Showing papers on "Transformation (genetics) published in 2019"

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice

Abstract: Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9) (CRISPR–Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9–guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR–Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9–gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol–calcium (PEG–Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9–gRNA RNPs into plant zygotes. Cas9–gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14–64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species. Efficient genome editing using DNA-free systems remains challenging in most plant species. Now, a new genome-editing system achieves efficient DNA- and selectable-marker-free gene editing by direct delivery of Cas9–guide RNA ribonucleoproteins into rice zygotes.

An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat ( Triticum aestivum L.)

Abstract: Despite wheat being a worldwide staple, it is still considered the most difficult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and effective wheat transformation system is still greatly needed. We have developed and optimised a reproducible Agrobacterium-mediated transformation system for the spring wheat cv ‘Fielder’ that yields transformation efficiencies of up to 25%. We report on some of the important factors that influence transformation efficiencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for independent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efficiencies might be even higher than those reported. We reported here a high-throughput, highly efficient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR–Cas9 based genome editing.

Genome-Scale Sequence Disruption Following Biolistic Transformation in Rice and Maize

Abstract: Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell's genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

Agrobacterium tumefaciens-Mediated Transformation of Tomato.

Abstract: Tomato is both an important food crop and serves as a model plant species that is used for various research investigations including understanding gene function. Transformation is commonly utilized to facilitate these investigations in combination with all the extensive genetic and genomic resources available for tomato. The transformation protocol routinely used in our laboratory has been applied to many different tomato genotypes and relies on Agrobacterium tumefaciens infection of young cotyledon sections. We have used vector systems for overexpression, RNA interference for gene silencing, and CRISPR/Cas9 for genome editing. Vectors used to design gene constructs contained selectable marker genes that conferred resistance to kanamycin, hygromycin, and the herbicide component, bialaphos. The protocol we follow for Agrobacterium-mediated transformation of both cultivated and wild species of tomato is detailed in this chapter.

Development of an efficient root transgenic system for pigeon pea and its application to other important economically plants.

Abstract: For non-model plants, functional characterization of genes is still hampered by lack of efficient stable transformation procedures. Here, we report a simple, fast and efficient transformation technique with Agrobacterium rhizogenes for generating stable transgenic roots in living plants to facilitate functional studies in vivo. We showed that injection of A. rhizogenes into stems of various plant species lead to stable transgenic root generation, which can sustain plant growth after the original, non-transgenic roots were cut off. A transformation system was established for pigeon pea, a major woody food crop, after optimizing the selection of A. rhizogenes strains, bacterium concentration, injection position and seedling age. RT-PCR and fluorescence observation indicated a transgenic root induction efficiency of about 39% in pigeon pea. Furthermore, induction of hairy roots was achieved in nine out of twelve tested economically important plants at an efficiency of 15-39%. As proof of concept, bimolecular fluorescence complementation (BiFC) assay was applied to test the interaction between CcCIPK14 and CcCBL1/2 in pigeon pea. Additionally, ectopic expression of the bZIP transcription factor MdHY5 from apple confirmed the utility of the transformation technique for engineering anthocyanin synthesis in roots. Taken together, we show that this method allows fast in vivo studies of gene function in a wide range of plant species.

Widespread occurrence of natural genetic transformation of plants by Agrobacterium

Abstract: Naturally transgenic plant species occur on an unexpectedly large scale. Agrobacterium-mediated gene transfer leads to the formation of crown galls or hairy roots, due to expression of transferred T-DNA genes. Spontaneous regeneration of transformed cells can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of bacterial origin. This particular type of horizontal gene transfer (HGT) could play a role in plant evolution. However, the material available today is not enough for generalizations concerning the role of Agrobacterium in HGT from bacteria to plants. In this study, we searched for T-DNA-like genes in the sequenced genomes of dicots and monocots. We demonstrate the presence of cT-DNAs in 23 out of 275 dicot species, within genera Eutrema, Arachis, Nissolia, Quillaja, Euphorbia, Parasponia, Trema, Humulus, Psidium, Eugenia, Juglans, Azadirachta, Silene, Dianthus, Vaccinium, Camellia, and Cuscuta. Analysis of transcriptome data of 356 dicot species yielded 16 additional naturally transgenic species. Thus, HGT from Agrobacterium to dicots is remarkably widespread. Opine synthesis genes are most frequent, followed by plast genes. Species in the genera Parasponia, Trema, Camellia, Azadirachta, Quillaja, and Diospyros contain a combination of plast and opine genes. Some are intact and expressed, but the majority have internal stop codons. Among the sequenced monocot species, Dioscorea alata (greater yam) and Musa acuminata (banana) also contain T-DNA-like sequences. The identified examples are valuable material for future research on the role of Agrobacterium-derived genes in plant evolution, for investigations on Agrobacterium strain diversity, and for studies on the function and evolution of cT-DNA genes in natural transformants.

Creation of Early Flowering Germplasm of Soybean by CRISPR/Cas9 Technology.

Abstract: Soybean is an important economic crop and a typical short-day crop, sensitive to photoperiod, and has narrow geographical adaptative region, which limit the creation of transgenic materials and reduce the breeding efficiency of new varieties. In addition, the genetic transformation efficiency of soybean is lower than that of many other crops, and the available receptor genotypes are limited. In this study, Agrobacterium-mediated transformation were used to introduce the CRISPR/Cas9 expression vector into soybean cultivar Jack and generated targeted mutants of E1 gene controlling soybean flowering. We obtained two novel types of mutations, 11 bp and 40 bp deletion at E1 coding region, respectively, and frameshift mutations produced premature translation termination codons and truncated E1 proteins, causing obvious early flowering under long day condition. In addition, no off-target effects were observed by predicting and analyzing the potential off-target sites of E1 targets. Significant decreased E1 gene expression of two novel mutants showed that the truncated E1 protein disinhibited GmFT2a/5a and increasing GmFT2a/5a gene expressions resulted obvious early flowering. Homozygous trans-clean mutants without T-DNA elements were also obtained and showed early flowering under long day condition. The photo-insensitive soybean transformation receptor we created laid a foundation for breeding excellent transgenic receptors suitable for high latitudes.

Identification of a Novel Plasmid Carrying mcr-4.3 in an Acinetobacter baumannii Strain in China.

Abstract: Here, we identified mcr-4.3 in Acinetobacter baumannii, which had not been previously observed to carry an mcr gene. The mcr-4.3-harboring A. baumannii strain AB18PR065 was isolated from pig feces from a slaughterhouse in Guangdong Province of China. The mcr-4.3-carrying pAB18PR065 is 25,602 bp in size and could not be transferred in conjugation, transformation, and electroporation experiments, as we did not find any conjugation-related genes therein. pAB18PR065 harbors two copies of type II toxin-antitoxin systems, which are functional in plasmid stabilization and maintenance. pAB18PR065 shares similarity only with one recently identified plasmid, pAb-MCR4.3 (35,502 bp), from a clinical A. baumannii strain. It is likely that the emergence of pAb-MCR4.3 was due to the insertion of an 11,386-bp, ISAba19-based, composite transposon into pAB18PR065. These data indicate that mcr-4.3 was captured by an A. baumannii-original plasmid via horizontal gene transfer.

Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea

Abstract: Chickpea transformation is an important component for the genetic improvement of this crop, achieved through modern biotechnological approaches. However, recalcitrant tissue cultures and occasional chimerism, encountered during transformation, hinder the efficient generation of transgenic chickpeas. Two key parameters, namely micro-injury and light emitting diode (LED)-based lighting were used to increase transformation efficiency. Early PCR confirmation of positive in vitro transgenic shoots, together with efficient grafting and an extended acclimatization procedure contributed to the rapid generation of transgenic plants. High intensity LED light facilitate chickpea plants to complete their life cycle within 9 weeks thus enabling up to two generations of stable transgenic chickpea lines within 8 months. The method was validated with several genes from different sources, either as single or multi-gene cassettes. Stable transgenic chickpea lines containing GUS (uidA), stress tolerance (AtBAG4 and TlBAG), as well as Fe-biofortification (OsNAS2 and CaNAS2) genes have successfully been produced.

Comparison of regeneration capacity and Agrobacterium-mediated cell transformation efficiency of different cultivars and rootstocks of Vitis spp. via organogenesis.

Abstract: The success of in vitro plant regeneration and the competence of genetic transformation greatly depends on the genotype of the species of interest. In previous work, we developed a method for the efficient Agrobacterium-mediated genetic transformation via organogenesis of V. vinifera cultivar Thompson Seedless, by using meristematic bulk (MB) as starting tissue. In this study, we applied this method for the regeneration and transformation of MBs obtained from the Italian cultivar Ciliegiolo and two of the commonly used Vitis rootstocks, 110 Richter and Kober 5BB, in comparison with Thompson Seedless. The A. tumefaciens strain EHA105, harbouring pK7WG2 binary vector, was used for the transformation trials, which allowed selection through the enhanced-green fluorescent protein (eGFP) and the neomycin phosphotransferase (nptII) gene. Putative transformed tissues and/or shoots were identified by either a screening based on the eGFP expression alone or its use in combination with kanamycin in the medium. MBs obtained from Thompson Seedless showed the highest regeneration and transformation cell competence, which subsequently allowed the recovery of stably transformed plants. Ciliegiolo, 110 Richter, and Kober 5BB, produced actively growing transgenic calli showing eGFP fluorescence, more consistently on selective media, but had no regenerative competence.

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

Abstract: The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide-insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low-level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co-transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high-throughput genetic transformation of plants and algae.

Diversity in Natural Transformation Frequencies and Regulation across Vibrio Species.

Abstract: In Vibrio species, chitin-induced natural transformation enables bacteria to take up DNA from the external environment and integrate it into their genome. Expression of the master competence regulator TfoX bypasses the need for chitin induction and drives expression of the genes required for competence in several Vibrio species. Here, we show that TfoX expression in Vibrio campbellii strains DS40M4 and NBRC 15631 enables high natural transformation frequencies. Conversely, transformation was not achieved in the model quorum-sensing strain V. campbellii BB120 (previously classified as Vibrio harveyi). Surprisingly, we find that quorum sensing is not required for transformation in V. campbellii DS40M4 or Vibrio parahaemolyticus in contrast to the established regulatory pathway in Vibrio cholerae in which quorum sensing is required to activate the competence regulator QstR. Similar to V. cholerae, expression of both QstR and TfoX is necessary for transformation in DS40M4. There is a wide disparity in transformation frequencies among even closely related Vibrio strains, with V. vulnificus having the lowest functional transformation frequency. Ectopic expression of both TfoX and QstR is sufficient to produce a significant increase in transformation frequency in Vibrio vulnificus To explore differences in competence regulation, we used previously studied V. cholerae competence genes to inform a comparative genomics analysis coupled with transcriptomics. We find that transformation capability cannot necessarily be predicted by the level of gene conservation but rather correlates with competence gene expression following TfoX induction. Thus, we have uncovered notable species- and strain-level variations in the competence gene regulation pathway across the Vibrio genus.IMPORTANCE Naturally transformable, or competent, bacteria are able to take up DNA from their environment, a key method of horizontal gene transfer for acquisition of new DNA sequences. Our research shows that Vibrio species that inhabit marine environments exhibit a wide diversity in natural transformation capability ranging from nontransformability to high transformation rates in which 10% of cells measurably incorporate new DNA. We show that the role of regulatory systems controlling the expression of competence genes (e.g., quorum sensing) differs throughout both the species and strain levels. We explore natural transformation capabilities of Vibrio campbellii species which have been thus far uncharacterized and find novel regulation of competence. Expression of two key transcription factors, TfoX and QstR, is necessary to stimulate high levels of transformation in Vibrio campbellii and recover low rates of transformation in Vibrio vulnificus.

Observability, redundancy and modality for dynamical symmetry transformations

Abstract: I provide a fairly systematic analysis of when quantities that are variant under a dynamical symmetry transformation should be regarded as unobservable, or redundant, or unreal; of when models related by a dynamical symmetry transformation represent the same state of affairs; and of when mathematical structure that is variant under a dynamical symmetry transformation should be regarded as surplus. In most of these cases the answer is `it depends': depends, that is, on the details of the symmetry in question. A central feature of the analysis is that in order to draw any of these conclusions for a dynamical symmetry it needs to be understood in terms of its possible extensions to other physical systems, in particular to measurement devices.

New Insights on the Role of the pLMST6 Plasmid in Listeria monocytogenes Biocide Tolerance and Virulence.

Abstract: Listeria monocytogenes the causative agent of listeriosis is an important public health concern and food safety challenge. Increased tolerance of this bacterium to benzalkonium chloride (BC), an antibacterial agent widely used in industrial settings, is a growing issue. Plasmid pLMST6 harboring the gene of the multidrug efflux pump protein EmrC has been recently linked to enhanced BC tolerance and meningitis due to L. monocytogenes ST6 strains. In this study, occurrence and contribution of this plasmid to BC tolerance was examined using PCR, plasmid curing and transformation, RT-qPCR and proteome analysis, respectively. Furthermore, the substrate specificity of the pLMST6 associated EmrC efflux pump and the impact of the plasmid on L. monocytogenes virulence were investigated. pLMST6 was detected in 7 (1.6%) of 439 L. monocytogenes strains isolated from different sources. A phenotypic role of this plasmid in conferring increased BC tolerance was confirmed by showing that plasmid cure increases BC susceptibility whereas plasmid complementation and transformation increased BC tolerance in different L. monocytogenes genetic backgrounds and L. innocua. RT-qPCR showed that BC stress exposure strongly induces the expression of mRNAs associated with pLMST6 genes for EmrC and a TetR transcription regulator. A full proteome analysis in a plasmid harboring L. monocytogenes strain revealed that the pLMST6 encoded putative TetR family transcription regulator protein is the most upregulated protein in response to BC stress exposure. An investigation into the EmrC efflux pump's substrate spectrum showed that while pLMST6 confers increased tolerance to other quaternary ammonium compounds (QACs) based disinfectants it has no impact on the sensitivity of L. monocytogenes to non-QAC disinfectants as well as on antibiotics such as ampicillin, tetracycline and gentamicin. A reduction in the survival of zebrafish embryos infected with pLMST6 plasmid harboring L. monocytogenes strains was observed when compared with plasmid cured variants of the same strains suggesting that some pLMST6 harbored genes might contribute to increased virulence capacity. Overall these results confirm the phenotypic contribution of pLMST6 plasmid in promoting and dissemination of BC tolerance in L. monocytogenes as well as provide new insights on different molecular levels of pLMST6 associated genes in response to BC stress.

Optimization of Agrobacterium-mediated transformation in spring bread wheat using mature and immature embryos.

Abstract: Wheat is the most widely grown staple food crop in the world and accounts for dietary needs of more than 35% of the human population. Current status of transgenic wheat development is slow all over the world due to the lack of a suitable transformation system. In the present study, an efficient and reproducible Agrobacterium-mediated transformation system in bread wheat (Triticum aestivum L.) is established. The mature and immature embryos of six recently released high yielding spring bread wheat genotypes were used to standardize various parameters using Agrobacterium tumefaciens strain EHA105 harbouring binary vector pCAMBIA3301 having gus and bar as marker genes. The optimum duration for embryo pre-culture, inoculation time and co-cultivation were 2 days, 30 min and 48 h, respectively. The bacterial inoculum concentration of OD of 1 at 600 nm showed 67.25% transient GUS expression in the histochemical GUS assay. The filter paper based co-cultivation limits the Agrobacterium overgrowth and had 82.3% explants survival rate whereas medium based strategy had 22.7% explants survival only. The medium having picloram 4 mg/l along with antibiotics (cefotaxime 500 mg/l and timentin 300 mg/l) was found best suitable for initial week callus induction. The standardized procedure gave overall 14.9% transformation efficiency in immature embryos and 9.8% in mature embryos and confirmed by gene-specific and promoter-specific PCR and southern analysis. These results indicate that the developed Agrobacterium-mediated transformation system is suitable for diverse wheat genotypes. The major obstacle for the implication of the CRISPR-based genome editing techniques is the non-availability of a suitable transformation system. Thus, the present system can be exploited to deliver the T-DNA into the wheat genome for CRISPR-based target modifications and transgene insertions.

Diverse conjugative elements silence natural transformation in Legionella species.

Abstract: Natural transformation (i.e., the uptake of DNA and its stable integration in the chromosome) is a major mechanism of horizontal gene transfer in bacteria. Although the vast majority of bacterial genomes carry the genes involved in natural transformation, close relatives of naturally transformable species often appear not competent for natural transformation. In addition, unexplained extensive variations in the natural transformation phenotype have been reported in several species. Here, we addressed this phenomenon by conducting a genome-wide association study (GWAS) on a panel of isolates of the opportunistic pathogen Legionella pneumophila. GWAS revealed that the absence of the transformation phenotype is associated with the conjugative plasmid pLPL. The plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination. We identified a small RNA (sRNA), RocRp, as the sole plasmid-encoded factor responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded sRNA RocR which controls the transient expression of the DNA uptake system. Assisted by the ProQ/FinO-domain RNA chaperone RocC, RocRp acts as a substitute of RocR, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species. Their low to high prevalence may result in the lack of transformability of some isolates up to the apparent absence of natural transformation in the species. Generally, our work suggests that conjugative elements obscure the widespread occurrence of natural transformability in bacteria.

Rapid and high efficiency transformation of Chlamydomonas reinhardtii by square-wave electroporation

Abstract: Chlamydomonas reinhardtii, the unicellular green algae, is the model organism for studies in various physiological processes and for bioindustrial applications. To explore the molecular mechanisms underlying physiological processes or to establish engineered cell lines, the exogenous DNA needs to be integrated into the genome for the insertional mutagenesis or transgene expression. However, the amount of selected marker DNA is not seriously considered in the existing electroporation methods for mutants library construction. Here, we reported a rapid-and-high-efficiency transformation technique for cell-walled strains using square-wave electroporation system. The final yield with this electroporation method was 2-6 × 103 transformants per μg exogenous DNA for cell-walled strains in a strain-dependent manner. In general, this electroporation technique was the easy and applicable way to build a mutant library for screening phenotypes of interest.

Event-trigger-based adaptive fuzzy hierarchical sliding mode control of uncertain under-actuated switched nonlinear systems.

Abstract: In this technical note, we present an adaptive fuzzy hierarchical sliding mode control method to deal with the control problem of under-actuated switched nonlinear systems. For the system under consideration, both the issues of unknown uncertain functions and aperiodically updating input are taken into account, which are of practical importance. A bounded time-varying function is employed to make a linear transformation of the control input, leading to a transformed system that can be applied to the control design. By introducing the so-called hierarchical structure, a top layer hierarchical sliding surface containing all the system states' information is obtained. Furthermore, by carrying out fuzzy logic systems' universal approximation, the problem caused by unknown system uncertainties is tackled. The approximation errors together with the measurement error resulted from the effects of the triggering event are lumped into a function, and its upper bound is estimated on-line. Based on these, the boundedness of all the signals are verified by combining the Lyapunov theory and projection algorithm. To testify the validity of our control scheme, a simulation example is carried out.

Stable transformation of the green algae Acutodesmus obliquus and Neochloris oleoabundans based on E. coli conjugation

Abstract: Microalgae are an ideal platform for the production of high-value chemicals, nutritional products and biofuels. Genetic engineering could speed up the development of microalgae derived products and reduce the overall production costs. Genetic methods such as particle bombardment, electroporation, Agrobacterium tumefaciens mediated transformation (ATMT), and agitation with glass beads and silicon carbide whiskers have been developed for the genetic transformation of microalgae. However, the transformation efficiency is species dependent, so a variety of transformation methods are required to engineer a wide range of microalgae species. The oleaginous microalgae Acutodesmus obliquus and Neochloris oleoabundans have a great potential as production platforms due to their ability to produce large amounts of triacylglycerol (TAG). Genetic modification techniques however are required to increase TAG levels further or to modify the fatty acid composition. Recently, a conjugation-based method for the delivery of episomes from bacteria to diatom microalgae has been reported. In this study, we have achieved the successful transformation of green oleaginous microalgal strains by transferring an expression vector via conjugation from E. coli. Since delivery of exogenous DNA into the microalgae cells is only the first step in obtaining transgenic microalgae, we further analyzed transformation efficiencies by PCR and expression of the Clover fluorescent protein in the targeted species.

Effective Agrobacterium -mediated transformation protocols for callus and roots of halophyte ice plant ( Mesembryanthemum crystallinum )

Abstract: Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols. To establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL−1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33–100% transient transformation rates, respectively. The transient transformation assays in ice plant callus and seedlings demonstrated that the concentrations of Agrobacteria, the durations of co-incubation time, and the plant growth stages were three important factors affecting the transient transformation efficiencies. Additionally, pot-grown juvenile plants were syringe-injected with two A. rhizogenes strains A8196 and NCPPB 1855, to establish transformed roots. After infections, ice plants were grown hydroponically and showed GUS expressions in transformed roots for 8 consecutive weeks. Our Agrobacterium-mediated transformation protocols utilized hypocotyl-derived callus and seedlings as plant materials, which can be easily obtained in large quantity. The average successful transient transformation rates were about 2.4–3.0% with callus and 33.3–100.0% with seedlings. We also developed a rapid and efficient protocol to generate transgenic roots by A. rhizogenes infections without laborious and challenging tissue culture techniques. This protocol to establish composite ice plant system demonstrates excellent improvements in efficiency, efficacy, and ease of use over previous ice plant transformation protocols. These Agrobacterium-mediated transformation protocols can be versatile and efficient tools for exploring gene functions at cellular and organ levels of ice plants.

HtrA-mediated selective degradation of DNA uptake apparatus accelerates termination of pneumococcal transformation.

Abstract: Natural transformation mediates horizontal gene transfer, and thereby promotes exchange of antibiotic resistance and virulence traits among bacteria. Streptococcus pneumoniae, the first known transformable bacterium, rapidly activates and then terminates the transformation state, but it is unclear how the bacterium accomplishes this rapid turn-around at the protein level. This work determined the transcriptomic and proteomic dynamics during the window of pneumococcal transformation. RNA sequencing revealed a nearly uniform temporal pattern of rapid transcriptional activation and subsequent shutdown for the genes encoding transformation proteins. In contrast, mass spectrometry analysis showed that the majority of transformation proteins were substantially preserved beyond the window of transformation. However, ComEA and ComEC, major components of the DNA uptake apparatus for transformation, were completely degraded at the end of transformation. Further mutagenesis screening revealed that the membrane-associated serine protease HtrA mediates selective degradation of ComEA and ComEC, strongly suggesting that breakdown of the DNA uptake apparatus by HtrA is an important mechanism for termination of pneumococcal transformation. Finally, our mutagenesis analysis showed that HtrA inhibits natural transformation of Streptococcus mitis and Streptococcus gordonii. Together, this work has revealed that HtrA regulates the level and duration of natural transformation in multiple streptococcal species.

Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

Abstract: 'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Novel Pollen Magnetofection System for Transformation of Cotton Plant with Magnetic Nanoparticles as Gene Carriers.

Abstract: Plant genetic transformation systems have facilitated insights into molecular plant biology and revolutionized commercial agriculture. Unfortunately, agrobacterium-mediated transformation was still the main method although its subsequent regeneration process from tissue culture in cotton remained arduous even after 30 years of technological advances. At the same time, a number of transformation methods based on pollen grains have been developed, such as electroporation, biolistic bombardment, ultra-sonication, and Agrobacterium incubation in plant pollens. But these pollen-based techniques have not been widely adopted. In this chapter, we have presented a protocol of pollen-based transformation technology in cotton, "pollen magnetofection," to directly produce transgenic seeds without tissue culturing. In this system, magnetic nanoparticles loaded with pure plasmid DNA carrying functional genes were delivered into pollens via pollen apertures in the presence of a magnetic field. Afterward, these magnetofected pollens were used for pollination, to produce transformed seeds. Pollen magnetofection is a genotype-independent transformation system; in addition, exogenous DNA was successfully integrated into the genome and stably expressed in the successive generations. We emphasize that pollen magnetofection is a most efficient platform for genetic transformation of cotton and other crops with high-throughput and efficient potential infield operation.

Agrobacterium-Mediated Transformation of Barley Immature Embryos

Abstract: Barley transformation is an essential tool for a range of functional genomics studies as well as for future crop improvement. The demand for efficient crop transformation systems continues to grow, with new genome editing technologies adding to that demand. Here we describe an efficient and routine transformation protocol for the spring barley Golden Promise, based on Agrobacterium-mediated inoculation of immature embryos. This protocol has been widely used for overexpression and RNAi applications and more recently for CRISPR/Cas9 mediated genome editing. Average transformation efficiencies of 25% can be easily achieved.

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector.

Abstract: Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.

Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer.

Abstract: Storage, manipulation and delivery of DNA fragments is crucial for synthetic biology applications, subsequently allowing organisms of interest to be engineered with genes or pathways to produce desirable phenotypes such as disease or drought resistance in plants, or for synthesis of a specific chemical product. However, DNA with high G+C content can be unstable in many host organisms including Saccharomyces cerevisiae. Here, we report the development of Sinorhizobium meliloti, a nitrogen-fixing plant symbioticα-Proteobacterium, as a novel host that can store DNA, and mobilize DNA to E. coli, S. cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. To achieve this, we deleted the hsdR restriction-system in multiple reduced genome strains of S. meliloti that enable DNA transformation with up to 1.4 x 105 and 2.1 x 103 CFU μg-1 of DNA efficiency using electroporation and a newly developed polyethylene glycol transformation method, respectively. Multi-host and multi-functional shuttle vectors (MHS) were constructed and stably propagated in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum. We also developed protocols and demonstrated direct transfer of these MHS vectors via conjugation from S. meliloti to E. coli, S. cerevisiae, and P. tricornutum. The development of S. meliloti as a new host for inter-kingdom DNA transfer will be invaluable for synthetic biology research and applications, including the installation and study of genes and biosynthetic pathways into organisms of interest in industry and agriculture.

Efficient Agrobacterium-Mediated Transformation of the Commercial Hybrid Poplar Populus Alba × Populus glandulosa Uyeki.

Abstract: Transgenic technology is a powerful tool for gene functional characterization, and poplar is a model system for genetic transformation of perennial woody plants. However, the poplar genetic transformation system is limited to a number of model genotypes. Herein, we developed a transformation system based on efficient Agrobacterium-mediated transformation for the hybrid poplar Populus Alba × Populus glandulosa Uyeki, which is a fast-growing poplar species that is suitably grown in the northern part of China. Importantly, we optimized many independent factors and showed that the transformation efficiency was improved significantly using juvenile leaf explants. Explants were infected by an Agrobacterium suspension with the OD600 = 0.6 for 15 min and then co-cultured in dark conditions for 3 days. Using the improved transformation system, we obtained the transgenic poplar with overexpression of β-glucuronidase (GUS) via direct organogenesis without callus induction. Furthermore, we analyzed the GUS gene in the transgenic poplars using PCR, qRT-PCR, and GUS staining. These analyses revealed that the GUS gene was efficiently transformed, and it exhibited various expression levels. Taken together, these results represent a simple, fast, and efficient transformation system of hybrid poplar plants. Our findings may facilitate future studies of gene functions in perennial woody plants and tree breeding via transgenic technology assisted design.

Nuclear transformation of the versatile microalga Euglena gracilis

Abstract: Euglena gracilis is a unicellular microalga studied for the production of nutraceuticals, cosmeceuticals and biofuel. Full exploitation of the organism requires the development of genetic engineering tools including a method for obtaining genetically stable transformants. In this work, Agrobacterium mediated transformation, biolistic bombardment and electroporation were explored to obtain stable nuclear E. gracilis transformants. Two 3′UTR fragments of the E. gracilis gapC gene were added at the 5′and 3′ ends of the pCambia1302 T-DNA to promote homologous integration of the transforming DNA into the genome. E. gracilis transformants growing on hygromycin plates and expressing the mgfp5 gene coding for green fluorescent protein were obtained from all three approaches. Maintenance of the transforming DNA in the nucleus was confirmed by PCR. Agrobacterium-mediated transformation yielded 10 transformants, biolistic bombardment seven and electroporation one transformant per 10,000 cells plated. Transformants from biolistic bombardment and electroporation were able to transiently express the hptII gene based on their growth on hygromycin containing plates, but this property was lost during repeated rounds of cultivation suggesting lack of (stable) integration of the transforming DNA into the Euglena genome. In contrast, Agrobacterium-mediated transformation produced stable nuclear transformants growing on hygromycin plates even after 12 rounds of cultivation. This work will pave the way for further improvement of E. gracilis strains for the production of valuable compounds.