. The effect of salinity on plant growth is further complicated by the interactions of environmental conditions with plant water and ion uptake. This thesis examines the osmotic and toxic effects of salinity on wheat (Triticum aestivum L.) and chickpea (Cicer arietinum L.), with particular focus on plant water availability, effects of Na and Cl toxicity, and temperature and humidity effects. While considerable research has been undertaken into the physiological response of plants to NaCl, our understanding of the capacity of plants to extract water from saline soils has remained largely theoretical. Total plant available water is largely determined by the matric potential of the soil. Presence of sodium chloride would have an additional osmotic effect, and previous theory stated that the salt tolerance of the plant determined the extent to which this osmotic potential reduced plant available water. The capacity of wheat and chickpea to extract water from saline soils was examined in a soil experiment where water stress was imposed on established plants, which were then grown until permanent wilting point (PWP) was reached. Wheat extracted to lower soil water potentials (-1.5 MPa), than chickpea (-900 kPa) in 0 NaCl treatments. Where salinity was low to moderate, plants were able to extract water to a PWP determined by the combined total of matric and osmotic potentials. Wheat extracted water to PWP in salinity treatments up to soil ECse of 5.3 dS/m, and chickpea to 2.9 dS/m. As salinity increased, toxic effects of salinity dominated, and water extraction by plants was significantly lower than that determined by total soil water potential. Solution culture experiments investigated the comparative toxic effects of Na, Cl and salt mixtures. Growth of wheat was reduced by Na toxicity, but not Cl toxicity, with Na causing a small, but significant additional reduction in growth, compared to high Cl or a salt mixture. Reductions in growth of 50% from control treatments occurred at -500 kPa for the Na treatment, and -630 kPa for the Cl and mixed salt treatments. In contrast, growth of chickpea was significantly reduced by both Na and Cl toxicity, with a large difference in growth compared to the salt mixture. Growth reductions of 50% occurred at -330 kPa for the Cl treatments, and -450 kPa for the Na treatment. A 50% growth reduction was not observed in the mixed salt treatment. Tolerance of saline conditions is reduced under stressful environmental conditions, such as high temperature and low relative humidity. Hot and dry conditions were shown to reduce the tolerance of saline conditions by both wheat and chickpea, compared to cool or humid conditions. Tissue concentrations of Na in wheat were disproportionately high in treatments with high evaporative demand, while tissue Cl was not related to evaporative demand. Tissue concentrations of Na in chickpea increased with temperature, but not relative humidity, while tissue Cl concentrations were highly correlated with evaporative demand. The relationships between NaCl salinity, plant water use, and environmental conditions were examined, allowing further development of the two phase salinity model. In particular, the transition point between the osmotic and toxic salinity effects. While the concentration of NaCl in the soil remains the primary factor, soil water status, environmental stresses and presence of other salts may dictate whether salinity be tolerated by the plant or not. The ability of the plant to extract water to PWP, as determined by total matric and osmotic potential has been identified as a useful indicator of salinity tolerance. The point at which toxicity of Na and/or Cl is observed is associated with a rapid increase in Na and Cl uptake by the shoot tissue, and a decrease in the amount of water the plant is able to extract from the soil. Within the osmotic region of salinity stress, the plant is able to extract water to PWP, but as NaCl becomes toxic the plant is unable to utilize this water.

O amido resistente (AR) é a fração do amido que não sofre a ação das enzimas digestivas, apresentando comportamento semelhante ao da fibra dietética. O objetivo do presente trabalho foi avaliar o teor e as características dos AR obtidos a partir dos amidos de ervilha e de grão-de-bico por diferentes processos de redução de massa molecular. Os amidos naturais ou gelatinizados foram submetidos a processos de hidrólise ácida (HCl 2 M por 2,5 h) ou enzimática (pululanase, 40 U/g por 10 h) previamente a um processo controle, que constou de tratamento hidrotérmico (autoclavagem a 121 °C por 30 min), refrigeração (4 °C por 24 h) e liofilização. O material produzido foi caracterizado quanto ao aspecto geral por microscopia eletrônica de varredura (MEV), teor de AR, teor de fibra dietética total (FDT), índice de absorção de água (IAA), índice de solubilidade em água (ISA), padrão de cristalinidade (difração de raios X), viscosidade (RVA - Rapid Visco Analyser) e propriedades térmicas (DSC - Differential Scanning Calorimeter). O teor de AR para o amido natural de ervilha e grãode- bico foi de 39,8 e 31,9 %, respectivamente. Nos amidos processados esse teor variou de 38,5 a 54,6 % para a ervilha e de 16,4 a 32,3 % para o grão-de-bico. Os melhores tratamentos para elevar o teor de AR foram o tratamento ácido (amido natural e gelatinizado) para o amido de ervilha e o tratamento enzimático (amido gelatinizado) para o amido de grão-de-bico. Os teores de FDT dos amidos processados de ervilha variaram de 22,9 a 37,1 % e foram superiores aos do grão-de-bico, que variaram entre 7,2 e 15,7 %. A MEV revelou a presença de grânulos inteiros ou não fragmentados nos amidos de ervilha dos diferentes tratamentos, enquanto os amidos de grão-de-bico tratados apresentaram apenas massa amorfa, sem a evidência de grânulos. Os padrões de cristalinidade dos amidos naturais foram tipo B e C, respectivamente para os amidos de ervilha e grão-de-bico, entretanto, os amidos processados de ambos apresentaram padrão do tipo B. Os amidos naturais apresentaram endotermas entre 56 e 90 °C, enquanto os amidos processados apresentaram endotermas em temperaturas superiores, entre 131 e 171 °C. A entalpia de gelat inização (H) dos amidos de ervilha processados foi superior à dos amidos de grão-de-bico. Os amidos natural e processados de ervilha, de modo geral, apresentaram viscosidade baixa (< 20 RVU) em RVA. O amido de grão-de-bico natural apresentou viscoamilograma bem definido, característico da fonte. Os amidos resistentes obtidos por hidrólise, de ambas as fontes, apresentaram redução na viscosidade em comparação com o controle. A viscosidade foi inversamente proporcional ao teor de AR nas amostras. A hidrólise e o processamento térmico promoveram o aumento no IAA e ISA dos amidos tratados. Os processos de hidrólises dos amidos de ervilha e de grão-de-bico podem elevar o teor de AR se comparados com o método controle. / Resistant starch (RS) is the fraction of starch that does not suffer the action of digestive enzymes, showing similar behavior to that of dietary fiber. The aim of this study was to evaluate the amounts and characteristics of RS obtained from chick-pea and high amylose pea starches using different processes of molecular weight reduction. The natural or pregelatinized starches were submitted to acid (2 M HCl for 2.5 h) or enzymatic (pullulanase, 40 U / g per 10 h) hydrolysis prior to a control process, which consisted of hydrothermal treatment (autoclaving at 121 ° C for 30 min), refrigeration (4 ° C for 24 h) and liophilization. The material was characterized as to the general appearance by scanning electron microscopy (SEM), RS content, total dietary fiber (TDF) content, water absorption index (WAI), water solubility index (WSI), crystallinity pattern (X-ray diffraction), viscosity (RVA - Rapid Visco Analyzer) and thermal properties (DSC - Differential Scanning Calorimeter). The RS content in pea and chick-pea natural starch was 39.8 and 31.9%, respectively. The processed starches showed contents ranging from 38.5 to 54.6% for pea and from 16.4 to 32.3% for chick-pea starch. The best treatments to raise the level of RS were the acid treatment (natural and gelatinized starch) for pea and the enzymic treatment (gelatinized starch) for chick-pea starch. The FDT amounts in processed pea starches ranged from 22.9 to 37.1% and were higher than those of chick-pea, which ranged between 7.2 and 15.7%. The photomicrographs in SEM revealed the presence of whole grains (not fragmented) in pea starches treatments, while the treated chick-pea starches showed only amorphous mass, without the evidence of granules. The cristallinity patterns of natural starches were B and C types, respectively for pea and chick-pea starch. Both processed starches, however, presented B type pattern. The natural starches showed endotherms between 56 and 90°C, while the processed starches showed endotherms at higher temperatures (131 and 171°C). The processed pea starches gelatinizati on enthalpy (H) was higher than those of processed chick-pea starches. The natural and processed pea starches, in general, showed low viscosity (<20 RVU) in RVA. The natural chick-pea starch presented viscoamylogram well defined, typical of this botanical source. The resistant starches obtained by hydrolysis showed in both sources, a decrease in viscosity compared to the control treatment. The viscosity was inversely proportional to the RS content in the samples. The hydrolysis and the thermal processing promoted an increase in WAI and WSI of treated starches. The hydrolysis process of pea and chickpea starches may raise the level of RS when compared to the control process.

MSCAGR (Plant Production) / Department of Plant Production / Chickpea (Cicer arietinum L) is adapted to cool-seasons and its organs are of high nutritive value and serve as cheap sources of protein, especially in developing countries. Chickpea crop is mainly grown for human consumption, animal feed and for medicinal purposes. The introduction and promotion of chickpea to especially small-scale South African crop farmers has multiple objectives including the improvement of soil fertility. Small-scale farmer’s flounder to afford Nfertilizers, coupled with the challenges faced by programmes aimed at assisting them about soil fertility in their cropping fields that are still without enough N concentration to meet N demand. It is therefore important that other alternatives that can help improve the N status of soils be explored. The shoot δ13C is an indicator of WUE in C3 plants. However, shoot-WUE is affected by a variety of factors including genotypes, phosphorus fertilizer application and availability of native or introduced rhizobial bacteria. However, not much is known on whether application of phosphate fertilizer, seed inoculation with rhizobial strain affect the shoot C/N ratio of chickpea genotypes in South Africa. Therefore, field experiments were established at Thohoyandou and Syferkuil in Limpopo to assess the role of phosphorus fertilization and rhizobial inoculation on C assimilation, C/N ratio and shoot-WUE of chickpea genotypes. Field experiments were conducted during winter season in 2016 and 2017 (April to August). Treatments consisted of a factorial combination of two rates of phosphorus fertilizer (0 and 90 kg P ha-1 ), four desi chickpea genotypes (ACC#1, ACC#2, ACC#3 and ACC#5) and two rhizobial inoculation levels (bradyrhizobium strain and without rhizobial strain. In Thohoyandou, ACC#1 showed greater grain yield in 2016 and 2017. Which was associated with more branches and greater plant height. Furthermore, the interaction between genotypes, phosphorus fertilizer and rhizobial inoculation had significant effect on grain yield in 2016. ACC#1, 3 and 5 of chickpea genotypes fixed the most N compared to that of ACC#2. In addition, ACC#5 had the highest soil N-uptake in both seasons followed by ACC#3, while ACC#1 had the least value of soil N-uptake in both seasons. Phosphorus fertilizer application increased the fixation of N by 36.8% (P≤0.01), and similarly in soil N-uptake by difference of 59.9% compared to control in 2016. Furthermore, rhizobial inoculation increased N-fixed in 2016 and soil N-uptake in both seasons. ACC#5 had the highest N fixed at phosphorus-fertilized with bradyrhizobuim across two locations in both seasons. ACC#5 depended more on soil N-uptake than fixing its own N as compared to ACC#1. N fixation differed across seasons; however, ACC#3 had greater N-fixed in both locations. Moreover, chickpea genotype that fixed more N had least δ15N. This finding indicates that N fixation is exhibited by the genotypes that depend less on δ15N, because N2 fixation is inhibited by high soil N concentration or δ15N. Furthermore, ACC#2 and ACC#3 had greater δ13C at Thohoyandou in 2017; chickpea genotypes had significant effect on δ13C at P≤0.05 at Thohoyandou, 2016. The results showed that ACC#1 with phosphorus fertilizer application and no bradyrhizobium strain showed greater δ13C. Also, δ13C increased with a decrease in N-fixed (r=.1000), this indicates that there was a functional relationship between plant WUE and N fixation in chickpea, probably because improved water use in legumes supports N fixation. / NRF

Thesis (M. A. (Agricultural Management)) -- University of Limpopo, 2020 / Field experiments were conducted at two locations; University of Limpopo (Syferkuil) and University of Venda (Thohoyandou) during 2015 and 2016 winter cropping seasons. The objectives of this study were to determine; the effect of genotype (ACC# 1, 3, 4 and 7) and planting density (33, 25 and 20 plants/m2) on four chickpea genotypes having contrasting growth patterns and also to determine the effect genotype and planting density on water use and water use efficiency of four chickpea genotypes having contrasting growth patterns. The experimental design was randomized complete block design in factorial arrangement with three replications. Plant height, number of primary and secondary branches, grain yield and yield components (number of pods per plant, number of seeds per pod, Harvest Index and 100 seed weight [100-SW] and above ground biomass, and were determined at different growth stages. Data obtained was subjected to analyses of variance using the general linear model of Genstat 17th edition. Significant differences between the treatments means were compared using the standard error of difference (LSD) of the means at 5% level. Correlation analyses were performed to assess the relationship between parameters. Plant height varied with genotype from 41 cm (84 DAE) to 44 cm (118 cm) at Syferkuil and 41 (56 DAE) to 44 cm (63 DAE) at Thohoyandou. Primary branches was not significantly affected by genotype and planting density at both locations and seasons. Planting density had significant effect on number of secondary branches, greater number was recorded at low (32, 6) density at Syferkuil in 2016. Above ground biomass was significantly affected by planting density at Syferkuil during in 2015 (5344 kg ha-1) and 2016 (3701 kg ha-1) growing seasons. Genotype and planting density did not affect number of pods plant-1, number of seeds plant-1, 100 SW (100 seed weight), and Harvest index were not significant at both locations and seasons. Grain yield was significantly affected by planting density at Syferkuil in 2015 and Thohoyandou in 2016. Grain yield increased with the increase in planting density at both locations. Two field experiments were conducted at University of Venda (Thohoyandou) during 2015 and 2016 winter cropping seasons. This study aimed at assessing the effect of genotype v and planting density on water use efficiency of four chickpea genotypes with contrasting growth patterns. Crop water use (WU) was determined by monitoring soil water content at 7-day intervals using a neutron probe and, water use efficiency (WUE) was determined as a ratio of crop biomass and grain yield to WU. Genotype and planting density had no significant effect on WU in 2015 and 2016. Genotype and planting density had no significant effect on biomass production (WUEb) and grain yield production (WUEg) in 2015. In contrast, WUEb and WUEg was significantly affected by planting density in 2016. WUEb was 43.2% greater at high density compared to low density. Similarly, WUEg was 39.3% greater at high density compared to low density. WUEb and WUEg increased with the increase with planting density. Therefore, manipulation of management practices such as planting density may increase chickpea production. Keywords: Planting density, genotype, grain yield and yield components, water use efficiency. / National Research Foundation (NRF) and University of Venda Capacity Development

The plasmids pDN211 and pDNll, isolated from the gene bank of the Rhizobium japonicum strain I-110, have been reported to complement two different Nif+ Hup· (nitrogen fixation positive and hydrogen uptake negative) mutants. A 5.9-kb Hindiii DNA fragment of the cosmid pHU52, isolated from the gene bank of R. japonicum strain 122DES, has been reported to code for the two polypeptide subunits of uptake hydrogenase. To determine homology between the structural genes of uptake hydrogenase of the two strains, a Southern blot of the Hindiii restriction fragments of the plasmids pDN211 and pDN11 was hybridized to the 5.9-kb Hindiii fragment. A 6.0-kb HindIII DNA fragment of pDN11 was observed to be homologous to the hup DNA probe. Thus, the hup genes of the two Rhizobium strains are conserved. Colony hybridization with the 5.9-kb DNA as the probe was used to detect the homologous hup genes in alfalfa-, chickpea- and pigeonpea- Rhizobium species. These Rhizobium species were also successfully derepressed for uptake hydrogenase in free living conditions. It was found that 30% of the alfalfa-, 30% of the chickpea- and 21% of the pigeonpea- Rhizobium strains tested were Hup+ as determined by the methylene blue (MB) reduction assay. All but one strain of alfalfa- (Celpril Ind. 3623) and one strain of pigeonpea- Rhizobium (IC3282) that showed strong homology to the hup DNA probe also exhibited MB reduction activity. The Hup+ strains of alfalfa- and pigeonpea- Rhizobium produced significantly higher yields as compared to the Hup- strains, whereas those of the chickpea-Rhizobium strains produced significantly lower yields as compared to the Hup- strains. Two of the alfalfa-Rhizobium strains, USDA1024 and CmRm~, exhibited Hup activities greater than any reported previously for this bacterial species. The cosmid-borne hup genes of R. japonicum were successfully expressed in all strains tested but the enzyme activities were very low in alfalfa-Rhizobium compared to those in chickpea- and pigeonpea-Rhizobium species. The relative efficiency of N2-fixation was significantly increased by the transfer of hup genes into the chickpea- and pigeonpea- Rhizobium strains.