Improve Transformation Efficiency Research Articles

Evaluation of influence of different strains of Agrobacterium rhizogenes on efficiency of hairy root induction in Rauwolfia serpentina

An improved protocol was established for hairy root induction in Rauwolfia serpentina using two strains of Agrobacterium rhizogenes MTCC 532 and 2364. Hairy root induction efficiency was standardized based on infection time, co-cultivation period, OD600 and acetosyringone concentration. Higher transformation efficiency was established using MTCC 532 (31%) and MTCC 2364 (24%) with 30 min infection time, 72 h co-cultivation period and 0.6 OD600. Transformation efficiency was further enhanced to 55 % with 125 μM acetosyringone. MTCC 532 was proven a better strain over the MTCC 2364 in all the tested factors. Putative transgenic hairy roots were confirmed by polymerase chain reaction using rolA specific primers. Biomass of randomly selected 5 hairy root lines was also significantly enhanced. No significant difference of growth was recorded among the lines except line no 4. In the present study, an enhanced system for Agrobacterium rhizogenesmediated hairy root culture was established which offer an effective means to attain improved transformation efficiency and ultimately beneficial for the industrial scale in vitro production of the secondary metabolite.

Online Surge Detection Method Based on Axial Displacement Sensor of MSCC

Magnetically suspended centrifugal compressor (MSCC) can get higher speed with no friction and improve transformation efficiency. However, with speed increases, air flows more easily to separate from the blade and cause the gas pressure unsteadily, which leads to surge occur, causing the system destroyed. Aiming at the problem that existing surge detection methods usually increase complexity or cannot detect surge in time, this paper proposes an online surge detection method and corresponding surge judgment criteria of MSCC system using the signal of axial displacement sensor based on adaptive interference canceller (AIC) and average phase forward linear predictor (APFLP). At first, the system model of the MSCC is established and the transfer function between surge pressure signal and rotor displacement signal is derived. On these basis, an AIC is designed to eliminate the synchronous vibration signal, which has serious interference to surge detection and an APFLP is designed to estimate the frequency of the surge signal. Finally, the simulation and experimental research of the new method are carried out. The simulation and experimental results show that the method can detect surge quickly and effectively.

Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus

Miscanthus species, including M. sacchariflorus, are important bioenergy crops, used also as industry feedstocks and in environmental projects. M. sacchariflorus is also utilised as a donor in interspecific crossings to generate new forms of M. × giganteus. Research on Miscanthus species and their application potential may be considerably extended using methods of genetic modification. However, to date there are scare reports on miscanthus transformation. To improve transformation efficiency, new expression cassettes of a selectable marker gene were constructed and used for biolistic transformation of M. sacchariflorus. The sequence of the bacterial hpt gene encoding hygromycin phosphotransferase in the initial pCAMBIA vector was optimised according to codon usage characteristic of Zea mays, which is closely related to Miscanthus sp., and placed under control of 35S CaMV or the Zea mays ubiquitin promoter, resulting in the pCA35Sohpt and pCAUBQohpt vectors. Transformation rate using optimised vectors significantly increased in comparison to pCAMBIA, and reached 4.2–5.0% vs 2.5%. In respective transgenic plants, significantly higher transcription of optimised hpt was determined while the activity of the reporter GUS enzyme was similar. These results constitute basis for specific miscanthus transformation in a variety of research and practical applications.

EASI Transformation: An Efficient Transient Expression Method for Analyzing Gene Function in Catharanthus roseus Seedlings.

The Catharanthus roseus plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for C. roseus necessitates a fast and reliable method for studying gene function. In this study, we developed an Agrobacterium-mediated transient expression method to enable the functional study of genes rapidly in planta, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age, Agrobacterium density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and GUS genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 – 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the A. tumefaciens strain with the mutated VirGN54D or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 – 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (Renilla luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the CroSTR1 promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in C. roseus. Our efficient Agrobacterium-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of C. roseus cotyledons and provides a timely tool for the community to rapidly assess the function of genes in planta, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.

Optimisation of regeneration parameters improves transformation efficiency of recalcitrant tomato

Genetic transformation of tomato was first accomplished around 30 years ago. However, variability in transformation efficiency of distinct cultivars exists and to some extent remains a bottleneck for transgenic research. This study reports strategies to improve transformation efficiency in tomato and investigates regeneration capacity of transgenic plants under different selection regimes and hormonal applications. Tomato cv. Rio Grande was used as plant material and hygromycin and phosphinothricin (PPT) were used as selection agents. We found that cv. Rio Grande inherently produced a significant number of abnormal (“blind”) shoots lacking an apical meristem. Replacing cytokinin zeatin riboside with 6-benzylaminopurine (BAP) reduced the number of blind shoots although it slightly prolonged regeneration time. Survival rate of calli and shoots was very low using PPT as selection, whereas regeneration was achieved using hygromycin. Delayed application of hygromycin selection following co-cultivation with Agrobacterium tumefaciens improved the overall callus and shoot production. In vivo GFP fluorescence was detected to investigate the development of transgenic tissues using different hygromycin selection regimes. Higher transformation frequency was achieved when explants were continuously exposed to selection agents immediately following the pre-selection stage. Reducing the selection period followed by a non-selection stage increased the number of shoots, but these shoots were mostly non-transgenic. Thus, although less stringent selection, as expected, encouraged regeneration of shoots from calli, it did not improve transformation efficiency. Omitting selection altogether greatly reduced the efficiency of transformation. It was concluded that BAP is more suitable for normal shoot development, and that delayed selection followed by continuous selection results in higher transformation frequency. A significant improvement in regeneration of transgenic tomato through identification of key regeneration factors and robust screening is described, enabling high throughput genetic engineering in tomato.

Infection of Embryonic Callus with Agrobacterium Enables High-Speed Transformation of Maize.

Several approaches have recently been adopted to improve Agrobacterium-mediated transformation of maize; however, about eight months of in vitro culture are still required to isolate transgenic plants. Furthermore, genetic transformation of maize depends on immature embryos, which greatly increases costs. Here, we report a method that ensures the competency of an embryogenic callus secondary culture under laboratory conditions for Agrobacterium-mediated transformation. Moreover, pretreatment of the cell wall with a mixed lytic enzyme solution prior to Agrobacterium infection, significantly improved transformation efficiency and stability. Average stable transformation efficiency was approximately 30.39%, with peaks of 94.46%. Expression and phenotypic analysis of the Rsc reporter gene were tested in the T0 generation of transgenic plants. Using this system, we successfully regenerated transgenic maize plantlets within three months of the emergence of the embryogenic callus. Additionally, we reduced somaclonal variation accompanying prolonged culture of maize cells in the dedifferentiated state, thus facilitating the molecular breeding of maize.

Genome alterations associated with improved transformation efficiency in Lactobacillus reuteri

BackgroundLactic acid bacteria (LAB) are one of the microorganisms of choice for the development of protein delivery systems for therapeutic purposes. Although there are numerous tools to facilitate genome engineering of lactobacilli; transformation efficiency still limits the ability to engineer their genomes. While genetically manipulating Lactobacillus reuteri ATCC PTA 6475 (LR 6475), we noticed that after an initial transformation, several LR 6475 strains significantly improved their ability to take up plasmid DNA via electroporation. Our goal was to understand the molecular basis for how these strains acquired the ability to increase transformation efficiency.ResultsStrains generated after transformation of plasmids pJP067 and pJP042 increased their ability to transform plasmid DNA about one million fold for pJP067, 100-fold for pSIP411 and tenfold for pNZ8048. Upon sequencing of the whole genome from these strains, we identified several genomic mutations and rearrangements, with all strains containing mutations in the transformation related gene A (trgA). To evaluate the role of trgA in transformation of DNA, we generated a trgA null that improved the transformation efficiency of LR 6475 to transform pSIP411 and pJP067 by at least 100-fold, demonstrating that trgA significantly impairs the ability of LR 6475 to take-up plasmid DNA. We also identified genomic rearrangements located in and around two prophages inserted in the LR 6475 genome that included deletions, insertions and an inversion of 336 Kb. A second group of rearrangements was observed in a Type I restriction modification system, in which the specificity subunits underwent several rearrangements in the target recognition domain. Despite the magnitude of these rearrangements in the prophage genomes and restriction modification systems, none of these genomic changes impacted transformation efficiency to the level induced by trgA.ConclusionsOur findings demonstrate how genetic manipulation of LR 6475 with plasmid DNA leads to genomic changes that improve their ability to transform plasmid DNA; highlighting trgA as the primary driver of this phenotype. Additionally, this study also underlines the importance of characterizing genetic changes that take place after genome engineering of strains for therapeutic purposes.

CRISPR-Mediated Genome Editing and Gene Repression in Scheffersomyces stipitis.

Scheffersomyces stipitis, renowned for its native xylose-utilizing capacity, has recently demonstrated its potential in producing health-promoting shikimate pathway derivatives. However, its broader application is hampered by the low transformation efficiency and the lack of genetic engineering tools to enable sophisticated genomic manipulations. S. stipitis employs the predominant non-homologous end joining (NHEJ) mechanism for repairing DNA double-strand breaks (DSB), which is less desired due to its incompetence in achieving precise genome editing. Using CRISPR technology, here a ku70Δku80Δ deficient strain in which homologous recombination (HR)-based genome editing appeared dominant for the first time in S. stipitis is constructed. To build all essential tools for efficiently manipulating this highly promising nonconventional microbial host, the gene knockdown tool is also established, and repression efficiency is improved by incorporating a transcriptional repressor Mxi1 into the CRISPR-dCas9 platform. All these results are obtained with the improved transformation efficiency, which is 191-fold higher than that obtained with the traditional parameters used in yeast transformation. This work paves the way for advancing a new microbial chassis and provides a guideline for developing efficient CRISPR tools in other nonconventional yeasts.

Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.

Non-capsulated mutants of a chemical-producing Klebsiella pneumoniae strain.

To investigate the outcomes of capsule lost on cell transformation efficiency and chemicals (1,3-propanediol, 2,3-butanediol, and 2-ketogluconic acid) production by Klebsiella pneumoniae. The cps gene cluster showed low sequence homology with pathogenic strains. The wza is a highly conserved gene in the cps cluster that encodes an outer membrane protein. A non-capsulated mutant was constructed by deletion of wza. Phenotype studies demonstrated that non-capsulated cells were less buoyant and easy to sediment. The transformation efficiency of the non-capsulated mutant reached 6.4×105 CFUμg-1 DNA, which is 10 times higher than that of the wild strain. 52.2g 1,3-propanediol L-1, 30.7g 2,3-butanediol L-1, and 175.9g 2-ketogluconic acid L-1 were produced by non-capsulated mutants, which were 10-40% lower compared to wild strain. Furthermore, viscosities of the three fermentation broths decreased to approximately 1.3 cP from the range of 1.8-2.2 cP. Non-capsulated K. pneumoniae mutants should allay concerns regarding biological safety, improve transformation efficiency, lower viscosity, and subsequently ameliorate the financial burden of the downstream process of chemicals production.

First production of wild hemmer (Triticum turgidum ssp. dicoccoides) transgenic plants

Plant genetic transformation and regeneration has become a valuable research tool for functional genomics. A successful transformation event involves the transfer of the target gene into a suitable explant, the integration and expression of the transgene into the host genome and the regeneration of the fertile transgenic plants from the transformed tissues. Wheat is considered as a recalcitrant species even if many efforts have been done in recent years to improve transformation efficiency. The transformation of its progenitors has never been attempted, even though the possibility to transform wild hemmer represents a valuable tool to evaluate structural and functional variability occurring in wild hemmer and explaining its higher adaptation to abiotic stresses. In this paper we report, as far as we know, for the first time, the microparticle transformation of immature embryos of the wild hemmer Triticum dicoccoides with the Tapgip1 gene. The transformation method was successfully transferred from durum wheat and several transgenic lines were obtained. Its application for the exploitation of wheat progenitors for molecular breeding is of great relevance for genomic and functional genomics studies. This result, indeed, opens new perspectives in complementation studies for the comprehension of durum and bread wheat adaptation mechanisms to stresses.

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms.

Mutation breeding of high 9α-hydroxy-androst-4-ene-3,17- dione transforming strains from phytosterols and their conversion process optimization

In order to improve transformation efficiency of phytosterols into 9α-hydroxylation of 4-androstene-3,17-dione (9α-OH-AD) by Mycobacterium sp. LY-1, we studied the strains breeding using atmospheric and room temperature plasma (ARTP) technology and optimized their conversion process. A high production strain named C33 with a good genetic stability was selected and the product molar yield reached to 15.5%, 34.8% higher than that of original strain with 15 g/L phytosterols. Furthermore, the fermentation medium was optimized through the design of orthogonal experiment. Besides, oil-water bidirectional transformation system was set up to improve the 9α-OH-AD molar yield of mutant strain C33. With adding 12 mL soybean oil to each 1 g phytosterols, the molar yield of 9α-OH-AD reached 47.0%, which increased twice than that of control (15.5%).

Production of transgenic diploid Cucumis melo plants

Melon (Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid.

Transformation of Cm-ADH gene to melon genotype

Alcohol dehydrogenases (ADH) participate in the biosynthetic pathway of aroma volatiles in fruit by interconverting aldehydes to alcohols and providing substrates for the formation of esters. Two highly divergent ADH genes of cantaloupe Charentais melon (Cucumis melo var. cantalupensis 'Vedrantais') have been isolated. This research is aimed at identifying the role of the Cm-ADH genes (Cm-ADH1 and 2) in the synthesis of aroma volatiles via the RNAi method. Different regeneration media were investigated to improve transformation efficiency. The best regeneration result was obtained from MS medium supplemented with 2.2 μM BAP and 0.54 μM NAA. Regeneration protocol has been applied to obtain transformed plants, using Agrobacterium tumefaciens which has a vector RNAi pFGC5941-(11406 bp) carrying genes of resistance to kanamycine and Bar. Transformed plants were characterized by PCR analysis.

Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower β-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

A Perspective on Hypericum perforatum Genetic Transformation.

Hypericum perforatum (St John's wort) is a reservoir of diverse classes of biologically active and high value secondary metabolites, which captured the interest of both researchers and the pharmaceutical industry alike. Several studies and clinical trials have shown that H. perforatum extracts possess an astounding array of pharmacological properties. These properties include antidepressant, anti-inflammatory, antiviral, anti-cancer, and antibacterial activities; and are largely attributed to the naphtodianthrones and xanthones found in the genus. Hence, improving their production via genetic manipulation is an important strategy. In spite of the presence of contemporary genome editing tools, genetic improvement of this genus remains challenging without robust transformation methods in place. In the recent past, we found that H. perforatum remains recalcitrant to Agrobacterium tumefaciens mediated transformation partly due to the induction of plant defense responses coming into play. However, H. perforatum transformation is possible via a non-biological method, biolistic bombardment. Some research groups have observed the induction of hairy roots in H. perforatum after Agrobacterium rhizogenes co-cultivation. In this review, we aim at updating the available methods for regeneration and transformation of H. perforatum. In addition, we also propose a brief perspective on certain novel strategies to improve transformation efficiency in order to meet the demands of the pharmaceutical industry via metabolic engineering.

Regeneration selection improves transformation efficiency in narrow-leaf lupin

Weed management is a critical issue in the production of narrow-leaf lupin (NLL) (Lupinus angustifolius L.). The gene pool has limited herbicide tolerance, early vigour or short season productivity, hampering conventional breeding approaches. Previous transformation of NLL used the bar gene encoding tolerance to the herbicide glufosinate, for selection. However that methodology achieved <1 % success at T1, with one confounding factor being chimerism of the T0 shoots. Therefore glyphosate tolerance was examined as a potential alternative selectable marker with possible commercial application. Agrobacterium-mediated gene transfer using four vectors with two promoter and two transit peptide (tp) sequences in factorial combination was followed by three methods of selection post-transformation, following failure to achieve transformation by the published glufosinate selection method. An overall heritable transformation efficiency of 0.14 % was achieved from a total of 36,607 inoculated explants. Transformation with constructs containing the ubiquitin 3 promoter had a higher success rate (0.20 %) than with the CaMV promoter (0.07 %) however no difference between the two tp sequences was observed. The highest rate of transformation (0.45 %) was obtained by glyphosate selection during early phase of growth on regeneration media. This regeneration selection method more frequently produced highly tolerant events, with more heritable expression stability in T2 generation than alternative methods. Single insertion, single copy events were obtained. However these were recovered with significantly lower frequency than previously observed with the glufosinate selection method. Nonetheless such events have potential to contribute weed management options for broad-acre NLL production.

Development of a plasmid-based expression system in Clostridium thermocellum and its use to screen heterologous expression of bifunctional alcohol dehydrogenases (adhEs)

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis.

Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8×10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available.