The agrobacterium tumefaciens C58-6b gene confers resistance to N(6)-benzyladenine without modifying cytokinin metabolism in tobacco seedlings

The 6b gene of Agrobacterium tumefaciens has been demonstrated to modify the activity of the plant growth regulators, auxin and cytokinin. To study the possible mode of action of the gene, tobacco (Nicotiana tabacum L. cv. Samsun) plants were transformed with the A. tumefaciens C58-6b gene. Seeds obtained from morphologically normal transgenic as well as wild-type plants were germinated on media supplemented with growth-inhibitory levels of cytokinin, N(6)-benzyladenine (BA). The transgenic seedlings showed increased resistance to cytokinins, as reflected by continuous shoot development, whereas further growth of the wild-type plants beyond the cotyledonary stage was inhibited. Concurrently, the levels of 6b gene transcripts in transgenic seedlings increased greatly upon BA treatment. Since glucosylation of BA represents the main inactivation mechanism of the hormone, we analyzed BA glucoside formation during the early stages of seedling growth. Intracellular levels of the major BA metabolite, N(6)-benzyladenine-7-glucoside (80-92%), as well as other BA-derived components were found to be comparable in transgenic and wild-type seedlings. Therefore, increased resistance of the C58-6b transgenic seedlings to cytokinins could not be directly attributed to enhanced BA glucosylation and subsequent hormone inactivation.

[The safety and usefulness of transgenic plants]. / Bezpecnost a prospesnost transgenních rostlin pro cloveka.

Transgenic crop plants, used in food and feed production, carry different beneficial transgenes, mostly for resistance to pests, herbicides and diseases. All new transgenic plant varieties, the genes they carry and their products have been thoroughly tested before released for agriculture and even more for marketing. Genetically modified organisms carry the same risk as any other organism. Food derived from genetically modified organisms due to legal regulation is most controlled and therefore most safe food ever placed on the market. In future, transgenic plants offer many new possibilities also for medical use, like plant vaccines, antibiotics and rare proteins of clinical importance produced by plants.

Construction and use of Agrobacterium tumefaciens binary vectors with A. tumefaciens C58 T-DNA genes.

Five plant morphoregulatory genes were isolated from the Agrobacterium tumefaciens Ti plasmid and binary plasmid vectors for plant transformation with these genes were constructed. All vectors have a similar structure with T-DNA borders, RK2 origin of replication and chimeric kanamycin resistance gene for the selection of transformed plant tissues. Over twenty vectors with single and combined morphoregulatory genes were constructed and their effects after tobacco tissue transformation studied.

Modification of T-DNA of nopaline Ti plasmid by intermediary vector and utilization of agrocin 84 sensitivity as simple criterion of conjugation transfer of modified Ti plasmid.

The chloramphenicol resistance gene from pSa was introduced into T-DNA of pTi T37 of Agrobacterium tumefaciens by cointegration with intermediary plasmid based on pBR322. The resulting intermediary vector was mobilized to A. tumefaciens T37 by conjugative plasmid pRK2. The RK2 plasmid also forms contegrates with pTi due to the Tn3 transposon which was used for the mobilization of modified pTi into plasmid-less A. tumefaciens strain. Transconjugants were selected on the basis of their antibiotic resistance markers and tested for agrocin sensitivity as proof of Ti plasmid transfer. Agrocin sensitivity of tranconjugants together with chloramphenicol resistance was shown to be a sufficient and simple criterion of transfer of modified Ti plasmids. Agrobacterium strains with modified Ti plasmids showed decreased virulence in consequence of the presence of additional borderline sequence inside their T-DNA.

The influence of calf thymus DNA and deoxyribonucleosides on the induction of different mutation types in drosophila.

The influence of exogenous DNA on the induction of mutations by X-rays was compared with the influence of an equimolar mixture of four deoxyribonucleosides. Pretreatment and posttreatment with calf thymus DNA did not influence the frequency of mutations at the specific loci dp, b, cn and bw as well as Minute mutations induced in Drosophila sperm by X-radiation. Pretreatment with the equimolar mixture of four deoxyribonucleosides increased the frequency of the Minutes, but it did not affect the frequency of mutations at the loci dp, b, cn, bw. Equimolar mixture of nucleosides alone induced a low frequency of Minute mutations in Drosophila sperm. DNA alone induced a low frequency of recessive lethals. These lethals arose as mosaics of small sectors of the gonads of F1 females and they were revealed as late as the F3 generation.