Efficient Genetic Transformation Method Research Articles

Exploring an economic and highly efficient genetic transformation and genome-editing system for radish through developmental regulators and visible reporter.

Radish (Raphanus sativus L.) is one of the most important root vegetable crops worldwide. However, gene function exploration and germplasm innovation still face tremendous challenges due to its extremely low transformation efficiency. Here, an economic and highly efficient genetic transformation method for radish was explored by Agrobacterium rhizogenes-mediated transformation with the help of combining special developmental regulator (DR) genes and the visual identification reporter. Firstly, the RUBY gene, a betalain biosynthesis system, could result in a visual red-violet color used as a convenient and effective reporter for monitoring transgenic hairy roots screening of radish. However, the hairy roots-to-shoots conversion system of radish still stands as a barrier to the obtainment of whole transgenic plants, although different hormone combinations and various culture conditions were tried. Following, two DR genes including Wuschel2 (Wus2) and isopentenyl transferase (ipt), as well as their combination Wus2-ipt were introduced for the shoot regeneration capacity improvement. The results showed that the transgenic shoots could be directly generated without externally supplying any hormones in the presence of a Wus2-ipt combination. Then, Wus2-ipt along with the RUBY reporter was employed to establish an efficient genetic transformation system of radish. Moreover, this system was applied in generating gene-edited radish plants and the phytoene desaturase (RsPDS) gene was effectively knockout through albino phenotype observation and sequencing analysis. These findings have the potential to be widely applied in genetic transformation and genome-editing genetic improvement of other vegetable species.

Highly Efficient CRISPR/Cas9 Mediated Gene Editing in Ocimum basilicum 'FT Italiko' to Induce Resistance to Peronospora belbahrii.

Ocimum basilicum (sweet basil) is an economically important aromatic herb; in Italy, approximately 1000 ha of "Genovese-type" basil are grown annually in greenhouses and open fields and are subjected to Downy Mildew (DM) disease, caused by Peronospora belbahrii, leading to huge crop losses. Mutation of the Susceptibility (S) gene DMR6 (Downy Mildew Resistant 6) has been proven to confer a broad-spectrum resistance to DM. In this work, an effective Genome Editing (GE) approach mediated by CRISPR/Cas9 in O. basilicum 'Italiko', the élite cultivar used to produce "Pesto Genovese D.O.P", was developed. A highly efficient genetic transformation method mediated by A. tumefaciens has been optimized from cotyledonary nodes, obtaining 82.2% of regenerated shoots, 84.6% of which resulted in Cas9+ plants. Eleven T0 lines presented different type of mutations in ObDMR6; 60% were indel frameshift mutations with knock-out of ObDMR6 of 'FT Italiko'. Analysis of six T1 transgene-free seedlings revealed that the mutations of T0 plants were inherited and segregated. Based on infection trials conducted on T0 plants, clone 22B showed a very low percentage of disease incidence after 14 days post infection. The aromatic profile of all in vitro edited plants was also reported; all of them showed oxygenated monoterpenes as the major fraction.

Generation of Composite Rosa roxburghii Plants with Transgenic Roots by Agrobacterium-Mediated Transformation

Rosa roxburghii Tratt. is an emerging fruit endemic to China, which has the reputation of being the “King of Vitamin C” because of its abundance of vitamin C. However, it is also a recalcitrant species that imposes severe limitations on the transformation and whole-plant regeneration processes, restricting the verification of the functional genes. Therefore, developing a feasible and efficient genetic transformation method for R. roxburghii is an urgent requirement. Herein, K599 with eGFP was used as the Agrobacterium strain to optimize the genetic transformation from four factors: bacterial concentration, seedling age, infection site, and method. First, the original roots of 5-day-old seedlings were excised, and then the slant cuts of the remaining hypocotyls with 0.5 cm length were placed in K599 at an OD600 of 0.4. Subsequently, the explants were planted in a moistened sterile vermiculite after the beveled site was stained with a clump of bacteria. The results showed that the transformation efficiency of this cutting method was almost 28% at 30 days post-inoculation, while the transformation efficiency obtained by injecting 5-day-old seedlings 0.5–1.0 cm away from the primary root with K599 at an OD600 of 0.4 was only about 7%. Taken together, the current findings provide evidence that Agrobacterium-mediated transformation is a simple, fast, and efficient approach for generating composite R. roxburghii plants. Thus, this method has a broad application to analyze the gene functions in R. roxburghii and other related plant species.

Establishment of an efficient genetic transformation system: A case study of RNAi-induced silencing of the transcription factor MeGI in Diospyros oleifera Cheng seedlings

Persimmon (Diospyros) is an important genus of temperate fruit trees that originated in East Asia. High-efficiency genetic transformation in Diospyros spp. remains challenging, limiting studies of gene function and the use of modern technology, such as gene editing, in breeding programs. In this study, we established an efficient genetic transformation method based on the infection of hypocotyls and radicles obtained from immature D. oleifera seeds. In particular, we focused on optimising methods for infection and induction of calluses and adventitious buds. RNA interference vectors targeting the transcription factor MeGI, which has been reported to have a feminising effect, were transformed into the hypocotyls and radicles of D. oleifera, producing five positively transformed seedlings. Compared with the wild-type controls, the positively transformed seedlings exhibited dwarfed adventitious buds and narrower leaves. The phenotypic characteristics of the flowers of these positively transformed seedlings will be observed at a later date; this will not only confirm the function of MeGI in Diospyros spp. but also potentially induce the development of male floral buds in gynoecious species and further promote the development of breeding methods. The proposed genetic transformation protocol will be valuable for investigations into gene function and the development of persimmon breeding programs.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies. In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate on average. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5–4.0 months. Average transformation frequency is up to 10.83%. The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

A method for the preparation of electrocompetent cells to transform unicellular green algae, Coccomyxa (Trebouxiophyceae, Chlorophyta) strains Obi and KJ

Unicellular green algae, Coccomyxa spp. strains Obi and KJ are capable of growing at broad pH ranges, and accumulate lipids at levels higher than 50% of the dry cell weight under nitrogen-depleted conditions. An efficient genetic transformation method applicable to the two strains is required for their genetic improvement to facilitate their application in commercial production of biofuels. After an extended trial and error period, we fortuitously determined conditions required for the transformation of the strains through electroporation. First, the growth of the strains in isotonic medium e.g., minimal salt medium containing 0.6 M sorbitol or mannitol was required. Second, the growth under a light/dark cycle was critical. In the dark period in the cycle, cells contained two or four endospores surrounded by a mother-cell wall. Within half an hour after the beginning of the light period, the mother-cell wall was cleaved, and unicellular autospores that were electrocompetent were released. The application of a single electroporation pulse at an electric-field strength of 10 kV cm−1 to the autospores yielded transformants at a frequency of >10−6 per input cell for strain KJ and >10−7 per input cell for strain Obi. The transformation method is relatively simple, and provided several to several dozen higher transformation frequencies than those applying the biolistic bombardment method that we previously developed. The present study also provides insights that could facilitate the development of genetic transformation systems for microalgae, which reproduce by endospore formation.

Toward a genetic system in the marine cyanobacterium Prochlorococcus.

As the smallest and most abundant primary producer in the oceans, the cyanobacterium Prochlorococcus is of interest to diverse branches of science. For the past 30 years, research on this minimal phototroph has led to a growing understanding of biological organization across multiple scales, from the genome to the global ocean ecosystem. Progress in understanding drivers of its diversity and ecology, as well as molecular mechanisms underpinning its streamlined simplicity, has been hampered by the inability to manipulate these cells genetically. Multiple attempts have been made to develop an efficient genetic transformation method for Prochlorococcus over the years; all have been unsuccessful to date, despite some success with their close relative, Synechococcus . To avoid the pursuit of unproductive paths, we report here what has not worked in our hands, as well as our progress developing a method to screen the most efficient electroporation parameters for optimal DNA delivery into Prochlorococcus cells. We also report a novel protocol for obtaining axenic colonies and a new method for differentiating live and dead cells. The electroporation method can be used to optimize DNA delivery into any bacterium, making it a useful tool for advancing transformation systems in other genetically recalcitrant microorganisms.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions.The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

Using green fluorescent protein sheds light on Lupinus angustifolius L. transgenic shoot development

Narrow-leaf lupin (NLL) is the main legume crop grown in rotation with wheat and other cereals in Western Australia. Efforts to improve NLL germplasm by use of genetic technologies have been hampered by the lack of an efficient genetic transformation method, an issue that is in common with dominant crop legumes globally. Prior research has primarily used the bar gene for phosphinothricin (PPT) resistance. The aim of recent research has been to investigate alternative selection methodologies, in order to determine whether the limitations of low frequency of transgenic shoots, combined with chimerism at T0 could be overcome. Investigation of hygromycin resistance as a selectable marker compared to PPT is reported here. The results suggested that hygromycin resistance was a more suitable selectable marker for NLL transformation than PPT. Surprisingly, from investigation of transformation using the GUS reporter gene, it was also observed that transformation frequency was greater when selection treatment was reduced or delayed, compared to the existing protocols. To further investigate this observation, an eGFP expressing construct was prepared. Observations within the first week after Agrobacterium exposure of lupin explants demonstrated that transformation of NLL explant cells was not a rate-limiting step. Instead, the results indicated that the current selection methodology was killing the cells that were competent to regenerate into transgenic shoots. It was concluded that further research on the development of the treated explants should focus on delayed selection and exposure to Agrobacterium of cells below the apical meristem.

Comprehensive screening of influential factors in the Agrobacterium tumefaciens-mediated transformation of the Himalayan elixir: Ajuga bracteosa Wall. ex. Benth

Ajuga bracteosa is a valuable medicinal plant producing many important compounds, including withanolides, phytoecdysteroids, neo-clerodane-di and triterpenes, iridoid glycosides, flavonoids, etc. These compounds possess a broad spectrum of biological, pharmacological and medicinal properties, but their yield is very low in wild-type plants, and chemical synthesis is not viable. Metabolic engineering strategies offer a promising solution for the bulk production of these natural products. However, these strategies usually require the establishment of an efficient genetic transformation method, which to date has not been reported for A. bracteosa. Therefore, the current study was conducted by employing Agrobacterium tumefaciens C58C1 harboring the binary plasmid p35SGUSint with GUS as the reporter gene and the NTPII gene as the selectable marker. The influential factors, e.g. explant type, optical density of agrobacterial culture, inoculation period, co-cultivation duration and acetosyringone concentration, were systematically investigated and the optimal transformation conditions were established by 72 independent transformation experiments. Putative transformants were selected on kanamycin 100mg/L. MS medium supplemented with 3.6mg/L BA resulted in maximum regeneration frequency of the transformed explants. The expression of the GUS gene was confirmed by histochemistry and polymerase chain reaction. We established that nodal explants of A. bracteosa precultured for 3days, inoculated with a culture of A. tumefaciens with an OD of 1.0 for 20min and co-cultivated for 2days in MS medium with 200μM acetosyringone at media pH 5.8 showed 100% transformation induction. This is the first report of a successful A. tumefaciens-mediated transformation of A. bracteosa.

An improved method for the electrotransformation of lactic acid bacteria: A comparative survey

An efficient method for genetic transformation of lactic acid bacteria (LAB) by electroporation is presented in this work. A comparative survey with other electrotransformation methods already published showed that the method proposed here yields the higher electrotransformation efficiency in the 12 LAB strains tested, which could make the method applicable to other LAB species or genera.

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

A novel and highly efficient method for genetic transformation of fungi employing shock waves

Genetic transformation of filamentous fungi is an essential tool in many areas such as biotechnology, medicine, phytopathology and genetics. However, available protocols to transform fungi are inefficient, laborious and have low reproducibility. We report the use of underwater shock waves as a novel method to transform filamentous fungi. An experimental piezoelectric shock wave generator was designed to expose fungal conidia to heterologous DNA. The device was successfully tested in Aspergillus niger, Fusarium oxysporum, Trichoderma reesei and Phanerochaete chrysosporium. The transformation frequency per number of conidia was between two and four orders of magnitude higher in comparison to previously published methods. For example, the frequency of transformation in A. niger was improved up to 5400-fold as compared with Agrobacterium protocols. Transformation was verified by expression of the green fluorescent protein, PCR and Southern blot. Our method offers new possibilities for fast, easy and efficient genetic manipulation of diverse fungal species.

Agrobacterium-mediated transformation of kabocha squash (Cucurbita moschata Duch) induced by wounding with aluminum borate whiskers

An efficient genetic transformation method for kabocha squash (Cucurbita moschata Duch cv. Heiankogiku) was established by wounding cotyledonary node explants with aluminum borate whiskers prior to inoculation with Agrobacterium. Adventitious shoots were induced from only the proximal regions of the cotyledonary nodes and were most efficiently induced on Murashige–Skoog agar medium with 1 mg/L benzyladenine. Vortexing with 1% (w/v) aluminum borate whiskers significantly increased Agrobacterium infection efficiency in the proximal region of the explants. Transgenic plants were screened at the T0 generation by sGFP fluorescence, genomic PCR, and Southern blot analyses. These transgenic plants grew normally and T1 seeds were obtained. We confirmed stable integration of the transgene and its inheritance in T1 generation plants by sGFP fluorescence and genomic PCR analyses. The average transgenic efficiency for producing kabocha squashes with our method was about 2.7%, a value sufficient for practical use.

Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper

In order to establish a reliable and highly efficient method for genetic transformation of pepper, a monitoring system featuring GFP (green fluorescent protein) as a report marker was applied to Agrobacterium-mediated transformation. A callus-induced transformation (CIT) system was used to transform the GFP gene. GFP expression was observed in all tissues of T0, T1 and T2 peppers, constituting the first instance in which the whole pepper plant has exhibited GFP fluorescence. A total of 38 T0 peppers were obtained from 4,200 explants. The transformation rate ranged from 0.47 to 1.83% depending on the genotype, which was higher than that obtained by CIT without the GFP monitoring system. This technique could enhance selection power by monitoring GFP expression at the early stage of callus in vitro. The detection of GFP expression in the callus led to successful identification of the shoot that contained the transgene. Thus, this technique saved lots of time and money for conducting the genetic transformation process of pepper. In addition, a co-transformation technique was applied to the target transgene, CaCS (encoding capsaicinoid synthetase of Capsicum) along with GFP. Paprika varieties were transformed by the CaCS::GFP construct, and GFP expression in callus tissues of paprika was monitored to select the right transformant.

Quick and efficient method for genetic transformation of biopolymer‐producing bacteria

AbstractIn order to genetically modify microorganisms capable of producing polyhydroxyalkanoate (PHA) biopolymers, a simple and rapid method to prepare freshly plated Pseudomonas cells for transformation via electroporation was developed. This method can be used to transfer both replicative plasmids and linear DNA to knock out genes into the cells. The transformation efficiencies were in the range of ≥107 transformants µg−1 DNA for replicative plasmids and ≥106 transformants µg−1 DNA for linear DNA, which are comparable with commercially available competent cells. Furthermore, this transformation procedure can be performed in less than 10 min, saving a great deal of time compared with traditional methods. Knockout mutants of several Pseudomonas species were generated by transformation of linear DNA and these mutations were verified by PCR and analysis of PHA content. Copyright © 2009 Society of Chemical Industry

Analysis of morphological traits of bird's foot trefoil plants cv. Bokor transformed with Agrobacterium rhizogenes

An efficient method for genetic transformation and shoot regeneration was achieved in bird's foot trefoil cv. Bokor using A. rhizogens. The transformed shoots were regenerated on hairy root segments in high frequency. After rooting and acclimation, transformed To plants were grown in experimental field. Analysis of morphological traits and chemical content in ten unintentionally chosen To bird's foot trefoil plants (genotypes no. 2 and no. 5) was performed. They were compared to those of control non-transformed plants. The traits as a number of stems per plant, length of internodes in longest stem, number of flowers per plant and plan high were very significant differed than the same traits in control plants, while there were no significant differences in the leaf area. No signs of the rol genes genotype and "T" phenotype were present. The transformed plants had significantly higher content of cellulose, while the protein and nitrogen contents of are in the range of control plants.

Optimization of electroporation procedure to transform B. polymyxa SCE2 and other nitrogen-fixing Bacillus

An efficient method for genetic transformation of different nitrogen-fixing Bacillus polymyxa strains by electroporation is presented. Various parameters on plasmid transformation of b. polymyxa SCE2 with plasmid pC194 were investigated to enhance transformation efficiency: voltage, buffer strength and type of electroporation buffer, plasmid DNA concentration, purity of plasmid preparation and plasmid source. Electroporation of B. polymyxa SCE2 resulted in a transformation efficiency as high as 3.2 × 10 5 transformants/μg of pC194 when the optimized procedure was used. Stability of different plasmids (pE194, pBD64, pBC16 and pFT30) transformed to B. polymyxa SCE2-43 and/or SCE2-43 was analysed. pE194 showed high frequency of curing while pFT30 and pBC16 were stable after 10 transfers in non-selective medium. Plasmid DNA isolated from transformants had not undergone detectable deletions. Although the electroporation protocol presented here can be used to transform B. azotofixans, lower efficiencies of transformations were achieved. No transformants were detected in other nitrogen-fixing strains belonging to B. macerans.

High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at -85 degrees C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 x 10 transformants per mug of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method.

Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning

In this report, we demonstrate the utility of electroporation as an efficient method for genetic transformation of Leuconostoc paramesenteroides. We optimized several factors which determine the transformation frequency, resulting in transformation efficiencies of up to 4 x 10(3) transformants per micrograms of pNZ12 DNA, which contains the promiscuous Lactococcus lactis pSH71 replicon. Slightly lower efficiencies were obtained with a deletion derivative of the broad-host-range plasmid pAM beta 1. These plasmids could be stably maintained in L. paramesenteroides NZ6009 for more than 100 generations, even in the absence of selective pressure. In order to show the use of the developed host-vector system, we cloned the Lactococcus lactis gene encoding phospho-beta-galactosidase in L. paramesenteroides. Expression of this heterologous gene in L. paramesenteroides under control of Lactococcus lactis expression signals was evident from the presence, in transformants, of phospho-beta-galactosidase activity and a specific phospho-beta-galactosidase protein band on Western blots (immunoblots). In addition, we transformed a lactose-deficient derivative of L. paramesenteroides with a plasmid carrying a Lactococcus lactis-Escherichia coli lacZ gene fusion. The resulting transformants synthesized high levels of beta-galactosidase, indicating the efficiency of heterologous gene expression signals in L. paramesenteroides.