Agrobacterium Tumefaciens-mediated Transformation Research Articles

Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae).

This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis. Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1mg/L 1-Naphthaleneacetic acid (NAA) and 2mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1mg/L NAA and 0.1mg/L 6-BA and that high transformation efficiency required 100µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK applicationand endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Thyrostroma carpophilum insertional mutagenesis: A step towards understanding its pathogenicity mechanism

Thyrostroma carpophilum, a causal agent of shot hole disease of stone fruits, cause severe loss in economically important fruit crops of Kashmir. Understanding its pathogenesis at molecular level will aid in devising a better management strategy. In this study, we optimized Agrobacterium tumefaciens mediated transformation (ATMT) conditions for T. carpophilum using PBIF2-EGFP construct. Using this protocol, we obtained 328 positive transformants per 104 spores and subsequent sub-culturing of transformants on selective and non-selective media resulted in stable T-DNA integration. Southern blot analysis revealed that most of the transformants embodied single T-DNA integration. Using this method, we obtained a small-scale transformant library (2050 transformants). Among this pool, we tested 1005 transformants for their pathogenicity; out of which 185 showed complete pathogenicity loss, 35 displayed reduced virulence and 785 were pathogenically similar to wild type. Out of this experimental stock, three transformants from each category were randomly selected to dissect the infection assay. The findings deciphered that transformants with complete pathogenicity loss failed to penetrate the host tissue and a few transformants failed to sporulate in laboratory. Transformants from reduced category could not form appressorium and occasionally sporulated. Transformants similar to wild type were morphologically and pathogenically similar to wild type because of un-alteration in their modus operandi. Our work provides a new platform to understand the pathogenicity mechanism of T. carpophilum. The optimized ATMT protocol will help in developing large transformant library that can help to identify the virulence arsenals necessary for the pathogen to cause disease.

Induction of competent cells for Agrobacterium tumefaciens-mediated stable transformation of common bean (Phaseolus vulgaris L.).

Stable transformation of common bean (Phaseolus vulgaris L.) has been successful, to date, only using biolistic-mediated transformation and shoot regeneration from meristem-containing embryo axes. In this study, using precultured embryo axes, and optimal co-cultivation conditions resulted in a successful transformation of the common bean cultivar Olathe using Agrobacterium tumefaciens strain EHA105. Plant regeneration through somatic embryogenesis was attained through the preculture of embryo axes for 12 weeks using induced competent cells for A. tumefaciens-mediated gene delivery. Using A. tumefaciens at a low optical density (OD) of 0.1 at a wavelength of 600 nm for infection and 4-day co-cultivation, compared to OD600 of 0.5, increased the survival rate of the inoculated explants from 23% to 45%. Selection using 0.5 mg L-1 glufosinate (GS) was effective to identify transformed cells when the bialaphos resistance (bar) gene under the constitutive 35S promoter was used as a selectable marker. After an 18-week selection period, 1.5% -2.5% inoculated explants, in three experiments with a total of 600 explants, produced GS-resistant plants through somatic embryogenesis. The expression of bar was confirmed in first- and second-generation seedlings of the two lines through reverse polymerase chain reaction. Presence of the bar gene was verified through genome sequencing of two selected transgenic lines. The induction of regenerable, competent cells is key for the successful transformation, and the protocols described may be useful for future transformation of additional Phaseolus germplasm.

Transformation using RNAi technology for developing potato lines resistance to late blight (Phytophthora infestans)

Phytophthora infestans (Mont.) de Bary causes late blight, a major disease of potato and tomato that causes production loss at about 10–100% in Indonesia. Breeding for late blight resistance in potato has been carried out in this country to minimize fungicide application. The objective of this study was to generate potato lines resistant to P. infestans through RNAi method. A series of studies were conducted, including confirmation of RNAi construct insertion in Agrobacterium tumefaciens and PCR analysis, transformation of two potato cultivars using A. tumefaciens carrying the RNAi fragment, in addition to selection and molecular analysis of selected plantlet using Polymerase Chain Reaction (PCR). The RNAi plasmid construct was confirmed via PCR analysis using specific primers for 35s and Tnos fragment, and resulted in 500 bp and 250 bp for fragment of 35s and Tnos, respectively. Transformation was performed on 733 Granola’s internode explants and 569 Atlantic’s internode explants. The transformation process produced 282 explants from Atlantic, while Granola did not produce any transformants. The level of transformation efficiency of Atlantic on selection medium containing hygromycin was 49.61%. Following regeneration step, the 282 selected explants produced 167 plantlets. Based on PCR reaction using specific primers for hpt gene, 14 plantlets were PCR positive and contained hpt fragment. Overall, Agrobacterium tumefaciens-mediated transformation on potato internode explants was successful. Therefore, the selected transformants should be further tested using bioassay for resistance to P. infestans.

Agrobacterium tumefaciens-Mediated Transformation of Three Milkweed Species (Asclepias hallii, A. syriaca, and A. tuberosa: Apocynaceae).

Milkweeds have ecological significance for insect herbivores that rely on them as hosts for either part of or the entirety of their life cycles. Interesting interactions, some of which are not completely understood, have evolved over time. To develop these species as models to elucidate the interplay with insect herbivores, we established Agrobacterium tumefaciens-mediated transformation approaches for Asclepias hallii (Hall's milkweed), A. syriaca (common milkweed), and A. tuberosa (butterflyweed). The method is based on infection of stem internodal explants, which were more amenable to transformation than leaf explants. We found that addition of freshly prepared dithiothreitol was critical to prevent browning of stem explants. Depending on the species, the time from infection to the regeneration of transgenic lines ranges from 2 to 4 months. Transformation efficiency for A. hallii was 9%, whereas efficiencies for A. syriaca and A. tuberosa were 6% and 13%, respectively. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Agrobacterium tumefaciens-mediated transformation of Asclepias internodal stem explants Basic Protocol 2: Preparation of Agrobacterium glycerol stocks containing gene constructs.

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond.

Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate-based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host-specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi-a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal-specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium-mediated transformation. We also describe the use of a set of web-based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Software-assisted modular DNA assembly of a two gene expression-cassette with GB Basic Protocol 2: Agrobacterium tumefaciens-mediated transformation of filamentous fungi Basic Protocol 3: Software-assisted modular DNA assembly of a gene disruption-cassette using GB Basic Protocol 4: Obtaining disruption transformants.

Screening of Secreted Proteins of Sporisorium reilianum f. sp. zeae for Cell Death Suppression in Nicotiana benthamiana.

Sporisorium reilianum f. sp. zeae (SRZ) is a biotrophic fungus causing head smut in maize. Maize infection with SRZ leads to very little cell death suggesting the presence of cell-death suppressinpg effectors. Several hundred effector proteins have been predicted based on genome annotation, genome comparison, and bioinformatic analysis. For only very few of these effectors, an involvement in virulence has been shown. In this work, we started to test a considerable subset of these predicted effector proteins for a possible function in suppressing cell death. We generated an expression library of 62 proteins of SRZ under the control of a strong constitutive plant promoter for delivery into plant cells via Agrobacterium tumefaciens-mediated transient transformation. Potential apoplastic effectors with high cysteine content were cloned with signal peptide while potential intracellular effectors were also cloned without signal peptide to ensure proper localization after expression in plant cells. After infiltration of Nicotiana benthamiana leaves, infiltration sites were evaluated for apparent signs of hypersensitive cell death in absence or presence of the elicitin INF1 of Phytophthora infestans. None of the tested candidates was able to induce cell death, and most were unable to suppress INF1-induced cell death. However, the screen revealed one predicted cytoplasmic effector (sr16441) of SRZ that was able to reliably suppress INF1-induced cell death when transiently expressed in N. benthamiana lacking its predicted secretion signal peptide. This way, we discovered a putative function for one new effector of SRZ.

Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

Breeding of the transgenic chrysanthemum (Chrysanthemum morifolium Ramat.) carrying aphid-resistance gene, Pinellia ternata agglutinin (PTA)

Plasmid pCAMBIA2201-pta, containing the selectable marker neomycin phosphotransferase gene (npt II), Pinellia ternata agglutinin (PTA) gene and β-glucuronidase gene, were transformed into leaf discs of Chrysanthemum (Chrysanthemum morifolium Ramat.) via Agrobacterium tumefaciens-mediated transformation. It was confirmed by GUS (β-glucuronidase) and PCR analysis that the PTA gene had been successfully inserted into the plant genome. Hemagglutination assay was carried out and the expression of PTA gene in transgenic plants in vitro and the aphid-resistance assay result showed that the inhibition rates of the transformed plants were improved than the control. T1 progenies generated through the cuttage breeding from T0 generation had preserved the aphid-resistance and the inhibition rates of T1 generation had no significant difference with T0 generation (P > 0.05). The results suggest that PTA gene can improve aphid resistance of chrysanthemum and its aphid resistance can be generated to T1 generation stably.

Identification of the genes involved in growth characters of medicinal fungus Ophiocordyceps sinensis based on Agrobacterium tumefaciens-mediated transformation.

Ophiocordyceps sinensis, one of the well-known and precious fungal species in the world, parasitizes soil-dwelling larvae of ghost moths on the Tibetan Plateau. The genetic intractability of this extremely psychrophilic and slow-growing O. sinensis fungus is a major limitation on the molecular study. In this study, an Agrobacterium tumefaciens-mediated genetic transformation (ATMT) system for this fungus was established. ATMT procedure was optimized based on the fungal recipient, Agrobacterium strains, and different co-cultivation conditions. Blastospores were ideal recipients for this system. Acetosyringone (AS) was not essential for the transformation of O. sinensis. Sixty to 100 hygromycin B-resistant transformants per 1 × 106 blastospores were obtained. Southern blot analysis indicated the presence of a random single-copy integration of T-DNA into the O. sinensis genome. The insertional transformants with altered growth characters such as colony, blastospore, and fruiting body production were selected to identify the T-DNA flanking sequences by modified hiTAIL-PCR and FPNI-PCR techniques. Eight genes, including genes for an MFS transporter, a 2-oxoglutarate dehydrogenase, a DNA-directed RNA polymerase III complex subunit Rpc37, a cytochrome oxidase subunit I, a mitochondrial import inner membrane translocase subunit tim54, a cytidine deaminase, a phosphoribosylaminoimidazole carboxylase, and a histone H3-like centromeric protein cse-4 were identified. This ATMT system provides a useful tool for gene discovery and characterization of O. sinensis and contributes to the better understanding of the mysterious life cycle of O. sinensis and the molecular interaction between this fungus and its host insects.

Enhancement of cardenolide production in transgenic Digitalis purpurea L. by expressing a progesterone-5β-reductase from Arabidopsis thaliana L.

Metabolic engineering of Digitalis purpurea L. has emerged as a powerful biotechnological tool to enhance in vitro cardenolide biosynthesis. Thereto, Agrobacterium tumefaciens-mediated transformation protocols have been developed using nptII as selectable marker gene and uidA as reporter gene. In this work, the Arabidopsis thaliana L. VEP1 gene, encoding progesterone-5β-reductase, was expressed in cardenolide-producing D. purpurea plants. Resistance to hygromycin B was efficiently used as a selectable marker in callus induction and multiplication media. Successful transformation was confirmed in nine transgenic lines by PCR analyses using primers for the selectable marker gene (hpt) and the VEP1 gene. The number of copies of the transgene construct was estimated between one and three in different lines by quantitative PCR and inverse PCR. VEP1 expression was confirmed in eight transgenic lines by reverse transcription-quantitative PCR. Digitoxin and digoxin content were increased up to 3.8-fold (757 μg/gDW) and 2.2-fold (199 μg/gDW) respectively in transgenic plants cultivated in vitro. However, for plants grown in the greenhouse, digitoxin content was not significantly different from non-transgenic plants and digoxin production was enhanced up to 1.8-fold (87 μg/gDW). Genetic transformation thus allowed to enhance cardenolide production in vitro with higher efficiency than with other biotechnological strategies reported so far.

A Na+/H+ antiporter, K2-NhaD, improves salt and drought tolerance in cotton (Gossypium hirsutum L.).

Overexpression of K2-NhaD in transgenic cotton resulted in phenotypes with strong salinity and drought tolerance in greenhouse and field experiments, increased expression of stress-related genes, and improved regulation of metabolic pathways, such as the SOS pathway. Drought and salinity are major abiotic stressors which negatively impact cotton yield under field conditions. Here, a plasma membrane Na+/H+ antiporter gene, K2-NhaD, was introduced into upland cotton R15 using an Agrobacterium tumefaciens-mediated transformation system. Homozygous transgenic lines K9, K17, and K22 were identified by PCR and glyphosate-resistance. TAIL-PCR confirmed that T-DNA carrying the K2-NhaD gene in transgenic lines K9, K17and K22 was inserted into chromosome 3, 19and 12 of the cotton genome, respectively. Overexpression of K2-NhaD in transgenic cotton plants grown in greenhouse conditions and subjected to drought and salinity stress resulted in significantly higher relative water content, chlorophyll, soluble sugar, proline levels, and SOD, CAT, and POD activity, relative to non-transgenic plants. The expression of stress-related genes was significantly upregulated, and this resulted in improved regulation of metabolic pathways, such as the salt overly sensitive pathway. K2-NhaD transgenic plants growing under field conditions displayed strong salinity and drought tolerance, especially at high levels of soil salinity and drought. Seed cotton yields in transgenic line were significantly higher than in wild-type plants. In conclusion, the data indicate that K2-NhaD transgenic lines have great potential for the production of stress-tolerant cotton under field conditions.

Overexpression of the OvBAN gene enhances the proanthocyanidin content in transgenic alfalfa (Medicago sativa L.)

Sainfoin (Onobrychis viciaefolia Scop) is a forage crop in the Leguminosae family. The leaves of this plant and other tissues have a high level of proanthocyanidins (PAs). The BAN gene encodes anthocyanidin reductase (ANR), which is a key enzyme in PA biosynthesis. This report describes cloning of a BAN homolog gene (viz., OvBAN) from local sainfoin and overexpression of this gene in alfalfa (Medicago sativa L.), a globally important forage crop. The open reading frame (ORF) of OvBAN contains 1020 bp, which is deduced to encode 339 amino acids. Alignment with amino acid sequences showed that OvBAN had 99%, 87%, and 85% identity to homologs from another Onobrychis viciaefolia variety, known as Lotus corniculatus, and Medicago sativa, respectively. The ORF of OvBAN was cloned into a binary vector under the control of the CaMV35S promoter, in which the nptII gene was used as a selectable marker. The resulting binary vector was introduced to alfalfa using Agrobacterium tumefaciens-mediated transformation. Kanamycin-resistant plants were obtained and grown in potting soil. PCR and Southern blot analyses demonstrated the integration of the OvBAN transgene in transgenic plants. Quantitative real-time PCR analysis revealed that OvBAN was highly expressed in transgenic plants. Crude enzyme was extracted from transgenic and wild-type plants. Enzyme tests showed higher ANR activity in the transgenic samples than in the wild-type. Furthermore, butanol-HCl-based cleavage analysis showed that the PA content was higher in transgenic plants than in wild-type plants. These results demonstrate that OvBAN is an appropriate gene for the enhancement of PAs in alfalfa.

An optimized Agrobacterium tumefaciens-mediated transformation system for random insertional mutagenesis in Fonsecaea monophora

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Agrobacterium-mediated Transformation of Sweet Basil (Ocimum basilicum).

Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we present a detailed protocol for a highly efficient Agrobacterium tumefaciens-mediated transformation method for sweet basil, which was established based on a previously reported method by other researchers, with modifications on several aspects, including growth of sweet basil, age of plants used for explants, preparation and concentration of Agrobacteria. This protocol allows researchers in academia and agroindustry to generate transgenic sweet basil plants in an easy, quick and highly reproducible manner. In addition, this protocol may be applicable to transform other species within the genus Ocimum.

An improved Agrobacterium-mediated transformation method for cotton (Gossypium hirsutum L. ‘KC3’) assisted by microinjection and sonication

An efficient and reproducible Agrobacterium tumefaciens-mediated transformation method was developed for Gossypium hirsutum L. ‘KC3’ using a shoot apex explant with the combination of microinjection and sonication. To optimize the transformation efficiency, preculture conditions, acetosyringone concentration, A. tumefaciens cell density, and precision microinjection with sonication were evaluated. A high-throughput A. tumefaciens-mediated genetic transformation method was developed using the uidA gene. The shoot apex explants were microinjected with an A. tumefaciens strain EHA105 carrying the uidA gene and co-cultivated for 3 d on mMS medium fortified with 100 mM acetosyringone. Among the different parameters and conditions tested, preculturing explants for 24 h, microinjecting three times with an A. tumefaciens cell suspension of OD600 nm = 0.6 containing 100 mM acetosyringone, and sonicating explants for 20 s produced the maximum transformation efficiency of 20.25%. Putative transformants were transferred to regeneration medium supplemented with 0.8 mg L−1 1-naphthaleneacetic acid, 2 mg L−1 thidiazuron, 16% (v/v) of an Oscillatoria acuminata extracellular extract, and 50 mg L−1 kanamycin. Initially, the transgenic shoots were evaluated by GUS histochemical analysis, and integration was confirmed by polymerase chain reaction and Southern blot analysis. Plantlets transferred from an artificial growth chamber to greenhouse conditions showed a higher survival rate of 90% compared to the directly transferred plantlets. This simple and efficient transformation system could be valuable to transfer any gene of interest into cotton with high transformation efficiency in a short time.

A Procedure for Agrobacterium tumefaciens Mediated Genetic Transformation of Sweet Potato

Many sweet potato plants have been successfully transformed but the transgenic plants regenerated, however, have been limited to a few genotypes. Reported in this paper is a procedure in which several explants of three sweet potato genotypes from Papua New Guinea (PNG) were used to transform and regenerate transgenic plants. To achieve stable transformation, an efficient shoot regeneration system for different explants was developed. The shoot regeneration protocol developed enabled for a reproducible stable transformation mediated by Agrobacterium tumefaciens strain 1065. The plasmid pVDH65 contains the npt II gene for kanamycin (km) resistance, hpt gene for hygromycin resistance and Gus-intron reporter gene (GUS) for β-glucuronidase. Explants inoculated with the bacterial strain were co-cultured for 3, 5 and 7 days (d) in the dark on Murashige and Skoog (MS) medium without growth hormones. After co-cultivation, the explants were washed in liquid MS medium containing 500 mg L-1 cefotaxime, rinsed in sterile, deionised water for 10 mins and cultured on km selection medium containing 100 mg L-1 km followed by transfer of explants to 125 mg L-1 km after 14 d of culture. The km-resistant shoots selected on the former km concentration were transferred to the latter for double selection. Km-resistant shoots obtained at 125 mg L-1 were rooted on MS based medium also containing 0.008 mg L-1 IAA, 0.03 mg L-1 kinetin and 0.001 mg L-1 folic acid. This double selection method led to effective elimination of escapes (up to 75%) and successful recovery of transgenic plants from stem explants at more than 25%, leaf discs 10% and petioles 13.3% of each sweet potato cultivar. Polymerase chain reaction (PCR) analysis of the three km-resistant and GUS-positive plants revealed the presence of the expected fragment for npt II. This is the first report of successfully transforming sweet potato plants with bacterial strain 1065 and selection of transgenic plants at km concentrations higher than 100 mg L-1.

A novel electroporation procedure for highly efficient transformation of Lipomyces starkeyi

Microbial lipids produced by oleaginous microorganisms as raw materials for the production of oleochemicals and biodiesel are sustainable while avoiding competition with food products. The oleaginous yeast Lipomyces starkeyi is an excellent lipid producer with a great industrial potential that is suitable as a valuable host to improve lipid production through genetic engineering modifications. However, genetic tools, including effective transformation methods, for L. starkeyi are insufficient for improvement of lipid production and analysis of lipid production mechanisms. We previously developed a polyethylene glycol (PEG)-mediated spheroplast transformation method that significantly improved the homologous recombination efficiency of L. starkeyi strain ∆lslig4. Although other transformation methods, including lithium acetate (LiAc)-mediated transformation and Agrobacterium tumefaciens-mediated transformation, have been reported, a more efficient and convenient transformation method for L. starkeyi is desired. In this study, we developed a novel electroporation transformation method that was first applied for integration of drug-resistance gene markers into the genome of L. starkeyi strain ∆lslig4 at the 18S ribosomal DNA locus of a multiple-copy gene, which yielded approximately 60 transformants/μg of DNA. Optimization of five parameters (i.e., cell growth phase, cell density, osmotic stabilizers, pretreatment agents, and electric conditions) enhanced the efficiency of transformation to approximately 1.5 × 104 transformants/μg of DNA. As compared with those of LiAc-mediated transformation and PEG-mediated spheroplast transformation, the efficiency of the proposed transformation method was increased by about 111- and 7-fold, respectively. Additionally, the transformation efficiency of our proposed electroporation method targeting a single-copy gene locus yielded 273 transformants/μg of DNA. To our knowledge, this is the first report of a successful electroporation method to accelerate analysis of lipid production by L. starkeyi.

Establishment of an Agrobacterium tumefaciens-mediated transformation system for Tilletia foetida

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 μg/ml, cefotaxime sodium concentrations with 200 μg/ml, Acetosyringone (AS) concentration at 200 μmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.