Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana

In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment. MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant. A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator. The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato. Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes. However, no transgenic somatic embryos were recovered from selection on 50-100 micromol/L HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos. Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues. Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed.     

Repetitive somatic embryogenesis from peanut cultures in liquid medium

Summary A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BA 6-benzylaminopurine - MSO Modified Murashige and Skoog basal medium - EM embryogenic masses     

Polyphenols from peanut skins and their free radical-scavenging effects

Separation of the water-soluble fraction of peanut skins led to the isolation of five proanthocyanidins. Based on the spectroscopic investigation and partial acid catalyzed degradation, their structures were determined to be epicatechin-(2beta-->O -->7, 4beta -->6)-[epicatechin-(4beta-->8)]-catechin (1), epicatechin-(2beta-->O -->7, 4beta-->8) epicatechin-(4beta-->8)-catechin-(4alpha-->8)-epicatechin (2), and procyanidins B2 (3), B3 (4) and B4 (5). The absolute configuration of the new compounds was determined from their circular dichroism curves and the (1)H NMR spectra of analysis of flavan-3-ols formed by thiolytic degradation of 1 and 2 in the presence of a chiral dirhodium complex (dirhodium tetra-(R)-(trifluoromethyl) phenyl acetate).     

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     

Tolerance of peanut to excess boron

The tolerance ofArachis hypogaea cv. Shulamit to high concentrations of B in nutrient solution, [B]o, was determined under greenhouse conditions that promoted the production of vegetative dry matter. Plants grew in large containers in which a root zone of nutrient solution was separated from a pod zone of soil. Grain yield was reduced at a calculated [B]o-threshold of 0.29 mM, which was associated with a concentration of B in the vegetative shoots that was approximately four times larger than the control. Symptoms of B toxicity occurred on leaves as young as the third unfolded leaf from the shoot apex before the [B]o-threshold. Excess B caused a relatively larger decrease in pod number than in vegetative shoot weight, which was high in all treatments (78 g d.wt/plant) and it did not decrease single grain weight. It was suggested that the tolerance of grain development to excess B was a consequence of the high ratio of vegetative matter to pod number.     

Somatic embryogenesis from mature embryo-derived leaflets of peanut (Arachis Hypogaea L.)

Summary Embryogenic masses were obtained from immature leaves of peanut (Arachis hypogaea L.) cultured on a medium containing 20 mg/l 2,4-D. Somatic embryos developed from these masses following transfer to a medium containing 3 mg/l 2,4-D. The embryo morphology was quite variable. Following transfer to hormone-free medium, these embryos germinated. Shoot elongation was obtained in 25% of the embryos following transfer to a medium supplemented with 0.5 mg/l each of BAP and Kn. The plants grown in vitro by this method survived in sand:soil mixture and were grown to maturity.Abbreviations ABA abscisic acid - BAP 6-benzyl amino purine - 2,4-D 2,4 dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kn kinetin - NAA 1-naphthaleneacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - Z zeatin     

Comparison of anionic with cationic peroxidase from cultured peanut cells

A cationic and an anionic peanut peroxidase were isolated to purity as shown by 2D electrophoresis. Amino acid analysis offered evidence for differences. Variations between the isozymes were also noted in a slight difference in the heme absorption maxima, specific enzyme activity and particularly in the relative amount of each in the suspension medium measured by the heme absorption. In contrast the two isozymes were at least partially similar in their structure as demonstrated by the crossreaction with the antisera. The percent crossreactions were used in turn to amend the calculation for the synthetic rate of each isozyme. In spite of the difference in amount secreted in the suspension medium, the in vivo biosynthetic rate of the two isozyme measured cellularly is much the same.     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

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)     

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.     

Development of an RFLP linkage map in diploid peanut species

An RFLP linkage map of peanut has been developed for use in genetic studies and breeding programs aimed at improving the cultivated species (Arachis hypogaea L.). An F₂ population derived from the interspecific hybridization of two related diploid species in the sectionArachis (A. stenosperma ×A. cardenasii) was used to construct the map. Both random genomic and cDNA clones were used to develop the framework of the map. In addition, three cDNA clones representing genes coding for enzymes involved in the lipid biosynthesis pathway have been mapped in peanut. Of the 100 genomic and 300 cDNA clones evaluated, 15 and 190, respectively, revealed polymorphisms among the parents of our mapping population. Unfortunately, a large number of these produced complex banding patterns that could not be mapped. Of the 132 markers analyzed for segregation, 117 are distributed among 11 linkage groups, while 15 have not yet been associated with any other marker. A total map distance of approximately 1063 cM has been covered to-date.     

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     

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 .     

Evaluation of peanut (Arachis hypogaea L.) leaflets from mature zygotic embryos as recipient tissue for biolostic gene transfer

Leaflets from mature peanut embryos are a useful recipient tissue for biolistic DNA transfer. Fertile plants were regenerated from leaflets from genotypes representing all botanical types of peanut. Regeneration frequency was strongly influenced by genotype. NPT II and GUS chimaeric gene fusions, driven by the CaMV 35S promoter, were expressed transiently following biolistic delivery to unexpanded leaflets. Bombardment conditions affecting transient expression frequency were determined using a prototype of the Bio Rad PDS 1000/He helium-powered particle acceleration apparatus. Stably transformed calli were derived routinely from leaflet tissue bombarded with the NPT II gene and subsequently cultured on kanamycin. Several plants have been regenerated from treated explants under kanamycin selection. Thus far, none of these has been stably transformed. The occurrence of escapes suggests that kanamycin is an inefficient selective agent for the recovery of transgenic peanuts from this explant. Experiments designed to regenerate plants using published regeneration protocols from stably transformed calli, devoid of primary explant tissue, have been unsuccessful.     

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.     

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.     

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.     

Localization of mannose- and galactose-binding lectins in an effective peanut nodule

Summary Mannose/glucose- and galactose-binding lectins (ML and GL respectively, were located by immunogold labelling in tissues of a peanut (Arachis hypogaea) nodule induced by an effectiveBradyrhizobium sp. strain. Light and electron microscopic examination of silver-enhanced semithin and ultrathin sections, respectively, revealed that both lectins were widely distributed throughout the cortex and bacteroidal zones although ML was more abundant. The lectins were predominantly in the vacuoles of cortical cells but GL was absent from, or at low concentration in, a two-cell-thick layer of cortical cells surrounding the bacteroidal region. Only ML was detected in cells of the vascular bundle endodermis and in central vascular bundle cells; neither lectin was found in pericycle cells. Bacteroidal cells contained abundant ML in the nuclei and cytoplasm surrounding bacteroids while GL was mainly located in the central vacuoles of these cells. Neither lectin was associated with bacteroid surfaces, peribacteroid membranes, plant cell walls or cell organelles and membranes. The above observations indicate that the nodule lectins are not symbiotic cell recognition determinants and suggest that they have protein storage functions.Abbreviations BSA bovine serum albumin - GL galactose-binding lectin - ML mannose-binding lectin - PBS phosphate-buffered saline - PBST phosphate-buffered saline plus Tween     

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.     

花生茎叶酚性成分研究

运用大孔树脂对花生茎叶提取液进行富集,不同浓度乙醇洗脱,硅胶、RP-18、Sephadex LH-20等多种材料进一步分离纯化,研究花生茎叶化学成分,并通过理化方法和光谱分析对化合物进行结构鉴定。结果表明:从花生茎叶大孔树脂10%乙醇洗脱部位中分离并鉴定了10个化合物,分别为邻苯二甲酸二异丁酯(1)、水杨酸(2)、儿茶酚(3)、对羟基苯甲酸(4)、(反)-3,4-二羟基苯丙烯酸(5)、对羟基苯酚(6)、邻苯二甲酸二丁酯(7)、3,4-二羟基苯乙醇(8)、对羟基苯乙醇(9)、3,4-二羟基苯甲酸(10)。除化合物1、2和4外,其余均为首次从该植物中分离得到。