Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.

• MeSH
• Agrobacterium tumefaciens genetika   MeSH
• Bacillus thuringiensis genetika   MeSH
• bakteriální proteiny genetika   MeSH
• endotoxiny genetika   MeSH
• genetická transkripce MeSH
• genetické vektory MeSH
• geneticky modifikované rostliny * MeSH
• hemolyziny genetika   MeSH
• jedle embryologie   genetika   MeSH
• promotorové oblasti (genetika) MeSH
• semena rostlinná genetika   růst a vývoj   MeSH
• somatická embryogeneze rostlin MeSH
• transformace genetická MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant's leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.

• MeSH
• adenin analogy a deriváty   metabolismus   farmakologie   MeSH
• antivirové látky metabolismus   farmakologie   MeSH
• biotransformace MeSH
• Brassica metabolismus   virologie   MeSH
• Caulimovirus účinky léků   růst a vývoj   MeSH
• DNA virů analýza   MeSH
• ELISA metody   MeSH
• kyseliny fosforité metabolismus   farmakologie   MeSH
• polymerázová řetězová reakce metody   MeSH
• virová nálož MeSH
• virové proteiny analýza   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A new method was developed for testing antiviral compounds against plant viruses based on rapidly growing brassicas in vitro on liquid medium. This method enables exchange of media containing tested chemicals in various concentrations and simultaneous evaluation of their phytotoxicity and antiviral activity. While using ribavirin as a standard for comparison, phytotoxicity and ability of the acyclic nucleotide analogues (R)-PMPA, PMEA, PMEDAP, and (S)-HPMPC to eliminate ssRNA Turnip yellow mosaic virus (TYMV) were evaluated by this method. Double antibody sandwich ELISA and real-time PCR were used for relative quantification of viral protein and nucleic acid in plants. Ribavirin had the most powerful antiviral effect against TYMV. On the other hand, (R)-PMPA and PMEA had no antiviral effect and almost no phytotoxicity compared to the control. (S)-HPMPC and PMEDAP showed moderate antiviral effect, accompanied by higher phytotoxicity. The tested compounds can be screened within 6-9 weeks in contrast to the 6 months for traditionally used explants on solid medium. The method enables large-scale screening of potential antivirals for in vitro elimination of viruses from vegetatively propagated crops and ornamentals.

• MeSH
• acyklické uhlovodíky farmakologie   terapeutické užití   MeSH
• antivirové látky farmakologie   terapeutické užití   MeSH
• Brassica účinky léků   růst a vývoj   virologie   MeSH
• hydroponie metody   MeSH
• kultivační média MeSH
• mikrobiální testy citlivosti metody   MeSH
• nemoci rostlin terapie   virologie   MeSH
• nukleosidy farmakologie   terapeutické užití   MeSH
• replikace viru účinky léků   MeSH
• ribavirin farmakologie   terapeutické užití   MeSH
• Tymovirus účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• Caulimovirus genetika   metabolismus   MeSH
• Cytomegalovirus genetika   MeSH
• finanční podpora výzkumu jako téma MeSH
• glukuronidasa genetika   metabolismus   MeSH
• lidé MeSH
• promotorové oblasti (genetika) imunologie   MeSH
• Solanum tuberosum genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• chromozomy genetika   MeSH
• restrikční mapování využití   MeSH
• rostliny genetika   MeSH
• Solanum lycopersicum genetika   MeSH
• Publikační typ
• srovnávací studie MeSH