André Hoekema

NewAgrobacterium helper plasmids for gene transfer to plants

We describe the construction of new helper Ti plasmids forAgrobacterium-mediated plant transformation. These plasmids are derived from three differentAgrobacterium tumefaciens Ti plasmids, the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying thevir genes that will complement binary vectorsin trans. Data are included that demonstrate strain utility. The advantages ofAgrobacterium strains harbouring these ‘disamed’ Ti plasmids for plant transformation viaAgrobacterium are discussed.

Meristem transformation of sunflower via Agrobacterium.

SummaryFor transformation of sunflower (Helianthus annuus L. cv. Zebulon), shoot apical meristems were dissected from seeds and cocultivated with a disarmed Agrobacterium tumefaciens strain harboring a binary vector carrying genes encoding GUS- and NPTII-activity. The influence of the media conditions, the time of cocultivation and the stage of the developing seed on shoot development and meristem transformation was analysed. Transformants were selected by their ability to grow on kanamycin. Transformation was confirmed by assays for GUS and NPTII. GUS-positive shoots were rooted on rockwool and transferred to soil. Transformation of shoot meristem cells occurred at low frequencies. Chimaeric expression of the two genes was observed in transformed plants. Integration of the foreign DNA in the sunflower genome was confirmed with the polymerase chain reaction.

Factors influencing transformation frequency of tomato (Lycopersicon esculentum).

We developed an efficient procedure for transformation and regeneration of L. esculentum cv. Moneymaker from cotyledon explants. The effect of two parameters on the transformation frequency was investigated in detail. The use of feeder layers during cocultivation proved to be critical. In addition, it was found that Agrobacterium strains harbouring a L,L-succinamopine type helper plasmid yielded significantly higher transformation frequencies than those with octopine or nopaline type helper plasmids. The optimized protocol was used to obtain transformation frequencies averaging 9%. Of the plants produced approximately 80% proved to be diploid, of which 67% contained the transgene(s) on a single locus.

Engineering virus resistance in agricultural crops.

Plant viral genomes are relatively small and in the past decade many have been characterized at the molecular level. This has prompted research into the development of virus resistance based on interference with the viral multiplication cycle by the introduction of viral sequences into the plant genome. Several strategies have been tested. The most successful one so far involves the constitutive expression of the coat protein gene of the virus against which resistance is desired. In this review we describe progress made in engineering virus resistance into potato, an important agricultural crop. To this end the molecular structure of the potato viruses X and Y and leafroll is discussed as well as the introduction of resistance against potato virus X into potato. In addition, we address the question of preservation of cultivar-specific characteristics, an important prerequisite for commercial application. Finally, recent investigations for alternative forms of virus resistance are described against the background of the results of coat protein-mediated protection.

Non-oncogenic plant vectors for use in the agrobacterium binary system.

SummaryAgrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.

Studies on the structure of cointegrates between octopine and nopaline Ti-plasmids and their tumour-inducing properties

Stable cointegrates between incRh-1 octopine (Ach5) and nopaline (C58) Ti-plasmids, present in ten independently isolated Agrobacterium tumefaciens strains, showed identical restriction endonuclease patterns. Each cointegration event had taken place in the common sequence between the T-regions of both Ti-plasmids. This illustrates a high preference for this region when used in the formation of cointegrates. Four crown gall tissues, obtained after transformation of Nicotiana tabacum cells by one of the mutants, were analysed by using Southern blot analysis for their T-DNA structure. The borders of T-DNA frequently appeared to differ from T-DNA borders previously detected in tumour tissues that had been induced by Agrobacterium strain C58 or Ach5. Therefore, it was concluded that possibly a less stringent mechanism exists for the integration into plant DNA of T-DNA, derived from a composite (octopine/nopaline) T-region than for integration of T-DNA from a normal (octopine or nopaline) T-region.

Direct screening for high-level expression of an introduced α-amylase gene in plants

A method is described for obtaining transgenic plants with a high level of expression of the introduced gene. Tobacco protoplasts were transformed with an expression construct containing a translational fusion between mature α-amylase from Bacillus licheniformis and the signal peptide of the tobacco PR-S protein. A total number of 5200 transformed protoplasts was cultured to microcalli and screened for α-amylase expression by incubation on media containing starch followed by staining with iodine. The calli were divided into four classes, based on the resulting halo sizes on the plates. The halo sizes were found to correlated with the expression levels in transgenic plants regenerated from the calli. The expression levels varied between 0 and 0.5% of soluble leaf protein in the regenerated transgenic plants. Wider implications of this method are discussed.

Efficient production of active industrial enzymes in plants

Abstract An industrial bulk enzyme, Bacillus licheniformis α-amylase was produced in transgenic tobacco at a maximum level of about 0.3% of total soluble protein. The molecular weight of the enzyme produced in tobacco differed from the bacterial enzyme due to complex-type carbohydrate chains attached to the protein. Milled transgenic seeds containing α-amylase were successfully applied in the liquefaction of starch. The resulting hydrolysis products were virtually identical to those obtained from degradation with B. licheniformis α-amylase. No effect was observed of the presence of the enzyme on endogenous starch. Homogenization of transgenic leaves, followed by incubation at 95°C, resulted in degradation of the endogenous starch. Different approaches to the concept of production and application of industrial enzymes in plants are presented that show the efficacy of transgenic plants as a new source of active industrial enzymes.

Gene Organization of the Ti-Plasmid

The phenomenon tumor formation on plants has intrigued biologists for a long time. More than 75 years ago, it was reported that a particular type of plant tumor, known as crown gall, was caused by bacteria (Smith and Townsend, 1907). The present taxonomic name of this soil bacterium is Agrobacterium tumefaciens. Crown gall is characterized by unlimited cell proliferations, which may lead to regression or even to death of the plant. The disease and its causative agent have been studied intensively, first to uncover concepts underlying the tumorous state, secondly because it is the cause of losses of economically important plants in Europe (Kerr and Panagopoulos, 1977), North-America (Kennedy and Alcorn, 1980), and Australia (New and Kerr, 1972), and thirdly, more recently, to develop plant gene vectors.

Transgenic Potato Cultivars Resistant to Potato Virus X

Extensive potato breeding programs over the years have yielded improved varieties with a whole set of proven valuable traits. The process of potato breeding, however, is laborious and time-consuming due to the tetraploid character of the potato genome. Techniques for the genetic engineering of plants present a new tool to improve potato via the introduction of traits presently missing in the existing cultivars. An essential element in the successful application of these new techniques is the preservation of the existing, desired traits of the cultivars. So far, no systematic studies on the actual performance of engineered plants have been performed. In this paper we report the engineering of resistance to potato virus X in the two commercial potato cultivars Bintje and Escort. Also, the results of two years of field trials testing the performance of these transgenic lines are discussed.