Binary Vector System Research Articles

Agrobacterium tumefaciens: Biology and application in genetic engineering

Agrobacterium tumefaciens is a rod-shaped soil bacterium renowned for its unique ability to transfer tumour-inducing plasmid (Ti plasmid) segments to plant cells. This mechanism has been extensively exploited in plant genetic engineering. This review delves into the intricate biological interactions between A. tumefaciens and plant cells, including the critical steps of bacterial attachment, activation of virulence (Vir) genes, generation and transport of the T-complex, and integration of T-DNA into the plant chromosome. Furthermore, the review examines the engineering of A. tumefaciens as a transformation tool, focusing on the modifications of Ti plasmids to create binary and co-integrative vector systems, which have significantly improved the efficiency and versatility of transformation protocols. The paper also highlights applications of Agrobacterium-mediated transformation in the production of edible vaccines. By providing a detailed examination of the biological, technical, and practical aspects of Agrobacterium-mediated transformation, this review aims to offer insights into optimizing this technique for various plant biotechnology applications. Ultimately, understanding and improving Agrobacterium-mediated transformation is crucial for advancing plant biotechnology.

Practical use of tobravirus-based vector to produce SARS-CoV-2 antigens in plants

A plant-based heterologous expression system is an attractive option for recombinant protein production because it is based on a eukaryotic system of high feasibility, and low biological risks. Frequently, binary vector systems are used for transient gene-expression in plants. However, plant virus vector-based systems offer advantages for higher protein yields due to their self-replicating machinery. In the present study, we show an efficient protocol using a plant virus vector based on a tobravirus, pepper ringspot virus, that was employed for transient expression of severe acute respiratory syndrome coronavirus 2 partial gene fragments of the spike (named S1-N) and the nucleocapsid (named N) proteins in Nicotiana benthamiana plants. Purified proteins yield of 40–60 µg/g of fresh leaves were obtained. Both proteins, S1-N and N, showed high and specific reactivities against convalescent patients’ sera by the enzyme-linked immunosorbent assay format. The advantages and critical points in using this plant virus vector are discussed.

The Basic Experimental Setup for the Vacuum-assisted, Agrobacterium-mediated Transient Expression in Tomato

Several methods are available for use, to deliver the gene of interest into plants. Among these, Agrobacterium-mediated transformation utilizes binary vector systems to achieve the stable transformation of plants. Alas, this process is labor-intensive and time-consuming as several months are needed to obtain a true transgenic plant. Transient gene expression (e.g., vacuum-infiltration) systems were offered as an alternative over stable transformation, specifically to overcome time-related drawbacks. However, this method requires expensive equipment such as vacuum chambers. In this study, we report a vacuum-infiltration protocol for the transient expression of a reporter gene, modified green fluorescent protein (mGFP), in tomato seedling. With a basic experimental setup (including a custom-built growth chamber) with affordable equipment, we showed that the entire leaf can be infiltrated by applying a mere 200 millibar vacuum for 6 minutes, and thus the transient expression can be achieved in tomato plant, evidenced by PCR-based detection of the T-DNA, detection of mGFP both analytically with SDS-page analysis, and visually by the images acquired by fluorescence microscopy. Furthermore, different Agrobacterium tumefaciens strains were tested for their transformation efficiency, and we showed that LBA4404 was the most effective strain to use in the vacuum-assisted transient expression.

Development of a miniaturized Ti-plasmid and helper plasmid system for Agrobacterium-mediated plant transformation

Tumor-inducing (Ti) plasmid is the requisite for Agrobacterium-mediated plant transformation. Over decades, continuous efforts have been made to improve the efficiency of Agrobacterium-mediated plant transformation and most of them focused on the binary vector system. A binary vector system comprises of a binary vector of which transferred DNA (T-DNA) resided on and a Ti plasmid to carry those essential virulence genes. In this study, we constructed a miniaturized helper Ti plasmid, designated as pYL102, with the aim to enhance the overall Agrobacterium-mediated transformation rate. The size of pYL102 was reduced to ~60% of the original plasmid pCAMBIA5105. Subsequently, pYL102 was coupled with the broad host range (BHR) bacterial expression vector, pYL101C, of which the key regulatory virulence gene, virG-N54D, was cloned in and expressed under the control of a strong constitutive PINTc promoter. To test the functionality of the constructed vector system, A. tumefaciens C58C1 carrying pYL102, pYL101C::virG-N54D and the transformation vector pGWB2::e35S-sfGFP was used to transform Nicotiana benthamiana leaves by agroinfiltration. Green fluorescence was observed in spots infiltrated with Agrobacterium carrying the test plasmids. The fluorescence intensity from the test agroinfiltrated leaves was significantly higher than those of the mock-infiltrated leaves (p<0.01), indicating the vector system can be used for plant transformation.

Development of an efficient marker-free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1.

SummaryWe have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium‐derived vir and T‐DNA‐mediated transformation. Oh H1 is a unique, non‐phytopathogenic species, categorized as a BSL‐1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1‐8 strain containing a binary vector system. Oh H1‐8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy‐. Oh H1‐8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1‐8 generated high‐quality, transgenic events with single‐copy, plasmid backbone‐free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock‐inducible excision system resulting in marker‐free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null‐segregant lines to wild‐type controls. We have demonstrated that Oh H1‐8, combined with spectinomycin selection, is an efficient, rapid, marker‐free and yield‐neutral transformation system for elite soybean.

An efficient genetic transformation system for Chinese medicine fungus Tolypocladium ophioglossoides

Tolypocladium ophioglossoides is a rare and valuable fungus extensively used in Chinese medicine for relieving postmenopausal syndrome in women yet its bioactive molecules are unknown. To explore its molecular mechanisms, we have developed a reliable Agrobacterium-mediated transformation system using the selective marker: the chlorimuron ethyl-resistance gene sur. For this purpose, we firstly constructed a T-DNA binary vector system and then improved the transformation efficiency by optimizing conditional parameters including the Agrobacterium tumefaciens concentration, the conidia number of T. ophioglossoides, the co-culture time and the concentration of acetosyringone. Furthermore, we have knocked-out the ku70 gene，which is a key gene in non-homologous end joining (NHEJ) DNA repair pathway，and the effect of the length of the homologous arms (HA) on the genetic transformation efficacy was also examined, which increased by 60% when HA was about 3 kb in length. Our results suggest that the genetic transformation system is efficient and feasible for the truffle-parasite fungus T. ophioglossoides, which can further be used in large-scale experiments for characterization of genes of interest in future work.

An optimized double T-DNA binary vector system for improved production of marker-free transgenic tobacco plants.

In the plant transformation process, marker genes play a vital role in identifying transformed cells from non-transformed cells. However, once transgenic plants have been obtained, the presence of marker genes may provoke public concern about environmental or biosafety issues. In our previous study, a double T-DNA vector system has been developed to obtain marker-free transgenic plants, but the T-DNA left border (LB) and right border (RB) of the vector showed an RB-LB-RB-LB pattern and led to high linkage integration between the selectable marker gene (SMG) and the gene of interest (GOI). To improve this double T-DNA vector system, we inverted the first T-DNA direction such that a LB-RB-RB-LB pattern resulted to avoid transcriptional read-through at the LB and the subsequent linkage transfer of the SMG and GOI. We separately inserted the green fluorescent protein (GFP) gene as the GOI and the neomycin phosphotransferase II (NPTII) gene as the SMG in both optimized and original vectors and carried out Agrobacterium-mediated tobacco transformation. Statistical analysis revealed that the linkage frequency was 25.6% in T0 plants transformed with the optimized vector, which is a 42.1% decrease compared with that of the original vector (44.2%). The frequency of obtaining marker-free transgenic plants was 66.7% in T1 plants transformed with the optimized vector, showing a 33.4% increase compared with that of the original vector (50.0%). Our results demonstrate that the optimized double T-DNA binary vector system is a more effective, economical and time-saving approach for obtaining marker-free transgenic plants.

A zero-background CRISPR binary vector system for construction of sgRNA libraries in plant functional genomics applications

Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing is a ground-breaking biotechnology for agricultural applications such as precision breeding in crop plants. Agrobacterium-mediated CRISPR delivery has been successfully adapted for gene knockout applications for basic research and agricultural technology development. However, selecting proper single-guide RNA (sgRNA) for CRISPR binary constructs to induce double-strand break in certain target genes has presented difficulties mainly due to unpredictable in vivo sgRNA activities. Therefore, more than three independent CRISPR constructs, each harboring different sgRNAs, are often applied to ensure the desired CRISPR-induced knockout alleles. Here, we report a zero-background CRISPR binary vector platform, featuring ccdB conjugation within sgRNA cloning cassette, which is later removed by AarI endonuclease, that allows positive survival selection for bona-fide sgRNA clones and effective exclusion of uncut or self-ligated ‘background’ negative clones. We demonstrate the advantage of using the zero-background CRISPR binary platform in a high-throughput pooled cloning strategy of multiple different sgRNAs which produced Agrobacteria containing multiple sgRNAs without any background. We also tested the integrity of pooled CRISPR sgRNA construct libraries during extended bacterial culture and during the transfer between Escherichia coli to Agrobacterium, and verified that the fidelity of sgRNA species representation was faithfully maintained during library generation.

Live-cell assays reveal selectivity and sensitivity of the multidrug response in budding yeast

Pleiotropic drug resistance arises by the enhanced extrusion of bioactive molecules and is present in a wide range of organisms, ranging from fungi to human cells. A key feature of this adaptation is the sensitive detection of intracellular xenobiotics by transcriptional activators, activating expression of multiple drug exporters. Here, we investigated the selectivity and sensitivity of the budding yeast (Saccharomyces cerevisiae) multidrug response to better understand how differential drug recognition leads to specific activation of drug exporter genes and to drug resistance. Applying live-cell luciferase reporters, we demonstrate that the SNQ2, PDR5, PDR15, and YOR1 transporter genes respond to different mycotoxins, menadione, and hydrogen peroxide in a distinguishable manner and with characteristic amplitudes, dynamics, and sensitivities. These responses correlated with differential sensitivities of the respective transporter mutants to the specific xenobiotics. We further establish a binary vector system, enabling quantitative determination of xenobiotic-transcription factor (TF) interactions in real time. Applying this system we found that the TFs Pdr1, Pdr3, Yrr1, Stb5, and Pdr8 have largely different drug recognition patterns. We noted that Pdr1 is the most promiscuous activator, whereas Yrr1 and Stb5 are selective for ochratoxin A and hydrogen peroxide, respectively. We also show that Pdr1 is rapidly degraded after xenobiotic exposure, which leads to a desensitization of the Pdr1-specific response upon repeated activation. The findings of our work indicate that in the yeast multidrug system, several transcriptional activators with distinguishable selectivities trigger differential activation of the transporter genes.

The piggyBac-based double-inducible binary vector system: A novel universal platform for studying gene functions and interactions

Eukaryotic inducible overexpression systems, including Tet-On and mifepristone-inducible systems, have been widely used to study gene functions by reverse genetics. Among the transposon systems reported to date, the piggyBac transposon system is one of the most efficient in cultured mammalian cells. Here, we report a piggyBac-based double-inducible system that combined the advantages of previous systems. To create this system, the trans- and cis-elements of the Tet-On and mifepristone-inducible systems were cloned into a piggyBac-based trans-vector and cis-vector, respectively. The coding regions of two splicing variants of RUNX1, RUNX1a and RUNX1b, were inserted into the cis-vector to test its ability to express foreign genes along with fluorescent marker proteins. Transgenic 293 T cells were established, and the system was tested by inducing expression of foreign genes with DOX and/or mifepristone; GFP and/or mCherry were used as reporter genes. The system efficiently and stringently induced expression of GFP/mCherry and their co-expressed genes without significant mutual interference, as determined by qRT-PCR and Western blot. This piggyBac-based double-inducible system represents a new genetic tool for studying gene functions and interactions in vitro and in vivo in almost all organisms.

Gateway-compatible inducible vector set for the functional analysis of transcription factors in plants.

The inducible vectors pER8-Gateway-3Flag and pER8-Gateway-3Flag-SRDX have been subjected to considerable research in terms of the function of transcription factors (TFs) via transcription activity and repression, respectively. Approximately 1500 TFs have been identified in Arabidopsis thaliana genome. To identify their functions, loss-of-function and gain-of-function mutants were generated to analyze the phenotype. However, many loss-of-function mutants did not show any evident phenotype because of the functional redundancy within the TF family. The constitutive misexpression of some TFs may result in lethality or sterility. To overcome these problems, we produced a Gateway-compatible inducible binary vector system that facilitates fast and reliable DNA cloning and biological function identification. The vector can be used for the inducible expression of protein fusions to a polypeptide protein tag named 3xFLAG tag. This vector system can also be used to generate an inducible transcription inhibition of protein fusion to an Ethylene-Responsive Factor-associated amphiphilic repression (EAR) motif. The EAR motif makes it possible to get rid of redundancy within a TF family, thereby facilitating studies on loss of function. With these Gateway vectors, conventional subcloning technology that depends on restriction digestion and ligation is avoided. Thus, these Gateway vectors should be useful not only for the rapid analysis of the functions of redundant plant TFs, but also for the manipulation of TF overexpression, resulting in plant lethality or sterility, via an inducible promoter.

BiBAC Modification and Stable Transfer into Maize (Zea mays) Hi-II Immature Embryos via Agrobacterium-Mediated Transformation.

Binary Bacterial Artificial Chromosomes (BiBAC) are large insert cloning vectors that contain the necessary features required for Agrobacterium-mediated transformation. However, the large size of BiBACs and low-copy number in Escherichia coli (DH10B) and Agrobacterium tumefaciens make cloning experiments more difficult than other available binary vector systems. Therefore, a protocol that outlines preparation, modification, and transformation of high-molecular weight (HMW) constructs is advantageous for researchers looking to use BiBACs in plant genomics research. This unit does not cover the cloning of HMW DNA into BiBAC vectors. Researchers looking to clone HMW DNA into BiBACs can refer to Zhang etal. (2012; doi: 10.1038/nprot.2011.456). © 2017 by John Wiley & Sons, Inc.

New Hairpin RNAi Vector with Brassica rapa ssp. pekinensis Intron for Gene Silencing in Plants

Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpinbased double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR ), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops

Development of an R4 dual-site (R4DS) gateway cloning system enabling the efficient simultaneous cloning of two desired sets of promoters and open reading frames in a binary vector for plant research.

Vast numbers of proteins work cooperatively to exert their functions in various cells. In order to understand the functions and molecular mechanisms of these proteins in plants, analyses of transgenic plants that concomitantly express two protein-coding genes are often required. We developed a novel Gateway cloning technology-compatible binary vector system, the R4 dual-site (R4DS) Gateway cloning system, which enables the easy and efficient cloning of two desired sets of promoters and open reading frames (ORFs) into a binary vector using promoter and ORF entry clones. In this system, C-terminal fusions with 17 kinds of tags including visible reporters and epitope tags are available for each ORF, and selection by four kinds of resistance markers is possible. We verified that the R4DS Gateway cloning system functioned well in Arabidopsis thaliana by observing the expression and localization patterns of fluorescent proteins fused with organelle-targeting signals and driven by stomatal-lineage specific promoters. We also confirmed that the two cloning sites in the R4DS Gateway cloning system were equivalent and independently regulated. The results obtained indicate that the R4DS Gateway cloning system facilitates detailed comparisons of the expression patterns of two promoters as well as co-localization and interaction analyses of two proteins in specific cells in plants.

Generation of Marker- and/or Backbone-Free Transgenic Wheat Plants via Agrobacterium-Mediated Transformation.

Horizontal transfer of antibiotic resistance genes to animals and vertical transfer of herbicide resistance genes to the weedy relatives are perceived as major biosafety concerns in genetically modified (GM) crops. In this study, five novel vectors which used gusA and bar as a reporter gene and a selection marker gene, respectively, were constructed based on the pCLEAN dual binary vector system. Among these vectors, 1G7B and 5G7B carried two T-DNAs located on two respective plasmids with 5G7B possessing an additional virGwt gene. 5LBTG154 and 5TGTB154 carried two T-DNAs in the target plasmid with either one or double right borders, and 5BTG154 carried the selectable marker gene on the backbone outside of the T-DNA left border in the target plasmid. In addition, 5BTG154, 5LBTG154, and 5TGTB154 used pAL154 as a helper plasmid which contains Komari fragment to facilitate transformation. These five dual binary vector combinations were transformed into Agrobacterium strain AGL1 and used to transform durum wheat cv Stewart 63. Evaluation of the co-transformation efficiencies, the frequencies of marker-free transgenic plants, and integration of backbone sequences in the obtained transgenic lines indicated that two vectors (5G7B and 5TGTB154) were more efficient in generating marker-free transgenic wheat plants with no or minimal integration of backbone sequences in the wheat genome. The vector series developed in this study for generation of marker- and/or backbone-free transgenic wheat plants via Agrobacterium-mediated transformation will be useful to facilitate the creation of “clean” GM wheat containing only the foreign genes of agronomic importance.

A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.

CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.

A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

One of the major intent of metabolic engineering in cell culture systems is to increase yields of secondary metabolites. Efficient transformation methods are a priority to successfully apply metabolic engineering to cell cultures of plants that produce bioactive or therapeutic compounds, such as Vitis vinifera and Taxus x media. The aim of this study was to establish a reliable method to transform non-embryogenic cell cultures of these species. The V. vinifera cv. Gamay/cv. Monastrell cell lines and Taxus x media were used for Agrobacterium-mediated transformation using the Gateway-compatible Agrobacterium sp. binary vector system for fast reliable DNA cloning. The Taxus x media and Vitis cell lines were maintained in culture for more than 4 and 15 months, respectively, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernible effect on cell growth, or led to extracellular accumulation of phytoalexin trans-Resveratrol (t-R) in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA) in the grapevine transgenic cell lines compared to the parental control. The method described herein provides an excellent tool to exploit exponentially growing genomic resources to enhance, optimize or diversify the production of bioactive compounds generated by grapevine and yew cell cultures, and offers a better understanding of many grapevine and yew biology areas.

Gateway®-compatible plant transformation vectors.

Studies in functional genomics and crop improvement programs often rely on the introduction and expression of transgenes in plants. There are two essential components required for in planta transgene expression, a plasmid vector on which the transgene sequence is carried and a delivery system capable of transferring the vector to the target cells. Agrobacterium-mediated plant transformation and the binary plasmid vector system is the preferred method of transgene delivery. The cloning technologies used for DNA manipulation underpin many of these studies. Increased demand for efficient high-throughput transformation systems is driving forward improvements in gene cloning techniques. This chapter gives an overview of Gateway(®)-compatible binary vectors for use in Agrobacterium-mediated transformation systems. It describes a fast, efficient, and robust cloning protocol for the production of an over-expression binary vector using Gateway(®) recombinational cloning.

Agrobacterium tumefaciens-mediated genetic transformation of haptophytes (Isochrysis species).

Isochrysis galbana and Isochrysis sp. are economically important microalgae from the division of haptophytes. Here, we report Agrobacterium-mediated stable DNA transfer into their nuclear genomes. Initial studies were performed to standardize co-cultivation media and determine the sensitivity of the microalgae to selective agents. Up to 1 mg/ml of the antibiotic hygromycin did not inhibit growth, whereas both the haptophytes bleached in artificial seawater (ASW) medium containing micromolar concentrations of the herbicide norflurazon. Co-cultivation of Isochrysis sp. and I. galbana with Agrobacterium tumefaciens strain LBA 4404 harboring the binary vector pCAMBIA 1380-pds-L504R yielded norflurazon-resistant (NR) colonies visible on selective plates after 20-30 days. pCAMBIA 1380-pds-L540R was constructed by cloning a mutated genomic phytoene desaturase (pds) gene from Haematococcus pluvialis as a selectable marker gene into the binary vector system pCAMBIA 1380. Co-cultivation of Isochrysis sp. with A. tumefaciens in ASW medium containing 200 μM of acetosyringone for 72 h produced the highest number of NR cells. For I. galbana, 100 μM of acetosyringone, ASW medium, and 48 h co-cultivation period appeared to be optimum co-cultivation parameters. The NR colonies kept their resistance phenotype for at least 24 months, even in the absence of selective pressure. The transfer of the pds gene in NR cells was shown by PCR amplification of the T-DNA sequences from the genomic DNA of NR cells and Southern blot analysis using T-DNA sequences as probes. The genetic manipulation described here will allow metabolic engineering and a better understanding of several biochemical pathways in the future.

Regeneration and transformation of Polish cultivars of potato

The article presents the results of regeneration and transformation experiments of 12 Polish cultivars of potato. The cultivars Brda, Bzura, Elipsa and Irga regenerated the highest number of shoots from leaf explants (60% to 100% of explants regenerated 4.9 to 16.5 shoots per explant). The cultivars Brda, Bzura, Elipsa and Irys were the source of the best regenerating tuber explants (66% to 100% of explants regenerated 6.2 to 11.9 shoots from one explant). Both types of explants (from leaves and tubers) were used for transformation experiments. <i>Agrobacterium tumefaciens</i> strains used for transformation contained binary vector system: LBA 4404 (pAL 4404:pBI 121) and C58C1 (pGV2260:pVU104). There was a strong correlation between regeneration ability of tested cultivars and transformation efficiency. GUS-positive, kanamycin resistant and well rooted plants from leaf explants in cultivars Bzura, Brda and from tuber explants in Bzura and Elipsa were obtained. Northern blot analysis confirmed the presence of β-glucuronidase mRNA in transgenic plants.