Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1^-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I^-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I^-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I^-1 2,4-d.Abbreviations B5 Gamborg et al. medium (Gamborg et al. 1968) - 2,4-d 2,4-dichlorophenoxyacetic acid - FN Finer & Nagasawa medium (Finer & Nagasawa 1968) - MS Murashige & Skoog medium (Murashige & Skoog 1962)     

Effect of genotype on somatic embryogenesis from axes of mature peanut embryos

Genotypes representing the three botanical varieties of peanut (Arachis hypogaea L.) were assessed for somatic embryogenesis and subsequent plant conversion from mature zygotic embryo axes. Explants were initially cultured on Murashige and Skoog medium supplemented with 12.42 M 4-amino-3,5,6-trichloropicolinic acid. Individual somatic embryos wer isolated from explant tissue and used to initiate repetitive liquid cultures. There were significant differences among genotypes and varieties for somatic embryo formation and plant regeneration using a single media sequence. Botanical variety fastigiata had a lower embryogenic frequency and produced significantly fewer embryos than either hypogaea or vulgaris, which were similar in response.Abbreviations EA zygotic embryo axes - MS Murashige and Skoog (1962) medium - picloram 4-amino-3,5 - 6 trichloropicolinic acid     

The effect of mineral elements on the maturity of peanut seed

A greenhouse study was conducted to determine the effect, of certain nutrient elements, on the maturing peanut. Peanut fruits were grown in fruiting zones, which contained a complete nutrient medium, for 15 days. Individual plants were then cultured to maturity whilst allowing the fruit to develop in a nutrient medium which contained the complete nutrient (N, P, K, Ca, Mg and B) from which one element had been excluded. Except in the ‘minus B’ treatment, the basal seed weighed more than 500 mg. In the head seed the “minus Ca” treatment produced the lowest number of seeds which weighed 500 mg or more and P, K and B deficiencies produced not quite such low numbers of seeds above 500 mg. When basal and head seeds were grouped into 3 grades of fresh weights, those from Ca and K deficiencies produced smaller dry weights in seeds harvested on the 80th day. Seeds from a Ca deficient medium had a smaller lipid content and an increased sugar content. The starch content of the seed was decreased by K deficiency.     

Diallel and generation means analyses for the components of resistance to Cercospora arachidicola in peanut

Summary The inheritance of the components of partial resistance to Cercospora arachidicola Hori in peanut (Arachis hypogaea L.) was examined in two five-parent diallels and in the six generations of two single crosses in greenhouse tests. The Griffing (1956) analysis indicated general combining ability (GCA) to be of most importance, yet large ratios of SCA/GCA sum of squares suggested nonadditive genetic variance as well. Reciprocal effects were found for lesion area and lesion number/10 cm² leaf area. The importance of nonadditive genetic variance was substantiated by the lack of fit for the additive-dominance model in the Hayman's analysis (1954 a, b). Further evidence from the Hayman's analysis indicated that epistasis may be important in determining the inheritance of some of the components of resistance. Additive gene effects alone accounted for the genetic variability observed among the generation means from two single crosses for all components of resistance except latent period. There was evidence that epistasis was an important mode of gene action for the inheritance of latent period.Paper No. 10172 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27601, USA     

Soil amelioration effects on peanut growth,yield and quality

Summary In order to study the reasons for poor peanut (Arachis hypogaea L.) performance on an Avalon medium sandy loam, a three year field study was undertaken to investigate the effects of lime and gypsum applications on growth, yield and quality. Rates of up to 2,400 kg agricultural lime/ha/annum significantly increased soil pH (1N KCl) and exch. Ca and decreased levels of exch. Al and Al saturation in the soil (0–150 mm). The effect of the same rates of gypsum was much less marked, only exch. Ca increasing and Al saturation decreasing to any substantial extent. In the absence of lime (i.e. even where gypsum was applied). nodulation was poor and the plants developed a general chlorosisc. 90 days after planting. Liming markedly improved nodulation whereas annual applications of gypsum had the opposite effect. Liming significantly increased the hay, pod and kernel yields by up to 73, 105 and 117%, respectively. On average, gypsum applications had no significant effects. Liming increased shelling percentage, the percentage mature pods, 100-kernel mass and protein concentration in the kernel, and decreased the incidence of pops and kernels with black plumule. Applications of gypsum had little effect on quality except for a decreased incidence of black plumule. It appeared that the improved crop performance with liming resulted from a reduction in Al toxicity which improved nodulation. Calcium deficiency did not appear to be a major cause of poor peanut growth and quality in the unameliorated soil.     

Appearance of storage lipid (triglycerides) in somatic embryos of peanut (arachis hypogaea L.)

Summary We estimated the level of triglycerides (triacylglycerol) at intervals of 2 wk during somatic embryogenesis in peanut. The initial triglyceride content in the leaflet explants was depleted during the formation of embryogenic tissue. It increased with the onset of somatic embryogenesis. Concentration of triglyceride in a fully developed embryo increased further if incubated in the same dehydrated medium for a longer period of time. Transferring these embryos to fresh medium led to germination of somatic embryos with a depletion of storage lipids.     

Effect of selected soil factors on chlorosis of peanut plants grown in calcareous soils in Cyprus

In 1986 and 1987 surveys were conducted of 34 (1986) and 35 (1987) peanut (Arachis hypogaea L.) fields in which the plants showed various degrees of chlorosis. In the areas concerned, plant appearance was classified according to a chlorotic index and corresponding soil samples were taken and analysed for CaCO₃, pH, NO₃–N and DTPA-extractable Fe in 1986 and for CaCO₃, NO₃–N and active lime in 1987.Regression analyses showed that CaCO₃, active lime and NO₃–N were positively correlated, while DTPA-extractable Fe was negatively correlated, with the chlorosis problem. The critical levels above which plants were chlorotic were 20 to 25% CaCO₃ and 10% active lime. Plants began to be chlorotic when DTPA-extractable Fe was below 2.5 mg·kg^–1. The soil factors examined explained about 60% of the variability in plant chlorosis.     

The influence of zinc and sulphur deficiency on oil-filling in peanut (Arachis hypogaea L.) kernels

In the developing peanut (Arachis hypogaea L.) kernels, the period between 15 and 35 days after podding (DAP) was identified as the active period of oil-filling. The period of active oil-filling was associated with a decrease in the starch, soluble sugars and proteins so as to make available the energy and carbon skeleton for the synthesis of oil. The oil content in the mature kernels decreased by 11, 12 and 25 per cent with Zn, S and Zn+S deficiency, respectively. In addition, proteins and starch content decreased significantly while that of soluble sugars increased slightly. The activity of malate dehydrogenase and glucose-6-phosphate dehydrogenase also decreased due to Zn as well as S deficiency. The deficiency treatments resulted in a decrease in phospholipids, free fatty acids and triacylglycerols in mature kernels. Further the proportion of 16∶0 and 18∶2 decreased while that of 18∶1 increased in developing kernels.     

Diversity and compatibility of peanut (Arachis hypogaea L.) bradyrhizobia and their host plants

A high degree of genetic diversity among 125 peanut bradyrhizobial strains and among 32 peanut cultivars collected from different regions of China was revealed by using the amplified fragment length polymorphism (AFLP) technique. Eighteen different peanut bradyrhizobial genotypes and six peanut cultivars were selected for symbiotic cross-inoculation experiments. The genomic diversity was reflected in the symbiotic diversity. The peanut cultivars varied in their ability to nodulate with the strains used. Some cultivars had a more restricted host range than the others. Also the strains displayed a range of nodulation patterns. In yield formation there were clear differences between the plant cultivar/bradyrhizobium combinations. There was good compatibility between some peanut bradyrhizobial strains and selected cultivars, with inoculation resulting in well-nodulated, high-yielding symbiotic combinations, but no plant cultivar was compatible with all strains used. The strains displayed a varying degree of effectiveness, with some strains being fairly effective with all cultivars and others with selected ones. The AFLP genotypes of the strains did not explain the symbiotic behavior, whereas the yield formation of the plant cultivars was more related to the genotype. It is concluded that to obtain optimal nitrogen fixation efficiency of peanut in the field, compatible plant cultivar-bradyrhizobium combinations should be selected either by finding inoculant strains compatible with the plant cultivars used, or plant cultivars compatible with the indigenous bradyrhizobia.     

Direct somatic embryogenesis from axes of mature peanut embryos

Summary Plant regeneration via somatic embryogenesis was obtained in peanut (Arachis hypogaea L.) from axes of mature zygotic embryos. The area of greatest embryogenic activity was a 2-mm region adjacent to and encircling the epicotyl. Somatic embryogenesis was evaluated on Murashige and Skoog media supplemented with a variety of auxin treatments. Maximum production occurred on medium supplemented with 3 mg · liter⁻¹ 4-amino-3,5,6-trichloropicolinic acid. Explant cultures were transferred to half-strength medium supplemented with 1 mg · liter⁻¹ gibberellic acid for somatic embryo germination and early plantlet growth. Plantlets, transferred to soil, were placed in a greenhouse and grown to maturity.     

Role of exogenous nitric oxide in alleviating iron deficiency-induced peanut chlorosis on calcareous soil

This study examined the effects of exogenous nitric oxide (NO) on physiological characteristics of peanut (Arachis hypogaea L.) growing on calcareous soil. Sodium nitroprusside (SNP), a NO donor, was root application (directly; slow-release bag; slow-release capsule; slow-release particle) and foliar application. The results showed that SNP application alleviated iron (Fe) deficiency-induced chlorosis, increased the yield of peanut and increased the Fe concentration in peanut grain. SNP, especially supplied by slow-release particle improved the available Fe in soil by reducing pH of soil and increasing available Fe of soil. Furthermore, SNP application significantly increased the H⁺-ATPase and Fe³⁺ reductase activities and increased the total Fe concentration in the leaves. Meanwhile, SNP application, especially foliar application enhanced the availability of Fe in the plant by significantly increasing the active Fe content and chlorophyll content in the leaves. In addition, SNP also increased the antioxidant activities, but decreased the superoxide anion (O₂^??) generation rate and malondialdehyde content, which protected peanut against the Fe deficiency-induced oxidative stress. Therefore, these results support a physiological action of SNP on the availability, uptake and transport of Fe in the plant and foliar application SNP had the best effects in leaves and SNP supplied by slow-release particle had the best effects in roots. In addition, on the whole, the effects of SNP supplied by slow-release ways were better than directly supplied into the soil.     

Characterization of two class II chitinase genes from peanut and expression studies in transgenic tobacco plants

Two different genes encoding class II chitinases from peanut (Arachis hypogaea L. cv. NC4), A.h.Chi2;1 and A.h.Chi2;2, have been cloned. In peanut cell suspension cultures, mRNA levels of A.h.Chi2;2 increased after ethylene or salicylate treatment and in the presence of conidia from Botrytis cinerea. The second gene, A.h.Chi2;1, was only expressed after treatment with the fungal spores. Transgenic tobacco plants containing the complete peanut A.h.Chi2;1 gene exhibited essentially the same expression pattern in leaves as observed in peanut cell cultures. Expression characteristics of transgenic tobacco carrying a promoter-GUS fusion of A.h.Chi2;1 are described.     

Growth and nutrient uptake of peanut inoculated with the mycorrhizal fungusGlomus fasciculatum compared with non-inoculated ones

Summary In a greenhouse study, inoculation with the mycorrhizal fungusGlomus fasciculatum enhanced peanut growth and increased its dry matter more than 2-fold compared with the non-inoculated control, in both sterilized and non-sterilized soil. It also significantly increased uptake of phosphorus and micronutrients such as zinc, copper, manganese and iron.Present address: International Crops Research Institute for the semi-arid tropics (ICRIAST), Patancheru 502 324, A.P. India.     

Effect of silver nitrate on multiple shoot formation of Virginia-type peanut through shoot tip culture

Summary A protocol for micropropagation of Virginia-type peanut plants, an ancient crop of the New World, is reported. This study was conducted to explore the effect of silver nitrate (AgNO₃), alone or in combination with growth regulators, on multiple shoot formation from shoot tip culture. Incorporation of AgNO₃ into the medium, without growth regulators, induced regeneration of the explants (which did not develop at all in the AgNO₃-free medium), and stimulated the emergence of axillary shoots. When AgNO₃ was added in combination with cytokinins and α-naphthaleneacetic acid (NAA), maximum average shoot number per regenerating explant was recorded (6.3) in Murashige and Skoog (MS) medium containing 33 μM 6-benzyladenine, 5.3 μM NAA, and 23.54 μM AgNO₃. Moreover, AgNO₃ showed a positive and marked effect on both shoot elongation and the reduction of callus proliferation from the basal ends of shoot tips. Following a period of elongation, the shoots were rooted in hormone-free Ms medium, showing no residual effects due to the long-term culture in AgNO₃-containing media. Acclimatization was easily obtained after plantlets were transferred to pots under greenhouse conditions, with 90% survival.     

Sulphur fractionation in calcareous soils and bioavailability to plants

Sulphur fractionation and availability to plants are poorly understood in calcareous soils. Sixty-four calcareous soils containing varying amounts of CaCO₃ were collected from ten provinces in China and their S fractions determined. Organic S was the predominant fraction of S, accounting for on average 77% of the soil total S. The amounts of adsorbed sulphate were found to be negligible. 1 M HCl extracted substantially more sulphate than either 0.01 M CaCl₂ or 0.016 M KH₂PO₄, indicating the existence of water-insoluble but acid-soluble sulphate, probably in the form of sulphate co-precipitated with CaCO₃. The concentrations of water-insoluble sulphate correlated positively with the contents of CaCO₃ and accounted for 0.03–40.3% (mean 11.7%) of soil total S. To test the bioavailability of water-insoluble sulphate, a sulphate-CaCO₃ co-precipitate labelled with ³⁵S was prepared and added to a calcareous soil in a pot experiment with either NH₄⁺ or NO₃^– as the N source. In 29 days, wheat plants took up 10.6% and 3.0% of the ³⁵S added to the soil in the NH₄⁺ and NO₃^– treatments, respectively. At the end of the pot experiment, the decrease of water-insoluble, acid-soluble, sulphate was more apparent in the NH₄⁺ than in the NO₃^– treatment. The results indicate that sulphate co-precipitated with CaCO₃ in calcareous soils may become partly available for plant uptake, depending on rhizosphere pH, if the field precipitate is similar to the laboratory prepared sample studied.     

Organization and evolution of resistance gene analogs in peanut

The scarcity of genetic polymorphism in Arachis hypogaea (peanut), as in other monophyletic polyploid species, makes it especially vulnerable to nematode, bacterial, fungal, and viral pathogens. Although no disease resistance genes have been cloned from peanut itself, the conserved motifs in cloned resistance genes from other plant species provide a means to isolate and analyze similar genes from peanut. To survey the number, diversity, evolutionary history, and genomic organization of resistance gene-like sequences in peanut, we isolated 234 resistance gene analogs (RGAs) by using primers designed from conserved regions of different classes of resistance genes including NBS-LRR, and LRR-TM classes. Phylogenetic and sequence analyses were performed to explore evolutionary relationships both among peanut RGAs and with orthologous genes from other plant taxa. Fifty-six overgos designed from the RGA sequences on the basis of their phyletic association were applied to a peanut BAC library; 736 hybridizing BAC clones were fingerprinted and contigs were formed in order to gain insights into the genomic organization of these genes. All the fingerprinting gels were blotted and screened with the respective overgos in order to verify the authenticity of the hits from initial screens, and to explore the physical organization of these genes in terms of both copy number and distribution in the genome. As a result, we identified 250 putative resistance gene loci. A correlation was found between the phyletic positions of the sequences and their physical locations. The BACs isolated here will serve as a valuable resource for future applications, such as map-based cloning, and will help improve our understanding of the evolution and organization of these genes in the peanut genome. Electronic Supplementary Material Supplementary material is available for this article at .     

Differences between Bradyrhizobium strains NC92 and TAL1000 in their nodulation and nitrogen fixation with peanut in iron deficient soil

The effects of Bradyrhizobium (strains NC92 and TAL1000) and Fe supply on nodulation and nitrogen fixation of two peanut (Arachis hypogaea L.) cultivars (cv. Tainan 9 (Fe inefficient) and cv. 71-234 (Fe efficient)) grown under Fe deficient conditions (imposed by adding 40% CaCO₃ to a ferruginous soil) were examined in a glasshouse experiment. When inoculated with TAL1000 without Fe, both cultivars had low shoot N concentration, very low nodule numbers and weight and no measurable acetylene reduction activity per plant. Inoculation with NC92 without Fe increased all these parameters substantially; addition of Fe with NC92 had no further effect on N concentration but doubled nodule number, weight and acetylene reduction activity per plant. Addition of Fe with TAL1000 increased all parameters to the same level as Fe+NC92, indicating that the poorer nodulation and N₂ fixation of TAL1000 in the absence of Fe, resulted from a poorer ability in getting its Fe supply from the alkaline soil. The nodules from all treatments with measurable activity had the same specific acetylene reduction activity suggesting that Fe deficiency limited nodule development.The results support previous suggestions that Bradyrhizobium strains differ greatly in their ability to obtain Fe from soils and that selection of Fe efficient strains could complement plant breeding in the selection of legume crops for Fe deficient soils.     

Effect of TDZ and 2, 4-D on peanut somatic embryogenesis and in vitro bud development

Failure of peanut somatic embryos to convert into plantlets is attributed to the abnormal development of the plumule. Thidiazuron (TDZ) was effective in the conversion of peanut somatic embryos to plantlets by triggering morphogenetic activity in the abnormal plumules of the rooted somatic embryos. The present study aimed to induce normal embryo differentiation by culturing the embryogenic masses in embryo development medium containing 2,4-D and various concentrations of TDZ. Although this was not achieved due to restricted somatic embryo development in the presence of TDZ, bud-like projections appeared in the embryogenic masses when these were cultured in media containing combinations of 2,4-D and TDZ. These projections developed into buds, which subsequently formed shoots and plantlets. The response varied with the concentration and exposure of TDZ. At lower concentrations, the buds appeared in a defined row in the equatorial region of the explant, and with extended incubation, more and more buds appeared in rows alongside the initial row. Induction of multiple buds in a defined row in this specific site (equatorial region) suggested the presence of potent cells around this region. At higher concentrations, these projections appeared in large numbers spread over the whole upper part of the embryogenic mass starting from the equatorial region. The ability of embryogenic mass to convert into organogenic mass and to produce large number of organogenic buds provides an excellent system for basic studies and for the genetic transformation of peanut.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine