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.     

Quantification of the geocarposphere and rhizosphere effect of peanut (Arachis hypogaea L.)

Roots and pods of field-grown peanut (groundnut) (Arachis hypogaea L.) were sampled at the R3, R5, and R7 developmental stages and examined in comparison to root- and pod-free soil for microbial population densities to assess the geocarposphere and rhizosphere effects. G/ S (no. geocarposphere microorganisms/no. soil microorganisms) and R/S (no. rhizosphere microorganisms/no. soil microorganisms) ratios were calculated for total fungi,Asperigillus flavus, spore-forming bacilli, coryneform bacteria, fluorescent pseudomonads, and total bacteria isolated on low- and high-nutrient media. A clear geocarposphere effect was evidenced by increased population densities of bacteria and fungi associated with developing pods compared to soil. G/S and R/S ratios were generally greater than 1.0 for all groups of microorganisms except bacilli. G/S ratios were greater for total bacteria than for total fungi at two of the three sample times, suggesting that bacteria were stimulated more than fungi in the zone around developing pods. In contrast, R/S ratios, were higher for total fungi than for total bacteria at two of three sample times. The preferential association of fungi and bacteria with early developmental stages of the pod indicates that some microorganisms are particularly well adapted for colonization of the peanut geocarposphere. These microorganisms are logical candidates for evaluation as biological control candiates forA. flavus.     

Shoot organogenesis from cultured seed explants of peanut (Arachis hypogaea L.) using thidiazuron

Summary Thidiazuron (TDZ) was utilized to induce adventitious shoot formation from the hypocotyl region of cultured seed explants of peanut (Arachis hypogaea L.). Excision of the radicle from seed explants was more stimulatory to shoot initiation than removal of the epicotyl alone. Removal of both the radicle and the epicotyl from seeds resulted in a 37-fold increase in the frequency of shoot production when compared to intact seeds. Half seed explants with epicotyl and radicle removed produced the greatest number of shoots per explant. Explants from mature seeds were more responsive to TDZ than immature seed-derived explants. A 1-wk exposure to 10 μM TDZ was sufficient to stimulate the initiation of adventitious shoots that subsequently developed into plants. High frequency of shoot initiation was readily induced in a variety of genotypes ofA. hypogaea and a wild peanut (A. glabrata). Plants regenerated from shoots induced by TDZ were phenotypically normal and fertile.     

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.     

Efficient transformation and regeneration of diverse cultivars of peanut (Arachis hypogaea L.) by particle bombardment into embryogenic callus produced from mature seeds

Peanut, one of the world's most important oilseed crops, has a narrow germplasm base and lacks sources of resistance to several major diseases. The species is considered recalcitrant to transformation, with few confirmed transgenic plants upon particle bombardment or Agrobacterium treatment. Reported transformation methods are limited by low efficiency, cultivar specificity, chimeric or infertile transformants, or availability of explants. Here we present a method to efficiently transform cultivars in both botanical types of peanut, by (1) particle bombardment into embryogenic callus derived from mature seeds, (2) escape-free (not stepwise) selection for hygromycin B resistance, (3) brief osmotic desiccation followed by sequential incubation on charcoal and cytokinin-containing media; resulting in efficient conversion of transformed somatic embryos into fertile, non-chimeric, transgenic plants. The method produces three to six independent transformants per bombardment of 10 cm2 embryogenic callus. Potted, transgenic plant lines can be regenerated within 9 months of callus initiation, or 6 months after bombardment. Transgene copy number ranged from one to 20 with multiple integration sites. There was ca. 50% coexpression of hph and luc or uidA genes coprecipitated on separate plasmids. Reporter gene (luc) expression was confirmed in T1 progeny from each of six tested independent transformants. Insufficient seeds were produced under containment conditions to determine segregation ratios. The practicality of the technique for efficient cotransformation with selected and unselected genes is demonstrated using major commercial peanut varieties in Australia (cv. NC-7, a virginia market type) and Indonesia (cv. Gajah, a spanish market type).     

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.     

Agrobacterium tumefaciens mediated gene transfer in peanut (Arachis hypogaea L.)

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring -glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - NOS nopaline synthase - NPT II neomycin phosphotransferase II - SDS Lauryl sulfate     

Effect of microspore stage and media on anther culture of peanut (Arachis hypogaea L.)

This study was designed to study the effects of stage of microspore development and culture medium on androgenic response in peanut (Arachis hypogaea L.). Anthers of various developmental stages were cultured for 7 days, then fixed and observed cytologically. Three sets of media, involving different basal media, growth regulators, sucrose levels and glutamine concentrations, were tested. In all experiments, the stage of development of the microspores at the time of culture was highly significant. The early uninucleate microspores stage was identified as producing the highest anther response rating. The effect of media was nonsignificant in all experiments. However, the stepwise modification of the media through the course of the study resulted in an almost 8 x increase in anther response rating. Numerically, the best media tested was N₆ basal medium with 1 mg 1^-1 NAA, 0.1 mg 1^-1 BA, 5.5% sucrose, and 3.5 g 1^-1 glutamine. While no haploids were obtained, four-nucleate cells were observed, indicating the potential in peanuts for an androgenic reponse.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

花生转基因研究进展

花生是世界上重要的油料和经济作物之一,是人们生活的植物脂肪和蛋白质来源。现代生物技术的不断发展为花生育种和种质创新提供了新的技术手段, 它可以直接将来自不同种属的异源目的基因插人到花生基因组, 使花生表达目标性状, 实现花生品种的遗传改良。近年来, 国内外花生转基因研究取得了重大进展。文章综述了花生转基因在抗虫、抗病、抗非生物逆境和品质改良等方面的最新进展,并总结了近年来人们对农杆菌介导法、基因枪法和不依赖组织培养的转化法等主要的花生遗传转化方法的改进和探索。     

Expression of a synthetic cry1EC gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.)

The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). S. litura is susceptible to the chimeric delta-endotoxin Cry1EC reported earlier. De-embryonated cotyledon explants of peanut were transformed using Agrobacterium tumefaciens strain EHA101 harboring a synthetic cry1EC gene driven by the CaMV 35S promoter. Transgenic plants of peanut with a single copy insertion of cry1EC were selected in the T(0) generation by Southern blot hybridization. Real-time PCR, Western blot and ELISA analysis indicated that expression of the cry1EC gene was higher in single copy T(1) plants. Immunoassay showed expression of Cry1EC up to 0.13% of total soluble protein in T(1) plants. Leaf feeding bioassay on highly expressing transgenic lines showed 100% killing of larvae at the 2(nd) instar stage of S. litura. This is the first report of transgenic peanut plants with resistance to S. litura.     

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.     

Light, dark and growth regulator involvement in groundnut (Arachis hypogaea L.) pod development

Gynophore elongation and pod formation were studied in peanut plants (Arachis hypogaea L.) under light and dark conditions in vivo. The gynophores elongated until pod formation was initiated. Pod (3–20 mm length) development could be totally controlled by alternating dark (switched on) and light (switched off) conditions, repeatedly. Gynophore elongation responded conversely to light/dark conditions, compared to pods. In this study we aimed to correlate the light/dark effects with endogenous growth substances. The levels of endogenous growth substances were determined in the different stags of pod development. Gynophores shortly after penetration into the soil, white gynophores, released twice the amount of ethylene as compared to the aerial green ones, or to gynophores bearing pods. Ethylene inhibitors had no effect on the percent of gynophores that developed pods, but affected pod size which were smaller compared to the control. A similar level of IAA was extracted from gynophore tips of green gynophores, white gynophores and pods. ABA levels differed between the three stages and were highest in the green gynophores and lowest in the pods.Abbreviations ABA abscisic acid - AOA aminooxyacetic acid - ELISA enzyme linked immunosorbent assay - Ethrel 2-chloroethanephosphonic acid - GC gas chromatography - HPLC High Performance Liquid Chromatography - IAA indole-3-acetic acid - NAA naphthalene acetic acid - RIA radioimmunoassay - STS silver thiosulfhate - TIBA 2,3,6-triiodobenzoic acid     

Uptake and partitioning of cadmium by cultivars of peanut (Arachis hypogaea L.)

Cadmium has been found to accumulate in peanut (Arachis hypogaea) kernels to levels exceeding the current maximum permitted concentration in Australia of 0.1 mg kg^-1. Little is known of the mechanisms of Cd uptake into kernels by cultivars of peanut, so the aims of the experiments reported here were to determine if Cd is absorbed directly through the pod wall or via the main root system, and if differences exist between cultivars in this respect. Split-pot soil and sand/nutrient solution experiments were performed with two cultivars of peanut (cv. NC7 and Streeton) known to accumulate Cd to different levels in the kernel. The growth medium was separated into pod and root zones with Cd concentrations in each zone varied. In confirmation of previous field trial results, cv. NC7 had higher concentrations of Cd in kernels, given the same Cd levels in the external medium (solution or soil). Despite total Cd uptake by cv. NC7 being similar to cv. Streeton, cv. NC7 appeared to retain more Cd in the roots and translocate less Cd to shoots. Results from both soil and sand/solution culture indicated that the dominant path of Cd uptake by peanut was via the main root system, with direct pod uptake contributing less than 5% of the total Cd in the kernel. There was little difference between cultivars in this characteristic. This indicates that unlike Ca nutrition of peanuts, agronomic techniques to manage Cd uptake will require modification of soil to the full depth of root exploration, rather than just the surface strata where pods develop. Cadmium concentrations in testa were up to an order of magnitude higher than in the kernel, indicating that blanching of kernels would be effective in reducing Cd in the marketed product. This revised version was published online in June 2006 with corrections to the Cover Date.     

Factors influencing the Agrobacterium tumefaciens-mediated transformation of carrot (Daucus carota L.)

To develop an efficient procedure for Agrobacterium tumefaciens-mediated genetic transformation of carrot (Daucus carota L.) the effects of several factors were studied. Parameters which significantly affected the transformation frequency were the variety, the explant type, and the co-cultivation period. Under optimal conditions, using the A. tumefaciens C58C1 containing either pGSTRN943 or pGSGluc1 and 3 days of co-cultivation, the frequency of transformation of petiole explants of the variety Nanco was greater than 45%. This procedure does not require acetosyringone or prolonged precultivation period. Using kanamycin (100 mg l^-1) for selection, a large number of transgenic plantlets developed from the embryogenic calli within 8–10 weeks of culture on hormone-free medium. Transformation was confirmed by histochemical detection of -glucuronidase activity in the transformed cells, by the ability of petiole segments to produce embryogenic calli in presence of kanamycin, and by Southern hybridization analyses.     

Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile peanut (Arachis hypogaea L.) plants

Summary An efficient regeneration system was developed by culturing immature cotyledons and embryo axes of Arachis hypogaea L. cv. Georgia Green on Murashige and Skoog basal medium (MS) supplemented with various concentrations of thidiazuron (TDZ; 1, 5, 10, and 15 μM). Highly morphogenic callus was produced from 100% of the explants comprising the cotyledon with attached embryo axis when cultured in the dark on 10 μM TDZ. Upon excision and continued culture in the dark on 10 μM TDZ, morphogenic callus grew repetitively during monthly subcultures and retained its regeneration potential. For organogenesis, a gradual reduction in TDZ concentration and exposure to light were necessary before transfer to MS basal medium. Inclusion of indole-3-butyric acid in liquid MS medium favored rooting of recovered shoots. A distinct feature of this investigation is the induction of highly morphogenic callus by TDZ and regeneration of morphologically normal, fertile peanut plants after 8 months of callus subculture.     

农杆菌介导的小麦遗传转化几个影响因素的研究

采用携带gus和（或）bar基因双元表达载体（p3301,pBTAaB)的3个根癌农杆菌（Agrobacterium tumefaciens)菌株（AGL-1,EHA105和LBA4404）对普通小麦（Triticum aestivumL.)冬性栽培品种农大170和农大146的幼胚及幼胚愈伤组织进行了遗传转化,结果表明,菌液浓度OD6001.0和侵染时间1h对外植体的生存和转化最为有利;侵染前对外植体进行高渗处理较明显地提高了抗性愈伤获得率;乙酰丁香酮（AS）对小麦转化的作用随菌株和外植体的不同而异;菌株/质粒组合,受体基因型及外植体的类型,年龄和生理状态对转化效率有很大的影响,条件优化后,得到大量具有PPT抗性的愈伤和一些抗性植株,抗性愈伤的GUS染色阳性率在50%-60%之间,所检测的抗性苗呈GUS阳性,对6株抗性苗的PCR和Southern检测初步证明,外源基因已经整合到其中3株的基因组中。     

The transformation of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): applicable methods of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.     

Diallel analysis in groundnut (Arachis hypogaea L.)

Summary An 8 × 8 full diallel experiment based on 4 bunch plus 4 spreading types of groundnut (Arachis hypogaea L.) was conducted over three environments. For both number of pods and pod yield, additive, nonadditive and reciprocal cross effects were detected and these were also influenced by changes in environments. For number of pods additive genetic variance was predominant whereas it was approximately equal to non-additive genetic variance for pod yield. Graphical analysis revealed the presence of strong non-allelic interaction for number of pods whereas for pod yield absence of dominance and/or presence of non-allelic interaction was evident.Part of Ph.D. Thesis of the first author     

Genetic transformation of peanut (Arachis hypogaea L.) using cotyledonary node as explant and a promoterlessgus::nptII fusion gene based vector

We have generated putative promoter tagged transgenic lines inArachis hypogaea cv JL-24 using cotyledonary node (CN) as an explant and a promoterless gus::nptII bifunctional fusion gene mediated byAgrobacterium transformation. MS medium fortified with 6-benzylaminopurine (BAP) at 4 mg/l in combination with 0.1 mg/l α-napthaleneacetic acid (NAA) was the most effective out of the various BAP and NAA combinations tested in multiple shoot bud formation. Parameters enhancing genetic transformation viz. seedling age,Agrobacterium genetic background and co-cultivation periods were studied by using the binary vector p35SGUSINT. Genetic transformation with CN explants from 6-day-old seedlings co-cultivated withAgrobacterium GV2260 strain for 3 days resulted in high kanamycin resistant shoot induction percentage (45%); approximately 31% transformation frequency was achieved with p35S GUSINT in Β-glucuronidase (GUS) assays. Among thein vivo GUS fusions studied with promoterless gus::nptII construct, GUS-positive sectors occupied 38% of the total transient GUS percentage. We have generated over 141 putative T₀ plants by using the promoterless construct and transferred them to the field. Among these, 82 plants survived well in the green house and 5 plants corresponding to 3.54% showed stable integration of the fusion gene as evidenced by GUS, polymerase chain reaction (PCR) and Southern blot analyses. Twenty-four plants were positive for GUS showing either tissue-specific expression or blue spots in at least one plant part. The progeny of 15 T₀ plants indicated Mendelian inheritance pattern of segregation for single-copy integration. The tissue-specific GUS expression patterns were more or less similar in both T₀ and corresponding T₁ progeny plants. We present the differential patterns of GUS expression identified in the putative promoter-tagged transgenic lines in the present communication.