Transmission of tobacco rattle virus (TRV) via seed potatoes.

Susceptibility of potato varieties for tobacco rattle virus (TRV) and sensitivity for spraing in potato tubers both depend on the interaction between cultivar and virus strain. Of the six potato cultivars investigated in this research, cultivar Santana was the most susceptible to TRV, the cultivars Roxy and Saturna were the least susceptible. In general, potato cultivars were most susceptible to the virus type transmitted by P. pachydermus and least for the virus type transmitted by T. primitivus. Potato cultivars showed a large difference in susceptibility for various TRV types. This was the most obvious for cultivar Wilja. Wilja showed high virus concentrations (susceptible) for a virus type transmitted by P. pachydermus but very low concentrations (resistant) for virus types transmitted by P. teres and T. primitivus. Cultivar Santana was the most sensitive for spraing, cultivar Wilja was the least sensitive. As Wilja is very susceptible to the virus strain transmitted by P. pachydermus, but is insensitive for spraing caused by this strain, this cultivar is called a "symptomless carrier" for this type of TRV. All six investigated potato cultivars can pass on TRV via seed potatoes to the next generation (secondary infection) but the degree of this virus transmission depended on potato cultivar, virus type and the interaction between cultivar and TRV and seems to be connected with the susceptibility for TRV. Transmission of the TRV type of P. pachydermus via seed potatoes was the highest in the cultivars Santana and (to a lesser degree) Santé and in cultivar Wilja. Even in the very sensitive cultivar Santana, TRV was passed on via seed potatoes. The prevailing theory that in sensitive potato cultivars TRV particles are immobilized in the necrotic 'spraing' tissue, therefore seems to be inaccurate. Since virus-vector combinations show specific interactions with cultivars, it is recommended to do potato variety research in several fields with different viruliferous trichodorid species.

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens.

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca(2+)concentration was lowered by ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.

T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum.

Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells. Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous. Detailed analysis of T-DNA organization showed that TL- or "core" T-DNA was always present at one or two copies per diploid genome. However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated. TR-DNA was not detected. The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived.

Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens.

The molecular basis of complementation by a mixture of two different types of octopine T-region mutants (LBA4060 and LBA4210) was studied. Six randomly chosen cellular clones derived from a tumor obtained after mixed infection were analyzed for their T-DNA content via Southern blot hybridization. The clones appeared to contain T-DNA that originated from each of both mutants, indicating that they developed from doubly infected single cells. Genetic complementation, therefore, might explain at least in part the observed complementation phenomenon. However, complementation as a result of cross-feeding between separately transformed cells could not be excluded. Following protoplast isolation, small aggregates might have formed that developed into the clones analyzed.

Differential expression of crown gall tumor markers in transformants obtained after in vitro Agrobacterium tumefaciens-induced transformation of cell wall regenerating protoplasts derived from Nicotiana tabacum.

To obtain transformation of plant cells, we incubated 3-day-old cell wall-regenerating protoplasts from tobacco with Agrobacterium tumefaciens harboring tumor-inducing plasmids. Putative transformed tobacco cells were selected by phytohormone autotrophic growth and were shown to be transformed by the detection of the tumor cell specific enzymes lysopine dehydrogenase or nopaline dehydrogenase. This was substantiated by the detection, in transformed tumor tissues, of DNA sequences homologous to sequences in the tumor-inducing plasmid. Segregation of tumor markers has been observed among the transformants and it is suggested that this happened during the initiation of the transformation. The stable character of the transformed state was shown by the retention of tumor markers in subcloning of primary transformants under nonselective conditions. Suppression of the neoplastic state of transformants could take place, resulting in the development of transformed shoots. Indications were obtained for the inheritance of tumor markers through meiosis from seedlings obtained from seeds of flowering transformed plants that still expressed nopaline synthesis.

Retention of tumor markers in F1 progeny plants from in vitro induced octopine and nopaline tumor tissues.

Tumorous tobacco shoots have been derived from callus tissues produced by Agrobacterium tumefaciens--induced transformation of tobacco protoplasts and by fusion of normal protoplasts with those from crown gall tumors. The continued presence of T-DNA sequences in shoots is directly demonstrated by Southern blotting and is also revealed by the presence of the tumor markers octopine and nopaline. When grafted onto normal tobacco plants, both octopine- and nopaline-type shoots (including those from somatic hybrids) produced flowers and set seed. Germination of these seeds gave F1 progeny that showed retention of morphological markers of their parental shoots, and one seedling retained the ability to synthesize nopaline. The data demonstrate that T-DNA markers can be retained during meiosis and are expressed in F1 plants.

The expression of tumour markers in intraspecific somatic hybrids of normal and crown gall cells from Nicotiana tabacum.

Following fusion of protoplasts from crown gall tumour calli, characterized by hormone independent growth, and protoplasts from normal tissues of a streptomycin-resistant mutant, SR1, we selected hormone independent streptomycin-resistant calli in Nicotiana tabacum. The tumour line, B6S3, lost the ability to form shoots. Some of the selected lines, similar to SR1, however, are morphogenic. Both calli and shoots contained the tumour specific enzyme lysopinedehydrogenase. The hybrid shoots are resistant to Agrobacterium infection and do not root. These tumorous properties are dominantly expressed in the somatic hybrids.