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).     

Salt tolerance of pigeon pea (Cajanus cajan (L.) Millsp.) at three growth stages

Salt tolerance of pigeon pea (Cajanus cajan (L.) Millsp.) was determined at three growth stages following observations by a number of workers that degree of salt tolerance of different crops varies with their ontogeny. The salt tolerance of three accessions, Local arhar, ICPL-151 and ICPL-850014 of pigeon pea was assessed at the germination, seedling and adult stages. There was no positive correlation between tolerance at the early growth stages and at the adult stage since no clear difference in salt tolerance of the three accessions was observed at the germination and the seedling stages, whereas accessions differed considerably at the adult stage. Although increasing salt concentrations adversely affected the growth of all three accessions, ICPL-151 was superior to the other two accessions in fresh and dry biomass, yield and yield components when tested at the adult stage. The tolerant accession, ICPL 151, accumulated significantly lower Na⁺and CI in shoots. By contrast it was higher in shoot and root K⁺, K/Na ratios, K vs Na selectivity, soluble sugars, free amino acids and proline compared with the other two accessions.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Characterization of a proteinase inhibitor from Cajanus cajan (L.)

A protein proteinase inhibitor (PI) has been purified from pigeonpea Cajanus cajan (L.) PUSA 33 variety by acetic-acid precipitation, salt fractionation and chromatography on a DEAE-Cellulose column. The content of inhibitor was found to be 15 mg/20 g dry weight of pulse. The molecular weight of the inhibitor as determined by SDS-PAGE under reducing conditions was found to be about 14,000. It showed inhibitory activity toward proteolytic enzymes belonging to the serine protease group, namely trypsin and alpha-chymotrypsin. The inhibitory activity was stable over a wide range of pH and temperatures. Estimation of sulfhydryl groups yielded one free cysteine and at least two disulfide linkages. N-terminal sequence homology suggests that it belongs to the Kunitz inhibitor family. Structural analysis by circular dichroism shows that the inhibitor possesses a largely disordered structure.     

Influence of cadmium stress and arbuscular mycorrhizal fungi on nodule senescence in Cajanus cajan (L.) Millsp

Cadmium (Cd) causes oxidative damage and affects nodulation and nitrogen fixation process of legumes. Arbuscular mycorrhizal (AM) fungi have been demonstrated to alleviate heavy metal stress of plants. The present study was conducted to assess role of AM in alleviating negative effects of Cd on nodule senescence in Cajanus cajan genotypes differing in their metal tolerance. Fifteen day-old plants were subjected to Cd treatments--25 mg and 50 mg Cd per kg dry soil and were grown with and without Glomus mosseae. Cd treatments led to a decline in mycorrhizal infection (MI), nodule number and dry weights which was accompanied by reductions in leghemoglobin content, nitrogenase activity, organic acid contents. Cd supply caused a marked decrease in nitrogen (N), phosphorus (P), and iron (Fe) contents. Conversely, Cd increased membrane permeability, thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), and Cd contents in nodules. AM inoculations were beneficial in reducing the above mentioned harmful effects of Cd and significantly improved nodule functioning. Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) increased markedly in nodules of mycorrhizal-stressed plants. The negative effects of Cd were genotype and concentration dependent.     

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     

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     

Genotype-Dependent morphogenetic potentiality of various explants of a food legume,the pigeon pea (Cajanus cajan L.)

Summary The morphogenetic response of various explants of seven different cultivars of a food legume, the pigeon pea (Cajanus cajan L.), has been studied. The stimulation and elongation of shoot buds into shoots derived from the mature embryo axis and intact seed on Murashige and Skoog’s medium supplemented with 2.32µM kinetin and 22.2µM benzyladenine was found to be optimum in Murashige and Skoog’s medium supplemented with 0.46µM kinetin, 0.53µM naphthalene acetic acid, and 0.29µM gibberellic acid. Even though the response of these two explants for formation of shoot buds in all the genotypes is 30–100% depending on media composition, subsequent growth and elongation of these shoot buds into plants is genotype dependent and is restricted to two genotypes. Cotyledon and epicotyl explants of pigeon pea cultivars on the other hand differentiated directly into four to eight and two to four shoots, respectively, depending on the media composition and genotype. In vitro rhizogenesis of regenerated shoots was 80% and the survival of these plantlets in the field was 70–80%. NCL Communication no.: 5667.     

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。     

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.     

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     

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.     

Seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) Effect of differential soil crusting,as induced by raindrop size,and depth of sowing

The effects of differential soil crusting, as achieved by varying the raindrop size, and depth of sowing on seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) were investigated. Three drop diameters: 2.75, 1.65 and 0.93 mm and four depths of sowing: 2, 4, 6, and 8 cm were studied. Mean time of emergence increased with increase in raindrop size and depth of sowing. Crops differed in their emergence capacity under adverse soil conditions from delay in the emergence of chickpea to complete failure in the case of pigeonpea. Pearl millet was not significantly affected by drop size but was found to be sensitive to depths beyond 4 cm. Within the chickpea crop, the variety H 208 was observed to have better emergence qualities than Pusa 209. Other parameters like epicotyl diameter, swelling index, and the area of crust broken by the emerging seedling were measured.     

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.     

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.     

Cajaflavanone and cajanone released from Cajanus cajan (L. Millsp.) roots induce nod genes of Bradyrhizobium sp.

A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium sp (F-4) nod DABC-lacZ translation fusion was constructed and used to monitor nod gene expression in response to pigeonpea root exudate. Two nod-inducing compounds were isolated and identified. Spectral analysis using ultraviolet absorption, infrared spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 5,4-dihydroxy-6-(3-methyl-2-butenyl)-2, 2-dimethyl pyrano-[5, 6:7, 8]-flavanone (cajaflavanone) and 2,4,5-trihydroxy-5-isopentenyl-6, 7-dimethylchromene iso-flavanone (cajanone). When pEA2-21 was introduced into Rhizobium trifolii and R. meliloti cajanone and cajaflavanone did not induce nod gene indicating that specificity of induction appears to be influenced by the host-strain genome.     

Influence of zinc on cadmium-induced toxicity in nodules of pigeonpea (Cajanus cajan L. Millsp.) inoculated with arbuscular mycorrhizal (AM) fungi

Arbuscular mycorrhizal (AM) fungi are known to alleviate heavy-metal stress in plants. The intent of the present work was to analyze accumulation of heavy metals (Cd and Zn) in nodules of two Cajanus cajan (L.) Millsp. genotypes and their subsequent impact on nitrogen fixation, oxidative stress, and non-protein thiols (glutathione and phytochelatins) with and without AM fungus Glomus mosseae. Accumulation of Cd and Zn in nodules resulted in sharp reduction in nodule number, nodule dry mass as well as nitrogen fixation (leghemoglobin and nitrogenase (N₂ase)), although Cd had more pronounced effects than Zn. Cd-induced lipid peroxidation, H₂O₂ accumulation, and electrolyte leakage were largely reversed by Zn supplementation. Zn application significantly altered the negative effects of Cd on the synthesis of non-protein thiols, suggesting antagonistic behaviour of Zn. Higher concentration of Zn was more effective in lessening the negative effects of Cd than its lower concentration. Remarkable genotypic variation was found, with more severe effects of both the metals in P792 than Sel 85N. Glomus mosseae attenuated the phytotoxic effects of metals in nodules by decreasing metal uptake, oxidative stress, and by enhancing defense system ultimately leading to better nitrogen-fixing potential of pigeonpea nodules.