Enzymes of ammonia assimilation and ureide biogenesis in developing pigeonpea (Cajanus cajan L.) nodules

Ammonia assimilatory and ureide biogenic enzymes were measured in the cytosol fraction of pigeonpea nodules during the period 15–120 days after sowing. The activity of enzymes involved in the initial assimilation of ammonia, i.e. glutamine synthetase, glutamate synthase, asparagine synthetase and aspartate aminotransferase, substantially increased activities during the period of plant growth and reached a maximum value around 105 days after sowing. These increases paralleled the increase in nodule mass, nitrogenase activity and ureide content in nodules. Though no regular pattern was obtained for their specific activities, yet these activities when expressed relative to the specific activity of nitrogenase were many fold higher at each stage of development. Similar increases were observed in the activities of enzymes associated with the formation of ureides from purines. In almost all cases, the activities were again maximum around 90–105 days after sowing. The specific activities of nucleotidase, nucleosidase, xanthine dehydrogenase, uricase and allantoinase, when expressed relative to the specific activity of nitrogenase at vegetative, flowering and podsetting stages were again many fold higher indicating the sufficiency of the levels of these enzymes for the biosynthesis of ureides. The data presented are consistent with the proposal that in ureide producing legumes, ammonia is initially assimilated into glutamine, aspartate, etc., which are metabolised for the denovo synthesis of purines. The purines are then utilised for the production of ureides by a group of enzymes investigated here     

Plant regeneration via somatic embryogenesis in pigeonpea (Cajanus cajan L. Millsp)

Efficient plant regeneration via somatic embryogenesis has been developed in pigeonpea. Cotyledon and leaf explants from 10-day-old seedlings produced embryogenic callus and somatic embryos when cultured on Murashige and Skoog (MS) medium supplemented with 10 μm thidiazuron (TDZ). Subsequent withdrawal of TDZ from the induction medium resulted in the maturation and growth of the embryos into plantlets on MS basal medium. The rooted plantlets were transferred and acclimatized on vermiculite where they showed normal morphological characters. Received: 23 December 1996 / Revision received: 22 July 1997 / Accepted: 2 August 1997     

Protease inhibitors of pigeonpea (Cajanus cajan) and its wild relatives

Plant protease inhibitors have been implicated in defense against insect pests. Podborer and pod fly are major pests of developing seeds of pigeonpea ( Cajanus cajan L. Millsp.). Therefore, we studied the presence of protease inhibitors in seeds of pigeonpea and its wild relatives. Seed extracts were analyzed for protease inhibitor activities by caseinolytic assay, and the number of protease inhibitors determined by polyacrylamide gel electrophoresis. Besides trypsin and chymotrypsin inhibitors, seed extracts contained weak papain inhibitor(s) but no bromelain inhibitor. Treatment of seed extract with bromelain generated new active forms of trypsin inhibitors. The relative amounts of different trypsin inhibitors and the total trypsin inhibitor activity varied with different extraction media. Trypsin inhibitors were not detectable in pigeonpea leaves. The profiles of trypsin and chymotrypsin inhibitors in almost all the cultivars of pigeonpea analyzed were similar; however, those in wild relatives were quite variable.     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Short Communication: Immobilization of urease from pigeonpea (Cajanus cajan L.) on flannel cloth using polyethyleneimine

Urease of pigeonpea has been immobilized on polyethyleneimine-activated cotton cloth followed by cross-linking with dimethyl suberimidate. Optimum immobilization (56%) was obtained at a protein loading of 1.2mg/5×5cm2 cloth piece. The immobilized enzyme stored in 0.1M Tris/acetate buffer, pH6.5, at 4°C had a t1/2 of 70 days. There was practically no leaching of the enzyme from the immobilization matrix in 15 days. The immobilized enzyme was used 7 times at an interval of 24h between each use with 75% residual activity at the end of the period. Blood urea analysis was carried out with immobilized urease for some clinical samples.     

Nodulation,nitrogen fixation and nitrogen uptake in pigeonpea (Cajanus cajan (L.) Millsp) of different maturity groups

Summary The seasonal patterns of nodulation, acetylene reduction, nitrogen uptake and nitrogen fixation were studies for 11 pigeonpea cultivars belonging to different maturity groups grown on an Alfisol at ICRISAT Center, Patancheru, India. In all cultivars the nodule number and mass increased to a maximum around 60–80 days after sowing and then declined. The nodule number and mass of medium- and late-maturing cultivars was greater than that of early-maturing cultivars. The nitrogenase activity per plant increased to 60 days after sowing and declined thereafter, with little activity at 100 days when the crop was flowering. At later stages of plant growth nodules formed down to 90 cm below the soil surface but those at greater depth appeared less active than those near the surface. All the 11 cultivars continued to accumulate dry matter until 140 days, with most biomass production by the late-maturing cultivars (up to 11 t ha⁻¹) and least by the early-maturing determinate cultivars (4 t ha⁻¹). Total nitrogen uptake ranged from 69 to 134 kg ha⁻¹. Nitrogen fixation by pigeonpea was estimated as the difference in total nitrogen uptake between pigeonpea and sorghum and could amount to 69 kg N ha⁻¹ per season, or half the total nitrogen uptake. Fixation by pigeonpea increased with crop duration, but there were differences within each maturity group. The limitations of the methods used for estimating N₂ fixation by pigeonpea are discussed. Submitted as J.A. No. 552 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT).     

Broad-based resistance to pigeonpea sterility mosaic disease in wild relatives of pigeonpea (Cajanus: Phaseoleae)

Sterility mosaic disease (SMD), an important biotic constraint on pigeonpea (Cajanus cajan) in the Indian subcontinent, is caused by Pigeonpea sterility mosaic virus (PPSMV) transmitted by the eriophyid mite, Aceria cajani. Distinct PPSMV isolates occur in different geographical regions and broad‐based resistance to all these isolates is scarce in cultivated pigeonpea germplasm. Wild relatives of pigeonpea, which are known to possess resistance to several pests and diseases, were evaluated for broad‐based SMD resistance. One hundred and fifteen wild Cajanus accessions from six species (C. albicans, C. platycarpus, C. cajanifolius, C. lineatus, C. scarabaeoides and C. sericeus) were evaluated against three PPSMV isolates prevailing in peninsular India. Evaluations were done under greenhouse conditions in endemic locations of each isolate through mite‐mediated virus inoculation. Fifteen accessions showed resistance to all three isolates: ICP 15614, 15615, 15626, 15684, 15688, 15700, 15701, 15725, 15734, 15736, 15737, 15740, 15924, 15925 and 15926. Most of the wild accessions did not support mite multiplication. The majority of the accessions resistant to PPSMV following inoculations with viruliferous mites were susceptible by graft inoculation, suggesting that vector resistance is conferring resistance to infection with PPSMV. The 15 accessions identified as being resistant to infection to all three virus isolates tested are cross compatible with pigeonpea by traditional breeding. They are therefore useful for exploitation in breeding programmes to increase both the level of SMD resistance and to diversify its genetic base in the cultivated pigeonpea gene pool.     

Resistance to cyst nematode (Heterodera cajani) in pigeonpea cultivars and in wild relatives of Cajanus*

Heterodera cajani is an important nematode pest of pigeonpea in India. Evaluation of 58 pigeonpea cultivars and 61 accessions of Cajanus acutifolius, C. cajanifolius, C. grandifolius, C. lanceolatus, C. lineatus, C. mollis, C. pla-tycarpus, C. reticulatus, C. scarabaeoides, C. sericeus, C. volubilis, Flemingia macrophylla, F. stricta, F. strobilifera, Rhynchosia aurea, R. bracteata, R. cana, R. densiflora, R. minima, R. rothii, R. suaveolens and R. sublobata revealed that the tested pigeonpea cultivars lacked resistance to H. cajani. Eight accessions of wild relatives were resistant and 20 accessions were moderately resistant. Based on the white cyst number on roots and low plant-to-plant variation, two accessions of C. scarabaeoides (ICPWs 111 and 128), three accessions of Flemingia spp. (ICPWs 194, 202 and 203), and one accession each of R. rothii (ICPW 257), R. densiflora (ICPW 224), and R. aurea (ICPW 210) were identified as resistant and promising for use in intergeneric hybridisation programmes.     

Laboratory screening of different Bacillus thuringiensis strains against certain lepidopteran pests and subsequent field evaluation on the pod boring pest complex of pigeonpea (Cajanus cajan)

Laboratory evaluation of different Bacillus thuringiensis subspecies revealed that B. thuringiensis subsp. kurstaki (NCIM 2514) at 10⁸ spores/ml concentration caused more than 85% mortality to the neonate larvae of the lepidopteran insects Spodoptera litura (F.) and Phthorimaea operculella (Z.). This strain at 10¹⁰ and 10⁸ spores/ml concentration was effective against the major lepidopteran pests comprising the pod boring complex of pigeonpea (Cajanus cajan), viz. Helicoverpa armigera (H.) and Exelastia atomosa (W.) under the field trials. Total grain yield from this treatment was at least 1.5 times more than the untreated control.     

Variability in nuclear DNA content within pigeonpea,Cajanus cajan (Fabaceae)

4C DNA values have been estimated in 16 cultivars ofCajanus cajan by cytophotometry. The values range between 6.19 pg to 7.97 pg, a 23.6% variation. The cultivars form four groups which differ significantly from each other but have insignificant difference within them. The implications of this variability with respect to the heterogeneity and origin of this legume crop are discussed.     

Sex allocation and mating systems in pigeonpea,Cajanus cajan (Fabaceae)

Sex allocation theory predicts that: (1) resources allocated to androecium should decrease with an increase in selfing, (2) a decrease in androecium biomass should be accompanied by an increase in the biomass of pistils, and (3) a decrease in androecium biomass should be coupled with a decrease in flower size, specifically corolla biomass. Another predicted change in reproductive traits associated with variation in selfing concerns seed to ovule ratios, but does not directly stem from sex allocation theory. It has been postulated that seed to ovule ratios should be positively correlated with the amount of selfing. These predictions were tested for six accessions of pigeonpea,Cajanus cajan L., that differed in selfing rates. The results were remarkably in accordance with the predictions. We conclude that sex allocation theory provides a powerful tool to understand the evolution of many reproductive traits in plants.     

Partial 16S rRNA gene sequence diversity and numerical taxonomy of slow growing pigeonpea (Cajanus cajan L Millsp) nodulating rhizobia

An investigation was carried out to determine the diversity of 30 isolates of slow growing pigeonpea nodulating rhizobia based on variations in partial sequences of the 16S rRNA gene and numerical analysis of 80 phenotypic traits. Phylogenetic analysis using molecular sequences of 23 isolates showed that ARPE1 separated from the other isolates at an average distance of >14% divergence level. The other isolates were all within 5% divergence from each other but separated into four main groups, with group 1 containing 16 of the 23 isolates. Comparisons to sequences of reference strains revealed that the group 1 isolates were phylogenetically closely related to the slow growing soybean nodulating rhizobia belonging to Bradyrhizobium elkanii, although only three of these isolates were able to nodulate soybean. Numerical analysis of phenotypic data of 19 isolates showed that 14 isolates clustered together in one branch of the phenogram, which included the group 1, group 2 and group 4 isolates from the phylogenetic analysis. The group 3 isolates were highly variable in the phenogram with similarity levels lower than 50% among these isolates.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

Biosystematic relationships among Cajanus,Atylosia, and Rhynchosia species and evolution of pigeonpea (Cajanus cajan (L.) Millsp.)

Summary Biosystematic studies encompassing morphocytological and electrophoretic analyses of Cajanus cajan, seven species of Atylosia and one of Rhynchosia revealed that A. cajanifolia is closest to C. cajan, followed by A. lineata, A. scarabaeoides, A. sericea, A. albicans, A. volubilis, A. platycarpa and R. rothii, in that order. A revision has been suggested for the taxonomic placement of the seven Atylosia species. Regarding the evolution of cultivated C. cajan, three possible alternatives have been suggested. Firstly, C. cajan could have evolved through gene mutation in A. cajanifolia; secondly, some of the Atylosia species and pigeonpea probably evolved from the same source; and thirdly, the pigeonpea might have developed from naturally occurring interspecific crosses of A. lineata and A. scarabaeoides.     

木豆毛状根的诱导及其培养条件的研究

利用发根农杆菌LBA9402对木豆叶片直接进行诱导产生毛状根。本实验研究出诱导木豆毛状根的最佳条件是,以木豆叶片为外植体,于1/2MS固体培养基上预培养2~4 d,菌液浓度OD₆₀₀=0.6~0.8,浸染20 min,共培养3 d,诱导率为60.00%。在分子水平用PCR检测表明,发根农杆菌9402Ri质粒上的T-DNA成功整合进木豆毛状根的基因组中。     

Evaluation of Cajanus cajan (pigeon pea) for phytoremediation of landfill leachate containing chromium and lead

Landfill leachates containing heavy metals are important contaminants and a matter of great concern due to the effect that they might have on ecosystems. We evaluated the use of Cajanus cajan to remove chromium and lead from landfill leachates. Eight-week-old plants were submitted to varied tests to select the experimental conditions. Water assays with a solution (pH 6) containing leachate (25% v/v) were selected; the metals were added as potassium dichromate and lead (II) nitrate salts. Soil matrices that contained leachate (30% v/v) up to field capacity were used. For both water and soil assays, the metal concentrations were 10 mg kg^?1. C. cajan proved able to remove 49% of chromium and 36% of lead, both from dilute leachate. The plants also removed 34.7% of chromium from irrigated soil, but were unable to decrease the lead content. Removal of nitrogen from landfill leachate was also tested, resulting in elimination of 85% of ammonia and 70% of combined nitrite/nitrate species. The results indicate that C. cajan might be an effective candidate for the rhizofiltration of leachates containing chromium and lead, and nitrogen in large concentrations.     

Influence of pod maturity and level of domestication on biochemical components in wild and cultivated pigeonpea (Cajanus cajan)

Variations in the trypsin inhibitors and lectin content in the developing pods of thirty accessions of Cajanus scarabaeoides , a wild relative of pigeonpea, from wide geographical locations and six cultivated genotypes were estimated at juvenile, immature and mature stages of pod development. Genotypes differed at all three stages for these two biochemical components. Total protein and trypsin inhibitor contents were higher in the wild accessions than in the cultivated genotypes. Although lectin content in the juvenile stage of pod development in the wild accessions ICPW 138 and ICPW 98 was highest, it was absent in the mature pods in both the cultivated and the wild genotypes. Very high broad-sense heritability estimates indicated the possibility of involvement of few genes in the inheritance of these biochemical components, which could be easily introgressed into the elite agronomic background.     

Enzymes of ammonia assimilation and ureide biosynthesis in soybean nodules: effect of nitrate

The effect of nitrate on N₂ fixation and the assimilation of fixed N₂ in legume nodules was investigated by supplying nitrate to well established soybean (Glycine max L. Merr. cv Bragg)-Rhizobium japonicum (strain 3I1b110) symbioses. Three different techniques, acetylene reduction, ¹⁵N₂ fixation and relative abundance of ureides ([ureides/(ureides + nitrate + α-amino nitrogen)] × 100) in xylem exudate, gave similar results for the effect of nitrate on N₂ fixation by nodulated roots. After 2 days of treatment with 10 millimolar nitrate, acetylene reduction by nodulated roots was inhibited by 48% but there was no effect on either acetylene reduction by isolated bacteroids or in vitro activity of nodule cytoplasmic glutamine synthetase, glutamine oxoglutarate aminotransferase, xanthine dehydrogenase, uricase, or allantoinase. After 7 days, acetylene reduction by isolated bacteroids was almost completely inhibited but, except for glutamine oxoglutarate aminotransferase, there was still no effect on the nodule cytoplasmic enzymes. It was concluded that, when nitrate is supplied to an established symbiosis, inhibition of nodulated root N₂ fixation precedes the loss of the potential of bacteroids to fix N₂. This in turn precedes the loss of the potential of nodules to assimilate fixed N₂.     

In vitro regeneration and transformation of pigeonpea [Cajanus cajan (L.) Millsp]

A requirement for generating transgenic pigeonpea [Cajanuscajan (L.) Millsp] plants is the development of a highly efficientin vitro regeneration procedure. This goal was achieved byusing germinated seedlings grown on B5 medium supplemented with 10 mgl^–1 6-benzylaminopurine, which induced differentiatingcallus formation in the cotyledonary node region. The calli were transferred onB5 medium with 0.2 mg l^–1 6-benzylaminopurine toobtain shoot induction. Elongated shoots were then further cultured on a B5hormone-free medium for rooting. Using this regeneration system transgenicpigeonpea plants were obtained both by particle bombardment andAgrobacterium tumefaciens-mediated gene transfer. Thepresence of the transgenes in the pigeonpea genome was confirmed by GUS assays,PCR and Southern hybridisation. The transgenic rooted plants were successfullytransferred to soil in the greenhouse. GUS and PCR assays of T1 progeniesconfirmed that the transgenes were stably transmitted to the next generation.This is the first report of successful use ofAgrobacteriumas well as particle bombardment for production of transgenic pigeonpea plants.