Sweet potato (Ipomoea batatas) trypsin inhibitors expressed in transgenic tobacco plants confer resistance against Spodoptera litura

A sweet potato (Ipomoea batatas cv. Tainong 57) trypsin inhibitor gene was introduced into tobacco plants (Nicotiana tabaccum cv. W38) by Agrobacterium tumefaciens– mediated transformation. From 30 independent transformants, three lines with high level of expression were further analyzed. The trypsin inhibitor gene, under control of the 35S CaMV promoter, led to the production of the trypsin inhibitor proteins up to 0.2% of the total protein. In insecticidal bioassays of transgenic tobacco plants, larval, growth of Spodoptera litura (F.), the tobacco cutworm, was severely retarded as compared to their growth on control plants. This observation implied that expression of sweet potato trypsin inhibitor can provide an efficient method for crop protection. Received: 29 July 1996 / Revision received: 15 November 1996 / Accepted: 8 December 1996     

A colony‐to‐lawn method for efficient transformation of Escherichia coli

Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation. Methods and Results: A single fresh colony from plasmid‐containing donor strain is picked up and suspended in 75% ethanol. Cells are pelleted and resuspended in CaCl₂ solution and lysed by repetitive freeze–thaw cycles to obtain plasmid‐containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid‐containing cell lysate for transformation. Additionally, a process based on colony‐to‐lawn transformation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation. Conclusions: With this method, a single colony from plasmid‐containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time‐saving. Significance and Impact of the Study: Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high‐throughput changing hosts of plasmids during mutant library creation.     

In vitro regeneration and genetic manipulation of grasses

Regeneration of plants from cultured cells is an important and essential component of plant biotechnology. Advances in the recovery of plants from cultured cells and protoplasts of grasses, and in genetic transformation provide challenging opportunities for the genetic manipulation and improvement of this most important group of food plants.     

Stable and invariant adjusted profile likelihood and directed likelihood for curved exponential models

For curved exponential models, the modified profile likelihoodand the modified directed likelihood are approximated, witherror 0(n^-1) under ordinary repeated sampling, by explicitlydefined and invariant quantities which do not require specificationof an ancillary and which are stable in the sense of havingthe appropriate conditionality property with respect to anyreasonable ancillary.     

Chemical diversity and variation of pyrrolizidine alkaloids of the senecionine type: biological need or coincidence?

Laboratory and field tracer experiments with ¹⁴C-labelled senecionine N–oxide (SO) and distant biosynthetic precursors such as [¹⁴C]putrescine revealed that pyrrolizidine alkaloid N-oxides (PAs) in Senecio vernalis Waldstr. & Kit. (Asteraceae) show no significant turnover over periods of up to 29 d. However, PAs are spatially mobile, they are continuously allocated, and labelled PAs are even detectable in leaves and capitula developed weeks after tracer application. Chemical diversification of SO, the common product of PA biosynthesis in roots, was studied in five Senecio species (i.e. S. vernalis Waldstr. & Kit., S. vulgaris L, S. inaequidens DC, two chemotypes of S. jacobaea L. and S. erucifolius L.). Tracer experiments revealed that shoots are capable of transforming [¹⁴ C]SO into the unique species–specific PA patterns. Within a plant, the transformation efficiency of SO can vary quantitatively and qualitatively between shoot organs (i.e. leaves, stems and inflorescences). All transformations proceed position-specifically and stereoselectively. They comprise simple one-step or two-step reactions such as hydroxylations, epoxidations, dehydrogenations, and O-acetylations, as well as the more complex conversion of the retronecine into the otonecine base moiety (e.g. SO into senkirkine). Taking all the evidence together, the qualitative and quantitative composition of the Senecio PA pattern is a dynamic and sensitive equilibrium between a number of interacting processes: (i) constant rate of de-novo synthesis of SO in roots, (ii) continuous long-distance translocation of SO into shoots, (iii) efficiency of SO transformations which may vary between plant organs, (iv) continuous allocation of PAs in the plant, and (v) efficiency and tissue selectivity of vacuolar storage. We suggest that in constitutive plant defence, without significant turnover of its components, such a highly plastic system provides a powerful strategy to successfully defend and possibly escape herbivory. Received: 27 March 1998 / Accepted: 19 May 1998     

Normality Testing in a Mixed Model of a Two-way Nested Classification

The paper presents a testing procedure verifying the hypothesis about the normal distribution of random components in a mixed model of a two-way nested classification. The basis for this application is a simple sample obtained from orthogonal and the O'Reilly-Quesenberry transformation to the residual vector from the method of least squares (MLS).     

Detection and characterization of natural transformation in the marine bacterium Pseudomonas stutzeri strain ZoBell

Soil isolates of Pseudomonas stutzeri have been shown previously to acquire genes by natural transformation. In this study a marine isolate, Pseudomonas stutzeri strain ZoBell, formerly Pseudomonas perfectomarina, was also shown to transform naturally. Transformation was detected by the Juni plate method and frequencies of transformation were determined by filter transformation procedures. Maximum frequencies of transformation were detected for three independent antibiotic resistance loci. Transformation frequencies were on the order of 4×10^-5 transformants per recipient, a frequency over 100 times that of spontancous antibiotic resistance. Transfer of antibiotic resistance was inhibited by DNase I digestion. Marine isolates achieved maximum competence 14 h after transfer of exponential cultures to filters on solid media, although lower levels of competence were detected immediately following filter immobilization. Like soil isolates, P. stutzeri strain ZoBell is capable of cell contact transformation, but unlike soil isolates where transformation frequencies are greater for cell contact transformation as compared to transformation with purified DNA, the maximum frequency of transformation achieved by cell contact in the marine strain was approximately 10-fold less than transformation frequencies with purified DNA. These studies establish the first marine model for the study of natural transformation.This paper is dedicated to John L. Ingraham, Professor Emeritus of Microbiology at the University of California, Davis. Professor Ingraham was the first person to recognize natural transformation in Pseudomonas stutzeri and has continued to contribute to our understanding of the process over the past eight years. This understanding of the genetics of P. stutzeri is only one of the many areas of microbiology to which Professor Ingraham has contributed in his exceptional career