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.     

Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants

Transformation of plants is a popular tool for modifying various desirable traits. Marker genes, like those encoding for bacterial β-glucuronidase (GUS), firefly luciferase (LUC) or jellyfish green fluorescent protein (GFP) have been shown to be very useful for establishing of efficient transformation protocols. Due to favourable properties such as no need of exogenous substrates and easy visualization, GFP has been found to be superior in to other markers in many cases. However, the use of GFP fluorescence is associated with some obstacles, mostly related to the diminishing of green fluorescence in older tissues, variation in fluorescence levels among different tissues and organs, and occasional interference with other fluorescing compounds in plants. This paper briefly summarizes basic GFP properties and applications, and describes in more detail the contribution of GFP to the establishment, evaluation and improvement of transformation procedures for plants. Moreover, features and possible obstacles associated with monitoring GFP fluorescence are discussed.     

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.     

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     

Routine utilization of green fluorescent protein as a visual selectable marker for cereal transformation

Summary Development of new selectable markers is needed to increase the efficiency and flexibility of plant transformation, and to overcome drawbacks sometimes associated with use of existing markers. A useful alternative to chemical-based selection systems would be a system using visual screening to obtain transgenic lines. Investigations were carried out to determine if the green fluorescent protein (gfp) gene could be utilized alone as a visual screenable marker to produce stably transformed, fertile oat plants. Twelve experiments were conducted in which gfp-based selection was utilized to obtain routinely stable transgenic lines in oat. A synthetic gfp gene under the control of the maize ubiquitin promoter was delivered into embryogenic oat callus via microprojectile bombardment. Cell clusters (1–3 mm) expressing gfp were visually identified using epifluorescence microscopy and physically isolated approximately 3 wk post-bombardment. Fertile, gfp-expressing T0 plants were regenerated from 78% of the glowing cell sectors placed on regeneration medium. T0 plants from 55% of the events produced gfp-expressing progeny in a 3∶1 Mendelian ratio. Southern blot and PCR analysis confirmed transgene integration and transmission to progeny. Expression of gfp did not reduce plant growth or fertility. Transgene copy number and integration patterns were similar to those in transgenic plants derived from chemical-based selection systems. The mean transformation frequency, based on fertile events obtained per bombarded plate, was 1.8%. Over 180 independent transgenic oat lines were produced, to date, using only visual screening for expression of gfp, demonstrating efficiency and repeatability of the selection system. Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the University of Wisconsin or the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Stable transformation of peanut callus viaAgrobacterium-mediated DNA transfer

Transformed callus was produced from peanut (Arachis hypogaea L. cv. Okrun) hypocotyl explants after four days of co-cultivation withAgrobacterium tumefaciens strains EHA101, LBA4404 or ASE1 carrying the binary vector pKYLX71GUS on a defined medium followed by selection with kanamycin (200 mg l^–1). Transformed calluses were cultured as independent cell lines potentially derived from a single transformation event. Stable integration and expression of foreign gene(s) in the callus was confirmed by Southern and western blot analyses and enzyme assays. A few cell lines showed a single insert of the foreign gene. Using the above protocol, transformed peanut callus expressing the peanut stripe virus coat protein gene was obtained.     

Green fluorescent protein as an efficient selection marker for Agrobacterium rhizogenes mediated carrot transformation

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot (Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants.     

Biological projectiles (phage, yeast, bacteria) for genetic transformation of plants

Summary Bacteriophage lambda particles, yeast cells, and bacterial cells were tested as projectiles to deliver marker/reporter genes into plant cells via the biolistic process. When phage particles were complexed to tungsten or gold particles and used to bombard tobacco cells, fewer than 15 cell clusters per plate transiently expressed β-glucuronidase (GUS). Cells of wildtype Saccharomyces cerevisiae were too large to be effective projectiles, but use of a reduced-size mutant resulted in a small number of transformants. Escherichia coli cells complexed with tungsten were the most effective projectile for plant transformation. Various methods to prepare E. coli were tested to reduce particle size, improve binding of bacteria to metal particles, and/or minimize particle clumping. In maize, the number of transformants was highest when bacteria/tungsten particles were air-dried onto macrocarriers from an aqueous solution. When maize cells were bombarded with bacteria/tungsten projectiles, rates of transient gene expression (2000 per plate) and stable transformation (50 per plate) were only two- to threefold lower than when purified DNA was used. Transformation of tobacco with E. coli projectiles was improved when the bacteria were treated with a series of ethanol and ether washes, then dried into a powder. Nevertheless, tobacco transformation was still 24- (transient) and 200-fold (stable) less than when purified DNA was used. Biological projectiles can be effective for plant transformation and are advantageous because once a DNA construct is made and put into the appropriate microorganism, the need to isolate and purify DNA for the biolistic process is eliminated, which saves time and lessens DNA shear. Such projectiles may be especially well suited where high molecular weight DNA constructs are needed.     

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.     

Exploratory integration of peanut genetic and physical maps and possible contributions from Arabidopsis

Arachis hypogaea is a widely cultivated crop both as an oilseed and protein source. The genomic analysis of Arachis species hitherto has been limited to the construction of genetic maps; the most comprehensive one contains 370 loci over 2,210 cM in length. However, no attempt has been made to analyze the physical structure of the peanut genome. To investigate the practicality of physical mapping in peanut, we applied a total of 117 oligonucleotide-based probes (overgos) derived from genetically mapped RFLP probes onto peanut BAC filters containing 182,784 peanut large-insert DNA clones in a multiplex experimental design; 91.5% of the overgos identified at least one BAC clone. In order to gain insights into the potential value of Arabidopsis genome sequence for studies in divergent species with complex genomes such as peanut, we employed 576 Arabidopsis-derived overgos selected on the basis of maximum homology to orthologous sequences in other plant taxa to screen the peanut BAC library. A total of 353 (61.3%) overgos detected at least one peanut BAC clone. This experiment represents the first steps toward the creation of a physical map in peanut and illustrates the potential value of leveraging information from distantly related species such as Arabidopsis for both practical applications such as comparative map-based cloning and shedding light on evolutionary relationships. We also evaluated the possible correlation between functional categories of Arabidopsis overgos and their success rates in hybridization to the peanut BAC library.Electronic Supplementary Material Supplementary material is available for this article at     

Use of the GUS gene as a selectable marker for Agrobacterium-mediated transformation of Rubus

A transformation system was established for red raspberry, blackberry and blackberry x raspberry hybrids, utilizing the binary vector system of Agrobacterium tumefaciens. Leaf discs or internodal stem segments were inoculated with Agrobacterium strain LBA4404 containing the binary vectors PBI121.X, which has the -glucuronidase (GUS) marker gene, or Bin 19, which has the neomycin phosphotransferase II (NPT II) gene. Regenerants were produced on media containing MS salts, 20 gl^-1 sucrose, 7 gl^-1 agar, 100 mgl^-1 inositol, 0.5 mgl^-1 nicotinic acid, 0.5 mgl^-1 pyridoxine-HCl, 0.1 mgl^-1 thiamine, and either 0.1 mgl^-1 IBA and 2 mgl^-1 BAP for leaf discs, or 0.2 mgl^-1 BAP and 0.2 mgl^-1 2,4-D for stem segments. Kanamycin sulphate, which was used as a selective agent for the NPT II gene, inhibited organogenesis at 50 mgl^-1 and was therefore unsuitable for use as a selectable marker gene in Rubus. All regenerants were assayed utilizing the fluorogenic assay procedure to determine if the GUS gene had been transferred into the material and could therefore cleave the substrate 4-methyl-umbelliferyl--D-glucuronide. Seven GUS-positive plantlets were obtained which confirmed that this marker gene had been transferred into Rubus. A dot blot assay was carried out on GUS-positive plant material to establish if the NPT II gene had also been transferred to the plant material.     

An improved method for Beauveria bassiana transformation using phosphinothricin acetlytransferase and green fluorescent protein fusion gene as a selectable and visible marker

An improved transformation method for the biocontrol agent, Beauveria bassiana, was developed. For convenience of transformation selection and detection, the coding regions of the genes for phosphinothricin acetyltransferase and green fluorescent protein were fused and an expression vector, pBFT, carrying this fusion was constructed. Under optimum conditions, over 60 transformants microg(-1) plasmid DNA were obtained. B. bassiana conidia frozen 1 month at -80 degrees C were fully competent for transformation. The method was significantly less laborious and more rapid than current methods for B. bassiana. The bar::egfp provides a selectable and visible marker which may expedite future genetic engineering of this fungus.     

Genetic variation for VA mycorrhiza-dependent phosphate mobilisation in groundnut (Arachis hypogaea L.)

Nine cultivars of groundnut (Arachis hypogaea L.) were grown in a soil poor in available N or P. There was clearly genetic variation of characteristics indicative of VA mycorrhiza-dependent phosphate mobilisation, namely, VA mycorrhiza fungal spore count (SC), percentage of infection (IF) by VA mycorrhizal fungi (VAMF) and acid and alkaline phosphatase activities. Among the cultivars, one was non-nodulating with low values for all characteristics and in another experiment, this non-nodulating cultivar, one of its parents (PI 259747), a national check (Robut 33-1) and the highest yielding cultivar among the original nine (NFG 7), were grown and investigated for various P-mobilising properties and yield. The linear regression coefficient of pod yield on % VAMF infection was significant in both the experiments. Additionally, many of the correlation coefficients of pod yield and VAM dependent characteristics were positive and significant. From consideration of published evidence, it seems possible to breed for the desirable reinforcing effects of infestation, by VAMF and Rhizobium that can ultimately improve the productivity of groundnuts.     

Comparative identification by fatty acid analysis of soil,rhizosphere, and geocarposphere bacteria of peanut (Arachis hypogaea L.)

Bacterial isolates were collected from the geocarposphere, rhizosphere, and root-free soil of field grown peanut (Arachis hypogaea L.) at three sample dates, and the isolates were identified by analysis of fatty acid methyl-esters to determine if qualitative differences exist among the bacterial microflora of these zones. Five bacterial genera were associated with isolates from soil, while pod and root isolates constituted 16 and 13 genera, respectively, indicating that bacterial diversity was higher in the rhizosphere and geocarposphere than in soil. The dominant (most frequently identified) genus across all three samples dates was Flavobacterium, for pods, Pseudomonas for roots, and Bacillus, for root-free soil. Sixteen bacterial taxa were only isolated from the geocarposphere, 7 only from the rhizosphere, and 5 only from soil. These results show that specific bacterial taxa are preferentially adapted to colonization of the geocarposphere and suggest that the soil, rhizosphere, and geocarposphere constitute three distinct ecological niches. Bacteria which colonize the geocarposphere should be examined as potential biological control agents for pod-invading fungi such as the toxigenic strains of Aspergillus flavus and A. parasiticus.     

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     

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     

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)     

Agrobacterium tumefaciens-mediated transformation of the legume Astragalus sinicus using kanamycin resistance selection and green fluorescent protein expression

To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l^–1 -naphthaleneacetic acid (NAA) and 1.0 mg l^–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml^–1 Km, 200 g ml^–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T₁ seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses.     

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.