Group leader: Györgyi Ferenc

Email: ferenc.gyorgyi[at]brc.hu

Group website:

Group members

Name

Title

Publications

CV

Dénes DUDITS

Professor Emeritus

publications

CV

Györgyi FERENC

Research associate

publications

CV

Bettina ZOMBORINÉ NAGY 

Research associate

publications

CV

András CSERI

Administrator Expert

publications

CV

Zoltán ZOMBORI

Administrator Expert

publications

CV

Feríz RÁDI

PhD student

publications

 

Bence VARGA

PhD student

publications

 

Károlyné TÖRÖK

Laboratory assistant

 

 


 

Research

Climate change and increased demand for food and energy have led to the need for fast and safe methods of plant breeding supported by basic research in plant sciences. Our aim is the better understanding the genetic and molecular regulation of plant development by optimizing highly precise oligonucleotide-directed mutagenesis and polyploidization methods. We expect that our projects will contribute to the progress of plant sciences and in long term to help economic and environment friendly food and energy production.

1. Oligonucleotide-directed mutagenesis (ODM) to improve stress tolerance in plants

Given the legislation banning GMOs, there is a need for precise genetic engineering methods that ensure the improvement of plant properties (e.g. drought tolerance) without the use of foreign DNA. Oligonucleotide directed mutagenesis is a promising, but for now low-efficient method. To enhance its efficiency, optimization of the chemical structure of oligonucleotides, their combination with CRISPR system, novel delivery methods, cell-cycle parameters and remodelling of chromatin structure are the targets of our research.

To detect the targeted mutation in a fast and simple way, transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) were produced. The result of the successful oligonucleotide-targeted mutagenesis was the recovery of the GFP expression, that was monitored by confocal fluorescence microscopy and quantified by flow cytometry (Fig.1). Pre-treatment of maize cells with sodium butyrate (5–10 mM) and nicotinamide (1–5 mM) as known inhibitors of histone deacetylases caused elevated chromatin sensitivity to DNase I. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques. (Tiricz et al., 2018)

Figure 1. Restoration of GFP function by correction of STOP codon in the mutant GFP gene. Confocal microscopy images of a GFP expressing single cell (a), a divided cell (b), a multicellular cluster (c) and an isolated protoplast (d) imaged at 36, 60 h, 10 days and 48 h post-bombardment, respectively. Sequence confirmation of gene editing using ion torrent sequencing of untreated control and 10 mM NaBt-treated samples (e). (Tiricz et al. 2018).

Previously, the oligonucleotide molecules coding the targeted nucleotide exchange were delivered into plant protoplasts or cell cultures. Unfortunately, given the difficulties of plant regeneration from cultured cells or generation of in vitro cultures from inbred lines, several attempts have been made to develop an in planta oligonucleotide-directed mutagenesis method. As one approach, solution of oligonucleotides was injected into the meristem region of haploid maize seedlings to silence the Phytoene Desaturase (PDS) gene. Albino regions appearing at developing leaves indicate the occurrence of a mutational event (see Figure 2. and Rádi et al. 2021).

Figure 2. Targeted mutation of Phytoene Desaturase (Pds) gene can lead to albinism after the treatment of shoot apical meristem of haploid maize seedlings with oligonucleotides (Rádi et al. 2021).

In the frame of collaboration with Kiskun Research Center Ltd. our aim is to produce a genetic material from their inbred maize lines that has an increased drought tolerance and since it does not carry a foreign gene, it can be used in their breeding program.

2. Genetic and developmental regulation of energy willow green and woody biomass yield

Energy willow (Salix viminalis) plants can play a key role in mitigating climate change processes, as they can be used as a feedstock for biogas production. Advantages of the willow plants is that they can be produced on poor quality or saline soils. A prerequisite for their successful application is to learn the genetic and developmental processes that underlie the desired functions. We have previously demonstrated that doubling the chromosome set of willow plants and producing tetraploid plants increases the efficiency of photosynthesis, CO2 uptake, the thickness of spleen and tree layers in woody stems, and better salt tolerance of tetraploid genotypes (Dudits et al. 2016, Cseri et al. 2020). To take advantage of the heterosis effect in the future, we want to increase the favourable properties by creating and detailed characterization of triploid willow cultivars.

2.1. Selective breeding of high biomass yield and low lignin energy willow cultivars for biogas production

Energy willow can be a good alternative to the silage corn used so far in biogas fermentation, because it has a short cutting cycle and can be grown in adequate yields in marginal areas. However, low lignin content is an important criterion in biogas fermentation using wood raw materials.

Within the framework of application (GINOP-2.2.1-15-2017-00081) we perform the selection of lines of our tetraploid and triploid hybrid populations. In the first round, based on the data of chemical analysis, we identified lines with reduced lignin content, which are compared in fermentation studies by the research group of Prof. Kornél Kovács at the Department of Biotechnology of USZ (Kakuk et al. 2021).

Given that the triploid heterosis (TH3 / 12) effect may be present after crossing diploid (Tordis cultivar) and tetraploid (PP-E7) plants (Fig. 3), These plants are characterized in detail both in the field and in the laboratory.

Figure 3. The prevalence of triploid heterosis in energy willow breeding materials.


 

Figure 4. Short rotation plantations of willow genotypes, harvested in vegetative growth phases, were tested as an alternative biomass for methane production. The substrate characteristics, maximal methane yields (K) and highest methane production rates (μmax) were determined. Leaves and stems from diploid Energo (EN) and tetraploid (PP) plants, harvested in June were superior methane sources to woody tissue. (Kakuk et al. 2021)

 

2.2. Identification of energy willow lines capable of phytoremediation

The new autotetraploid plants that we produced show several properties such as larger roots or intensive water metabolism that can contribute to increased detoxification. High NaCl content in soils can significantly limit the yield of energy crops, so the use of resistant genotypes is unavoidable. The detoxification ability of the individual cultivars, autotetraploid varieties, has not been characterized so far, therefore the characterization of salt tolerance and heavy metal sensitivity of energy willow cultivars with different genome sizes and their detoxification ability were performed by greenhouse and laboratory tests within GINOP-2.2.1-15-2017-00081 application.

2.2.1. Determination of salt tolerance of energy willow cultivars with different genome sizes

Improving the salt tolerance of high-yielding energy willow varieties can contribute to the expanded production of green energy in saline areas. Our autotetraploid willow genotypes show a moderate decrease in green biomass parameters under high saline stress. Doubling the size of the genome ensured the formation of roots that adapted well to the saline soil. The inhibitory effect of salt stress on photosynthetic assimilation rates was less significant in polyploid plants. Autotetraploid Salix genotypes can serve as breeding stock for selection of salt tolerant variants. (Cseri et al. 2020)

Figure 5. Tolerance of autotetraploid energy willow genotypes (PPE-2, PPE-7, PPE-13) in soils with high salt, NaCl (Cseri et al. 2020).

2.3.2. Determination of heavy metal tolerance of energy willow lines

From the heavy metals present in contaminated soils lead, cadmium and zinc are of the greatest importance, both in terms of their frequency and the extent of their biological harmful effects. The sensitivity to the applied heavy metal concentrations was determined by measuring the growth parameters of the shoot and the root system. Analytical studies were performed to determine the heavy metal accumulation capacity of each genotype. As a result of the experiments, lines with more efficient phytoremediation ability were identified.

3. Synthesis of chemically modified DNA and RNA oligonucleotides

Chemically synthesized DNA and RNA molecules are the essential gene specific tools of targeted gene editing methods. Our laboratory and the Laboratory of Nucleic Acid of Institute of Medical Chemistry, University of Szeged work as a unique team in Hungary to design and synthesize DNA and RNA oligonucleotides with novel structures to increase the precision and efficiency of the targeted mutagenesis.

 

Services

As a service, we offer custom synthesis of DNA and RNA oligonucleotides tailored for the need of our research partners, e.g. phosphorothioated, fluorescently labelled, incorporation of LNA nucleotides, 2´-OMe and 2´-MOE nucleotides among others.

 

Selected publications:

  1. Tiricz, Hilda ; Nagy, Bettina ; Ferenc, Györgyi ; Török, Katalin ; Nagy, István ; Dudits, Dénes; Ayaydin, Ferhan
    Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells
    JOURNAL OF PLANT RESEARCH 131 : 1 pp. 179-189. , 11 p. (2018)
  2. Rádi, Feríz ; Nagy, Bettina* ; Ferenc, Györgyi ; Török, Katalin ; Nagy, István ; Zombori, Zoltán ; Dudits, Dénes ; Ayaydin, Ferhan
    In planta test system for targeted cellular mutagenesis by injection of oligonucleotides to apical meristem of maize seedlings
    ACTA PHYSIOLOGIAE PLANTARUM 43 : 5 Paper: 79 (2021)

  3. Dudits, Dénes ; Torok, Katalin ; Cseri, András ; Paul, K ; Nagy, Anna Viktória ; Nagy, Bettina; Sass, László ; Ferenc, Györgyi ; Vankova, Radomíra ; Dobrev, Petre, Vass, Imre and Ayaydin, Ferhan.
    Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis L.
    PLANT PHYSIOLOGY 170 : 3 pp. 1504-1523. , 20 p. (2016)

  4. Cseri, András ; Borbély, Péter ; Poór, Péter ; Fehér, Attila ; Sass, László ; Jancsó, Mihály ; Penczi, András ; Rádi, Feríz ; Gyuricza, Csaba ; Digruber, Tamás et al.
    Increased adaptation of an energy willow cultivar to soil salinity by duplication of its genome size
    BIOMASS & BIOENERGY 140 Paper: 105655 , 9 p. (2020)

  5. Balázs Kakuk, Zoltán Bagi, Gábor Rákhely, Gergely Maróti, Dénes Dudits, Kornél L. Kovács, Methane production from green and woody biomass using short rotation willow genotypes for bioenergy generation,
    BIORESOURCE TECHNOLOGY, Volume 333, 2021, 125223, ISSN 0960-8524, https://doi.org/10.1016/j.biortech.2021.125223.

  6. Györgyi, Ferenc; Zoltán, Váradi; Zoltán, Kupihár; Gábor, Paragi; Lajos, Kovács
    Analytical and structural studies for the investigation of oxidative stress in guanine oligonucleotides
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 21: 14 Paper: 4981 , 27 p. (2020)

  7. Miklós Bege, Ilona Bereczki, Dénes J. Molnár, Máté Kicsák, Krisztina Pénzes-Daku, Zsuzsanna Bereczky, Györgyi Ferenc, Lajos Kovács, Pál Herczegh and Anikó Borbás
    Synthesis and oligomerization of cysteinyl nucleosides
    ORGANIC & BIOMOLECULAR CHEMISTRY, (2020), https://doi.org/10.1039/D0OB01890B

  8. Porkoláb, Gergő*; Mészáros, Mária*; Tóth, András; Szecskó, Anikó; Harazin, András; Szegletes, Zsolt; Ferenc, Györgyi; Blastyák, András; Mátés, Lajos; Rákhely, Gábor et al.
    Combination of Alanine and Glutathione as Targeting Ligands of Nanoparticles Enhances Cargo Delivery into the Cells of the Neurovascular Unit
    PHARMACEUTICS 12 : 7 p. 635 (2020)

  9. Tóth, Emese; Ungor, Ditta; Novák, Tibor; Ferenc, Györgyi; Bánhelyi, Balázs; Csapó, Edit; Erdélyi, Miklós; Csete, Mária
    Mapping Fluorescence Enhancement of Plasmonic Nanorod Coupled Dye Molecules
    NANOMATERIALS 10 : 6 Paper: 1048 , 17 p. (2020)

  10. Bugris, Valéria; Harmat, Veronika; Ferenc, Györgyi; Brockhauser, Sándor; Carmichael, Ian; Garman, Elspeth F.
    Radiation-damage investigation of a DNA 16-mer
    JOURNAL OF SYNCHROTRON RADIATION 26 pp. 998-1009. , 12 p. (2019)

  11. Nyerges, A ; Csorgo, B ; Draskovits, G ; Kintses, B ; Szili, P ; Ferenc, G ; Revesz, T ; Ari, E ; Nagy, I ; Balint, B et al.
    Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance.
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 115 : 25 pp. E5726-E5735. , 10 p. (2018)