Megnövelt antioxidáns kapacitású növények előállítása biotechnológiai módszerekkel fitoremediáció és rezisztencia-nemesítés céljára = Production of plants with increased antioxidant capacity for use in phytoremediation and resistance-breeding

A munkánk alapvető célja volt, hogy nagy antioxidáns kapacitású stressz-ellenálló növényeket állítsunk elő. Erre rendkívül alkalmas a genetikai transzformáció és az in vitro szelekció, habár a két módszer társadalmi elfogadottsága nagyban különbözik. Munkánk során mind a két módszert alkalmaztuk. A kísérleteket lúdfű (Arabidposis thaliana) és szürkenyár növényeken végeztük el Arabidopsis thaliana növényeket transzformáltunk a kukorica gstF4 génjével. A transzgénikus és kontroll növényeket többféle vegyszerrel, gyomirtó szerrel és nehézfémekkel kezeltük. A transzégnikus és kontroll növények között jelentős fenotípusos eltérést tapasztaltunk acetoklór, metolaklór, acifluorfén és kadmium esetében. A cinkkel paraquattal és hidrogén-peroxiddal történő kezelés során a növények reakciói között nem tapasztalunk különbséget. Ezek után szuperoxid-dizmutáz és kataláz enzimekkel is transzformáltunk Arabidopsis növényeket, amelyek értékelése jelenleg is tart. Paraquat toleráns szürkenyár növényeket állítottunk elő in vitro szelekcióval szubletális paraquat koncentráción. A táptalajon való ellenőrzést követően a klónokat mikroszaporítottuk, gyökereztettük és üvegházba kiültettük. A laboratóriumi tesztek során a paraquat toleráns növények toleranciát mutattak metolaklórral és acifluorfénnel szemben is. Azonban a paraquat toleráns a klón előnyös tulajdonságai nem biztos hogy szabadföldön is megjelennek, ezért ennek eldöntésére a szabadföldi kísérleteket is megkezdtük. | The aim of our work was to create crop plants with elevated antioxidant capacity and stress resistance. Genetic modification of and in vitro selection are powerful tools to produce plants with advantageous characteristics. In our experiments we used both methods to produce plants with elevated stress capacity. The experiments were carried out with Arabidopsis thaliana and Populus × canescens. Arabidopsis thaliana plants were transformed with Zea mays gstF4 gene to overexpress glutathione S-transferase. The transgenic and wild type plants were tested against different chemicals, herbicides and heavy metals. Strong phenotypic differences were observed when transgenic seeds were subjected to acetochlor, metolachor acifluorfen and cadmium-chloride. In contrast no differences were revealed between control and transgenic plants when treated with paraquat, hydrogen-peroxide and zinc After that Arabidopsis plants were transformed with superoxide-dismutase and catalase enzymes where the evaluation is in progress. Paraquat tolerant grey poplar clones were selected in in vitro nodal segment cultures at sublethal paraquat concentration. After testing on tissue culture media, regenerants were propagated, rooted and transplanted to a greenhouse. During laboratory experiments the plants showed cross tolerance to metolachlor and acifluorfen. In order to reveal the manifestation of the advantageous characteristics, field tests were started with paraquat tolerant poplar clones

Increased glutathione S-transferase activity in 35S(CaMV)-Zmgstf4 transgenic Arabidopsis thaliana

Clones of 35S-Zmgstf4 transgenic Arabidopsis thaliana expressing the glutathione S-transferase F4 gene of Zea mays was tested for stress-inductive GST (glutathione S-transferase) activity following treatments with the heavy metals Zn (150 and 1500 μM), Cd (20 and 30 μM) and chloroacetanilide herbicide metolachlor (2000 μM). The overexpression of Zmgstf4 gene in Arabidopsis resulted in an extreme resistance to all treatments. The GST activity of the transgenic plants was almost the double compared to the wild type plant in the untreated samples. After Cd (20 and 30 μM), and Zn (150 and 1500 μM) exposure the stress response activity of GSTs increased in both wild type and transgenic plants, however with significantly higher levels in transgenic plants with the extreme level at 20 μM CdSO4 treatment (0.24 in transgenic and 0.13 in wild-type). To compare GST responsivity, Zn treatments was less inductive compared to Cd. Metolachlor (200 μM) was totally tolerated by transgenic plants, compared to wild type plants, which died in 11 days

Response of Arabidopsis Clones to Toxic Compounds Released by Various Rhizoctonia Species

Response of 3 Arabidopsis clones to 41 strains of eight Rhizoctonia species was studied in model experiments. The seed germination was decelerated in most of the cases, although the inhibitory effect varied within large limits. The pre-emergence damping off and root neck rot leading to damping off were the most frequent symptoms of disease syndrome caused by toxic metabolites. The clone transformed with cDNA clone overexpressing gstf4 gene exhibited significantly improved tolerance as compared to parental one, meanwhile the sensitivity of D-mannose pyrophosphorylase/mannose-1-pyrophosphatase deficient clone dramatically increased. Strains of R. solani of AG-2, AG-4 and AG-7 and Athelia rolfsii produced the most toxic metabolites, however, no strict relationships were revealed between taxonomic position of Rhizoctonia strains and toxicity of their metabolites

Importance of herbicide-tolerant sunflower hybrids in suppressing common ragweed (Ambrosia artemisiifolia) pollen production. Minireview

Common ragweed (Ambrosia artemisiifolia) is the number one weed in Hungary: it covers ca. 5% of the arable land, causing huge losses in row crops, especially in sunflowers. In addition, because of the high allergenicity of its pollen, common ragweed is a heavy burden on the health care system. This minireview discusses the importance of use of herbicide-tolerant sunflower hybrids in eliminating common ragweed from sunflower fields, with special emphasis on the efficacy of common ragweed control of two acetolactate-synthase inhibitor postemergence herbicides (imazamox and tribenuron methyl) in several sunflower hybrids that carry the resistance gene against such herbicides. Common ragweed control by these herbicides was excellent: they suppressed the growth of the weed plant until the canopy closure of the crop (8-leaf stage). Common ragweed plants germinating after this date were unable to compete with the crop: although they survived, they remained small (ca. 70% reduction in height), produced ca. 90% less male inflorescences (source of the allergenic pollen), and caused no significant reduction in the crop yield. In order to stop the seed production by the few late-germinating weed plants we recommend a mechanical common measure (row-cultivator) in late August

Selective response of Ricinus communis seedlings to soil borne Rhizoctonia infection

Seedlings of Ricinus communis tolerated soil-borne Rhizoctonia infection in strain dependent manner. There was no connection revealed between pathogenicity of strains and their origin or taxonomic position, however, the castor plant proved to be susceptible to most strains highly pathogenic to other host plants as well. Rhizoctonia zeae (teleomorph: Waitea circinata), a species new for European flora, was less aggressive to R. communis as the most potent R. solani strains. The effect of Rhizoctonia infection on mass accumulation of hypocotyls was more prominent than that on cotyledons. The protein content and glutathione S-transferase (GST) activity increased in parallel with evolution of disease syndrome. Metalaxyl, an acetanilide type systemic anti-oomycete fungicide induced locally the GST activity in R. communis cotyledons with 24 hours lag phase, and this induction was altered in the seedlings grown in Rhizoctonia infested soil by strain dependent manner. It might be concluded, that the stress response related detoxication mechanisms of plants in tolerant host/parasite pairs take effect at higher level than in highly susceptible relationships

Gene up-regulation by DNA demethylation in 35S-gshI-transgenic poplars (Populus x canescens)

Gene expression levels of transgene 35S-gshI (γ-glutamylcysteine synthetase) cloned from E. coli, and the endogenous gene gsh1 of poplar (Populus x canescens) were upregulated by the DNA demethylating agent DHAC (5,6-dihydro-5'-azacytidine hydrochloride) (10-4 M for 7 days) in aseptic leaf discs cultures. Two 35S-gshI-transgenic (6lgl and 11ggs) and wild type (WT) poplar clones were used. The efficiency of gene upregulation was also analyzed under herbicide paraquat stress (4 x 10-7 M). Levels of gshI-mRNA and gsh1-mRNA were determined by RT-qPCR (reverse transcriptase quantitative PCR) after cDNA synthesis. For internal control, the constitutively expressed housekeeping poplar genes α-tubulin and actin were used, and the 2−HHCt method was applied for data analysis. In long term DHAC treatment (21 days), a morphogenetic response of de novo root development was observed on leaf discs in a wide concentration range of DHAC (10-8 to 10-6 M). Adventitious shoots (11ggs clone) also emerged from leaf discs after a combined treatment with DHAC (10-4 M) and paraquat (10-7 M). Shoots were dissected, rooted and transplanted in glass houses for further analyses for phytoremediation capacity. Since DNA methylation patterns are inherited (epigenetic memory), these poplar plants with increased gene expression levels of both transgene 35S-gshI and endogenous gene gsh1 provide novel plant sources for in situ application

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus canescens L.) in vitro

Clone stability and in vitro phytoextraction capacity of vegetative clones of R x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRI-MseI digestion. Four fragments generated by EcoAGT-MseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in R x canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10(-1) to 10(-5) m) incubated for 21 days on aseptic tissue culture media WPM containing 1 mu m Cu. Zn2+ caused phytotoxicity only at high concentrations (10(-1) to 10(- 2) m). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper