Direct DNA transfer using electric discharge particle acceleration (ACCELL™ technology)

Direct DNA transfer methods based on particle bombardment have revolutionized plant genetic engineering. Major agronomic crops previously considered recalcitrant to gene transfer have been engineered using variations of this technology. In many cases variety-independent and efficient transformation methods have been developed enabling application of molecular biology techniques to crop improvement. The focus of this article is the development and performance of electric discharge particle bombardment (ACCELL™) technology. Unique advantages of this methodology compared to alternative propulsion technologies are discussed in terms of the range of species and genotypes that have been engineered, and the high transformation frequencies for major agronomic crops that enabled the technology to move from the R&D phase to commercialization. Creation of transgenic soybeans, cotton, and rice will be used as examples to illustrate the development of variety-independent and efficient gene transfer methods for most of the major agronomic crops. To our knowledge, no other gene transfer method based on particle bombardment has resulted in variety-independent and practical generation of large numbers of independently-derived crop plants. ACCELL™ technology is currently being utilized for the routine transfer of valuable genes into elite germplasm of soybean, cotton, bean, rice, corn, peanut and woody species.     

外源DNA直接导入小麦及其在育种上的应用

本研究选用两个白粒小麦品种作供体,提取其总ＤＮＡ,采用花粉管直接携带法导入７５（１９８）红粒品种受体植株。ＤＮＡ导入的第一代（Ｄ１）,目的性状的转化频率为１．７５％和２．９４％。Ｄ２代变异率显著低于Ｄ１代。对Ｄ１,Ｄ２代所得目的性状变异后代,按照常规育种程序进行Ｄ３代观察与鉴定,得到已稳定遗传的后代,从中选取保持原品种其它优良性状而籽粒为的白色的变异类型混合脱粒,获得７５（１９８）改良新品系。     

Recovery and evaluation of soybean plants transgenic for aBacillus thuringiensis var.Kurstaki insecticidal gene

Summary Lepidopteran insects are major defoliating pests of soybean in the southeastern United States. Soybean plants transgenic for a nativecryIA(b) gene fromBacillus thuringiensis var.kurstaki HD-1 were obtained. Embryogenic cultures were induced by plating cotyledons on a Murashige and Skoog-based medium supplemented with 40 mg/liter of 2,4-dichlorophenoxyacetic acid (2,4-D). The embryogenic cultures were maintained in liquid medium containing 5 mg/liter 2,4-D. These cultures were subjected to microprojectile bombardment, followed by selection on 50 mg/liter hygromycin. Resistant embryogenic cell lines were transferred to growth regulator-free medium to permit recovery of mature somatic embryos. After a desiccation period, the somatic embryos were returned to growth regulator-free medium for conversion into plants. Southern hybridization analysis verified transformation. Feeding assays of T₁ plants from one cell line deterred feeding, development, and survival of velvetbean caterpillar at a level comparable to that of GatIR81-296, a soybean breeding line with a high level of insect resistance. Reduced feeding on T₁ plants correlated with the presence of the transgene.     

Kinetics of competitive inhibition and cometabolism in the biodegradation of benzene, toluene, and p-xylene by two Pseudomonas isolates

Two Pseudomonas species (designated strains B1 and X1) were isolated from an aerobic pilot-scale fluidized bed reactor treating groundwater containing benzene, toluene, and p-xylene (BTX). Strain B1 grew with benzene and toluene as the sole sources of carbon and energy, and it cometabolized p-xylene in the presence of toluene. Strain X1 grew on toluene and p-xylene, but not benzene. In single substrate experiments, the appearance of biomass lagged the consumption of growth substrates, suggesting that substrate uptake may not be growth-rate limiting for these substrates. Batch tests using paired substrates (BT, TX, or BX) revealed competitive inhibition and cometabolic degradation patterns. Competitive inhibition was modeled by adding a competitive inhibition term to the Monod expression. Cometabolic transformation of nongrowth substrate (p-xylene) by strain B1 was quantified by coupling xylene transformation to consumption of growth substrate (toluene) during growth and to loss of biomass during the decay phase. Coupling was achieved by defining two transformation capacity terms for the cometabolizing culture: one that relates consumption of growth substrate to the consumption of nongrowth substrate, and second that relates consumption of biomass to the consumption of nongrowth substrate. Cometabolism increased decay rates, and the observed yield for strain B1 decreased in the presence of p-xylene. (c) 1993 Wiley & Sons, Inc.     

Genetic Transformation in Helicobacter pylori

Genetic transformation in Helicobacter pylori was investigated by using its chromosomal and plasmid DNAs. Six out of the eight strains exhibited the natural competence for incorporation of H. pylori chromosomal DNA, and all the strains incorporated the donor DNA efficiently by washing and concentrating the cells, with a glycerol solution. The much higher frequency of transformation was obtained in each strain by means of electroporation. Electroporation experiments were also conducted by use of the recombinant DNAs consisting of the H. pylori and Escherichia coli plasmids as the donors, and the occurrence of the homologous recombination was demonstrated between the incoming H. pylori plasmid-derived region and the corresponding region of the originally residing plasmid in H. pylori.     

Regeneration of transgenic tamarillo plants

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.     

Enhanced transformation of tomato co-cultivated with Agrobacterium tumefaciens C58C1Rifr::pGSFR1161 in the presence of acetosyringone

Summary Explants of tomato (Lycopersicon esculentum Mill cv. Ailsa Craig) were co-cultivated with Agrobacterium tumefaciens C58C1Rif^r::pGSFR1161 in the presence of 20 (M acetosyringone). Transformed root clones were selected on kanamycin medium and the presence of the nptII gene in the plant DNA confirmed by the polymerase chain reaction. Root clones derived from acetosyringone treatment grew more vigorously in the presence of kanamycin and synthesized a greater amount of NPT-II enzyme. The conclusion is that acetosyringone treatment enhances the transformation process, possibly by stimulating multiple insertions of the T-DNA into the host genome.Abbreviations AS acetosyringone - CTAB hexadecyltrimethylammonium bromide - EDTA ethylenediaminetetraacetic acid - ELISA enzyme-linked immunosorbant assay - MS Murashige and Skoog (1962) medium - PCR polymerase chain reaction     

Semiparametric estimation of the transformation model by leveraging external aggregate data in the presence of population heterogeneity

Leveraging information in aggregate data from external sources to improve estimation efficiency and prediction accuracy with smaller scale studies has drawn a great deal of attention in recent years. Yet, conventional methods often either ignore uncertainty in the external information or fail to account for the heterogeneity between internal and external studies. This article proposes an empirical likelihood-based framework to improve the estimation of the semiparametric transformation models by incorporating information about the t-year subgroup survival probability from external sources. The proposed estimation procedure incorporates an additional likelihood component to account for uncertainty in the external information and employs a density ratio model to characterize population heterogeneity. We establish the consistency and asymptotic normality of the proposed estimator and show that it is more efficient than the conventional pseudopartial likelihood estimator without combining information. Simulation studies show that the proposed estimator yields little bias and outperforms the conventional approach even in the presence of information uncertainty and heterogeneity. The proposed methodologies are illustrated with an analysis of a pancreatic cancer study.     

Frog call transmission in forestry monocultures: Are there drawbacks?

Anuran males emit advertisement calls for the purpose of attracting females and repelling conspecific males. Call transmission is influenced by the acoustics of the propagation environment, including vegetation. Thus, forestry monocultures of non-native trees represent artificial environments that could modify call transmission. These monocultures have substituted large areas of Atlantic Forest in southern Brazil, representing a conservation challenge. Considering this context, we hypothesized that anurans have calls that are less attenuated in their native environment than in forest plantations. To test it, we performed sound transmission experiments using calls of three anuran species native to southern Brazil: Boana bischoffi, B. leptolineata, and Hylodes meridionalis. We compared sound attenuation between the native forest and forestry monocultures (Eucalyptus sp. and Pinus sp. forests), and included distance from the sound source, air temperature, humidity, and vegetation density as cofactors in linear mixed models. Our results show that the advertisement calls of the two tree frogs (Boana) attenuate less in tree plantations than in native forests, while the third species shows either no differences or less attenuation in native forests. Our results can be understood according to differences in natural history and microhabitat use among the studied species. The two tree frog species vocalize perched from vegetation so their calls become better transmitted in forestry monocultures, which have fewer trees than the native forest. On the other hand, H. meridionalis calls from rocks on streams and the propagation is finely tuned to this peculiar microhabitat. We discuss the conservation implications of our findings for anurans and for the Atlantic Forest. Abstract in Portuguese and Spanish are available with online material.