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Oryza sativa ssp. indica is not amenable to genetic modifications owing to its recalcitrant nature, rendering it difficult to transform and recover improved varieties to combat the persisting biotic and abiotic stress conditions. Therefore, in the present study, we have attempted to enhance transformation efficiency by applying polyamines and vacuum infiltration in Agrobacterium tumefaciens-mediated genetic transformation of Indica rice cv. IR64 seed-derived embryogenic calli. The highest number of regenerating calli, with maximum number of shoots per callus, was achieved in regeneration medium supplemented with 0.2 mM Spermidine, and the regenerated shoots displayed improved rooting when cultured on MS medium supplemented with 0.1 mM Putrescine. Further, vacuum infiltration of calli for 4 min resulted in the highest transformation efficiency of 8.1% as evident by GUS positive calli. The transformed calli were screened on the selection medium supplemented with hygromycin, and the integration and expression of the T-DNA in the rice genome were confirmed by GUS histochemical assay, PCR and Southern hybridization. The outcome of this study would be useful in micropropagation and genetic transformation studies of recalcitrant rice varieties. [ABSTRACT FROM AUTHOR]

One of the fundamental techniques in genetic engineering is the creation of Escherichia coli competent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5α competent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA‐deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA‐deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA‐deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET‐32a, and p1304, with enhancements of 2.23, 2.24, and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells. [ABSTRACT FROM AUTHOR]

The latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efficiencies which necessities screening of large number of transgenic plants to find the suitable edits. In the present study, we have simplified the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76–78 days. We also obtained about 90% regeneration efficiency for both Japonica and Indica cultivars. The transformation efficiency was about 97% in the case of Japonica and 69–83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efficiency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efficiency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars. [ABSTRACT FROM AUTHOR]

Mungbean, a socioeconomically important legume, is a key player in the pursuit of food security and sustainable agriculture demanding attention for developing a highly efficient transformation system to support functional genomics and translational research. This study was focused to fill in the gap using both Agrobacterium-mediated and biolistic methods by experimenting with variables of the procedure to attain higher regeneration and transformation efficiencies. The study found comparable regeneration and transformation efficiencies between direct and indirect methods of gene transfer under selection pressure. Biolistic transformation of embryonic axis explants yielded an average transformation efficiency of 14.22% alongwith the one step regeneration method, whereas the Agrobacterium-mediated method involving a selection regime achieved an average efficiency of 1.19%. Presence and transmission of transgene across two generations were confirmed by PCR and Southern blot analyses in five mungbean lines. Two southern positive homozygous transgenic lines (T3 generation), when under PEG (Polyethylene glycol) induced drought stress, revealed AtDREB1a transgene expression and tolerance to the stress that was evaluated by morpho biochemical parameters including relative water content (RWC), membrane stability index (MSI), proline concentration and total chlorophyll content of the investigated lines. This study documents a first report of use of biolistic in mungbean supported by a one-step regeneration method that minimizes losses incurred during in vitro based regeneration of transformants and thus enhances the transformation efficiency. It also reports generation of transgenic mungbean in cultivar Samrat that is tolerant to PEG induced drought stress.Key message: We compared the biolistic and Agrobacterium-mediated modes of genetic transformation in mungbean highlighting the bottlenecks of the latter. AtDREB1a overexpression conferred drought stress tolerance to T3 transgenic lines. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This article discusses the optimization of an ex vitro one-step hairy root transformation method in drug- and hemp-type cannabis using the RUBY system. Cannabis is a versatile plant species that has gained attention in medical research and industry due to recent legalization. The concentration of the cannabinoid Δ9‐tetrahydrocannabinol (THC) determines the legal categorization of cannabis as hemp-type or drug-type. The study presents a new method for transforming cannabis roots using the RUBY system, which allows for the visual identification of transformed roots. The results show that the transformation efficiency varied based on the bacterial strain and seed type, with drug-type cannabis exhibiting higher transformation rates. This ex vitro method offers simplicity, speed, and reduced contamination risk, making it suitable for the efficient production of secondary metabolites in cannabis. [Extracted from the article]

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However, the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated (ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches (one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings (Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%–51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin. The one-step method outperformed the two-step method; the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily, quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

[Objective] We aim to establish an efficient and stable genetic transformation system of Lycium ruthenicum and reduce the vitrification rate of the regenerated seedlings in order to promote gene function study and genetic improvement. [Methods] L. ruthenicum leaves were used as explants and Agrobacterium (LBA4404, EHA105) was used to transform L. ruthenicum. By adjusting the types of basic medium and adding plant hormones, we selected the optimal callus-induction medium, differentiation and selection medium, and rooting-induction medium. The transformation rate of L. ruthenicum was increased to over 65%, while the seedling vitrification rate was decreased to below 10%. This combined culture system laid a foundation for the molecular breeding of L. ruthenicum. [Results] (1) The optimal infection concentration of Agrobacterium (OD600) was 0.6 and the infection time was 25 min for the combined culture system of L. ruthenicum. Under this condition, the callus-induction rate was 78.2%-96%; (2) The optimal differentiation and selection medium contained: MS+inositol 50 mg/L+nicotinic acid 0.25 mg/L+vitamin B6 0.25 mg/L+Fe salt storage solution 1 mL/L+glycine 1.0 mg/L+ vitamin B1 0.05 mg/L+6-BA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L+Kanamycin 30 mg/L+Timentin 300 mg/L, pH 6.0. The optimal rooting medium contained: WPM+IBA 0.25 mg/L+sucrose 30 g/L+agar 6 g/L +Kanamycin 30 mg/L+Timentin 300 mg/L, pH 6.0. (3) On the optimal differentiation and selection medium, the seedling vitrification rate infected by Agrobacterium LBA4404-pBI121 was about 65%, while that infected by Agrobacterium EHA105-pBI121 was below 10%. (4) The rooting efficiency of the regenerated seedlings reached 81.2% using a low-salt WPM medium of woody plants. (5) The ratio of the positive callus to the total number of inoculated leaves was used to evaluate the transformation efficiency. Using the optimal transformation system, the transformation rates of Agrobacterium LBA4404-pBI121 and EHA105-pBI121 were 51% and 65.2%, respectively. [Conclusion] The combined culture system of L. ruthenicum leaves can improve transformation rate while reducing seedling vitrification rate.

Copyright of Chinese Journal of Bioprocess Engineering is the property of Chinese Journal of Bioprocess Engineering Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

One of the commonly employed methods in molecular biology is to utilize calcium chloride to treat Escherichia coli for the preparation of competent cells to facilitate foreign gene expression. However, the molecular mechanisms underlying Ca2+ mediation of competent cell formation and identification of the key genes involved in the process remain unclear. In previous studies, the combined analysis of transcriptomics and proteomics revealed bhsA as one of the crucial genes. The gene ontology functional annotation of bhsA identified it as a member of the YhcN family encoding an outer membrane protein that confers resistance to various stresses. The IPR0108542 domain found within the protein plays a significant role in stress response and biofilm formation in E. coli. Analysis of the STRING database showed that the proteins interacting with bhsA are primarily involved in biofilm formation and stress resistance. Using the RED homologous recombination method, a bhsA deletion strain was constructed to verify its role in E. coli genetic transformation. Although the mutant strain showed no significant differences in morphology or growth trend when compared to the wild‐type strain, its transformation efficiency decreased by 1.14‐ and 1.64‐fold with plasmids pUC19 and pET‐32a. Furthermore, the 1‐N‐phenylnaphthylamine assay indicated a 1.71‐fold reduction in cell membrane permeability in the mutant strain. [ABSTRACT FROM AUTHOR]

Abstract Background The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. Results A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. Conclusions Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Background: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. Results: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. Conclusions: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus. [ABSTRACT FROM AUTHOR]

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement. [ABSTRACT FROM AUTHOR]

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To analyze the endogenous plasmid pDX of Lactobacillus acidophilus, and construct the Escherichia coli-Lactobacillus acidophilus shuttle vector based on the plasmid. In this study, the endogenous plasmid pDX was isolated from Lactobacillus acidophilus XW118, its sequence determination and functional analysis were performed, and then the promotor of plasmid pDX was used to construct a vector that could shuttle between Escherichia coli and lactic acid bacteria. The activity of the endogenous plasmid replicon of L. acidophilus in E. coli and the host range, transformation efficiency and passage stability of the shuttle vector in lactic acid bacteria were studied. The results showed that the size of Lactobacillus acidophilus plasmid pDX was 2062 bp, the GC content was 38.2%, and the plasmid contained four open reading frames (ORFs), which was presumed to be a rolling loop replication plasmid. The copy number of plasmid pDX in L. acidophilus was 31.05. In this study, an E. coli-lactic acid bacteria shuttle vector based on the replicon and promoter of plasmid pDX was constructed. The transformation efficiency of the vector was 0.24×102 to 2.75×103 CFU/μg (plasmid weight), and the plasmid loss rate was 25.45% to 48.36%. In this study, a vector capable of shuttling between E. coli and lactic acid bacteria was constructed, and the promoter of the endogenous plasmid of lactic acid bacteria with activity in E. coli was obtained, which provided a new operational approach for genetic engineering of lactic acid bacteria and metabolic engineering of E. coli.

Abstract With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches.

Genetic transformation is widely used to improve target traits and to study gene function in wheat. However, transformation efficiency depends on the physiological status of the recipient genotype and that is affected by several factors including powdery mildew (PM) infection. The widely used recipient variety Fielder is very susceptible to PM. Therefore, it would be beneficial to develop PM resistant derivatives with high regeneration ability for use in genetic transformation. In the present study PM resistant lines CB037 and Pm97033 carrying genes Pm21 and PmV, respectively, were backcrossed to Fielder with selection for PM resistance. Five lines, NT89, NT90, NT154, and WT48 with Pm21 and line FL347 with PmV were developed, identified by molecular markers and genomic in situ hybridization (GISH) or fluorescent in situ hybridization (FISH), and further subjected to detailed assessment of agronomic traits and regeneration ability following genetic transformation capacity. Lines FL347, WT48, NT89 and NT154 assessed as being equal to, or superior, to Fielder in regeneration and transformation ability are recommended as suitable materials for the replacement of Fielder for wheat gene transfer and genome editing study.

Agrobacterium-based stable transformation is an imperative tool for functional genomics studies and crop improvement which is needed to maximize the benefits of emerging technologies like CRISPR/Cas9. In the present study, we report a highly efficient protocol capable of transforming different tomato cultivars. The three cultivated varieties of Solanum lycopersicum, Pusa Ruby, Arka Vikas, and Pusa early dwarf, and one wild species Solanum peruvianum were used. The CRISPR-based vectors pDIRECT and pMOD were used to prepare constructs for the transformation. We have compared effect of different types of explants, age of explants, different strains of A. tumefaciens, different cell densities of A. tumefaciens used for co-cultivation, and different temperatures of co-cultivation on regeneration and transformation. Among the growth hormones, zeatin (2 mg/l) and IAA (0.1 mg/l) were used as a source of cytokinin and auxin for regeneration. Enhanced rooting was observed with 0.1 mg/l IAA in the rooting medium. Among the cultivated varieties, S. lycopersicum cv. Arka Vikas showed maximum regeneration efficiency of 54.8% and wild species S. peruvianum displayed 65% efficiency. Pusa early dwarf showed the lowest regeneration efficiency of 17%; however, enough plantlets of this recalcitrant cultivar have been recovered proving the utility of the present method. More than 80% of the regenerated plantlets yielded PCR-positive transformed plants for all the varieties and species tested in the study. Copy number analysis using a novel qPCR method was also performed for transgenic plants. Here, we describe a protocol with minute details and care to be taken for efficient tomato transformation, with a single standard protocol capable of transforming any tomato variety. [ABSTRACT FROM AUTHOR]

To establish a system for rapid identification of millet gene functions, in this study, we compared the effects of explants from different foxtail millet cultivars, the concentrations of acetyleugenone, bacterial solution, and co-culture time on the hairy root induction mediated by Agrobacteria rhizogenes strain K599. When the shoot tips were infected with bacterial solution 0.5 optical density (OD) and containing 100 µmol L-1 acetyleugenone, and co-cultured for three days, the induction efficiency of hairy roots reached to 80.24%. Using this system, we analyzed the transformation efficiency of the hairy roots, the subcellular localization of SiDVL1and SiDVL3, and gene functions of SiNHX2, SiCBL4, and SiCBL7. The PCR results of GFP gene and GFP fluorescence microscopic observation indicated that the transformation efficiency of the hairy roots was over 70%. The subcellular localization of SiDVL1 and SiDVL3 was similar in millet hairy-root and tobacco epidermal cells. Simultaneously, the survival rates of SiNHX2, SiCBL4, and SiCBL7 transgenic millet were significantly higher than that of empty vector transformed millet. Therefore, our study established an efficient and rapid method to analyze the subcellular location and to identify the function of genes from foxtail millet. [ABSTRACT FROM AUTHOR]

Abstract Background Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. Results Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. Conclusions Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation.

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. [ABSTRACT FROM AUTHOR]

With the rising demand for new cultivars of carnation, efficient transformation protocols are needed to enable the bioengineering of new traits. Here, we established a novel and efficient Agrobacterium-mediated transformation system using callus as the target explant for four commercial carnation cultivars. Leaf-derived calli of all cultivars were inoculated with Agrobacterium tumefaciens strain LBA4404 containing the plasmid pCAMBIA 2301 harboring genes for β-glucuronidase (uidA) and neomycin phosphotransferase (nptII). Polymerase chain reaction (PCR) and histochemical assays confirmed the presence of uidA and β-glucuronidase (GUS), respectively in transgenic shoots. The effect on transformation efficiency of medium composition and the presence of antioxidants during inoculation and co-cultivation was investigated. The transformation efficiency was increased in Murashige and Skoog (MS) medium lacking KNO3 and NH4NO3, and also in MS medium lacking macro and micro elements and Fe to 5% and 3.1% respectively, compared to 0.6% in full-strength medium. Transformation efficiency was increased dramatically to 24.4% across all carnation cultivars by the addition of 2 mg/l melatonin to nitrogen-depleted MS medium. Shoot regeneration was also doubled in this treatment. The establishment of this efficient and reliable transformation protocol can advance the development of novel carnation cultivars through molecular breeding approaches. Highlights: Melatonin at 2 mg/l substantially increases the Agrobacterium-mediated transformation efficiency of carnation cultivars to 24%. Nitrogen-depleted inoculation and co-cultivation media can greatly enhance carnation transformation. Callus is a potential target explant for Agrobacterium transformation of carnation. Melatonin and lipoic acid can considerably promote the shoot regeneration from transformed callus. The established protocol for carnation transformation is cultivar independent. [ABSTRACT FROM AUTHOR]

Plant genetic transformation is a powerful tool that can facilitate breeding programs for disease tolerance, abiotic stress, fruit production, and quality by preserving the characteristics of fruit tree elite genotypes. However, most grapevine cultivars worldwide are considered recalcitrant, and most available genetic transformation protocols involve regeneration by somatic embryogenesis, which often requires the continuous production of new embryogenic calli. Cotyledons and hypocotyls derived from flower-induced somatic embryos of the Vitis vinifera cultivars Ancellotta and Lambrusco Salamino, in comparison with the model cultivar Thompson Seedless, are here validated for the first time as starting explants for in vitro regeneration and transformation trials. Explants were cultured on two different MS-based culture media, one having a combination of 4.4 μM BAP and 0.49 μM IBA (M1), and the other only supplemented with 13.2 μM BAP (M2). The competence to regenerate adventitious shoots was higher in cotyledons than in hypocotyls on both M1 and M2. M2 medium increased significantly the average number of shoots only in Thompson Seedless somatic embryo-derived explants. This efficient regeneration strategy, that proposes a combination of somatic embryogenesis and organogenesis, has been successfully exploited in genetic engineering experiments. Ancellotta and Lambrusco Salamino cotyledons and hypocotyls produced the highest number of calli expressing eGFP when cultured on M2 medium, while for Thompson Seedless both media tested were highly efficient. The regeneration of independent transgenic lines of Thompson Seedless was observed from cotyledons cultured on both M1 and M2 with a transformation efficiency of 12 and 14%, respectively, and from hypocotyls on M1 and M2 with a transformation efficiency of 6 and 12%, respectively. A single eGFP fluorescent adventitious shoot derived from cotyledons cultured on M2 was obtained for Ancellotta, while Lambrusco Salamino showed no regeneration of transformed shoots. In a second set of experiments, using Thompson Seedless as the model cultivar, we observed that the highest number of transformed shoots was obtained from cotyledons explants, followed by hypocotyls and meristematic bulk slices, confirming the high regeneration/transformation competences of somatic embryo-derived cotyledons. The independent transformed shoots obtained from the cultivars Thompson Seedless and Ancellotta were successfully acclimatized in the greenhouse and showed a true-to-type phenotype. The novel in vitro regeneration and genetic transformation protocols optimized in this study will be useful for the application of new and emerging modern biotechnologies also to other recalcitrant grapevine genotypes. [ABSTRACT FROM AUTHOR]

Agrobacteriumtumefaciens-mediated plant transformation has become routine work across the world to study gene function and the production of genetically modified plants. However, several issues hamper the transformation process in a profound way, both directly and indirectly. One of the major concerns is the overgrowth of Agrobacterium, which occasionally appears after the co-cultivation phase of the explant. This phenomenon is reported in several species and seems to spoil the whole transformation process. There are multiple approaches being employed to counter this unwanted growth of bacteria in a few plant species. In reality, once the overgrowth appears, it becomes nearly impossible to cure it. Hence, for the prevention of this phenomenon, numerous factors are regulated. These factors are: explant nature, A.tumefaciens strain, T-DNA vector, co-cultivation (time and condition), acetosyringone, washing medium, antibiotics (type, concentration, combination, incubation period), etc. In this article, we discuss these factors based on available reports. It can be of immense help in formulating viable strategies to control A.tumefaciens overgrowth. [ABSTRACT FROM AUTHOR]

In transgenic plant development, the low transformation efficiency of Agrobacterium with exogenous DNA is the major constraint, and hence, methods to improve its transformation efficiency are needed. Recently, nanoparticle-mediated gene transfer has evolved as a key transformational tool in genetic transformation. Since silver nanoparticles (AgNPs) can induce pores on the cell membrane, their efficacy in the improvement of conventional calcium chloride freeze-thaw technique of transformation of Agrobacterium was explored in this study. Agrobacterium cells in the exponential growth phase were exposed to different concentrations of AgNPs (0.01, 1, 5, 10, and 20 mg/l), and the half-maximal effective concentration (EC50) was determined via Probit analysis using the SPSS software. Transformation efficiency of AgNPs alone and in combination with calcium chloride was compared with that of the conventional calcium chloride freeze-thaw technique. AgNPs at a concentration of 0.01 mg/l in combination with calcium chloride (20 mM) showed a ten fold increase in the transformation efficiency (3.33 log CFU (colony-forming unit/microgram) of DNA) of Agrobacterium tumefaciens strain EHA 105 with plasmid vector pART27 compared with the conventional technique (2.31 log CFU/μg of DNA). This study indicates that AgNPs of size 100 nm can eliminate the freeze-thaw stage in the conventional (Agrobacterium transformation technique, with a 44% improvement in efficiency. The use of AgNPs (0.01 mg/l) along with 20 mM calcium chloride was found to be an economically viable method to improve the transformation of Agrobacterium with exogenous plasmid DNA.

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. [ABSTRACT FROM AUTHOR]

Background: Agrobacterium tumefaciens-mediated leaf disc genetic transformation is an important way to achieve transgenics or gene editing. Ensuring stable and efficient genetic transformation is still an important problem in modern biology. It is assumed that the difference in the development status of genetic transformation cells of receptor materials is the main reason for the difference and instability of genetic transformation efficiency; the stable and efficient genetic transformation rate can be obtained by defining the appropriate treatment period of the receptor material and applying genetic transformation in a timely manner. Results: Based on these assumptions, we studied and established an efficient and stable Agrobacterium-mediated plant transformation system with hybrid poplar (Populus alba × Populus glandulosa, 84 K) leaves, stem segments and tobacco leaves as the research objects. There were differences in the development process of leaf bud primordial cells from different explants, and the genetic transformation efficiency was significantly related to the cell development stage of the in vitro cultured materials. Among them, the genetic transformation rate of poplar and tobacco leaves was the highest on the 3rd and 2nd day of culture, reaching 86.6% and 57.3%, respectively. The genetic transformation rate of poplar stem segments was the highest on the 4th day of culture, reaching 77.8%. The best treatment period was from the development of leaf bud primordial cells to the S phase of the cell cycle. The number of cells detected using flow cytometry and 5-ethynyl-2ʹ-deoxyuridine (EdU) staining, the expression of cell cycle-related protein CDKB1; 2, CDKD1; 1, CYCA3; 4, CYCD1; 1, CYCD3; 2, CYCD6; 1, and CYCH; 1 of explants, and morphological changes of explants can be used as indicators to determine the appropriate treatment period for genetic transformation. Conclusions: Our study provides a new and universal set of methods and characteristics to identify the S phase of the cell cycle and apply genetic transformation treatments at the appropriate time. Our results are of great significance for improving the efficiency and stability of plant leaf disc genetic transformation. [ABSTRACT FROM AUTHOR]

The preparation of Escherichia coli competent cells by calcium chloride is a common method in molecular biology, but the mechanism is poorly understood. In a previous study, using transcriptomics and proteomics approaches, we found that the expression pattern of the gene loiP was upregulated by CaCl2. In order to investigate the function of the loiP gene in Ca2+- mediated formation of competent cells of E. coli DH5α, the loiP gene deletion strains were constructed by the lambda-derived Red homologous recombination technique. Then, the cell morphology, transformation efficiency, and cell membrane changes of the competent cells of the mutant strain were further explored. Compared with the wild-type E. coli DH5α, the mutant strains have no significant differences in the morphology, growth characteristics, and the permeability of the intracellular membrane. However, the transformation efficiencies of the mutant strains to plasmids of different sizes were significantly reduced, and the permeability of the outer membrane decreased by 2.94 times. These results indicate that the deletion of gene loiP may directly affect the transformation efficiency and outer membrane permeability of E. coli competent cells. [ABSTRACT FROM AUTHOR]

Paclitaxel (Taxol®) is a potent anticancer agent, but the widespread pharmaceutical use of paclitaxel is hampered by its limited availability due to low accumulation levels in the native yew (Taxus spp.) plants. Currently, hairy root culture is an emerging biotechnological tool that presents several advantages such as reduced costs and higher specialized metabolite production, therefore, its application to paclitaxel production can be of commercial and medicinal interest. The objective of present study was to induce hairy root in Taxus baccata L. by transformation with the wild type Rhizobium rhizogenes A4 strain. Thus, T. baccata was inoculated by three different inoculation methods: (a) ex vitro seedlings inoculation by direct injection of a liquid bacterial culture; (b) ex vitro needles inoculation by liquid co-culturing with bacteria; (c) ex vitro shoots inoculation by dipping liquid bacterial culture. Hairy roots were formed only from ex vitro seedlings inoculated by the direct inoculation method, with transformation efficiency of 14.3%. Formation of hairy roots was observed two months after inoculation. This project forms the basis for the establishment of hairy root cultures from T. baccata for the production of paclitaxel. [ABSTRACT FROM AUTHOR]

Plant genetic transformation is a powerful tool that can facilitate breeding programs for disease tolerance, abiotic stress, fruit production, and quality by preserving the characteristics of fruit tree elite genotypes. However, most grapevine cultivars worldwide are considered recalcitrant, and most available genetic transformation protocols involve regeneration by somatic embryogenesis, which often requires the continuous production of new embryogenic calli. Cotyledons and hypocotyls derived from flower-induced somatic embryos of the Vitis vinifera cultivars Ancellotta and Lambrusco Salamino, in comparison with the model cultivar Thompson Seedless, are here validated for the first time as starting explants for in vitro regeneration and transformation trials. Explants were cultured on two different MS-based culture media, one having a combination of 4.4 µM BAP and 0.49 µM IBA (M1), and the other only supplemented with 13.2 µM BAP (M2). The competence to regenerate adventitious shoots was higher in cotyledons than in hypocotyls on both M1 and M2. M2 medium increased significantly the average number of shoots only in Thompson Seedless somatic embryo-derived explants. This efficient regeneration strategy, that proposes a combination of somatic embryogenesis and organogenesis, has been successfully exploited in genetic engineering experiments. Ancellotta and Lambrusco Salamino cotyledons and hypocotyls produced the highest number of calli expressing eGFP when cultured on M2 medium, while for Thompson Seedless both media tested were highly efficient. The regeneration of independent transgenic lines of Thompson Seedless was observed from cotyledons cultured on both M1 and M2 with a transformation efficiency of 12 and 14%, respectively, and from hypocotyls on M1 and M2 with a transformation efficiency of 6 and 12%, respectively. A single eGFP fluorescent adventitious shoot derived from cotyledons cultured on M2 was obtained for Ancellotta, while Lambrusco Salamino showed no regeneration of transformed shoots. In a second set of experiments, using Thompson Seedless as the model cultivar, we observed that the highest number of transformed shoots was obtained from cotyledons explants, followed by hypocotyls and meristematic bulk slices, confirming the high regeneration/transformation competences of somatic embryo-derived cotyledons. The independent transformed shoots obtained from the cultivars Thompson Seedless and Ancellotta were successfully acclimatized in the greenhouse and showed a true-to-type phenotype. The novel in vitro regeneration and genetic transformation protocols optimized in this study will be useful for the application of new and emerging modern biotechnologies also to other recalcitrant grapevine genotypes.

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.

The molecular mechanism of the Ca2+-mediated formation of competent cells in Escherichia coli remains unclear. In this study, transcriptome and proteomics techniques were used to screen genes in response to Ca2+ treatment. A total of 333 differentially expressed genes (317 upregulated and 16 downregulated) and 145 differentially expressed proteins (54 upregulated and 91 downregulated) were obtained. These genes and proteins are mainly enriched in cell membrane components, transmembrane transport, and stress response-related functional terms. Fifteen genes with these functions, including yiaW, ygiZ, and osmB, are speculated to play a key role in the cellular response to Ca2+. Three single-gene deletion strains were constructed with the Red homologous recombination method to verify its function in genetic transformation. The transformation efficiencies of yiaW, ygiZ, and osmB deletion strains for different-size plasmids were significantly increased. None of the three gene deletion strains changed in size, which is one of the main elements of microscopic morphology, but they exhibited different membrane permeabilities and transformation efficiencies. This study demonstrates that Ca2+-mediated competence formation in E. coli is not a simple physicochemical process and may involve the regulation of genes in response to Ca2+. This study lays the foundation for further in-depth analyses of the molecular mechanism of Ca2+-mediated transformation. [ABSTRACT FROM AUTHOR]

Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens-based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., β-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes (virG and virE), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications. [ABSTRACT FROM AUTHOR]

Simple Summary: Glucoamylase is one of the most needed industrial enzymes in the food and biofuel industries. Aspergillus niger is a commonly used cell factory for the production of commercial glucoamylase. For decades, genetic manipulation has promoted significant progress in industrial fungi for strain engineering and in obtaining deep insights into their genetic features. However, genetic engineering is more laborious in the glucoamylase-producing industrial strains A. niger N1 and O1 because their fungal features of having few conidia (N1) or of being aconidial (O1) make them difficult to perform transformation on. In this study, we targeted A. niger N1 and O1 and successfully developed high-efficiency transformation tools. We also constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 editing marker-free system using an autonomously replicating plasmid to express Cas9 protein and to guide RNA and the selectable marker. By using the genetic tools developed here, we generated nine albino deletion mutants. After three rounds of sub-culturing under nonselective conditions, the albino deletions lost the autonomously replicating plasmid. Together, the tools and optimization process above provided a good reference to manipulate the tough working industrial strain, not only for the further engineering these two glucoamylase-hyperproducing strains, but also for other industrial strains. The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger. [ABSTRACT FROM AUTHOR]

Promoting the industrial transformation and upgrading of resource-based cities is an important condition for solving the dilemma of widening economic development differences and serious destruction of ecological resources between regions, and to achieve regional coordinated development strategy. From the new perspective of factor mismatch, this article discussed the economic transformation of resource-based cities. In theory, a growth model describing the factor input of resource-based cities was constructed, and the influence mechanism of factor mismatch on urban output efficiency, environmental efficiency and energy efficiency was investigated. In the empirical aspect, based on the panel data from 2005 to 2018 and using the improved Directional Distance Function (DDF), this article calculated the transformation efficiency of 114 resource-based cities in China from the five dimensions of labor, capital and energy input, expected output and non-expected output, and then tested the exact impact of factor mismatch on the transformation of resource-based cities. The results showed that whether it was over-allocation or under-allocation, factor mismatch had a significant inhibitory effect on the economic transformation efficiency of resource-based cities, and the inhibitory effect of labor mismatch was particularly obvious; after dealing with endogenous problems, changing analysis samples, replacing explained variables, and changing regression methods, the research conclusions still had strong robustness. Transformation efficiency was further decomposed in the three directions of output growth, energy consumption and environmental friendliness. It was found that the mismatch of various factors would hinder the improvement of energy efficiency and environmental efficiency, but different from labor mismatch and energy mismatch, the negative impact of capital mismatch on output efficiency was insignificant. According to the classification of resource-based cities in different geographical regions, it was found that the impact of factor mismatch on transformation efficiency would be significantly different in different geographic regions. Based on the problems in the transformation of resource-based cities, this article puts forward policy suggestions from the three levels of country, city and industry, and emphasizes the importance of improving the allocation efficiency of labor factor. This paper not only provides a new insight into the explanation of the transformation dilemma faced by resource-based cities, but also provides useful reference for strategy formulation and policy adjustment to promote the high-quality development of resource-based cities in the new era. [ABSTRACT FROM AUTHOR]

ABSTRACT: The antiphage ability is an important feature of fermentation strains in the dairy industry. Our previous work described the bacteriophage exclusion (BREX) system in the probiotic strain, Lactobacillus casei Zhang. The function of L. casei Zhang pglX gene in mediating 5′-ACRCm6AG-3′ methylation was also confirmed. This study aimed to further dissect the function of the BREX system of L. casei Zhang by inactivating its second methyltransferase gene (LCAZH_2054). The methylome of the mutant, L. casei Zhang Δ2054, was profiled by single-molecule real-time sequencing. Then, the cell morphology, growth, plasmid transformation efficiency, and stability of the wildtype and mutant were compared. The mutant did not have an observable effect in microscopic and colony morphology, but it reached a higher cell density after entering the exponential phase without obvious increase in the cell viability. The mutant had fewer 5′-ACRCm6AG-3′ methylation compared with the wildtype (1835 versus 1906). Interestingly, no significant difference was observed in the transformation efficiency between the 2 strains when plasmids without cognate recognition sequence (pSec:Leiss:Nuc and pG+host9) were transformed, contrasting to transforming cells with cognate recognition sequence-containing plasmids (pMSP3535 and pTRKH2). The efficiency of transforming pMSP3535 into the LCAZH_2054 mutant was significantly lower than the wildtype, whereas an opposite trend was seen in pTRKH2 transformation. Moreover, compared with the wildtype, the mutant strain had higher capacity in retaining pMSP3535 and lower capacity in retaining pTRKH2, suggesting an unequal tolerance level to different foreign DNA. In conclusion, LCAZH_2054 was not directly responsible for 5′-ACRCm6AG-3′ methylation in L. casei Zhang, but it might help regulate the function and specificity of the BREX system.

Developing an efficient transformation system is vital in genetically engineering recalcitrant crops, particularly trees. Here, we outline an Agrobacterium tumefaciens-based stable plant transformation methodology for citrus genetic engineering. The process was optimized to suit the requirements of fourteen citrus varieties by establishing appropriate infection, co-cultivation, selection, and culture media conditions. The procedure includes transforming seedling-derived epicotyl segments with an A. tumefaciens strain, then selecting and regenerating transformed tissues. Transgenic shoots were further identified by a visual reporter (e.g., β-glucuronidase) and confirmed by Northern and Southern blot analysis. Transgene integrations among the transgenic lines ranged between one to four. The methodology can yield transformation efficiencies of up to 11%, and transgenic plants can be recovered as early as six months, depending on the variety. In addition, we show that incorporating A. tumefaciens helper virulence genes (virG and virE), spermidine, and lipoic acid in the resuspension buffer before transformation improved the transformation efficiency of specific recalcitrant cultivars, presumably by enhancing T-DNA integration and alleviating oxidative stress on the explant tissues. In conclusion, the optimized methodology can be utilized to engineer diverse recalcitrant citrus varieties towards trait improvement or functional genetics applications.

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. [ABSTRACT FROM AUTHOR]

Streptococcus zooepidemicus, group C Streptococci, is currently used for the industrial production of hyaluronic acid (HA). However, genetic manipulation of S. zooepidemicus is severely limited by its low transformation efficiency, which might be in part due to the Restriction–Modification (R–M) systems. The complete genome sequence of S. zooepidemicus ATCC39920 revealed the presence of two putative R–M systems, type I and type II. The putative type I R–M system is encoded by three closely linked genes: hsdR (SeseC_01315), hsdS, hsdM (SeseC_01318), and the putative type II R–M system consists of two closely linked genes: SeseC_02360 and yhdJ (SeseC_02362). Inactivation of hsdR, encoding the restriction endonuclease (REase) of the type I R–M system, showed no apparent effects on transformation efficiency, implying that disarmament of the type I R–M system alone is not sufficient for increasing transformation efficiency. However, inactivation of SeseC_02360, encoding the REase of the type II R–M system, improved transformation efficiency by 4.97 folds, indicating that type II R–M system is the major barrier that restricts genetic transformation in S. zooepidemicus. Furthermore, S. zooepidemicus strains lacking either of the two R–M systems are phenotypically indistinguishable from the wild-type in terms of cell growth and HA production. In summary, our study revealed that the type II R–M system is the main barrier to genetic transformation in S. zooepidemicus ATCC39920, and that the deletion of the type II R–M system renders S. zooepidemicus more transformable, thus facilitating metabolic engineering of this industrially important microorganism. [ABSTRACT FROM AUTHOR]

In transgenic plant development, the low transformation efficiency of Agrobacterium with exogenous DNA is the major constraint, and hence, methods to improve its transformation efficiency are needed. Recently, nanoparticlemediated gene transfer has evolved as a key transformational tool in genetic transformation. Since silver nanoparticles (AgNPs) can induce pores on the cell membrane, their efficacy in the improvement of conventional calcium chloride freeze-thaw technique of transformation of Agrobacterium was explored in this study. Agrobacterium cells in the exponential growth phase were exposed to different concentrations of AgNPs (0.01, 1, 5, 10, and 20 mg/l), and the half-maximal effective concentration (EC50) was determined via Probit analysis using the SPSS software. Transformation efficiency of AgNPs alone and in combination with calcium chloride was compared with that of the conventional calcium chloride freeze-thaw technique. AgNPs at a concentration of 0.01 mg/l in combination with calcium chloride (20 mM) showed a ten fold increase in the transformation efficiency (3.33 log CFU (colony-forming unit/microgram of DNA) of Agrobacterium tumefaciens strain EHA 105 with plasmid vector pART27 compared with the conventional technique (2.31 log CFU/µg of DNA). This study indicates that AgNPs of size 100 nm can eliminate the freeze-thaw stage in the conventional Agrobacterium transformation technique, with a 44% improvement in efficiency. The use of AgNPs (0.01 mg/l) along with 20 mM calcium chloride was found to be an economically viable method to improve the transformation of Agrobacterium with exogenous plasmid DNA. [ABSTRACT FROM AUTHOR]

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.

Hairy root culture (HRC) represent a valuable biotechnological tool for the production of plant secondary metabolites. Secondary metabolome study of Arabidopsis thaliana may help to understand the biological roles of various secondary metabolites present in it. The present work deals with the establishment of Agrobacterium rhizogenes strains transformed HRC of A. thaliana with very high transformation frequency resulting in long term hairy root cultures grown in hormone free media. Optimization of culture medium and standardisation of co-cultivation period are the key role players in obtaining high frequency of hairy roots. Four days of preculture in CIM medium and five min of co-cultivation in the bacterial suspension were found to be optimal conditions for root induction. This protocol could become a powerful tool for transcriptomics, metabolomics and proteomics-based studies for different transgenic root lines of A. thaliana. Key message: Insertion of rol genes of Agrobacterium rhizogenes enhanced rhizogenic potential of excised explants in Arabidopsis thaliana. The optimization process of hairy root culture induction and establishment led to maintenance upto 4 years in hormone free media. [ABSTRACT FROM AUTHOR]

In the evolutionary "arms race" from prokaryotes to eukaryotes, some memories of foreign DNA have been conserved for defensive purposes. Shortly after invasion by the plasmid, pEGFP-N1, the conserved the defense gene, isg15, was activated in the zebrafish zygote and in mammalian cells. Based on the sequence similarity, we found three virus-derived sequences in pEGFP-N1 which share the 5ʹ-GTTTGTT-3ʹ core sequence, an epigenetic factor leading to increased expression of isg15. Mutation of the core sequence greatly reduces the degradation rate of the plasmid in E. coli cells or zebrafish embryos. We conclude that a conserved defense response, common to both eukaryotic and prokaryotic cells, allows identification and degradation of plasmids containing 5ʹ-GTTTGTT-3ʹ. [ABSTRACT FROM AUTHOR]

Soybean is one of the important food, feed, and biofuel crops in the world. Soybean genome modification by genetic transformation has been carried out for trait improvement for more than 4 decades. However, compared to other major crops such as rice, soybean is still recalcitrant to genetic transformation, and transgenic soybean production has been hampered by limitations such as low transformation efficiency and genotype specificity, and prolonged and tedious protocols. The primary goal in soybean transformation over the last decade is to achieve high efficiency and genotype flexibility. Soybean transformation has been improved by modifying tissue culture conditions such as selection of explant types, adjustment of culture medium components and choice of selection reagents, as well as better understanding the transformation mechanisms of specific approaches such as Agrobacterium infection. Transgenesis-based breeding of soybean varieties with new traits is now possible by development of improved protocols. In this review, we summarize the developments in soybean genetic transformation to date, especially focusing on the progress made using Agrobacterium-mediated methods and biolistic methods over the past decade. We also discuss current challenges and future directions. [ABSTRACT FROM AUTHOR]