Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

Protoplast Formation and Regeneration in Lactic Streptococci

We cloned the promoter of the 9-cis-epoxycarotenoid dioxygenase gene from Arachis hypogaea L. β-Glucuronidase (GUS) histochemical staining and GUS activity assay indicated that the activity of the promoter was exhibited predominantly in the leaves and enhanced by water and NaCl stresses, and by application of abscisic acid (ABA) and salicylic acid (SA) in transgenic Arabidopsis. Moreover, two novel ABRE-like (abscisic acid response element) elements were identified in the promoter region.     

稀土元素铈对花生幼苗生长与抗氧化保护酶系统的影响

采用溶液培养方法,研究不同浓度硝酸铈对花生(Arachis hypogaea)幼苗生长、开花数目及抗氧化酶过氧化物酶(POD)、超氧化物歧化酶(SOD)活性与丙二醛(MDA)含量的影响。结果表明,与对照相比,铈浓度低于20.0 mg·L–1能促进花生幼苗生长及开花,其中以10.0 mg·L–1铈的效果最为明显,其生物量和开花数分别约为对照的1.3倍和2.8倍;但高于20.0 mg·L–1则抑制花生幼苗生长,降低花朵数目;同时,低于20.0 mg/L铈可抑制花生幼苗过氧化物酶(POD)活性和降低其丙二醛(MDA)含量,其中以10.0 mg·L–1铈的抑制效果最明显,其POD活性和MDA含量约为对照的47.51%和20.76%;而低于20.0 mg·L–1铈能提高花生幼苗的超氧化物歧化酶(SOD)活性,其中以5.0 mg·L–1铈的促进效果最明显,其SOD活性约为对照的2.0倍。     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

Comparison of Methods for Assessing Resistance to Meloidogyne arenaria in Peanut

Use of resistant cultivars is a desirable approach to manage the peanut root-knot nematode (Meloidogyne arenaria). To incorporate resistance into commercially acceptable cultivars requires reliable, efficient screening methods. To optimize the resistance screening protocol, a series of greenhouse tests were done using seven genotypes with three levels of resistance to M. arenaria. The three resistance levels could be separated based on gall indices as early as two weeks after inoculation (WAI) using 8,000 eggs of M. arenaria per plant, while four or more weeks were needed when 1,000–6,000 eggs/plant were used. High inoculum densities (over 8,000 eggs/plant) were needed to separate the three resistance levels based on eggs per gram of root within eight WAI. A gall index based on percentage of galled roots could separate the three resistance levels at lower inoculum levels and earlier harvest dates than other assessment methods. The use of eggs vs. second-stage juveniles (J2) as inoculum provided similar results; however, it took three to five more days to collect J2 than to collect eggs from roots. Plant age affected gall index and nematode reproduction on peanut, especially on the susceptible genotypes AT201 and D098. The genotypes were separated into their correct resistance classes when inoculated 10 to 30 days after planting, but were not separated correctly when inoculated on day 40.     

C3和C4植物叶片对光氧化响应的日变化

田间生长的C3植物花生和C4植物玉米分别于晴天上午9：00、中午12：00、下午15：00取样。中午12：00花生叶片的Fv／Fm较早上9：见下降16％,出现了光抑制现象,玉米叶片的Fv／Fm则未下降。不同时间取样的花生和玉米叶片经甲基紫精（MV） 强光的人为光氧化处理,叶绿素和类胡萝卜素出现不同程度的氧化降解,中午12：00降解幅度最大,15时降幅最小。植物叶片的抗氧化能力与其SOD活性相关,而与PEPCase的活性没有明显的相关性。光氧化处理后,花生和玉米的叶绿素荧光参数FV／Fm、qp、pSII都下降,花生在12：00的降幅最小,玉米的降幅最大。光氧化引起花生的qN和热耗散系数（KD）上升,玉米则都下降．结果显示C3植物花生和C4植物玉米对光氧化的响应可能存在不同的机制。     

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.     

Bahiagrass,Corn, Cotton Rotations,and Pesticides for Managing Nematodes,Diseases, and Insects on Peanut

Florunner peanut was grown after 1 and 2 years of Tifton 9 bahiagrass, corn, cotton, and continuous peanut as whole-plots. Pesticide treatments aldicarb (3.4 kg a.i./ha), flutolanil (1.7 kg a.i./ha), aldicarb + flutolanil, and untreated (control) were sub-plots. Numbers of Meloidogyne arenaria second-stage juveniles in the soil and root-gall indices of peanut at harvest were consistently lower in plots treated with aldicarb and aldicarb + flutolanil than in flutolanil-treated and untreated plots. Percentages of peanut leaflets damaged by thrips and leafhoppers were consistently greater in flutolaniltreated and untreated plots than in plots treated with aldicarb or aldicarb + flutolanil but not affected by cropping sequences. Incidence of southern stem rot was moderate to high for all chemical treatments except those that included flutolanil. Stem rot loci were low in peanut following 2 years of bahiagrass, intermediate following 2 years of corn or cotton, and highest in continuous peanut. Rhizoctonia limb rot was more severe in the peanut monoculture than in peanut following 2 years of bahiagrass, corn, or cotton. Flutolanil alone or combined with aldicarb suppressed limb rot compared with aldicarb-treated and untreated plots. Peanut pod yields were 4,186 kg/ha from aldicarb + flutolanil-treated plots, 3,627 kg/ha from aldicarb-treated plots, 3,426 kg/ha from flutolanil-treated plots, and 3,056 kg/ha from untreated plots. Yields of peanut following 2 years of bahiagrass, corn, and cotton were 29% to 33% higher than yield of monocultured peanut.     

Effect of nontoxigenic Aspergillus flavus and A. parasiticus on aflatoxin contamination of wounded peanut seeds inoculated with agricultural soil containing natural fungal populations

Peanuts and other seed and grain crops are commonly contaminated with carcinogenic aflatoxins, secondary metabolites produced by Aspergillus flavus and A. parasiticus. Aflatoxin contamination of peanuts in the field can be reduced by 77–98% with biological control through the application of nontoxigenic strains of these species, which competitively exclude native aflatoxin-producing strains from developing peanuts. In this study, viable peanut seeds were artificially wounded and inoculated with field soil containing natural fungal populations that were supplemented with conidia of nontoxigenic A. flavus NRRL 21882 (niaD nitrate-nonutilizing mutant) and A. parasiticus NRRL 21369 (conidial color mutant). Increasing soil densities of applied nontoxigenic strains generally resulted in an increase in the incidence of seed colonization by applied nontoxigenic strains, a decrease in seed colonization by native A. flavus and A. parasiticus, and a decrease in aflatoxin concentration in seeds. Reduction of aflatoxins in peanut seeds depended on both the density and the aflatoxin-producing potential of native populations and on the fungal strain used for biological control. Wild-type strain A. flavus NRRL 21882 and its niaD mutant were equally effective in reducing aflatoxins in peanuts, indicating that nitrate-nonutilizing mutants, which are easily monitored in the field, can be used for evaluating the efficacy of biocontrol strains.     

Factors affecting cation-anion uptake balance and iron acquisition in peanut plants grown on calcareous soils

Dicotyledonous plants subjected to Fe-deficiency stress can decrease pH in the rhizosphere by proton excretion and reduce ferric iron by an activated reduction system in the plasma membranes of the root or by reductants released from the roots. The efficiency by which these plants take up Fe may strongly depend on their cation-anion balance. This study presents results of two experiments conducted to evaluate the effect of K, growth stage and cultivar on ionic balance and Fe acquisition of peanut (Arachis hypogaea L.) plants.Potassium applications to the high calcareous soil (30.3% CaCO₃) favoured proton release, but did not ameliorate plant Fe acquisition. At the earliest stages of plant growth, anion uptake exceeded cation uptake due to intensive N uptake. With time, a shift in the ionic balance was observed as a result of predominant cation uptake. It appears that the relationship between H/OH-ion release and Fe nutrition of peanut plants is actually a complex phenomenon under soil conditions and depends on some soil parameters, such as CaCO₃ content. Even by enhanced H-ion release Fe nutrition of plants can be impaired if soil CaCO₃ is too high.