Zeatin-induced shoot regeneration from immature chickpea (Cicer arietinum L.) cotyledons

For the purpose of developing an in vitro regeneration system for chickpea (Cicer arietinum L.), an important food legume, immature cotyledons approximately 5 mm long were excised from developing embryos and cultured on B5 basal medium supplemented with 1.5% sucrose and various growth regulator combinations. Only non-morphogenic callus was formed in response to concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid (NAA) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) previously reported to induce somatic embryogenesis on immature soybean cotyledons. However, 4.6, 13.7, and 45.6 M zeatin induced formation of white, cotyledon-like structures (CLS) at the proximal end of immature cotyledons placed with adaxial surface facing the agar medium. No morphogenesis, or occasional formation of fused, deformed CLS, was observed when zeatin was replaced with kinetin or 6-benzyladenine, respectively. The highest response frequency, 64% of explants forming CLS, was induced by 13.7 M zeatin plus 0.2 M indole-acetic acid (IAA). Within 20–40 days culture on zeatin, shoots formed at the base of CLS on approximately 50% of CLS-bearing explants, and proliferated upon subsequent transfer to basal medium with 4.4 M BA or 4.6 M kinetin. This regeneration system may be useful for genetic transformation of chickpea.     

Genotypical differences in responses to iron deficiency between sensitive and resistant cultivars of chickpea (Cicer arietinum)

Differences in responses to iron deficiency between two chickpea cultivars, NP-62 and K-850, were examined. The apical leaves of NP-62 quickly showed symptoms of iron-deficiency chlorosis when grown on an iron-free medium. By contrast, K-850 showed no visible symptoms on the same medium. Iron contents of the apical leaves of these two cultivars were similar during the first 7 days after they were transferred to the iron-free medium in spite of a marked difference in root-associated Fe³⁺-reduction activity. The susceptibility to iron-deficiency chlorosis observed in NP-62 was not attributable to the poor Fe³⁺-reduction activity of roots but to the inefficient utilization of iron within leaves under conditions when the supply of iron was limited.     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Effect of soil organic matter on chickpea inoculated withAzospirillum brasilense andRhizobium leguminosarum bv.ciceri

Azosprillum inoculated withRhizobium improved the nodulation of chickpea-Cicer arietinum. This interaction was further enhanced by organic matter present in the growth medium.     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

Gene action for cold tolerance in chickpea

Summary Six crosses were investigated using combining ability and generation mean analyses for reaction to cold tolerance in chickpea (Cicer arietinum L.). The combining ability variances revealed the significance of both additive and nonadditive gene effects, with preponderance of additive gene effects. The generation mean analysis revealed the presence of genie interactions in addition to additive and dominance gene effects. Among the interactions, additive×additive and dominance×dominance with duplicate epistasis were present. Cold tolerance was dominant over susceptibility to cold. Selection for cold tolerance would be more effective if dominance and epistatic effects were reduced after a few generations of selfing.Joint contribution from ICARDA and ICRISAT (International Crops Research Institute for the Semi-Arid Tropics), Patancheru P.O., A.P. 502 324, India. ICRISAT JA No. 1239.     

Ultrastructure of chickpea nodules

Summary Developing and senescing chickpea (Cicer arietinum L.) nodules formed byRhizobium sp. (Cicer) CC 1192 have been shown by light and electron microscopy to have general morphological and ultrastructural features that are characteristic of indeterminate nodules. These features included the presence of persistent meristematic tissue at the distal ends of the multi-lobed nodules, and a gradient of cells at different stages of development towards the proximal point of attachment of the nodules to the parent root. The cytoplasm of infected cells in the nitrogen-fixing region of the nodules was densely packed with symbiosomes, most of which contained a single bacteroid. Infection threads containing bacteria were noted in invaded cells from the nitrogen-fixing region of the nodules. Other features that were observed in chickpea nodules included the presence of electron-dense occlusions in intercellular spaces in the nitrogen-fixing region, and plasmodesmata that connected infected cells with other infected cells and with uninfected cells. No poly--hydroxybutyrate granules were noted in the bacteroids. In later stages of development, infected cells became enlarged and highly vacuolated, and eventually lost their contents. Uninfected cells in the central region were smaller than infected cells and were also highly vacuolated. Some of the degenerative processes that take place in senescing bacteroids were noted.     

Root exudates as mediators of mineral acquisition in low-nutrient environments

Plant developmental processes are controlled by internal signals that depend on the adequate supply of mineral nutrients by soil to roots. Thus, the availability of nutrient elements can be a major constraint to plant growth in many environments of the world, especially the tropics where soils are extremely low in nutrients. Plants take up most mineral nutrients through the rhizosphere where micro-organisms interact with plant products in root exudates. Plant root exudates consist of a complex mixture of organic acid anions, phytosiderophores, sugars, vitamins, amino acids, purines, nucleosides, inorganic ions (e.g. HCO₃ ^–, OH^–, H⁺), gaseous molecules (CO₂, H₂), enzymes and root border cells which have major direct or indirect effects on the acquisition of mineral nutrients required for plant growth. Phenolics and aldonic acids exuded directly by roots of N₂-fixing legumes serve as major signals to Rhizobiaceae bacteria which form root nodules where N₂ is reduced to ammonia. Some of the same compounds affect development of mycorrhizal fungi that are crucial for phosphate uptake. Plants growing in low-nutrient environments also employ root exudates in ways other than as symbiotic signals to soil microbes involved in nutrient procurement. Extracellular enzymes release P from organic compounds, and several types of molecules increase iron availability through chelation. Organic acids from root exudates can solubilize unavailable soil Ca, Fe and Al phosphates. Plants growing on nitrate generally maintain electronic neutrality by releasing an excess of anions, including hydroxyl ions. Legumes, which can grow well without nitrate through the benefits of N₂ reduction in the root nodules, must release a net excess of protons. These protons can markedly lower rhizosphere pH and decrease the availability of some mineral nutrients as well as the effective functioning of some soil bacteria, such as the rhizobial bacteria themselves. Thus, environments which are naturally very acidic can pose a challenge to nutrient acquisition by plant roots, and threaten the survival of many beneficial microbes including the roots themselves. A few plants such as Rooibos tea (Aspalathus linearis L.) actively modify their rhizosphere pH by extruding OH^– and HCO₃ ^– to facilitate growth in low pH soils (pH 3 – 5). Our current understanding of how plants use root exudates to modify rhizosphere pH and the potential benefits associated with such processes are assessed in this review.     

In vitro regeneration and propagation of chickpea (Cicer arietinum L.) from meristem tips and cotyledonary nodes

Summary Two representative cultivars ofCicer arietinum, the desi-type cv.Annigeri and the kabuli-type cv.ICCV6, were regenerated in vitro and clonally propagated from cotyledonary nodes and meristem tips. The explants were dissected from 1-wk-old seedlings aseptically germinated on WH medium. In both cultivars, all nodes cultured on B5 medium supplemented with 4.4μM 6-benzylaminopurine developed up to seven shoots per node within 3 wk. Meristem tips were much better suited for multiple shoot formation. Cultured on DKW-C-a medium supplemented with 4.4μM 6-benzylaminopurine and 0.05μM indole-3-butyric acid, 96% of the meristem tips produced up to 10 shoots per explant. A new method in improving clonal propagation was subdividing the meristem tips. Doing so, multiple shoot formation was considerably enhanced: up to 90 shoots per original explant could be obtained with cv.Annigeri, and up to 50 with cv.ICCV6. Indole-3-butyric acid proved to be the best rooting factor. From several media tested, the best root induction and development was achieved on WH medium supplemented with 2.5μ M indole-3-butyric acid: 72% rooting with cv.Annigeri and 68% rooting with cv.ICCV6. With both cultivars there were no differences in rooting capacity between shoots of nodal origin and those derived from meristem tips. The plantlets obtained were transferred into soil and kept under greenhouse conditions. The survival frequency was 28% with cv.Annigeri and 23% with cv.ICCV6. R₀ plants remained smaller than seed-grown controls and produced only a few fertile seeds. There was no difference between R₁ plants and controls in growth, development, and seed set.     

Multiple shoot induction by benzyladenine and complete plant regeneration from seed explants of chickpea (Cicer arietinum L.)

The efficacy of benzyladenine (BA) to induce multiple shoots from seed explants of chickpea (Cicer arietinum L.) was assessed. Shoot differentiation was influenced by the type of seed explant, genotype and concentration of BA. Orientation of the explant also strongly influenced the shoot regeneration response. The optimum BA concentration for shoot/shoot bud regeneration was genotype dependent. Two types of BA-induced response were observed: (1) at less than 7.5 gm BA, direct shoot differentiation (2 to 4-cm-long shoots) was observed within 30 days; (2) at higher BA concentrations (75–100 m), shoot/shoot bud differentiation was achieved in 45–90 days. A high BA concentration inhibited subsequent rooting of shoots. Roots, however, could be easily induced on shoots derived from <12.5 m BA. Following transfer to soil, 80% of the regenerants developed into morphologically normal and fertile plants.Abbreviations BA Benzyladenine     

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea (Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 µM thidiazuron (TDZ) and 2 mM lysine and 2 mM threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 µM TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l^-1 PPT. Increasing the concentrations of PPT to 15 mg l^-1 reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.Abbreviations AK: Aspartate kinase - CP: Chlorophenol red - GUS: -Glucuronidase - IBA: Indole-3-butyric acid - Kn: Kinetin (6-furfuryl aminopurine) - LT: Lysine plus threonine - MSB5: Murashige and Skoog salts with B5 vitamins - nptII: Neomycin phosphotransferase II - pat: Phosphinothricin-acetyltransferase - PPT: Phosphinothricin - TDZ: Thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-YL) urea]Communicated by P. LakshmananJ. Sen and N. Tewari-Singh have contributed equally to this article.     

Carbendazim alleviates effects of water stress on chickpea seedlings

Carbendazim (methyl-2-benzimidazole carbamate) promoted root growth of chickpea (Cicer arietinum L.) seedlings subjected to polyethylene glycol (PEG, osmotic potential −0.5 MPa) induced water stress. The relative water content, membrane stability index, 2,3,5-triphenyltetrazolium chloride reduction and contents of some osmolytes (proline, sucrose, glucose and fructose) enhanced significantly while the contents of lipid peroxides and hydrogen peroxide diminished effectively by addition of 0.05 % carbendazim into PEG solution. This revised version was published online in September 2005 with the corrected author information.     

Effects of supplemental nitrate and thermal regime on the nitrogen nutrition of chickpea (Cicer arietinum L.)

Summary Nodulated chickpea plants were grown in pots in a glasshouse programmed to simulate either hot (32.5°C day/18°C night) or warm (25°/18°C) thermal regimes characteristic of those experienced by crops grown in different seasons or locations in the semi-arid tropics. The plants were irrigated with nutrient solution either devoid of inorganic nitrogen or containing 0.71, 1.43 or 2.86 mM nitrate. Increasing concentrations of supplemental nitrate stimulated the rate of dry matter production by vegetative plants in both thermal regimes. Differences between vegetative dry weight of plants given nitrate and those relying exclusively on symbiotic dinitrogen fixation were greatest in the hot regime where the durations of vegetative growth were shorter. However, symbiotically-dependent plants and those given 0.71 mM nitrate continued to produce branches throughout the reproductive period, particularly in the warm regime. As they matured, these plants became more comparable in vegetative stature to those which had received greater concentrations of nitrate and had established final branch numbers earlier (i.e prior to main pod-fill). Potential seed yields were determined primarily by the number of potential reproductive sites (nodes) available (i.e. by the extent of branching) which largely determined the number of seeds harvested. Since final branch numbers in all nitrate treatments were greatest in the warm regime, yields were also larger than those at 32.5°C. The implications of these data for the nitrogen economy of chickpea crops is discussed.One of a series of papers resulting from a collaborative project with the International Crops Research Institute for the Semi-Arid Tropics, India; sponsored by the UK Overseas Development Administration.     

Effect of salinity on antioxidant responses of chickpea seedlings

The changes in the activity of antioxidant enzymes, like superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase, and growth parameters such as length, fresh and dry weight, proline and H₂O₂ contents, chlorophyll fluorescence (Fv/Fm), quantum yield of PSII and the rate of lipid peroxidation in terms of malondialdehyde in leaf and root tissues of a chickpea cultivar (Cicer arietinum L. cv. Gökçe) under salt treatment were investigated. Plants were subjected to 0.1, 0.2 and 0.5 M NaCl treatments for 2 and 4 days. Compared to controls, salinity resulted in the reduction of length and of the fresh and dry weights of shoot and root tissues. Salinity caused significant (P < 0.05) changes in proline and MDA levels in leaf tissue. In general, a dose-dependent decrease was observed in H₂O₂ content, Fv/Fm and quantum yield of photosynthesis under salt stress. Leaf tissue extracts exhibited three activity bands, of which the higher band was identified as MnSOD and the others as FeSOD and Cu/ZnSOD. A significant enhancement was detected in the activities of Cu/ZnSOD and MnSOD isozymes in both tissues. APX and GR activities exhibited significant increases (P < 0.05) in leaf tissue under all stress treatments, whereas no significant change was observed in root tissue. The activity of CAT was significantly increased under 0.5 M NaCl stress in root tissue, while its activity was decreased in leaf tissue under 0.5 M NaCl stress for 4 days. These results suggest that CAT and SOD activities play an essential protective role against salt stress in chickpea seedlings.     

Influence of inoculation with Ascochyta rabiei on the mineral contents of resistant and susceptible cultivars of chickpea

The determination of mineral contents of healthy as well as Ascochyta rabiei inoculated resistant, moderately resistant, moderately susceptible and susceptible cultivars revealed that the amount of N, P, Zn and Fe did not vary much in healthy plants of the resistant and susceptible cultivars. The amount of K and S was greater in the susceptible cultivars compared to the resistant cultivars while the reverse was true for Cu and Mn. Barring the recovery of Cu and Fe, the amount of all other elements (N, P, K, S, Zn and Mn) was enhanced upon inoculation of resistant, moderately resistant, moderately susceptible and susceptible cultivars. There was a noticeable increase in the amount of K in the resistant cultivars and the reverse was true for P, S and Mg contents after inoculation.     

Direct somatic embryogenesis and plantlet regeneration from immature leaflets in chickpea

Summary Direct somatic embryo formation and plantlet regeneration was achieved from immature leaflets of chickpea (Cicer arietinum L.). Optimal somatic embryogenesis was obtained when immature leaflets were exposed to media supplemented with 15 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for 7 d, to 2000 μM 2,4-D for 3 d, and to 50 μM 2,4-D for 10 d, followed by transfer onto Murashige and Skoog (MS) basal medium. Exposure of explants to high 2,4-D levels (200–2000 μM) for 3 d produced bottle-shaped embryos, while exposure to low 2,4-D levels (<50 μM) and 50–2000 μM for 10 d produced spherical-shaped embryos. Two percent of embryos converted into plants upon culture on MS medium containing 15 μM gibberellic acid and 1 μM 3-indolebutyric acid. All regenerated plants were phenotypically normal.