nptII Gene Research Articles

Obtaining of transgenic potato plants (Solanum tuberosum L.) that contain antisense sequence of prolindehydrogenase gene

Aim. The aim of our work was to obtain transgenic potato plants of Ukrainian varieties with the expression of a double-stranded RNA suppressor of proline dehydrogenase gene. We propose the decrease of proline degradation level and increase of overall proline concentration in obtained transgenic plants. Methods. The Agrobasterium tumefaciens-mediated method of genetic transformation to obtain transgenic plants of potato was used. Internodes of aseptic potato plants were transformed with a binary vector pBi2E containing an inverted repeats of two copies of proline dehydrogenase gene’s first exon and the gene of neomycin phosphotransferase II (nptII). Results. As a result of experiments kanamycin resistant transgenic potato lines of Deseiree, Belarusian 12 and Slavianka varieties were obtained. The transgenic nature of the obtained plants was confirmed by PCR with primers specific to the first exon of proline dehydrogenase and to nptII genes. Conclusions. The optimized conditions of genetic transformation and used agrobacterial strain allow to obtain the transgenic plants of a model potato variety Désirée, as well as varieties Belorussian 12 and Slovyanka which are of practical interest for cultivation in Ukraine. Keywords: transgenic plants, potatoes (Solanum tuberosum L.), stress resistance, proline.

Low risk of pollen-mediated gene flow in transgenic plants under greenhouse conditions

There is concern about the potential for transgene contamination in wild type (WT) plants via pollen-mediated gene flow from transgenic plants. In this study, we investigate pollen-mediated gene flow using tobacco transgenic lines carrying recombinant protein GA733-FcK, which is a putative candidate for producing colorectal cancer vaccine. Transgenic and WT plants were grown in greenhouse, with WT plants placed 0.3, 1, 5, 10, and 20 m away from transgenic plants. Seeds were harvested from randomly selected WT plants and sown on germination media supplemented with or without kanamycin. At 30 days after sowing, none of the WT seedlings produced true leaves and roots when grown on selective media. Polymerase chain reaction analysis revealed that GA733-FcK and nptII genes were expressed in shoots of transgenic plants but not of WT plants. Expression of GA733-FcK and nptII proteins was also abolished in WT leaves when compared to that of transgenic plants. Our findings suggested that there is low risk of pollen-mediated gene flow from transgenic plants expressing GA733-FcK when grown in greenhouse conditions.

Genetic engineering of sunflower (Helianthus annuus L.) for resistance to necrosis disease through deployment of the TSV coat protein gene

Necrosis disease incited by tobacco streak virus (TSV) causes significant yield losses in sunflower in the tropics and sub-tropics. Genetic engineering of sunflower through deployment of the coat protein gene of TSV (TSV-CP) was attempted along with neomycin phosphotransferase gene (nptII) for selection of putative transformants on kanamycin. Split cotyledons from decoated mature seeds were used as target tissues for Agrobacterium tumefaciens-mediated transformation and putative transformed shoots were obtained with an average frequency of 3.3%. Presence of the introduced transgene was identified by PCR; expression was determined through RT-PCR, northern blot analysis and quantitative Real-time PCR (qRT-PCR); stable integration was ascertained through Southern blot analysis of plants in T0 to T4 generations. Out of 102 positive T0 events, 20 events were carried to T1 generation from which five events (CP-S-237, CP-S-247, CP-S-481, CP-S-648 and CP-S-753) were advanced till T4 generation. Challenge inoculations of plants of event No 481 showed resistance to necrosis disease and plants grew to maturity in TSV-CP transgenics while control plants (untransformed) showed mortality within 1–2 weeks following inoculation. Expression analysis of the TSV-CP and nptII genes in different tissues at flowering and seed setting stages revealed constitutive expression of the transgene till seed maturation. One event (No 481) was selected for transfer of the TSV-CP gene into agronomically superior genotypes.

TRANSFER OF GLUCANASE GENE TO RESIST LATE BLIGHT DISEASE IN POTATO (Solanum tuberosum L.)

Potato late blight is a pandemic disease caused by the highly virulent (Phytophthora infestans) fungus. The regeneration capacity was done among three tested potato cultivars (Spunta, Diamont and Desiree). Two different types of explants (inter-node and leaf) were cultured for calli induction and plant regeneration .The highest value of leaf explants was for Desiree cultivar (80%), which induced from developed callus in the 2,4-D medium. The best value of maximum shoot regeneration was also for Desiree cultivar in the BA, IAA and GA3 media from leaf which proved to be more effective. Then cloning of glucanase gene in pRI plasmid which carrying kanamycin resistance (nptII) gene was perfomed and followed by transformation in Agrobacterium tumefaciens LBA4404 strain which used for plant transfection. Nucleotide and amino acid sequences of transformed Agrobacterium were analyzed. The putative transgenic plantlets genomes and cDNA of the isolated RNA for glucanase gene.

Over Expression of p5cs Gene to Increase Cold Stress Tolerance in Iranian Species of Petunia

In order to examine the relationship between proline and cold stress in Iranian (local) genotype of petunia, p5cs gene was transferred to petunia through Agrobacterium-mediated transformation. The leaf discs from four weeks and shoot apices from 7-day-old petunia in vitro plants were co-cultivated with Agrobacterium strain LBA4404 harboring a plasmid pBI121 as the vector system for transformation of petunia. pBI121 plasmid containing β-glucuronidase (gus) gene as a reporter gene and Δ1-pyrroline-5-carboxylate synthetase (p5cs) gene and neomycin phosphotransferase (nptII) gene were used as a selectable marker. The co-cultivated leaf discs and shoot apices were transferred to the selective medium and thereafter to proliferation medium respectively. To confirm transformation, regenerated plants were subjected to the polymerase chain reaction (PCR), GUS histochemical and proline assays. The results confirmed the presence of the gus and p5cs gene in the genome of all transformants and the transformed plants were more tolerant (674.87 μg/g fresh leaves) than the wild types under stress conditions.Plant Tissue Cult. & Biotech. 28(1): 35-44, 2018 (June)

Elimination of macro elements from inoculation and co-cultivation media enhances the efficiency of Agrobacterium-mediated transformation in Petunia.

In order to evaluate the effect of inoculation and co-cultivation media elements on transformation frequency in Petunia hybrida, modified MS media with different elements were tested on Alvan and Large Flower Alvan (LF Alvan), two local cultivars. Leaf explants of both cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 (pBI121) containing neomycin phosphotransferase (nptII) and an intron-containing β-glucuronidase (gus) genes. When medium lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used as inoculation and co-cultivation medium, a higher frequency of transformation for Alvan (22%) and LF Alvan (16%) was obtained. Kanamycin resistant plantlets were stained blue by GUS assay. Furthermore, polymerase chain reaction (PCR) analysis revealed the presence of both gus and nptII genes in all putative transformants. Finally, southern blot hybridization confirmed insertion of 1-4 copies of gus gene in transgenic plants.

PRICK AND SOAK Agroacterium tumefaciens-MEDIATED IN PLANTA TRANSFORMATION IN TOMATO (Lycopersicon esculentum Mill.)

One of the modern plant breedings through genetic engineering is Agrobacterium tumefaciens-mediated transformation. Agrobacterium tumefaciens-mediated transformation can be performed in vitro or in planta. In planta transformation arises from the weaknesses of the in vitro method such as need high hygiene standard, professional tissue culture experts, and more time to prepare explants and somaclonal variation. In planta transformation is a method to transfer the gene to the plant genome without any tissue culture stages. The aims of this research were to know the possibility of the prick and soak in planta method with the target of tomato seeds and to know the most suitable inoculation time for tomato seeds transformation by prick and soak method the transformation is done by pricking the seeds and soaking them in the A. tumefaciens suspension. The treatments in this study were 1 and 2 days inoculation time to test the efficacy of prick and soak in planta transformation method. Tomato seeds were pricked with a needle on the center once, and then soaked in A. tumefaciens strain LB4404 suspension carrying pKYS-SoSPS1 plasmid with Neomycin Phosphotransferase (NPTII) and Saccharum officinarum Sucrose Phosphate synthase (SoSPS1) genes. Visualization of tomato’s DNA samples after PCR showed that 1-day inoculation sample was positively integrated with NPTII gene and negative in the 2 days inoculation treatment.

"Cell grafting": a new approach for transferring cytoplasmic or nuclear genome between plants.

A new method based on mixing and wounding of callus tissue was used to transfer plastid or nuclear DNA between cells. Methods alternative to sexual hybridization can be powerful tools for crop improvement. We have developed a new hybridization technology based on wounding a mixed population of cells of two parents growing in vitro as callus ("cell grafting"), and have demonstrated the utility of this system for plastid or nuclear genome transfer. In our proof-of concept experiments, non-organized growing tissue (callus) from tobacco var. Samsun, carrying the nuclear marker genes nptII and uidA (GUS), and tobacco var. Petit Havana, carrying aadA and gfp genes in the plastid genome, were mixed together, wounded with a razor blade and placed for regeneration on selection medium containing both spectinomycin (aadA) and paromomycin (nptII). Plants with aadA and gfp positive plastids and nptII plus uidA positive nuclear background were produced. Molecular analysis confirmed the presence of all four genes in these plants. Morphology and ploidy level analysis confirmed the production of "diploid" plants similar to var. Samsun possessing transformed plastids from var. Petit Havana. Reciprocal crosses between the experimentally produced plants and wild type tobacco confirmed maternal inheritance of aadA and gfp and Mendelian inheritance of nptII and uidA. For transfer of nuclear traits between plants we used two nuclear-transformed parents with different selectable markers; one with nptII (paromomycin resistant), and another with aadA (spectinomycin resistant). Plants resistant to both antibiotics which also had different visible markers were produced.

A downstream box fusion allows stable accumulation of a bacterial cellulase in Chlamydomonas reinhardtii chloroplasts

BackgroundWe investigated strategies to improve foreign protein accumulation in the chloroplasts of the model algae Chlamydomonas reinhardtii and tested the outcome in both standard culture conditions as well as one pertinent to algal biofuel production. The downstream box (DB) of the TetC or NPTII genes, the first 15 codons following the start codon, was N-terminally fused to the coding region of cel6A, an endoglucanase from Thermobifida fusca. We also employed a chimeric regulatory element, consisting of the 16S rRNA promoter and the atpA 5′UTR, previously reported to enhance protein expression, to regulate the expression of the TetC-cel6A gene. We further investigated the accumulation of TetC-Cel6A under N-deplete growth conditions.ResultsBoth of the DB fusions improved intracellular accumulation of Cel6A in transplastomic C. reinhardtii strains though the TetC DB was much more effective than the NPTII DB. Furthermore, using the chimeric regulatory element, the TetC-Cel6A protein accumulation displayed a significant increase to 0.3% total soluble protein (TSP), whereas NPTII-Cel6A remained too low to quantify. Comparable levels of TetC- and NPTII-cel6A transcripts were observed, which suggests that factors other than transcript abundance mediate the greater TetC-Cel6A accumulation. The TetC-Cel6A accumulation was stable regardless of the growth stage, and the transplastomic strain growth rate was not altered. When transplastomic cells were suspended in N-deplete medium, cellular levels of TetC-Cel6A increased over time along with TSP, and were greater than those in cells suspended in N-replete medium.ConclusionsThe DB fusion holds great value as a tool to enhance foreign protein accumulation in C. reinhardtii chloroplasts and its influence is related to translation or other post-transcriptional processes. Our results also suggest that transplastomic protein production can be compatible with algal biofuel production strategies. Cells displayed a consistent accumulation of recombinant protein throughout the growth phase and nitrogen starvation, a strategy used to induce lipid production in algae, led to higher cellular heterologous protein content. The latter result is contrary to what might have been expected a priori and is an important result for the development of future algal biofuel systems, which will likely require co-products for economic sustainability.

Development of transgenic tobacco lines with pyramided insect resistant genes.

Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

PHENOTYPIC AND MOLECULAR CHARACTERIZATION OF MULTISHOOTS DEVELOPMENT IN TRANSGENIC Phalaenopsis amabilis (L.) BLUME HARBORING 35S::KNAT1 (KNOTTED-LIKE Arabidopsis thaliana 1)

Phalaenopsis amabilis (L.) Blume is one of Indonesian natural orchid which has an aesthetic flower and possesses high economic value. The low multiplication rate and long periods of life cycle are the main obstacles to conventionally propagate this orchid. The aims of this research were to analyze the stability of transgenic plant P. amabilis harboring 35S::KNAT1 based on morpho-genomic characterization. KNAT1 gene is reported as a gene that involved in the shoot formation, and it  had been successfully introduced into Phalaenopsis amabilis (L.) Blume genome. After seven times regeneration, the confirmation of the transgene existence in the genom is needed to ensure whether the plant could consistently maintain the transgene in its genome and to characterize the shoot development. The experiment was carried out in 3 steps: 1) Co-integration analysis of 35S::KNAT1 into P. amabilis genom; 2) Phenotypic analysis on the multiplication rate, morphological variation and venation pattern; and 3) Protein profile analysis of transgenic plants. The results showed that the survival rate of putative transgenic was 58.7% on NP0 medium and 62.5% on NP SIM medium. PCR analysis confirmed that 82.5% transgenic growth on NP0 and 93.33% on NP SIM contained DNA fragment of KNAT1 gene, NPTII gene and trnL-F intergenic spacer, indicating that those plants are positive transgenic. The 35S::KNAT1 transgenes and phytohormone were independently involved in multishoots formation of P. amabilis transgenic plants. The phenotypic of plantlets were classified into six main criteria, i.e. normal shape, lobed leaves, rosette, elongated stem, cup shoot and widened leaves. The normal type was the most abundant type of variation (± 29%) in both medium. Protein profile showed that all transgenic plants produced 45,8 kDa protein and that was equivalent with molecular weight of KNAT1 protein. Taken together, all those data indicated that 35S::KNAT1 transgene were consistently integrated into the transgenic plant genome.

Transformation and confirmation of GUS gene expression in Solanum melongena L. of PLR 1 cultivar

In the present study GUS gene transformation was carried out in eggplant using Agrobacterium strain with pBAL2 vector harboring gus gene and nptII as selection marker gene. The factors which are affecting (enhancing) the frequency of transient gus gene expression are different physical and biochemical variables has been carried out. It is observed that the 4 day precultured explants showed the minimum survival rate in the medium when compared with 2-day co cultivated medium. The explants which had undergone co-cultivation for 4 to 5 days showed GUS activity, the tissues were adversely affected due to the overgrowth of bacteria. The gene specific primers for nptII and gus gene were used for amplification and it has given 680bp and 1.9 kb amplified fragments respectively and recorded. The band was detected in the selected plants, but it was absent from the negative control (non-transformed) plant in the Southern hybridization. Our experiment showed 0.80-1.60 percentage of efficiency in transformation. With a total of 849 infected shoots were undergone confirmation tests which results 9 PCR positives (1.06% efficiency). The Transformant kept in the Environmental Growth Chamber and transferred to field condition subsequently.

Agrobacterium tumefaciens-mediated transformation of Jatropha curcas L. with a polyhydroxyalkanoate gene (phaC)

Polyhydroxybutyrate is a component of bioplastics that is synthesized under the control of enzymes encoded by pha multigenes. The genes are naturally present in Ralstonia eutropha. However, the production of bioplastics in bacteria is inefficient because the bacterial biomass is relatively small compared with plants or fungi. As such, engineering techniques have been developed that enable pha genes to be inserted into plant biomass, and then be expressed in the biomass of the plant to produce polyhydroxybutyrate. The objectives of this study were to transform the tissue of Jatropha curcas using the phaC gene (a pha gene), to regenerate the transformed plant, and to confirm the presence of the inserted genes with PCR. The genetic transformation of J. curcas was mediated by Agrobacterium tumefaciens strain GV3101 containing pARTC by dipping the cotyledon tissue of J. curcas in a suspension of the bacterium for 30 min, followed by cocultivation for 3 d on Murashige and Skoog (MS) medium. The tissue was then placed on a selection medium, i.e. MS medium containing 13.3 µM BAP and 0.05 µM IBA with the addition of 20 mg/L kanamycin. The results showed that 12.35% of the tissue survived and regenerated into a shoot after 1–2 months. Molecular analysis of the transformed tissue was performed using phaC and nptII primers, in order to detect the presence of the phaC and nptII genes. Specific bands were detected at 659 bp and 700 bp, corresponding to the nptII primer and phaC primer, respectively.

A survey of crop-derived transgenes in activated and digester sludges in wastewater treatment plants in the United States.

The use of transgenic crops has become increasingly common in the United States over the last several decades. Increasing evidence suggests that DNA may be protected from enzymatic digestion and acid hydrolysis in the digestive tract, suggesting that crop-derived transgenes may enter into wastewater treatment plants (WWTPs) intact. Given the historical use of antibiotic resistance genes as selection markers in transgenic crop development, it is important to consider the fate of these transgenes. Herein we detected and quantified crop-derived transgenes in WWTPs. All viable US WWTP samples were found to contain multiple gene targets (p35, nos, bla and nptII) at significantly higher levels than control samples. Control wastewater samples obtained from France, where transgenic crops are not cultivated, contained significantly fewer copies of the nptII gene than US activated and digester sludges. No significant differences were measured for the bla antibiotic resistance gene (ARG). In addition, a nested PCR (polymerase chain reaction) assay was developed that targeted the bla ARG located in regions flanked by the p35 promoter and nos terminator. Overall this work suggests that transgenic crops may have provided an environmental source of nptII; however, follow-up studies are needed to ascertain the viability of these genes as they exit WWTPs.

Agrobacterium-mediated transformation of Camelina sativa for production of transgenic plants.

Camelina sativa (C. sativa), an oilseed species rich in poly-unsaturated fatty acids, has gained great importance as an industrial oil platform crop in recent years. Despite the potential benefits of C. sativa for bioenergy applications, limited research has been conducted to improve its agronomic qualities. Hence, a simple and efficient technique for production of transgenic C. sativa plants is warranted. In the present study, shoot apical meristems of two C. sativa cultivars (Pl650159 and Pl650161) were transformed with Agrobacterium strain ‘EHA 105’ harboring the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase II (nptII) genes. After two days of co-cultivation in the dark, explants were transferred to selection medium. Transgenic shoots were identified on the basis of green fluorescence and kanamycin resistance. Shoots were then rooted and transferred to potting mix soil for acclimatization. This protocol describes an efficient method to generate transgenic C. sativa plants in as little as 4 weeks.

Agrobacterium- Mediated Transformation of Tobacco Plants using Walnut Polyphenol Oxidase Gene

Polyphenol oxidase (PPO) is a well-recognized copper-containing enzyme which can catalyze oxidation of a great variety of phenolic compounds. PPO is found in diverse microorganisms, plants, and animals. To examine the anti-pathogenic role of PPO in walnut (Juglans regia L.), Agrobacterium tumefaciens strain LBA4404 was used to transform tobacco (Nicotiana tobacum L.) explants. Recombinant binary vector pBI121, containing the walnut JrPPO gene and the nptII gene as a selectable marker, was incorporated into A. tumefaciens. MS medium supplemented with 50 mg/l of kanamycin and 200 mg/l of cefotaxime was used as a selection medium. Plantlets were regenerated from putatively transgenic calli and polymerase chain reaction and Southern blot analysis were performed to confirm the integration of JrPPO into the genome. To evaluate the function of PPO in pathogen resistance, transgenic tobacco plants were exposed to Pseudomonas syringae pv. tabaci. According to symptom progress and quantitative analyses, although there was no significant difference in transgenic tobacco, but mean comparison showed that disease severity of transgenic tobacco was less than wild plants. Finally, it may be concluded that PPO has a notable function in the resistance process in walnut, but tobacco transgenic plants might not be a suitable test plant to examine the resistance role of PPO in walnut.

Результаты пятилетнего лабораторного культивирования растений тополя берлинского, генетически модифицированных генами uidA и nptII

Результаты пятилетнего лабораторного культивирования растений тополя берлинского, генетически модифицированных генами uidA и nptII

Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.

Simplified methodology for large scale isolation of homozygous transgenic lines of lettuce

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays.

Genetic transformation of the white-rot fungus Dichomitus squalens using a new commercial protoplasting cocktail

D. squalens, a white-rot fungus that efficiently degrades lignocellulose in nature, can be used in various biotechnological applications and has several strains with sequenced and annotated genomes. Here we present a method for the transformation of this basidiomycete fungus, using a recently introduced commercial ascomycete protoplasting enzyme cocktail, Protoplast F. In protoplasting of D. squalens mycelia, Protoplast F outperformed two other cocktails while releasing similar amounts of protoplasts to a third cocktail. The protoplasts released using Protoplast F had a regeneration rate of 12.5% (±6 SE). Using Protoplast F, the D. squalens monokaryon CBS464.89 was conferred with resistance to the antibiotics hygromycin and G418 via polyethylene glycol mediated protoplast transformation with resistance cassettes expressing the hygromycin phosphotransferase (hph) and neomycin phosphotransferase (nptII) genes, respectively. The hph gene was expressed in D. squalens using heterologous promoters from genes encoding β-tubulin or glyceraldehyde 3-phosphate dehydrogenase. A Southern blot confirmed integration of a resistance cassette into the D. squalens genome. An average of six transformants (±2 SE) were obtained when at least several million protoplasts were used (a transformation efficiency of 0.8 (±0.3 SE) transformants per μg DNA). Transformation of D. squalens demonstrates the suitability of the Protoplast F cocktail for basidiomycete transformation and furthermore can facilitate understanding of basidiomycete gene function and development of improved strains for biotechnological applications.