Genetic Transformation Method Research Articles

A novel, simple, and stable mesoporous silica nanoparticle-based gene transformation approach in Solanum lycopersicum.

In this study, a novel and stable gene transformation system was developed under control of Maize Proteinase Inhibitor (MPI) as an inducible promoter using the Mesoporous Silica Nanoparticles (MSNs). The functionalized MSNs with a proper particle size were synthesized and attached to a recombinant construct (pDNA) containing cryIAb gene under the control of MPI promoter (pPZP122:MPI:cryIAb:MSN [pDNA: MSN]) following transformation of tomato plants through injection of the pDNA: MSN complex into tomato red fruit at early ripening stage and then, putative transgenic seeds were collected. As an initial selection, gentamicin-resistant seedlings of T1 (24.24%) and T2 (61.37%) plants were identified. The transgene integration and expression were confirmed through the PCR, RT-PCR, and western blot approaches in the selected seedlings. PCR analysis showed that transformation frequency was equal to 10.71% in T1 plants. Semi-quantitative RT-PCR analysis confirmed the transcript expression of cryIAb in all the T1 and T2 PCR-positive plants. Western blot analysis confirmed the existence of CryIAb protein in the leaves of T2 putative transgenic plants. Accordingly, the results demonstrated that the transgene has more likely integrated into the tomato genome through homologous recombination. Bioassay was carried out for further assessment of the plant responses to Tuta absoluta resulting in an enhanced tolerance of the plant. In conclusion, the MSN-mediated stable transformation system under the MPI as an inducible promoter can be used as a suitable alternative for conventional genetic transformation methods due to its biodegradability, biocompatibility, cost and time-effectiveness, and positive effect on the plant defense against pathogens and pests.

An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO3, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO3. Moreover, the application of AgNO3 promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO3 from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing. Genetic transformation of snapdragon was significantly improved by applying AgNO3 to induce indirect organogenesis, which will substantially facilitate its fundamental research as well as molecular breeding.

A hairy-root transformation protocol for Trigonella foenum-graecum L. as a tool for metabolic engineering and specialised metabolite pathway elucidation

The development of genetic transformation methods is critical for enabling the thorough characterization of an organism and is a key step in exploiting any species as a platform for synthetic biology and metabolic engineering approaches. In this work we describe the development of an Agrobacterium rhizogenes-mediated hairy root transformation protocol for the crop and medicinal legume fenugreek (Trigonella foenum-graecum). Fenugreek has a rich and diverse content in bioactive specialised metabolites, notably diosgenin, which is a common precursor for synthetic human hormone production. This makes fenugreek a prime target for identification and engineering of specific biosynthetic pathways for the production of triterpene and steroidal saponins, phenolics, and galactomanans. Through this transformation protocol, we identified a suitable promoter for robust transgene expression in fenugreek. Finally, we establish the proof of principle for the utility of the fenugreek system for metabolic engineering programs, by heterologous expression of known triterpene saponin biosynthesis regulators from the related legume Medicago truncatula in fenugreek hairy roots.

Optimization of genetic transformation method for an indica rice (Oryza sativa L.) variety Ratnagiri-711

A study for method optimization of Agrobacterium-mediated genetic transformation for insect resistance was carried out for a rice variety Ratnagiri-711 showing better regenerability. Three different cry genes viz. cry1Aabc, cry1Fa1 and cry2Aa with a vector system consisting of the disarmed hyper virulent Agrobacterium tumefaciens strain EHA-105 harboring pBinAR or BinBt3 were used. The respective genes were linked to CaMV35S promoter and nptII gene under control of nos promoter and terminator. Scutellum-derived callus bits and embryonic shoot apical meristem of germinating seeds were used as target tissues for callus-mediated and in planta transformation, respectively. Kanamycin screening and PCR analysis was employed for confirmation of presence of transgene. Among five methods of colonization and co-cultivation tried with three cry genes, a callus-mediated transformation method consisting of 20 minutes colonization and 3 days co-cultivation with cry2Aa gene recorded highest transformation frequency (13.79%) but minimum survival (5.27%). On the contrary, considerable transformation frequency (6.35%) with highest survival (79.42%) was observed in an in planta method employing mild injury to embryonic shoot apical meristem of germinating seeds followed by injection of Agrobacterium having cry2Aa gene followed by 15 minutes colonization and then directly sowing in pots. Among three cry genes used, the gene cry2Aa was found most effective showing more transformation frequency.

CRISPR/Cas mediated base editing: a practical approach for genome editing in oil palm.

The improvement of the yield and quality of oil palm via precise genome editing has been indispensable goal for oil palm breeders. Genome editing via the CRISPR/Cas9 (CRISPR-associated protein 9) system, ZFN (zinc finger nucleases) and TALEN (transcription activator-like effector nucleases) has flourished as an efficient technology for precise target modifications in the genomes of various crops. Among the genome editing technologies, base editing approach has emerged as novel technology that could generate single base changes i.e. irreversible conversion of one target base in to other in a programmable manner. A base editor (adenine or cytosine) is a fusion of catalytically inactive CRISPR-Cas9 domain (Cas9 variants) and cytosine or adenosine deaminase domain that introduces desired point mutations. However, till date no such genetic modifications have ever been developed in oil palm via base editing technology. Precise genome editing via base editing approach can be a challenging task in oil palm due to its complex genome as well as difficulties in tissue culture and genetic transformation methods. However, availability of whole genome sequencing data in oil palm provides a platform for developing the base editing technology. Here, we briefly review the potential application and future implications of base editing technology for the genetic improvement of oil palm.

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.

Transforming Candida hispaniensis, a promising oleaginous and flavogenic yeast.

Candida hispaniensis is an oleaginous yeast with a great potential for production of single cell oil according to its naturally high lipid accumulation capacity. Its unusual small genome size trait is also attractive for fundamental research on genome evolution. Our physiological study suggests a great potential for lipid production, reaching 224 mg/g of cell dry weight in glucose minimum medium. C. hispaniensis is also able to secrete up to 34.6 mg/L of riboflavin promising further riboflavin production improvements by cultivation optimization and genetic engineering. However, while its genome sequence has been released very recently, no genetic tools have been described up to now for this yeast limiting its use for fundamental research and for exploitation in an industrial biotechnology. We report here the first genetic modification of C. hispaniensis by introducing a heterologous invertase allowing the growth on sucrose using a biolistic transformation approach using a dedicated vector. The first genetic tool and transformation method developed here appear as a proof of concept, and while it would benefit from further optimization, heterogeneous expression of invertase allows for metabolism of an additional sugar and shows heterologous enzyme production capacity.

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga.

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

Simple and efficient genetic transformation of sorghum using immature inflorescences

Sorghum is an important cereal crop and has been grown in a wide range of environments from tropic to warm-temperate zones. Genetic improvement of this crop has been based on those traditional breeding methods, but there is a limited application of genetic transformation for sorghum improvement due to its recalcitrance to genetic manipulation in vitro. The current study aimed at development of immature inflorescences as a genetic transformation system for sorghum. Immature inflorescences of two sorghum elite lines were chosen as the explants for developing a new method for genetic transformation. Sorghum immature inflorescences were collected from plants at the flowering stage and co-cultured with virulent Agrobacterium tumefaciens strain LBA4404 containing one of the special gene constructs. Once co-cultivation was completed, the treated immature inflorescences were moved onto regeneration medium to induce development of shoot and root from the resulted embryogenic callus. This system was also amenable for the microprojectile bombardment (biolistic particle delivery system) method. Putatively transgenic plants were confirmed for the presence of transgene in the plants based on the molecular analysis of the T1 plants. All resulted transgenic plants were normal in morphology and fully fertile. The transformation system developed with the immature inflorescence is a simple and efficient method for sorghum transformation, which can speed up the process of engineering new varieties.

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.

An efficient protocol for Agrobacterium-mediated transformation and regeneration of Campanula medium (Canterbury bells) based on leaf disc explants

Campanula medium is an important ornamental plant known for its blue and bell-shaped flowers and a good model for studying flower colour formation and modification. The major outstanding obstacle preventing the developing of genetic modification and improvements in C. medium is a missing transformation method. For the first time, an effective and reliable in vitro regeneration and Agrobacterium-mediated transformation system has been established in this study. Different types and combinations of plant growth regulators (PGRs) were investigated for shoot regeneration by using leaf explants from three different cultivars. A high regeneration rate (50% for cv. ‘Blue double’, 58% for cv. ‘White double’ and 44% for cv. ‘Pink double’) was achieved on MS-based medium supplemented with 11.5 μM thidiazuron (TDZ) and 2.3 μM 1-naphthaleneacetic acid (NAA). In total, the Agrobacterium strains ABI, AGL1 and GV3101:pMP90, transformed with a binary plasmid harbouring NPTII and GFP gene cassettes, were evaluated for plant transformation, while various factors affecting the transformation efficiency were also identified. The highest transformation rate of about 22% was obtained from explants pre-cultured on regeneration medium and infected with AGL1. However, transgenic shoots were produced by all three Agrobacterium strains studied and confirmed by PCR and GFP fluorescence microscopy. Campanula medium regeneration and genetic transformation methods were established while various factors affecting the transformation efficiency were identified such as pre-culture treatment of leaf explants and different Agrobacterium strains.

Histological evaluation of pineapple transgenic plants following 8 years of field growth

Pineapple is among the most important fruits worldwide. However, the narrow genetic base of current commercial cultivars renders the industry at risk from environmental and other biological threats. Hence, there is a need to develop improved cultivars with tolerance to biotic and abiotic stresses. Our prior research resulted in the establishment of a method for genetic transformation of pineapple. The genetically modified pineapple plants generated were characterized following acclimatization and during the subsequent 8 years under field conditions. The current work highlights the results obtained for histological characterization of roots and leaves of transgenic pineapple plants. The four treatments investigated were macropropagated control plants (untransformed), micropropagated control plants (untransformed), and micropropagated genetically transformed plants (clones 90 and 98). Twenty indicators were measured in plants: two of them did not show statistically significant differences among the four groups of plants (2/20 = 10.0%); six showed “Low” overall coefficients of variation (OCV) (6/20 = 30.0%); 12 showed “Medium” OCVs (12/20 = 60.0%); and only two indicators showed “High” OCVs (2/20 = 10.0%). Consideration of medium OCVs showed that the biomass of transgenic plants was slightly greater than those of the non-transgenic controls. Statistically significant differences between the lines were recorded except in the central cylinder diameter of roots and the D leaf adaxial cuticle thickness. “High” OCVs were observed for root exo- and endodermis thickness where transgenic clones showed statistically significantly higher values. In terms of the roots, five out of nine histological indicators showed “Medium” OCVs: cortex, rhyzodermis, and parenchyma thicknesses, pith diameter, and thickness of the transversal root ratio. Again, genetically transformed materials showed statistically significantly higher averages. Only three out of seven histological indicators showed “Medium” OCVs in the leaves: D leaf abaxial cuticle thickness, chlorophyll parenchyma thickness, and D leaf aquifer parenchyma thickness. As noted above, statistically significantly higher values were recorded in the transgenic pineapple plants. Since the differences observed did not ultimately affect fruit yield (as shown in our previous work), they do not appear to be consequences of genetic transformation that impact on reproductive yield.

Cloning of the Arabidopsis thaliana poly (Adp-ribose) polymerase 2 cdna gene in Saccharomyces cerevisiae

 Poly(ADP-ribose)polymerase (PARP) catalyzes the synthesis of covalently attached ADP-ribose polymers to acceptor proteins. The donor of the residues of ADP-ribose is NAD+. The prominent roles of PARP is a DNA damage sensor. PARP1, in poly(ADP-ribosyl)ated form binds to DNA breaks and engages proteins of  DNA repair to this sites. Recently, it has been shown that PARP poly(ADP-ribosyl)ates both proteins and DNA breaks. However, there is no direct evidence of  poly(ADP-ribosyl)ated  DNA adducts in vivo in animals and plants. PARP activity is absent in Saccharomyces cerevisiae cells, despite the fact that it has been identified in organisms ranging from archaebacteria to mammals. Due to the simplicity of manipulation and genetic interpretation S. cerevisiae yeasts have been used to analyze the functions of proteins from animal and plant cells. Therein, the goal of our work is the cloning and heterologous expression of the AtPARP2 gene from the Arabidopsis thaliana in yeasts. By using genetic engineering  methods, we have constructed a recombinant plasmid with cDNA of the AtPARP2 gene under the control of the inducible promoter GAL10 from episomal vector pESC-LEU2. A recombinant S.cerevisiae strain capable for expressing the cDNA of the AtPARP2 gene was obtained by genetic transformation method. By using Western blotting with the applying of polyclonal anti-AtPARP2 antibodies, it was revealed that the product of gene expression in yeast is a globular protein weighing 72kDa, consisting  637 amino acids (pI 5.92). A slight inhibition of growth was shown in FF 18733 yeast cells which express AtPARP2. Induction of  AtPARP2 expression in yeast revealed PARylating  activity of protein in vivo. Auto-poly(ADP-ribosyl)ation activity of AtPARP2 was shown in yeasts in response to double-stranded  DNA breaks induced by bleomycin treatment of cells. 3-AB, inhibitor of PARP suppressed the amount of poly(ADP-ribosyl)ated protein products. Key words: Poly(ADP-ribose)polymerase, AtPARP2, poly(ADP-ribosyl)ation, Arabidopsis thaliana, Saccharomyces cerevisae.

Клонирование кднк гена поли(АДФ-рибоза) полимеразы 2 Arabidopsis thaliana в Saccharomyces cerevisiae

 Poly(ADP-ribose)polymerase (PARP) catalyzes the synthesis of covalently attached ADP-ribose polymers to acceptor proteins. The donor of the residues of ADP-ribose is NAD+. The prominent roles of PARP is a DNA damage sensor. PARP1, in poly(ADP-ribosyl)ated form binds to DNA breaks and engages proteins of  DNA repair to this sites. Recently, it has been shown that PARP poly(ADP-ribosyl)ates both proteins and DNA breaks. However, there is no direct evidence of  poly(ADP-ribosyl)ated  DNA adducts in vivo in animals and plants. PARP activity is absent in Saccharomyces cerevisiae cells, despite the fact that it has been identified in organisms ranging from archaebacteria to mammals. Due to the simplicity of manipulation and genetic interpretation S. cerevisiae yeasts have been used to analyze the functions of proteins from animal and plant cells. Therein, the goal of our work is the cloning and heterologous expression of the AtPARP2 gene from the Arabidopsis thaliana in yeasts. By using genetic engineering  methods, we have constructed a recombinant plasmid with cDNA of the AtPARP2 gene under the control of the inducible promoter GAL10 from episomal vector pESC-LEU2. A recombinant S.cerevisiae strain capable for expressing the cDNA of the AtPARP2 gene was obtained by genetic transformation method. By using Western blotting with the applying of polyclonal anti-AtPARP2 antibodies, it was revealed that the product of gene expression in yeast is a globular protein weighing 72kDa, consisting  637 amino acids (pI 5.92). A slight inhibition of growth was shown in FF 18733 yeast cells which express AtPARP2. Induction of  AtPARP2 expression in yeast revealed PARylating  activity of protein in vivo. Auto-poly(ADP-ribosyl)ation activity of AtPARP2 was shown in yeasts in response to double-stranded  DNA breaks induced by bleomycin treatment of cells. 3-AB, inhibitor of PARP suppressed the amount of poly(ADP-ribosyl)ated protein products. Key words: Poly(ADP-ribose)polymerase, AtPARP2, poly(ADP-ribosyl)ation, Arabidopsis thaliana, Saccharomyces cerevisae.

An Agrobacterium rhizogenes mediated hairy root transformation protocol for fenugreek

•This work describes a protocol for hairy root transformation of the medicinal crop legume fenugreek (Trigonella foenum-graecum L.). Hairy root plant transformation mediated by Agrobacterium rhizogenes is an established method for the rapid genetic transformation of various dicotyledonous plants. We have adapted a hairy root transformation protocol from the model legume Medicago truncatula for use in this metabolically rich non-model crop legume. Considering the great variety and abundance of phytochemicals in fenugreek and its established use in traditional medicine, we aim for this method to become a resource for metabolic pathway identification and for production of valuable specialised metabolites via metabolic engineering approaches.•Development rapid transformation (2.5–3 weeks) of fenugreek roots via A. rhizogenes.•Marker gene cassette with suitable promoter for visual detection of transformed fenugreek roots

OsRuvB transgene induces salt tolerance in pigeon pea

ABSTRACT Salinity of cultivable land is a growing global concern that has been affecting the yields of major crops worldwide such as pigeon pea. In the current study, transgenic pigeon pea plants were developed using an in-planta Agrobacterium-mediated genetic transformation method wherein OsRuvB, a rice DNA helicase gene, was incorporated to induce salt tolerance in pigeon pea plants. Transformation efficiency of 35.7% was achieved with stable insertion of OsRuvB in transgenic lines. When subjected to salinity stress induced by 75 mM NaCl increase in chlorophyll content, relative water content, peroxidase and catalase activity in transgenic lines was observed over the wild type plants. Membrane injury index and lipid peroxidation were significantly reduced in transgenic lines. Proline and Total Soluble Sugar content were enhanced in both transgenic plants and wild type strains. It was inferred that transgenic lines were tolerant to salinity stress and tolerance may be imparted through an alternative unknown pathway.

Genetic Transformation Methods for Crop Improvement- A Brief Review

Decades of documented successful pieces of evidence in agritech have shown us a clear picture of the importance of biotechnology in crop improvement. The production in agriculture should have steady growth and to achieve these objective conventional methods should go on parallel with biotechnological approaches. Genetic engineering which has revolutionized the path of crop improvement involves the identification and transfer of novel genes into the existing elite cultivars. Different methods of transferring the gene into plant cells have been developed and continuous efforts have been made to increase its efficiency. Both direct and indirect method of gene transfer has its own merits and demerits. Efforts have been made continuously to eliminate drawbacks and to develop an easy, elite and eco-friendly method to transfer genes. The transformation method which is a base of genetic engineering is vital and Agrobacterium-mediated gene transfer and gene gun have shown to be doing well in recent years. As a whole the methodology involved, merits and demerits of different methods have been briefly discussed.

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.

Genome editing in wheat microspores and haploid embryos mediated by delivery of ZFN proteins and cell-penetrating peptide complexes.

SummaryRecent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value‐added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell‐penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN‐treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

Agrobacterium-mediated genetic transformation of Chinese chestnut (Castanea mollissima Blume)

Chinese chestnut (Castanea mollissima) is an important germplasm resource for the breeding of Castanea species worldwide with vital ecological and economic value. Biotechnology overcomes the limitations of traditional breeding and accelerates germplasm improvement. However, a genetic transformation system for Chinese chestnut has not yet been established. In this study, a stable and efficient Agrobacterium-mediated genetic transformation method for Chinese chestnut is described. Embryogenic calli of C. mollissima cv. ‘Yanshanhongli’ were used as the target material. The sensitivity of embryogenic calli to kanamycin was determined, whereby the proliferation of non-transformed calli was completely inhibited at 180 mg/L. Antibiotic inhibition results for Chinese chestnut embryogenic calli showed that 50 mg/L cefotaxime and 500 μM timentin completely inhibited the growth of Agrobacterium tumefaciens but did not affect the normal growth of Chinese chestnut embryogenic calli. When embryogenic calli were co-cultured for 2 days with Agrobacterium tumefaciens strain AGL1 harboring the PBI121-EGFP plasmid, an embryogenic callus transformation efficiency of 4.55% was obtained, and two transgenic chimera were acquired. This Agrobacterium-mediated transformation system for Chinese chestnut provides a fundamental platform for genetic improvement of core germplasm and for further verification of gene function.