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Reasonable evaluation of the transformation efficiency of resource-based cities can provide a reliable basis for correcting factor misallocation and optimizing factor allocation. This study improves the directional distance function from the aspects of direction vector endogeneity, relative distance and exogenous weight. Based on the improved model, the data of China’s prefecture-level cities from 2003 to 2018 are used to measure and compare the transformation efficiency of resource-based cities and non-resource-based cities. By setting different exogenous weights, the transformation efficiency considering the total factor and the transformation efficiency only considering the energy factor are obtained. Further comparative analysis shows that the two transformation efficiencies of resource-based cities are lower than those of non-resource-based cities, and the two keep the same change trend. Whether it is a resource-based city or a non-resource-based city, the level of transformation efficiency that only considers energy factors is lower. Further, this study decomposes the transformation efficiency of resource-based cities according to the three dimensions of transformation efficiency and finds that the energy efficiency, output efficiency and environmental efficiency of China’s resource-based cities are different, and the transformation efficiency in the three dimensions of energy conservation, economic growth and environmental friendliness is also different.

Antibiotic resistance genes can be spread via gene horizontal transfer (GHT). Chlorination and UV irradiation are common disinfection methods used in wastewater treatment plants before the discharge of treated wastewater. This study aimed to elucidate the effects of disinfection on the transformation of naked DNA in the aquatic environment. The pUC19 plasmid possessing ampicillin-resistant beta-lactamase and subjected to different dosages of chlorine or UV irradiation was used for transformation in Escherichia coli to estimate the transformation efficiency and GHT in the environment after disinfection. The results showed that doses > 0.5 mg-Cl2/L can effectively decrease transformation efficiency (1.21 to 8.83-log10) based on pUC19 as the positive control. UV irradiation can decrease the efficiency (2.37 to 3.39-log10) following 10–60 min of treatment. PCR and qPCR detection have limitations for determining transformation efficiency because they provide approximate estimates damaged DNAs. Overall, these results indicate that proper disinfection management using chlorine and/or UV for treated wastewater before discharge from wastewater treatment plants can prevent the spread of antibiotic resistant bacteria and genes, by decreasing the efficiency of naturally occurring bacterial transformations in wastewater treatment plants.

Building strains of filamentous fungi for stable long-term heterologous expression of large biosynthetic pathways is limited by the low transformation efficiency or genetic stability of current methods. Here, we developed a system for targeted chromosomal integration of large biosynthetic gene clusters in Aspergillus nidulans based on site-specific recombinase-mediated cassette exchange. We built A. nidulans strains harboring a chromosomal landing pad for Cre/lox-mediated recombination and demonstrated efficient targeted integration of a 21 kb DNA fragment in a single step. We further evaluated the integration at two loci by analyzing the expression of a fluorescent reporter and the production of a heterologous polyketide metabolite. We compared chromosomal expression at those landing loci to episomal AMA1-based expression, which also shed light on uncharacterized aspects of episomal expression in filamentous fungi. This is the first demonstration of site-specific recombinase-mediated integration in filamentous fungi, setting the foundations for the further development of this tool.

The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Cassava genetic transformation has mostly been reported for African cassava varieties, but not for Asian varieties. This is the first report of cassava transformation in Asian elite varieties using friable embryogenic calli. Agrobacterium-mediated cassava transformation via friable embryogenic calli (FEC) has enabled the robust production of transgenic cassava. So far, mostly the model cassava variety 60444 and African varieties have been transformed because of their good production and regeneration from embryogenic tissues. It is important to develop transformation methods for elite Asian cassava varieties to meet the changing needs in one of the world's major cassava production areas. However, a suitable transformation method for the Asian elite variety Kasetsart 50 (KU50) has not been developed. Here, we report a transformation method for KU50, the cultivar with the highest planting area in Thailand and Vietnam. In cassava transformation, the preparation of FEC as the target tissue for transgene integration is a key step. FEC induction from KU50 was improved by using media with reduced nutrients and excess vitamin B1, and somatic embryo and plant regeneration optimized by manipulation of naphthalene acetic acid (NAA), and benzylamino purine (BAP). The transformation efficiency for KU50 was 22%, approximately half that of 60444 at 45%. Transcriptome analysis indicated that the expression of genes related to cell-wall loosening was upregulated in FEC from KU50 compared with 60444, indicating that cell-wall production and assembly were disproportionate in the Asian variety. The transformation system for KU50 reported here will contribute to the molecular breeding of cassava plants for Asian farmers using transgenic and genome-editing technologies.

Genetically transformed Bifidobacterium longum, a probiotic bacterium in the human intestines, has a high potential for in-situ delivery of therapeutic proteins. However, one limitation is its low transformation efficiency caused by its thick membrane as it is a Gram-positive bacterium. Here we present an excellent transformation efficiency of B. longum (up to 1 × 107 to 2.1 × 107 cells/μg of DNA), obtained when oxidized multi-walled carbon nanotubes are used in synergy with classic transformation methods. This approach opens a fruitful avenue for B. longum transformations that could increase its potential for in-situ delivery systems of recombinant proteins .

Genome editing technique is one of the methods for studying the expression of gene, eliminating unfavorable traits or phenotypes and generating the new characters of species. The pRGEB32 plasmid is one of the vectors that used in genome editing with carrying the Cas9 gene, restriction site of sgRNA (single guide RNA) and specific promoters that can be expressed in plants. The first step in the genome editing process is inserting pRGEB32 into Escherichia coli for propagation. The large size of the plasmid molecule becomes a challenge to determine the right method in the transformation process. This study aims to determine the temperature and time of heat shock transformation of plasmid pRGEB32 into E. coli. The transformation of pRGEB32 into plasmids was carried out with variations in temperature and time, 42℃ (30 seconds and 60 seconds) and 55℃ (30 seconds and 60 seconds). The results showed that a heat shock temperature of 55℃ with a time of 60 seconds was the best temperature for the transformation of pRGEB32 into E. coli. This optimization of heat shock condition will increase the transformation efficiency, which is in the range of 3322-10.989 cfu/µg.ÂÂ

A high-auxin medium, usually used for callus induction, was not effective for Indian short-day onion cv. Bhima super. In this study, we found that the onion seedling radicle was a better explant than shoot tip for embryogenic callus induction, and induction efficiency up to 85.33% along with high embryogenic calli weight was obtained in routinely used medium containing 1.0 mg/L 2,4-D, but specifically supplemented with 0.5 mg/L kinetin. MS medium supplemented with 1.5 mg/L kinetin and 0.125 mg/L ABA showed 73.15% shoot regeneration efficiency from the calli induced from seedling radicle. Geneticin and hygromycin B at 50 mg/L showed optimal selection pressure for 8-week-old onion calli. Agrobacterium-mediated transformation of 8-week-old friable embryogenic calli induced from seedling radicle resulted in phenotypically normal transgenic plants with 1% transformation efficiency. In this study, regeneration and transformation protocols were developed for a widely used Indian short-day onion cultivar, which is instrumental for the development of stable transgenics in this crop. The regeneration protocol is standardized for Indian short-day onion cultivar and Agrobacterium-mediated transformation of onion is established, following in vitro co-cultivation of calli induced from seedling radicle explant.

Resumen Introducción. La transformación del arroz (Oryza sativa L. ssp indica) mediada por Agrobacterium, representa una oportunidad para la investigación científica y el mejoramiento genético. Es necesaria la optimización del protocolo para obtener la mayor eficiencia de transformación. Objetivo. Evaluar diferentes factores que afectan la transformación genética en callos embriogénicos de arroz de la subespecie indica vía Agrobacterium tumefaciens. Materiales y métodos. Este estudio se realizó en San José, Costa Rica, entre 2012 y 2014. En seis tratamientos se evaluaron: el efecto de la edad del callo, la concentración de acetosiringona, condición luminosa, la presencia o ausencia de radícula y la cepa de Agrobacterium tumefaciens en la transformación genética de callos embriogénicos de arroz de la variedad CR5272 con el gen reportero gus. Se compararon la cepa de Agrobacterium LBA4404 con el plásmido pCAMBIA1305.2 y las cepas ATHV, GV3101 y LBA4404, con el plásmido pCAMBIA1303; mediante pruebas histoquímicas para la detección de la expresión transitoria del gen marcador que codifica para la β-glucuronidasa. Resultados. La evaluación de los seis tratamientos con la cepa LBA4404::pCAMBIA1305.2 resultó en expresión transitoria del gen GusPlus de 1,33-7,00 % para la variedad CR5272 y de 8,00 % para el control con la variedad Nipponbare (ssp. japonica). Las cepas con el plásmido pCAMBIA1303 presentaron una expresión transitoria del gen gusA entre el 100-65 % con un área promedio de 14,23 mm2 (ATHV), 8,81 mm2 (GV3101), y 8,83 mm2 (LBA4404), sin diferencias significativas entre ellas; sin embargo, sí hubo diferencias al compararlas con la cepa LBA4404::pCAMBIA1305.2 (85 %, 4,39 mm2). Conclusiones. La utilización de las condiciones: callos de seis días, concentración de acetosiringona de 76 µM, luz antes y después del cocultivo, presencia de radícula y la cepa ATHV::pCAMBIA 1303, mejoraron la eficiencia de transformación con Agrobacterium tumefaciens en la variedad de arroz CR5272. Abstract Introduction. Agrobacterium-mediated transformation of rice (Oryza sativa L. ssp indica) represents an opportunity for scientific research and genetic improvement. Optimization of the protocol is necessary to obtain the highest transformation efficiency. Objective. To evaluate different factors that affect the genetic transformation in embryogenic rice callus of subspecies indica through Agrobacterium tumefaciens. Materials and methods. This study was performed in San José, Costa Rica between 2012 and 2014. The following were evaluated in six treatments: the effect of callus age, acetosyringone concentration, lighting condition, the presence or absence of radicle, and Agrobacterium tumefaciens strain on the genetic transformation of embryogenic calli of rice variety CR5272 with gus reporter gene. Agrobacterium strains LBA4404 with pCAMBIA1305.2 plasmid, and strains ATHV, GV3101, and LBA4404 with the pCAMBIA 1303 plasmid were compared; by histochemical tests for the detection of transient expression of the β-glucuronidase reporter gene. Results. The evaluation of the six treatments with strain LBA4404::pCAMBIA 1305.2 resulted in transient expression of 1.33-7.00 % of the GusPlus gene for the CR5272 variety, and 8.00 % for the control with the Nipponbare (ssp. japonica) variety. The strains with the pCAMBIA1303 plasmid showed a transient expression of the gusA gene between 100-65 % with an average area of 14.23 mm2 (ATHV), 8.81 mm2 (GV3101), and 8.83 mm2 (LBA4404) with no significant differences between them; however, there were differences when compared with strain LBA4404::pCAMBIA1305.2 (85 %, 4.39 mm2). Conclusions. The use of the conditions: six-day callus, acetosyringone concentration of 76 µM, light before and after cocultivation, presence of radicle and the ATHV::pCAMBIA 1303 , improved the transformation efficiency with Agrobacterium tumefaciens in the rice variety CR5272.

The in vitro culture conditions plays a critical role in promoting callus and shoot induction in rice during Agrobacterium-mediated transformation. In this research, an attempt was made to optimize the key factors affecting the efficiency of Agrobacterium-mediated transformation in ADT 39 rice cultivar. Among the different combinations of the NB-Asmedia tried with varying concentrations of casein hydrolysate from 0.25 g l-1 to 1.25 g l-1, the media containing 0.5 g l-1 casein hydrolysate supplemented with 2 mg l-1 2,4-Dichlorophenoxyacetic acid (2,4-D), 1 mg l-16-Benzylaminopurie (6-BAP) and 1mg l-1 Naphthalene Acetic Acid (NAA) was found to be the best with callus induction frequency of 94.44 per cent. Incubation in NB-As media after infection for 20 min. considerably reduced the Agrobacterium overgrowth after co-cultivation. For shoot induction, regeneration media RNMH30 containing varied concentrations of 6-BAP from 2 mg l-1 to 4 mg l-1 was tried, the media containing 3.0 mg l-1 6-BAP added with 1 mg l-1 NAA recorded the highest shoot induction frequency of 71.11 per cent. The overall transformation efficiency in ADT 39 with the optimized culture conditions was found to be 7.96 per cent.

Explant contamination due to Agrobacterium overgrowth after the co-cultivation stage is a common problem in Agrobacterium-mediated plant transformation. In order to overcome this issue, this research undertook another approach by generating auxotrophic Agrobacterium tumefaciens AGL1 mutants to a specific amino acid by mini Tn5 transposon carrying spectinomycin resistance gene (spcR), and a total of 3315 AGL1 mutants were successfully constructed. Further screening identified 20 putative auxotrophs, and subsequently produced three mutants carried auxotroph properties to one specific amino acid. These mutants were AP5-2-51 threonine auxotroph, AP5-5-2 cysteine auxotroph, and AP5-7-27 tryptophan auxotroph. The mini Tn5 insertion position in the Agrobacterium genome showed that the insertion position of AP5-2-51 mutants was in the thrB gene (AAK86584.1; locus tag At1D132_04580), while the other two mutants were unable to be identified by TAIL-PCR technique. The effectiveness of these three mutants to transfer T-DNA (pCAMBIA1300-eGFP-hpt) was examined on fresh Nipponbare rice callus explants with AGL1 as control. Results showed that transformation efficiency of the three mutants was not significantly different from AGL1 (Tukey HSD, α = 0.05). The percentages of Agrobacterium overgrowth in control and samples (three mutants) were also measured. Interestingly, the AP5-2-51 mutant indicated the highest ability to prevent overgrowth by reducing Agrobacterium growth to 1.11%, while the other two mutants suppressed the overgrowth to 15.56% (AP5-5-2) and 12.22% (AP5-7-27).

The production of transgenic watermelon through Agrobacterium tumefaciens-mediated transformation with in vitro regeneration system is a time-consuming, labor-intensive and genotype-dependent process. To acquire large number of transgenic watermelons in a shorter period of time, the half-seed explants were infected with Agrobacterium strain EHA 105 harboring pCAMBIA1301 with bar and gus genes. The factors affecting in planta transformation efficiency, such as co-cultivation duration, acetosyringone concentration, sonication duration and vacuum infiltration were assessed in the present study. The half-seed explants were sonicated for 3 min and 2 min vacuum infiltrated in Agrobacterium suspension and co-cultivated for 3 days with 100 µM acetosyringone showed maximum transformation efficiency. The transformed watermelon plants were selected against BASTA® and GUS, PCR, Southern hybridization analysis confirmed the transgene integration. The amenability of this established protocol was analyzed on four genotypes, in which the response of all genotypes was positive, whereas Arka manik showed the higher transformation efficiency of 17%. The transformation protocol developed in the present study is efficient, economical and expeditious without the taking part of any tissue culture phases and produce a large number of transgenic lines within a short period of 56 days.

Summary The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'real‐world' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasound‐mediated DNA delivery (UDD) technique to introduce plasmid to established non‐competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 ± 0.3 × 10‐7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD‐treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD‐treated Shewanella oneidensis MR‐1 biofilms generated significantly (P, In this study, we applied an ultrasound‐mediated DNA delivery (UDD) technique to introduce plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway into established bacterial biofilms in microfluidic flow and microbial fuel cells (MFCs) respectively. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD‐treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD‐treated Shewanella oneidensis MR‐1 biofilms generated significantly greater (61%) bioelectricity production in MFC than a wild‐type control group. The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. This is the first study to report on a potentially scalable direct genetic engineering method for established non‐competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications.

Rose (Rosa hybrida) is widely used for cut flowers and as garden plants. Stable and efficient transformation system is required for functional genomics of rose. Here, we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus. Expanding rose leaves were used as explants to induce somatic embryos, which were subjected to transformation with A. tumefaciens strain GV3101 using Green Fluorescence Protein (GFP) as a marker gene. It took about 8 months to generate transgenic shoots from embryogenic callus. PCR, RT-PCR, Southern and Western blotting, as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome. According to our data, a transformation efficiency of up to 6% can be achieved by following this optimized protocol.

Background Protocols for Agrobacterium rhizogenes-mediated hairy root transformation of the model legume Lotus japonicus have been established previously. However, little efforts were made in the past to quantify and improve the transformation efficiency. Here, we asked whether effectors (nodulation outer proteins) of the nodule bacterium Sinorhizobium sp. NGR234 can promote hairy root transformation of L. japonicus. The co-expressed red fluorescent protein DsRed1 was used for visualization of transformed roots and for estimation of the transformation efficiency. Results Strong induction of hairy root formation was observed when A. rhizogenes strain LBA9402 was used for L. japonicus transformation. Expression of the effector gene nopP in L. japonicus roots resulted in a significantly increased transformation efficiency while nopL, nopM, and nopT did not show such an effect. In nopP expressing plants, more than 65% of the formed hairy roots were transgenic as analyzed by red fluorescence emitted by co-transformed DsRed1. A nodulation experiment indicated that nopP expression did not obviously affect the symbiosis between L. japonicus and Mesorhizobium loti. Conclusion We have established a novel protocol for hairy root transformation of L. japonicus. The use of A. rhizogenes LBA9402 carrying a binary vector containing DsRed1 and nopP allowed efficient formation and identification of transgenic roots.

Grapevine (Vitis spp.) is one of the most economically important fruit crops worldwide, and there is considerable interest in improving its major agronomic and enological traits in response to ever-changing agricultural environments and consumer demands. Molecular genetic techniques in particular, associated with rapid technological advancements, provide an attractive alternative to conventional breeding approaches for developing new grapevine varieties with enhanced yield performance, quality, stress tolerance and disease resistance. To date, several grapevine varieties have been transformed with genes associated with diverse functions through biolistic bombardment and/or Agrobacterium-mediated transformation, and transgenic grape lines have been obtained using established regeneration systems. Nevertheless, a wide range of factors, including genotype, explant source and culture medium, have been shown to affect the efficiency of plant regeneration. Moreover, the selection and use of acceptor materials, bacterial strain and cell density, selectable markers and selection methods also influence transformation efficiency. This paper provides an overview of recent advances in grapevine regeneration and genetic transformation and in-depth discussion of the major limiting factors, and discusses promising future strategies to develop robust plant regeneration and genetic transformation in grapevine.

Protoplasts, which lack cell walls, are ideal research materials for genetic engineering. They are commonly employed in fusion (they can be used for more distant somatic cell fusion to obtain somatic hybrids), genetic transformation, plant regeneration, and other applications. Cotton is grown throughout the world and is the most economically important crop globally. It is therefore critical to study successful extraction and transformation efficiency of cotton protoplasts. In the present study, a cotton callus protoplast extraction method was tested to optimize the ratio of enzymes (cellulase, pectinase, macerozyme R-10, and hemicellulase) used in the procedure. The optimized ratio significantly increased the quantity and activity of protoplasts extracted. We showed that when enzyme concentrations of 1.5% cellulase and 1.5% pectinase, and either 1.5% or 0.5% macerozyme and 0.5% hemicellulase were used, one can obtain increasingly stable protoplasts. We successfully obtained fluorescent protoplasts by transiently expressing fluorescent proteins in the isolated protoplasts. The protoplasts were determined to be suitable for use in further experimental studies. We also studied the influence of plasmid concentration and transformation time on protoplast transformation efficiency. When the plasmid concentration reaches 16 µg and the transformation time is controlled within 12–16 h, the best transformation efficiency can be obtained. In summary, this study presents efficient extraction and transformation techniques for cotton protoplasts.

Concern has been expressed on the control of agricultural biotechnology through patents that may adversely affect the development of competing crops. Soybean is one of the most important crops around the world (~287 million t per year), above potatoes (45 million t per year), tomatoes (23 million t per year), or wheat (116 million t per year), with prices for American producers ranging between USD 278.8 and USD 650.3 t-1. Soybean belongs to the Fabaceae family and has been genetically modified (GM) to improve its tolerance to herbicides, including glyphosate, its resistance to insect pests, and the quality of soy oil. Glyphosate-tolerant soybean has received a gene coding for the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). There are a number of variables that contribute to the development of a GM soybean event. Such variables include tissue culture, selection methods, cloning vectors, and Agrobacterium strains that affect transformation efficiency and can be associated with patents. Chlorine gas disinfection is the most appropriate technique for plant material. Production of explants with shoots and molecular and phenotypic features (e.g., antibiotic susceptibility) of bacterial strain must be assessed. A long-term glyphosate selection arrangement is the most suitable and a consistent approach for the selection of events of GM soybean with tolerance to glyphosate. Freedom-to-operate evaluation must be carried out to find the specific elements neccesary for GM plant development that do not infringe the rights of third parties. These rights come into effect from the patent application date for a definite geographical region involving construct design and its synthesis, transformation vector, bacterial strain, methods, or reporter gene. In this review, the protocols relating to experiments for the development of GM soybean using an epsps gene are included, and considerations relating to intellectual property rights are involved. The major elements associated with each stage of the development of patents are described including the following: the soybean genotype, seed disinfection, genetic construct design and its synthesis, tissue culture protocols, selection strategy without gene reporter, and Agrobacterium strain. This review is a guide for carrying out technical procedures when the desired product is the off-patent GM soybean with tolerance to glyphosate.

Transient gene expression is far superior to stable gene expression in terms of labor time efficiency and is well suited for the study of plant promoters, protein localization, protein–protein interactions, or binary vectors for genome editing. This study investigated the factors affecting the efficiency of transient gene expression in Panax ginseng by analyzing green fluorescent protein (GFP) fluorescence intensity under a confocal laser microscope. The influences of variations in Agrobacterium strain and suspension density, acetosyringone concentration, number of vacuum applications, infiltration buffer composition, and shoot developmental stage on transformation efficiency of P. ginseng leaf parenchyma cells were investigated. We found that the A. tumefaciens EHA105 strain is best suited for P. ginseng transformation, and the highest expression level was observed at the suspension density OD600 = 1.0. The addition of acetosyringone greatly increases transformation efficiency, but only if it does not exceed a concentration of 100 μM. A special mode of physical treatment, which includes five consecutive applications of a relatively deep vacuum, further increased the transformation efficiency. In addition, the use of MES buffer at pH 5.6 and mature leaves for transformation allowed us to achieve the highest level of transit expression, while more than 80% of leaf parenchyma cells were transformed. The optimized transient transformation method allowed us, for the first time, to evaluate the effect of rol gene expression on the accumulation of ginsenosides in P. ginseng leaves. Our study is the first work aimed at the optimization of transient ginseng transformation, and the developed protocol will be of particular importance for ginseng physiology research and modulation of valuable secondary metabolite biosynthesis. An optimized protocol for the transient transformation of Panax ginseng shoots is described, which allows fast transformation with high efficiency. The optimized method allowed us, for the first time, to evaluate the effect of rol gene expression on the accumulation of ginsenosides in P. ginseng leaves.

Genetic improvement of local cotton for tolerance against heat and other abiotic stresses is urgently needed due to climatic changes. Agrobacterium mediated transformation of G. hirsutum was done with heat shock protein gene (AsHSP70) and plants were analyzed for transgene expression at molecular, physiological and biochemical levels under heat stress. Genetic transformation revealed 1.9 % transformation efficiency. PCR amplified 1800 bp fragment of AsHSP70 in genomic DNA of transgenic plants. Polyamine oxidase induced the relative expression of AsHSP70 from 1.02 to 9.58 in transgenic plants as the duration of heat stress was increased. Expression of AsHSP70 was relatively higher in the leaves of transgenic plant as compared to root and stem under combined stress of heat/drought. Cell electrolyte leakage was 39.83 μS cm−1 and 63.43 μS cm−1 in transgenic and control plants respectively. Relative conductivity (4.05 %) was correlated with the membrane stability index (MSI) (97.19 %) in transgenic plants while it is 7.28 % when MSI is 90.19 % in control plants. Membrane injury was 19–52 % in control and 15.6–29 % in transgenic plants over the time of exposure to heat stress. Significant variation in chlorophyll, proline and soluble sugar content was observed in transgenic plants as compared to controls. Performance of transgenic plants was better for boll formation in the field. Single copy of transgene was detected in T1 progeny. Hence, transgenic cotton plants are tolerating heat and other abiotic stresses at physiological and biochemical levels. Single analyses of progeny may lead the selection of pure heat tolerant lines to be used in breeding program for cotton improvement under heat stress.

Sugarcane (Saccharum hybrid) is an important industrial crop worldwide. Its growth and sucrose contents are severely affected by drought stress. Genetic engineering offers a rapid solution to improve tolerance level of sugarcane against this stress. This study was designed to transform sugarcane with the Tomato ethylene responsive factor 1 (TERF1) gene through Agrobacterium. Embryogenic callus of sugarcane cv. XintaitangR22 was used for transformation with Agrobacterium strain LBA4404 harbouring the pROK2 vector containing the TERF1 gene driven by the CaMV 35S promoter. Highest regeneration efficiency (74%) was obtained with inoculum density (OD600) at 0.4 and co-cultivated for 4 days on MS-based medium; 5.4% transformation efficiency was acquired from the regenerated plants. Successful insertion of the TERF1 gene into sugarcane was indicated by PCR-positive plants (n = 4). Expression of TERF1 transcripts in transgenic lines at various levels was detected by reverse transcriptase-PCR. Under normal conditions, growth status of transgenic lines was similar to that of wild-type plants; by contrast, only transgenic lines were able to withstand water-deficit stress conditions, showing tolerance against drought stress. Physiological and biochemical assays revealed that TERF1-overexpressed plants showed not only increased accumulation of proline, soluble sugars and glycine betaine but also reduced malondialdehyde and H2O2 content in response to drought stress. Our results revealed that overexpression of TERF1 in sugarcane conferred drought tolerance through increased accumulation of osmo-protectant, decreasing reactive oxygen species and malondialdehyde content, which possibly resulted from activation of expression of stress-related genes by TERF1 under stress. These findings indicate that the gene might have a regulatory role in the response to drought stress in sugarcane.

Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

Wheat (Triticum aestivum L.) is a major food crop worldwide whose complex and very large genome hinders stable genetic transformation. An optimized method for transformation by particle bombardment is therefore greatly needed, both in general and for Korean commercial wheat varieties, which have not yet been successfully transformed. In this study, we describe the stable transformation of the Korean commercial wheat variety Keumkang and the DH20, which lacks the Glu-B3 and Gli-B1 loci (both related to gluten production), via particle bombardment of a vector harboring the enhanced green fluorescent protein (eGFP) gene. We confirmed the insertion of the eGFP transgene into the genome of primary (T0) transformants by genomic PCR, RT-qPCR, and fluorescence imaging. Our estimated average transformation efficiencies were 4.4% for Keumkang and 3.5% for DH20 in the T0 generation. We also validated the stable expression of the transgene in the T1 generation. We hypothesize that donor plant health, centrifugation, and high-sucrose pre-treatment increased the transformation efficiency reported here. Taken together, these results describe a useful method for transforming Korean commercial wheat varieties, including Keumkang and DH20, that will allow the manipulation of gene expression via such techniques as RNA interference, overexpression, and genome editing.

Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue culture independent protocol for floral transformation in bread wheat was developed. Three bread wheat genotypes namely, HD 2967, HD 3086 and Bobwhite were used for floral transformation. At pre-anthesis stage, clipped florets were sprayed with inoculation medium containing Agrobacterium tumefaciens strain EHA 105 culture harboring binary vector pCAMBIA1301-p35S:hptII-p35S:gus and allowed to grow till maturity. Positive transformants were confirmed through antibiotic selection, PCR analysis and southern blotting. The method led to generation of transformation events in Bobwhite and HD 2967 wheat genotypes. The transformation efficiency of T1 transgenic plants was higher in Bobwhite as compared to HD 2967. Floral spray method of transformation developed in the present study is efficient for development of transgenic wheat and can be used for improvement of wheat using transgenic approach.

Aim: The current study was carried out to develop transgenic cotton plantlets with glyoxalase I (gly I) gene using Agrobacterium-mediated transformation. Methodology: Seeds of cotton were inoculated on MS medium and the explants such as shoot tip (3-5 mm), hypocotyl and leaf were aseptically removed from in vitro plantlets. The pre-cultured and infected explants with Agrobacterium harboring gly I gene and the shoot tip were inoculated on MS media for shoot initiation and subcultured on elongation medium with growth hormones, and antibiotics. Healthy and well-grown shoots were subcultured on medium with indole butyric acid (IBA) (0.3 mgl-1) for root formation and the plantlets were hardened in plastic cups with sterile soil. The putative transgenic plantlets were analyzed histochemically for gus gene and the PCR analysis was performed for gly 1 gene. Results: The transformation efficiency of cotton ranged 48.57 to 64.53 %. The regenerated plantlets showed the presence of gus gene in terms of blue coloration in shoots, whole leaf and leaf segments. The PCR was performed in putative transgenic plant lets with both gus gene as well as gly I gene primers. The PCR results showed the presence of 1031 bp DNA band with gus gene primers and 800 bp DNA band with the gly I gene primers. Interpretation: The current study has proven the reproducible procedure for the Agrobacterium-mediated gene transfer and regeneration of Indian cotton varieties. The PCR results revealed the presence of glyoxalase I gene in the transformants. Key words: Cotton varieties, Glyoxalase I gene, PCR analysis, Regeneration, Transformation

Sugarcane is a prominent source of sugar and ethanol production. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, long breeding cycle, and recalcitrance to genetic transformation. The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis; however, quick and effective transformation systems are still lacking for sugarcane. Here, we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S. spontaneum. The yield of viable protoplasts was approximately 1.26 × 107 per gram of leaf material, and the transformation efficiency of 80.19% could be achieved under the optimized condition. Furthermore, using this approach, the nuclear localization of an ABI5-like bZIPs transcription factor was validated, and the promoter activity of several putative DNase I hypersensitive sites (DHSs) was assessed. The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays. A highly efficient S. spontaneum mesophyll cell protoplast isolation and transient transformation method was developed, and it shall be suitable for in vivo functional gene analysis in sugarcane.

Chlamydomonas reinhardtii is a model organism with three sequenced genomes capable of genetic transformation. C. reinhardtii has the advantages of being low cost, non-toxic, and having a post-translational modification system that ensures the recombinant proteins have the same activity as natural proteins, thus making it a great platform for application in molecular biology and other fields. In this review, we summarize the existing methods for nuclear transformation of C. reinhardtii, genes for selection, examples of foreign protein expression, and factors affecting transformation efficiency, to provide insights into effective strategies for the nuclear transformation of C. reinhardtii.

Moso bamboo (Phyllostachys edulis) and Ma bamboo (Dendrocalamus latiflorus Munro) are two major species, which have significant economic and research application value. At present, methods for interrogation of gene function in bamboo are limited and time consuming. Therefore, establishing an efficient transient expression system for bamboo is very useful for dissecting the function of candidate genes. In this study, we developed protocols for the transient transformation of protoplasts from different tissues in Moso bamboo and Ma bamboo and established the optimized protocol and materials for improving the efficiency of transient transformation. The results indicated that the transformation efficiency of protoplasts from 15-day-old etiolated seedlings was higher than other tissues, which were 44.7% in Moso bamboo and 35.2% in Ma bamboo. In addition, we also established an experimental approach for optimizing overexpression of external proteins using Agrobacterium-mediated transient transformation of bamboo leaves and whole seedlings. Finally, we utilized the RUBY reporter for monitoring successful transformation samples with red color for downstream experiments in Moso bamboo. In summary, this study provides optimized protocols for the over-expression of candidate genes in protoplasts or bamboo leaves and whole seedlings.

The recent advances in genome-wide transcriptome analysis have enabled researchers to identify new genes and manipulate the genome through novel genetic engineering methods to improve plant tolerance to different stresses. We conducted a genome-wide transcriptome analysis to determine tomato genes affecting the salt stress response. The results showed that among the 5784 genes that were responsive to salt stress, 103 genes (1.8%) encode transcription factors, of which 69 (1.2%) were upregulated and 34 (0.6%) were downregulated. The largest group of genes upregulated in response to salt stress (17 genes) is related to the ethylene response transcription factors (ERF) family. Specifically, it was found that the gene JERF1, encoding ERF in tomato, is upregulated in response to salt stress. In the present study we developed a new Agrobacterium-mediated transformation method to deliver the JERF1 gene to mature wheat embryos. It was investigated whether the factors vacuum infiltration, Agrobacterium cell density (OD600 1.0 and its dilutions 1:20, 2:20 and 3:20), the acetosyringone concentration (0, 200 and 400 μM) and the interaction between these factors affect transformation. To this end, Agrobacterium carrying pGWB14-JERF1 was injected into soaked wheat seeds, and T0 transgenic plants were obtained in the greenhouse. Molecular analysis of T0 plants was performed. The highest transformation efficiency was obtained under vacuum infiltration, 200 μM acetosyringone and 1:20 dilution. Notably, we used the in planta transformation method to overcome some of the problems of traditional wheat transformation methods e.g., sterile conditions, recalcitrant regeneration, time-constraints and somaclonal variations.

Violacein is a naturally occurring anticancer therapeutic compound with deep purple color. In this work, we harnessed the modular and combinatorial feature of a Golden Gate assembly method to construct a library of violacein producing strains in the oleaginous yeast Yarrowia lipolytica, where each gene in the violacein pathway was controlled by three different promoters with varying transcriptional strength. After optimizing the linker sequence and the Golden Gate reaction, we achieved high transformation efficiency and obtained a panel of representative Y. lipolytica recombinant strains. By evaluating the gene expression profile of 21 yeast strains, we obtained three colorful compounds in the violacein pathway: green (proviolacein), purple (violacein), and pink (deoxyviolacein). Our results indicated that strong expression of VioB, VioC, and VioD favors violacein production with minimal byproduct deoxyvioalcein in Y. lipolytica, and high deoxyviolacein production was found strongly associated with the weak expression of VioD. By further optimizing the carbon to nitrogen ratio and cultivation pH, the maximum violacein reached 70.04 mg/L with 5.28 mg/L of deoxyviolacein in shake flasks. Taken together, the development of Golden Gate cloning protocols to build combinatorial pathway libraries, and the optimization of culture conditions set a new stage for accessing the violacein pathway intermediates and engineering violacein production in Y. lipolytica. This work further expands the toolbox to engineering Y. lipolytica as an industrially relevant host for plant or marine natural product biosynthesis.

Whenever there is no adequate DNA replication in vitro, there are alternatives strategies to insert a piece of DNA into a convenient replicon such as small plasmids and bacteriophages. These are called vectors or cloning vehicles. These days, there is a high demand to manufacture recombinant and nanobodies which are required in biomedical research and for therapeutic and diagnostic purposes. In order to do so, E. coli, insect cells, or mammalian cells have been used to express and purify protein for nanobody production. This paper explains the experimental trials on the cloning of the Nanobody cDNA mock-up in pHEN6c plasmid vector from the subcultured E. coli sample taken from the lab. The concentration and purity of DNA plasmid were evaluated by UV spectrophotometer and agarose gel electrophoresis following plasmid DNA Purification from E. coli by alkaline lysis. Based on this, the concentration of the isolated pHEN6c plasmid DNA was found 44.7 ng/μl or 0.0447 μg/μl. Whereas, the purity at absorbance (A260/A280) was 0.893/0.501 = 1.78. Moreover, its yield was 2.235 μg. In addition, its transformation efficiency was 21.68 μg/μl. On the other hand, the molecular weight of the Nanobody and vector were 569 and 2717 respectively. Generally, most of the protocols used to clone a fragment of DNA, might not work very well with PCR products.

Escherichia coli strain K-12 MG1655 has been proposed as an appropriate host strain for industrial production. However, the direct application of this strain suffers from the transformation inefficiency and plasmid instability. Herein, we conducted genetic modifications at a serial of loci of MG1655 genome, generating a robust and universal host strain JW128 with higher transformation efficiency and plasmid stability that can be used to efficiently produce desired chemicals after introducing the corresponding synthetic pathways. Using JW128 as the host, the titer of isobutanol reached 5.76 g/L in shake-flask fermentation, and the titer of lycopene reached 1.91 g/L in test-tube fermentation, 40-fold and 5-fold higher than that of original MG1655, respectively. These results demonstrated JW128 is a promising chassis for high-level production of value-added chemicals.

Passiflora cincinnata stands out among Passifloraceae because of its medicinal properties and its resistance to pathogens, which promotes its use as a rootstock for other Passiflora species. A valuable strategy for obtaining disease-resistant passion fruit plants is represented by genetic transformation; however, this requires efficient regeneration. Here, we aimed to establish an efficient protocol for generating transgenic passion fruit plants using sonication-assisted Agrobacterium-mediated transformation of somatic embryos. The latter were obtained from anthers, sonicated, and exposed to an Agrobacterium suspension for 15 or 30 s. As a comparison, non-sonicated embryos were also exposed to the same bacterial treatment, whereas non-infected embryos were used as controls. Plant identity was confirmed by PCR, qPCR, and histochemical assay. Transgenic plants were obtained at higher rates in the treatments applying sonication. Overall, 171 plantlets were regenerated, 38 of which showed stable uidA reporter gene expression. Of these 38 transgenic plants, 22 (57.89%) and 13 (34.21%) were obtained by sonication-assisted Agrobacterium-mediated transformation for 30 s and 15 s, respectively, whereas the remaining 3 (7.89%) were exposed for 30 s but without prior sonication. Our results indicate that sonication-assisted Agrobacterium-mediated transformation for 30 s enhanced transformation efficiency in P. cincinnata. We believe that this system will allow for more efficient production of transgenic passion fruit plants.

Mosquitoes constitute the major living beings causing human deaths in the world. They are vectors of malaria, yellow fever, dengue, zika, filariases, chikungunya, among other diseases. New strategies to control/eradicate mosquito populations are based on newly developed genetic manipulation techniques. However, genetic transformation of mosquitoes is a major technical bottleneck due to low efficiency, the need of sophisticated equipment, and highly trained personnel. The present report shows the transgenerational genetic transformation of Aedes aegypti, using the particle inflow gun (PIG), by integrating the ecfp gene in the AAEL000582 mosquito gene with the CRISPR-Cas9 technique, achieving a mean efficiency of 44.5% of bombarded individuals (G0) that showed ECFP expression in their tissues, and a mean of 28.5% transformation efficiency measured on G1 individuals. The same transformation technique was used to integrate the egfp/scorpine genes cloned in the Minos transposon pMinHygeGFP into the Anopheles albimanus genome, achieving a mean efficiency of 43.25% of bombarded individuals (G0) that showed EGFP expression in their tissues. Once the technique was standardized, transformation of Ae. aegypti neonate larvae and An. albimanus eggs was achieved when exposed to gold microparticle bombardment. Integration of genes and heterologous protein expression were confirmed by PCR, sequencing, fluorescent microscopy, mass spectrometry, Western blot and dot blot analyses. Transgenerational inheritance of the transgenes was observed only on Ae. aegypti, as all transformed An. albimanus individuals died at the pupal stage of the G0 generation.

Stevia rebaudiana Bertoni is a commercially important zero calorie natural-sweetener herb which produce sweet compounds known as steviol glycosides. Rising demands of steviol glycosides by food and beverage industries has led to an increase in its cultivation in various countries. Unfortunately, stevia cultivation faces 2–25% yield penalty due to weeds which further adds to its cultivation cost. To resolve this major challenge, Agrobacterium-mediated genetic transformation of in vitro derived stevia-nodal explants using herbicide resistance gene (bar) has been optimized, for the production of stable transgenic stevia plants. Several parameters including explant type, pre-incubation duration, acetosyringone (As) concentration, Agrobacterium cell density, Agro-inoculation duration, co-cultivation duration, selection regime and plant growth regulators (PGRs) combination and concentration, have been successfully optimized. Among the two types of explants used, nodal explants showed a higher regeneration response of 82.85%, with an average of 25 shoots/explant. The best PGRs combination and concentration for shoot-induction, shoot-elongation and root-induction was found to be 6-benzyladenine (1.0 mg l−1) + naphthalene acetic acid (0.5 mg l−1), gibberellic acid (1.0 mg l−1), and half-strength MS medium, respectively. The two-step selection (phosphinothricin) regime resulted in an average transformation efficiency of 40.48% with nodal explants. Molecular characterization of putative transformants through PCR, RT-PCR, qRT-PCR and Southern-blot hybridization confirmed the presence, stability, expression as well as copy number of bar gene respectively. Compared to the non-transgenic plants, the T0 transgenic plants successfully tolerated 8 mg l−1 glufosinate ammonium sprays. Thus, the optimized protocol can be useful for the introduction of other genes (inter-kingdom transfer) into stevia genome.

Genetic transformation of recalcitrant species, like sugar beet, has been always a challenging task. In this study, a selectable marker gene (phosphomannose isomerase) and a reporter gene (synthetic green fluorescent protein) were transferred to sugar beet via Agrobacterium-mediated transformation by using sand-wounded shoot and petiole explants of two different diploid sugar beet genotypes. Considering the overall means of genotypes and explant types, a clear genotypic variation was evident as ELK345 produced a mean of 5.7% transient expression and 1.97% stable transformation efficiency while it was 4.3% and 1.28% for the genotype M1195. When explant types were compared independently from other parameters, it was shown that petiole explants were more productive than shoot explants, with an overall mean of 5.3% versus 4.7% and 1.77% versus 1.48% transient expressions and stable transformation efficiencies, respectively. On the other hand, when a comparison was made without taking into account the genotypes, the effect of wounding treatment on transient expression resulted in about 25% (4.7% vs. 5.9%) and 20% (4.3% vs. 5.1%) increases in petiole and shoot explants, respectively. Such a promoting effect of wounding was even more prominent when the stable transformation efficiencies were compared; petiole explants achieving almost a 60% increase (1.38% vs. 2.16%) while shoot explants doubled (0.98% vs. 1.97%) their efficiencies. The optimized protocol reported here can be employed for further increasing the transformation efficiency in recalcitrant plant species including sugar beet. The advantages of using GFP as a reporter and mannose as a positive selection are also discussed.

The objective of this research was introduction of chit42 to tuber mustard plants through Agrobacteriummediated transformation against white mold caused by Sclerotinia sclerotiorum. The binary plasmid pGisPEC1 was used in this study. Polymerase chain reaction analysis detected the transgene in 27 transformants with a transformation efficiency of 6.9%. Southern blot test was used to assess the copy number of transgene in tuber mustard plants. One, two, two, and two chit42-related bands were observed in the transformed lines TMB4, TMB7, TMB12, and TMB18, respectively. Enzymatic tests showed a significant increase in the activity of endochitinase in protein isolated from leaf tissues of chit42 transgenic 75-day tuber mustard lines. The pathogenicity of three pathogen isolates was tested on the leaves of transformed plans. The results of current study showed that expression of the gene chit42 in tuber mustard plants markedly reduced infection radius on the leaves 7 days after inoculation with the fungus.

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.

Collapsed sweet potato vines observed in Hangzhou fields in September 2019 were found to be due to Sclerotinia sclerotiorum based on its morphology and ITS rDNA sequence. Here, the chitinase gene chit42 from Trichoderma harzianum was introduced into Ipomoea batatas using Agrobacterium-mediated transformation to evaluate the effect against subsequent disease severity from S. sclerotiorum. PCR analysis detected the transgene in 23 transformants with a transformation efficiency of 5.1%. RT-PCR analysis showed transgene mRNA expression in all four selected lines harboring chit42. Enzymatic tests showed a significant increase in the activity of endochitinase in extracts from leaf tissues of chit42-transgenic 75-day sweet potato lines. Virulence of three genetically different isolates was markedly reduced on leaves of 75-day-old chit42-transgenic plants compared with the controls. To our knowledge, this is the first report of S. sclerotiorum on sweet potato plants in China.

Poly(ADP-ribose) polymerases (PARP) are involved in the regulation of plant growth and development, especially under stress conditions. These enzymes post-translationally modify target proteins by adding ADP-ribose polymers. This study presents the effect of PARP chemical inhibitor 3-methoxybenzamide (3 MB) on in vitro plant growth, microtuber formation, and Agrobacterium-mediated transformation of blue potato (Solanum tuberosum L. subsp. andigenum). The addition of 3 MB (0.2 to 0.6 mM) significantly increased the growth and microtuber formation of in vitro propagated plants. The highest gain in plant fresh weight, root mass, and microtuberization was observed in clonal propagation medium with 0.4 mM 3 MB. Transformation frequency of blue potato internodes increased to 43%, 2.7-fold over the non-treated control (16%), when 0.2 mM 3 MB was included in the culture medium during the whole process. The addition of 3 MB during Agrobacterium transformation did not affect transgene copy number in regenerated plants. Southern blot analyses and histochemical staining for s-glucuronidase activity confirmed the presence and expression, respectively, of the uidA transgene in plant tissues. From these results, it can be concluded that the inhibition of PARP activity can increase biomass production and transformation rates for genetic improvement of blue potato.

The glycoprotein of infectious hematopoietic necrosis virus (IHNV), the causative agent of acute disease in salmonids, is the only structural protein of the virus that can induce protective immunity in the fish host. Here, the reliability of bean (Phaseolus vulgaris) plant for the production of this viral protein was examined by the transient expression method. Using the syringe agroinfiltration method, leaves of bean plants were transformed with the expression construct encoding the full-length of IHNV glycoprotein (IHNV-G) gene. Furthermore, the transformation efficacy of two infiltration buffers including PBS-A (PBS+acetosyringone) and MMS-A (MES buffer + MgSO4 + sucrose + acetosyringone) was compared. The analysis of mRNA and dot-blot assay confirmed the transcription and translation of IHNV-G protein in bean leaves. Moreover, Western blotting verified the production of intact, full-length (∼57 kDa) IHNV-G protein in the agroinfiltrated plants. Of note, the production level of IHNV-G using MMS-A agroinfiltration buffer was approximately five times higher compared to PBS-A buffer (0.48 vs. 0.1% of total soluble protein), indicating the effect of infiltration buffer on the transient transformation efficiency. The recombinant protein was purified at the final yield of 0.35 μg/g of fresh leaf tissue, using nickel affinity chromatography. The present work is the first report describing the feasibility of the plant expression platform for the production of IHNV-G protein, which can be served as an oral vaccine against IHNV infection.

Scutellaria orientalis and S. araxensis are perennial species of genus Scutellaria (Lamiaceae) which produce some important pharmaceutical secondary metabolites. Four strains of Agrobacterium rhizogenes (A4, A13, ATCC15834 and MSU440), three types of explant (stem, petiole and leaf), six concentrations of acetosyringone (25–150 μM), two different co-cultivation methods (light and dark condition) and two co-cultivation media (full and half-strength MS) were used for optimization of A. rhizogenes mediated transformation efficiency. Moreover, two inoculation approaches including immersion and injection as well as three infection period (5, 7 and 10 min) were examined. The molecular confirmation analysis of putative transgenic roots was performed by PCR using specific primers of the rol B gene. The results revealed that the transformation efficiency was affected by bacterial strain, co-cultivation medium and type of explants. The highest transformation frequency of 93.3% was achieved on stem explants of S. orientalis with ATCC15834 strain, followed by MSU440, A13 and A4 with the frequency of 63.3%, 46.6% and 30%, respectively. In S. araxensis, the best transformation frequency of 76.6% and 43.3% were obtained on stem and petiole explants with MSU440 strain. 5 and 7 min for infection period significantly improved the transformation efficiency in S. orientalis and S. araxensis respectively. Transformation rates of stem explants induced by ATCC15834, A4 and A13 strains were 53.3%, 36.6% and 20% respectively. Transformation frequency was significantly higher in half-strength MS, regardless of A. rhizogenes strains.

Apple is one of precious fruit crop grown in temperate zone. In the post genomic era, the analysis of gene functions in horticultural crops such as apple is required for agricultural utilization. For analysis of such crops, the protocol establishment of tissue culture and transformation is essential. Although transformation efficiency in family Rosaceae is generally very low, some cultivars of Malus species have high transformation ability. Apple cultivars are usually clonally propagated by grafting on rootstocks, which can affect fruit quality and maturity and scion productivity. Apple rootstock cultivar Japan Morioka 2 (JM2) was produced at the Division of Apple Research, Institute of Fruit and Tea Science, NARO, in Japan. JM2, which was developed for dwarfing scions and improving disease resistance, is easily propagated by hardwood cutting. Furthermore, JM2 can be stably transformed at a high efficiency, which is better than other JM series rootstocks derived from the same parent. Leaflets of cultured shoots of JM2 have been transformed using Agrobacterium (Rhizobium) with a transducing gene. In this article, the JM2 transformation protocol is introduced in detail. Various genes and promoters have been confirmed to function as expected, with the resultant transformants exhibiting specific staining and fluorescent signals, and modified floral organ shapes, precious blooming and other characteristics. JM2 is thus a useful rootstock material for the enhancement of genetic research on apple and its relatives.

The tea plant (Camellia sinensis) contains various metabolic substances, including catechins and caffeine, for which genetic transformation techniques are essential for investigating the associated metabolic pathways. In this study, we sought to optimize the conditions and culture process for particle bombardment-mediated transformation of tea plant somatic embryos. We describe somatic embryo pretreatment for effective transient transformation in biolistic bombardment and the posttreatment conditions of somatic embryos for accelerating differentiation after bombardment. For the purpose of transformation, we used the somatic embryos of C. sinensis var. assamica ‘Tingamira normal,’ which were cultured in Murashige and Skoog (MS) medium containing 2 mg l−1 indole-3-butyric acid (IBA) and 4 mg l−1 6-benzyladenine (BA) at 25°C ±2°C under a 16-h photoperiod. With respect to the optimization of particle bombardment conditions for tea somatic embryos, we examined the effects of different Au colloid particle diameters and bombardment pressures, and accordingly established bombardment with 1.0-µm-diameter DNA-coated Au colloid at 1,100 psi as optimal conditions for introducing DNA for the transient expression of GUS. Additionally, we found that transplantation of tea somatic embryos from IBA/BA medium to a hormone-free medium prior to bombardment and incubation in the dark post-bombardment increased the frequency of secondary embryo production. Furthermore, osmotic treatment by culturing the somatic embryos in medium supplemented with 0.4 M mannitol improved transient transformation efficiency. After transformation, the culture of somatic embryos on filter papers or Kimwipes soaked in MS medium facilitated rapid and effective development of the somatic embryos.

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

Cabbage and cauliflower are extensively cultivated Cole vegetables all around the world. These are highly prone to the most notorious insect pest Plutella xylostella (diamondback moth), causing huge yield losses to the growers. Conventional breeding for host plant resistance against this pest is limited due to lack of resistance source in the available germplasm. The chemical control measures are cumbersome, costly, health hazardous and environment unfriendly. Therefore, using quaternary gene pool for incorporation of resistance genes from unrelated sources such as bacterial derived insecticidal Bt toxins through genetic engineering is a viable option. Genetic manipulation of plants through Agrobacterium-mediated transformation is limited by a multitude of factors resulting in poor transformation efficiency. Hence, optimization of plant transformation method suitable for a particular crop is highly essential. Here in we report the optimization of plant transformation protocol for cabbage and cauliflower by the manipulation of various key parameters, including the type of explants, Agrobacterium strains, bacterial cell density, age of the explant, preculture period, media formulations etc. The binary vector pPIPRA560 containing stacked Bt genes (Cry1B/Cry1C) and npt II/Basta as selectable marker was chosen for plant transformation. As a proof of concept, initially the effectiveness of the Bt genes against diamondback moth was validated in transgenic Arabidopsis. Insect bioassay revealed 100% mortality within 48 h of feeding. Based on the exciting results, the same construct was used for transforming Indian popular cultivars of cabbage (‘Golden Acre’) and cauliflower (‘Pusa Meghna’ and ‘Pusa Snowball K1’). The presence of the Bt genes in the transgenic lines was confirmed by PCR and expression through RT-qPCR. Insect bioassay revealed 100% mortality in some of the plants tested so far.

Electrotransformation was successfully transferred a 21.6 kb cosmid vector pLAFR1 into the selected Plant Growth-Promoting Rhizobacteria (PGPR) and Chromobacterium violaceum with maximum efficiency about 107 transformants/µg at electric field strength of 10kV/cm for shorter pulse length range between 4.1 to 4.4 milliseconds and about 108 transformants/µg at electric field strength of 5kV/cm for a longer pulse length range between 8.5 to 8.7 milliseconds. Longer incubation period will increase the transformation efficiency but continuing increasing the period after 20 hours will decrease the transformation efficiency.

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

A tissue culture independent in-planta transformation technique is an alternate option to generate transgenics in crop cultivars recalcitrant for regeneration. The T0 plants generated by in-planta technique are chimeric hence very large number of T1 seeds needs to be screened to identify the putative transformants. Besides, low transformation efficiency and stable integration is another lacuna, because the T0 seedling explants are not subjected for selection. To overcome these constraints, a modified in-planta technique was developed in this study to generate gain-in-function activation tagged mutants in the background of a recalcitrant rice genotype, KMP-153. A binary vector was developed with three glyphosate selectable marker genes and 4X CaMV 35S enhancer elements for developing rice plants. Simultaneous expression of Glyphosate insensitive CP4-EPSPS with two other glyphosate degrading enzymes, PsAKR1 (Aldo–Keto reductases) and GO (Glycine Oxidase) substantially improved tolerance up to 100 ppm of glyphosate in the seedling bioassay system. Selecting the seedling explants and T1 generation seeds at higher concentration (100 ppm) of glyphosate resulted in recovering fewer transformants but with stable integration and expression of all the transgenes compared to those screened at lower levels of glyphosate. Further, T2 generation plants from these transgenic lines grown in contained nethouse conditions were tolerant when sprayed up to 1500 ppm concentrations of glyphosate. These transgenics showed substantial lower levels of shikimic acid compared to wild type plants treated with glyphosate signifies the expression of the transgenes and less inhibition of shikimic acid pathway by glyphosate. The proteins extracted from the transgenic plants showed significant degradation of glyphosate. The stable integration was confirmed through flanking sequence information and event specific PCR information. Our results clearly demonstrate that the modified in-planta technique is a potential transformation protocol to generate stable transformants in rice cultivars.

Lithospermum erythrorhizon, a medicinal plant growing in Asian countries, produces shikonin derivatives that are lipophilic secondary metabolites. These red naphthoquinone pigments are traditionally used as a natural drug and a dye in East Asia. In intact L. erythrorhizon plants, shikonin derivatives are produced in the root epidermal cells and secreted into extracellular spaces. The biosynthetic pathway for shikonin derivatives remains incompletely understood and the secretion mechanisms are largely unknown. Understanding the molecular mechanisms underlying shikonin biosynthesis and transport in L. erythrorhizon cells requires functional analysis of candidate genes using transgenic plants. To date, however, standard transformation methods have not yet been established. This study describes an efficient method for L. erythrorhizon transformation using hairy roots by Rhizobium rhizogenes strain A13, present domestically in Japan. Hairy roots of L. erythrorhizon were generated from explants of the axenic shoots that were infected with R. rhizogenes strain A13. Integration into the genome was assessed by PCR amplifying a transgene encoding green fluorescent protein (GFP) and by monitoring GFP expression. This method enhanced transformation efficiency 50-70%. Although methods for the systematic stable transformation of L. erythrorhizon plants have not yet been reported, the method described in this study resulted in highly efficient stable transformation using hairy roots. This method enables the functional analysis of L. erythrorhizon genes.