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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]

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]

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]

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]

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]

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]

Purslane (Portulaca oleracea L.), a valuable medicinal herb, is used as a source of pharmaceutical components such as flavonoids, alkaloids, fatty acids, terpenoids, and sterols. Regeneration of transgenic purslane plantlets from transformed cells is a time-consuming procedure and needs hard work. In this study, in-planta transformation of purslane, using sonication and vacuum infiltration, is reported. The purslane seeds were infected through Agrobacterium tumefaciens strain LBA4404 having the uidA gene on pBI121 vector. Effective selection of transformants was performed by supplementing MS media with 250 mg l−1 kanamycin. Several factors affecting the in-planta procedure including pre-culture duration, acetosyringone dose, sonication, and vacuum infiltration duration were investigated. The results demonstrated that the highest number of GUS-positive purslane plantlets was obtained when the pre-cultured seeds were sonicated and vacuum-infiltered for 2 min in agro-bacterial cell suspension, and then co-cultivated in MS media having 100 µM acetosyringone. The integration and expression of uidA gene in transgenic purslane was successfully corroborated by southern blot and GUS histochemical analyses. [ABSTRACT FROM AUTHOR]

Key message: An efficient and improved transformation method for functional genetics studies in S. italica, being a boon for the Setaria scientific community and for crop improvement. Foxtail millet (Setaria italica) is a short life cycle C4 plant, with sequenced genome, and a potential model plant for C4 species. S. italica is also important on a global food security and healthiness context due to its importance in arid and semi-arid areas. However, despite its importance, there are just few transformation protocols directed to this species. The current protocols reached about 5.5–9% of efficiency, which do not make it a valuable model organism. Different types of explants were used in the above mentioned methods, such as immature and mature inflorescence and shoot apex. However, these techniques have many limitations, such as unavailability of explants throughout the year and a crucial, laborious and considerable time-consuming selection. Aiming a simplified and efficient methodology, we adopted dry mature seeds as explants, which are available in abundance, are constant along the year and well responsive to tissue culture, in addition to a differentiated approach that reaches on an average 19.2% transformation efficiency of S. italica. Thus, we propose a protocol that optimizes the transformation efficiency of this cereal crop allowing a high increase on transformation and regeneration rates. Our transformation protocol provides an interesting tool for Setaria community research as well as enables new strategies for breeding enhanced productivity in the species. [ABSTRACT FROM AUTHOR]

In the Agrobacterium tumefaciens-mediated transformations of maize immature embryos (IEs), the common co-culturing media used are MS or N6-based (MC). Here, we used a novel co-culturing method in which maize 'Qi319' IEs inoculated with Agrobacterium-harboring target vector were placed on dry filter paper (DC) in a petri dish. To compare the effects of the DC and MC co-culturing methods on transformation efficiency, we designed three experiments: (1) A. tumefaciens strain AGL1 independently carrying two plasmids, pXQD12 and pXQD70; (2) two A. tumefaciens strains, AGL1 and EHA105, carrying pXQD12; and (3) strains AGL1 and EHA105 each independently inoculated with pXQD12 and pXQD70 for different infiltration periods, 5, 10, 15, 20 and 25 min. We used A. tumefaciens to inoculate IEs derived from maize ears 9-15 d after pollination, and then IEs were placed in petri dishes for co-culturing. The DC treatment significantly increased the percentage of IEs expressing green fluorescence protein (%GFP), indicating positive transformants. DC-treated IEs had ~ 3 to 4 times the %GFP compared with MC-treated IEs at 8 d after inoculation (3 d co-culture and 5 d restoration). The average regeneration frequency (%GFP positive regenerated calli of infected IEs) and stable transformation frequency (%GFP positive T0 plants of infected IEs) significantly increased with the DC treatment. Thus, the DC method may be used to develop a more efficient Agrobacterium-mediated transformation method for maize IEs. [ABSTRACT FROM AUTHOR]

RNA silencing is a sequence-specific form of epigenetic regulation that targets invasive nucleic acids. RNA-dependent RNA polymerase6 (RDR6) converts target RNA molecules, such as transgene transcripts, into double-stranded RNAs (dsRNAs) during posttranscriptional gene silencing (PTGS). Then, these dsRNAs are processed into small RNAs that guide sequence-specific RNA degradation. T-DNA-derived small RNAs are generated during the transfer of T-DNA from Agrobacterium to plant cells and compromise the function of the genes in the T-DNA. In the present study, we produced selection-marker-free transgenic tobacco plants using the MAT vector system, and expression of the tobacco RDR6 gene (NtRDR6) was suppressed using inverted-repeat-induced PTGS. Reduced expression of the NtRDR6 gene improved the transient expression of the transgene in the agroinfiltrated leaves and enhanced the production of hairy roots after infection with Agrobacterium containing a root-inducing T-DNA. The expression level of the sense transgene was determined in individual hairy roots, and knockdown of the NtRDR6 gene did not affect the distribution of the expression levels in individual transformants. These results indicate that NtRDR6 partially inhibited T-DNA function during T-DNA transfer but did not affect the expression of the transgene in stable transformants, except in transformants showing sense-transgene-induced PTGS. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a genetic locus of prokaryotes and contain highly conserved direct repeats, spacers, and CRISPR-associated genes. Spacers in CRISPRs are known as adaptive immune markers and reveal what types of phage or foreign DNA have been introduced in the past. The primary objective of this study was to analyze spacer sequences in CRISPR arrays of 15 Salmonella enterica subspecies and to determine if Salmonella CRISPRs are indeed involved in resistance to foreign DNAs. Using a bioinformatics algorithm, the CRISPR arrays of 15 subspecies of S. enterica were predicted. The transformation efficiencies of the wild-type and mutant strains lacking a space were determined using the plasmid harboring the same sequences with the space. Analysis of the CRISPR arrays indicated that S. Typhimurium encoded three possible CRISPR regions in the genome. Notably, 48 or 55 spacers were predicted in the genomes of S. Typhimurium 14028 and LT2 strains, respectively, and 39 were precisely identical. To confirm this prediction, the predicted CRISPR regions of S. Typhimurium 14028 were sequenced using the specific primers. Interestingly, a homology search of individual spacers found that the 2nd spacer of CRISPR 2 was nearly identical to a partial genome region of phage FSL SP-016. The mutant strain showed two to threefold increased transformation efficiency compared to that of the wild-type strain. These results demonstrate that the spacer sequence is dependent on genetic relations, especially for adaptive immunity against phage or foreign DNAs. [ABSTRACT FROM AUTHOR]

Objectives: To improve the quality of mutagenesis libraries in directed evolution strategy.Results: In the process of library transformation, transformants which have been shown to take up more than one plasmid might constitute more than 20% of the constructed library, thereby extensively impairing the quality of the library. We propose a practical transformation method to prevent the occurrence of multiple-plasmid transformants while maintaining high transformation efficiency. A visual library model containing plasmids expressing different fluorescent proteins was used. Multiple-plasmid transformants can be reduced through optimizing plasmid DNA amount used for transformation based on the positive correlation between the occurrence frequency of multiple-plasmid transformants and the logarithmic ratio of plasmid molecules to competent cells.Conclusions: This method provides a simple solution for a seemingly common but often neglected problem, and should be valuable for improving the quality of mutagenesis libraries to enhance the efficiency of directed evolution strategies. [ABSTRACT FROM AUTHOR]

The orchid Dendrobium catenatum Lindl. has both ornamental and medicinal value. Studies on genetic transformation of other Dendrobium species have been reported; however, due to the heterogeneity among the Dendrobium species, an independent transformation system is required for optimum D. catenatum transformation. Agrobacterium tumefaciens-mediated transformation system for D. catenatum is reported herein. Three A. tumefaciens strains, GV3101, LBA4404, and EHA105, were tested for their ability to transform D. catenatum protocorms. It was found that infection with strain GV3101 at an OD600 of 0.6 yielded the highest transformation frequency of 56.5%. Addition of 0.001% (v/v) Triton™ X-100 or pretreatment with ultrasound (300 W, 40 kHz, 3 min) increased the transformation frequency by up to 2-fold. After 1 year, both green fluorescent protein (GFP) imaging and glucuronidase (GUS) histochemical analysis of transgenic plants demonstrated that DcObg-GFP and pDcObg-GUS construct DNA was transferred into the D. catenatum and expressed successfully. Functional and gene expression level analyses of transgenic plants containing a D. catenatum chloroplast-targeted homolog of Spo0B-associated GTP-binding protein (DcObgC) RNA interference construct (DcObgC-RNAi) further demonstrated that this transformation system was reliable and efficient for D. catenatum. The A. tumefaciens-based transformation protocol established and optimized in this current report could provide a powerful and efficient tool for academic research and genetic engineering-based breeding of D. catenatum. [ABSTRACT FROM AUTHOR]

Rosellinia necatrix: causing root rot disease is a very destructive pathogen of woody plants and is responsible for yield losses to a large number of fruit trees. The genetic analysis of this pathogen has not been picked up because of difficulty in generating mutations in Rosellinia necatrix for many reasons. A number of methods have been proposed for inducing mutations in Rosellinia necatrix but none of them proved worth because of very low transformation efficiencies. Here, we propose an efficient method for Rosellinia necatrix protoplast production, where protoplasts in the tune of 10 per ml can be easily generated. We also propose a restriction enzyme-mediated integration (REMI)-based methods for efficient transformation of Rosellinia necatrix. In the present study, an approximate of 800 transformants was obtained from 5 μg of linearized plasmid. Out of 47 single spored transformants analyzed, only 33 showed hygromycin gene amplification using PCR and only 19 transformants showed single gene integration in southern hybridization, which accounted for single gene integration percentage of 42%, highest amongst all the previous reports on Rosellinia necatrix transformations. Some of the transformants studied for pathogenicity phenotype also showed a marked reduction in pathogenicity. Thus, in the present investigation, 42% single gene integrations among the transformed colonies can be considered as excellent transformation efficiency. [ABSTRACT FROM AUTHOR]

One of small accessory genes between pol and env is tat gene encoding TAT protein. This research was aimed to optimize the expression of Jembrana TAT (JTAT) protein with preparing Escherichia coli ( E. coli ) in advance using adopted methods of M1 (MgCl 2 + CaCl 2 ) and M2 (CaCl 2 + Glycerol). The best transformation efficiency resulting from a better transformation method was used to subsequent expression of JTAT protein. A synthetic tat gene encoding protein JTAT was previously cloned into pBT- hisC . Concentration of 200; 400; 600 µM IPTG was induced to a small volume culture (200 ml; OD 600 = 4), incubated for 3 h. Pellets were harvested by centrifugation ( 4000 rpm; 4 °C; 15 min ) . Buffer B (10 mM Immidazole) was added into pellets, lysed by freeze-thaw followed by sonication. Supernatant was collected by centrifugation (10,000 rpm; 4 °C; 20 min) and purified using Ni-NTA Agarose resin, released by elution buffer (E) containing 400 mM Immidazole to collect purified protein twice (E1, E2). The protein was characterized by SDS-PAGE and Western Blot (WB), quantified (at λ 595 nm) with BSA standard method in prior. The result showed that transformation efficiency was better in M2 (2.53 × 10 6 ) than M1 (3.10 × 10 5 ). The JTAT protein was expressed at a right size of 11.8 kDa. Concentration of 200 µM IPTG produced a significantly better protein yield (1.500 ± 0.089 mg/ml; P < 0.05) than 600 µM IPTG (0.896 ± 0.052 mg/ml) and not different to 400 µM IPTG (1.298 ± 0.080 mg/ml). This research indicated that transformation efficiency needs to be taken account in prior of optimization of the protein expression. [ABSTRACT FROM AUTHOR]

Melon ( Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid. [ABSTRACT FROM AUTHOR]

Key message: The choice of promoter regulating the selectable marker gene impacts transformation efficiency, copy number and the expression of selectable marker and flanking genes in maize. Abstract: Viral or plant-derived constitutive promoters are often used to regulate selectable marker genes. We compared two viral promoters, cauliflower mosaic virus (CaMV 35T) and sugarcane bacilliform virus (SCBV) with two plant promoters, rice actin1 (OsAct1) and maize ubiquitin 1 (ZmUbi1) to drive aryloxyalkanoate dioxygenase ( aad-1) selectable marker gene in maize inbred line B104. ZmUbi1- and OsAct1-containing constructs demonstrated higher transformation frequencies (43.8 and 41.4%, respectively) than the two viral promoter constructs, CaMV 35T (25%) and SCBV (8%). Interestingly, a higher percentage of single copy events were recovered for SCBV (82.1%) and CaMV 35T (59.3%) promoter constructs, compared to the two plant-derived promoters, OsAct1 (40.0%), and ZmUbi1 (27.6%). Analysis of protein expression suggested that the viral promoter CaMV 35T expressed significantly higher AAD-1 protein (174.6 ng/cm) than the OsAct1 promoter (12.6 ng/cm) in T leaf tissue. When measured in T callus tissue, the two viral promoters both had higher expression and more variability than the two plant-derived promoters. A potential explanation for why viral promoters produce lower transformation efficiencies but higher percentages of low copy number events is discussed. In addition, viral promoters regulating aad-1 were found to influence the expression of upstream flanking genes in both T leaf and T callus tissue. [ABSTRACT FROM AUTHOR]

The present study reports the successful production of human pre-miR-29b both intra- and extracellularly in the marine phototrophic bacterium Rhodovulum sulfidophilum using recombinant RNA technology. In a first stage, the optimal transformation conditions (0.025 μg of plasmid DNA, with a heat-shock of 2 min at 35 °C) were established, in order to transfer the pre-miR-29b encoding plasmid to R. sulfidophilum host. Furthermore, the extracellular recovery of this RNA product from the culture medium was greatly improved, achieving quantities that are compatible with the majority of applications, namely for in vitro or in vivo studies. Using this system, the extracellular human pre-miR-29b concentration was approximately 182 μg/L, after 40 h of bacterial growth, and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 h. At the end of the fermentation, it was verified that almost 87 % of cells were viable, indicating that cell lysis is minimized and that the extracellular medium is not highly contaminated with the host intracellular ribonucleases (RNases) and endotoxins, which is a critical parameter to guarantee the microRNA (miRNA) integrity. These findings demonstrate that pre-miRNAs can be produced by recombinant RNA technology, offering novel clues for the production of natural pre-miRNA agents for functional studies and RNA interference (RNAi)-based therapeutics. [ABSTRACT FROM AUTHOR]

Artemisia annua L. is currently the only practical source of artemisinin, an effective antimalarial agent. Development of an efficient gene delivery system is crucial for genetic manipulation of this medicinal plant to enhance artemisinin production. Here, we report a protocol for efficient Agrobacterium-mediated transformation and plant regeneration of A. annua using young inflorescence as explants. We evaluated the influence of explant types and Agrobacterium strains on transformation and regeneration efficiency. Young inflorescence and leaf explants were co-cultivated with Agrobacterium tumefaciens strains, EHA105 or LBA4404, carrying the pCAMBIA2301 binary vector containing a ß-glucuronidase reporter. Shoot regeneration was observed when explants were incubated on modified Murashige and Skoog (MS) medium supplemented with 0.5 mg L 6-benzylaminopurine and 0.3 mg L α-naphthalene acetic acid. Shoot bud development from young inflorescence explants was observed within 7 wk of culture whereas those from the leaf explants regenerated after 9 wk. PCR and Southern blot analyses showed that 95% of the putative transgenic lines were positive for the GUS reporter gene. The maximum transformation frequency of 17% was achieved by co-cultivation of A. tumefaciens strain EHA105 with young inflorescence, representing a twofold improvement compared to using leaf explants. Molecular and genetic analysis of T progeny further confirmed the inheritance of the introduced gene. The transformation protocol using young inflorescence of A. annua reduces the transformation time and increases the efficiency; hence, this represents a practical platform for metabolic engineering of this important medicinal plant. [ABSTRACT FROM AUTHOR]

A high-efficiency Agrobacterium tumefaciens mediated transformation system was established through testing and optimising a range of variables using soybean cotyledonary as explants and glufosinate as selective agent. Seven cultivars (Heihe 19, Heihe 25, Heinong 37, Ha 03-3, YC-1, YC-2, and Zhonghuang 39) were used to screen the optimal cultivars. As a result, cultivars YC-1 and YC-2 were screened, which had the highest transient GUS expression percentage and shoot induction frequency. The cell density of Agrobacterium was found to be related with the transient GUS expression percentage; its optimal cell density was OD 0.8-1.0. It was beneficial to A. tumefaciens infection at 23 °C and dim light co-cultivation condition. Interestingly, the infection time showed no relation to the GUS transient percentage. Three types of antioxidant solutions were tested on their effects of alleviating cotyledonary explant browning and necrosis. The results showed that addition of 20 μM lipoic acid on solid CCM had the lowest cotyledonary explant necrosis and a high transient GUS expression rate. Following the optimized A. tumefaciens mediated transformation system, the bar gene was successfully transferred into soybean with the transformation efficiency reaching 14.71 %, more than 50 times increase from the un-optimized procedure. Southern and T progeny analysis showed the exogenous gene had been integrated into the soybean genome and could be inherited next progeny. [ABSTRACT FROM AUTHOR]

The osmotolerant yeast, Trichosporonoides oedocephalis, is an excellent producer of erythritol, which has wide industrial applications. In this study, we developed an efficient transformation method for T. oedocephalis. To evaluate the T. oedocephalis transformation, we constructed a DNA fragment (loxP-Kan-loxP/Cre system) that was targeted to the mitogen-activated protein kinase HOG1 gene. Transformants were selected on plates containing G418 and response surface methodology was employed to obtain optimum transformation conditions. Optimal transformation could be achieved at an incubation time of 40 min, when the concentration of zymolyase-100T was 30 µg/mL, and when 100 mM CaCl was added to the mixture. The predicted optimal transformation efficiency was 133 transformants per µg of DNA. This novel method will facilitate studies in synthetic biology and metabolic engineering of T. oedocephalis. [ABSTRACT FROM AUTHOR]

Abiotic stresses such as drought and high salinity unfavorably affect the growth and productivity of crop plants. Therefore, the development of stress-tolerant crops is essential for the affected cultivable areas. It has been shown in the current study that the overexpression of stress-responsive NAC1 ( SNAC1) transcription factor (TF) significantly increases salinity and drought tolerance in a farmer-popular high-yielding, transgenic rice. The indica rice variety BRRIdhan 55, which was poorly responsive to tissue culture was transformed with the SNAC1 TF from the rice landrace Pokkali by the in planta method. Addition of acetosyringone in the Agrobacterium suspension and co-culture media increased previously reported transformation efficiencies by four-folds. Integration of foreign genes into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression ( SNAC1) was also quantified by real-time PCR. Genetic segregation ratio for T progenies was calculated and found to follow the law of Mendelian inheritance. Phenotypic screening was conducted at T and T seedling stages where the transgenic lines exhibited much better tolerance compared to their control non-transgenic plants at 120 mM (NaCl) salt as well as drought stress implemented by withholding water for 20 days. [ABSTRACT FROM AUTHOR]

Over the past decade, Medicago truncatula has been adopted as a model legume species for a range of 'omic' studies. The availability of different transformation techniques has greatly advanced functional genomic studies in this species. In the present work, an efficient procedure for Agrobacterium-mediated transformation of M. truncatula cv . 'Jemalong 2HA' through a cell suspension culture was developed. This procedure resulted in transformed single cells or cell clusters, giving rise to stable transgenic plants within 4 mo. Transformation experiments were performed with a vector carrying two marker genes: β-glucuronidase (GUS) and green fluorescent protein (GFP) under the control of endogenous gene promoters from LIKE AUX1 3 ( LAX3) and GRAS transcription factor (named after GD3BERELLIC ACID INSENSITIVE [ GAI], REPRESSOR OF GA1 [ RGA], and SCARECROW [ SCR]), as well as with a binary destination vector for overexpressing the cyclin-like F-box gene fused to GFP. Maximum transformation efficiency was achieved under the following experimental conditions: acetosyringone at a concentration of 25 μM, bacterial suspension with an optical density of 0.3 at 600 nm, inoculation under agitation at 100 rpm for 24 h, co-cultivation periods of 48 h, and an uninterrupted selection with 50 mg/L kanamycin. Selection of positive transformation events was imposed early in the regeneration stage (after 48 h co-cultivation), following a large-scale screening for GFP activity. Histochemical GUS and GFP reporter activity was detected in single cells, embryogenic zones, emerging embryos, in vitro plantlets, and T progeny seedlings. The transgenic nature of transformed plants was further confirmed by nptII-specific PCR amplification of T and T plant lines. The transgenic plants grown under standard greenhouse conditions displayed a wild-type phenotype and the obtained progeny segregated in a classical Mendelian manner. The fundamental steps in the transformation procedure are outlined and discussed. [ABSTRACT FROM AUTHOR]

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43–69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19–46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5–1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8–34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93–100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies. [ABSTRACT FROM AUTHOR]

The biolistic method is reliable for delivering genes of interest into various species, but low transformation efficiency can be a limiting factor in its application. To test various conditions that could improve peanut transformation via particle bombardment, embryogenic tissues of the peanut cultivar Georgia Green were co-bombarded with two plasmids: one containing a green fluorescent protein gene and one containing a gene of interest plus a selectable marker. The fluorescence in bombarded embryogenic tissues was measured to evaluate transformation efficiency. A 4.6-fold improvement of transformation efficiency was achieved in stably transformed peanut lines by introducing protamine instead of conventional spermidine in a bombardment mixture with 70 ng/shot plasmid DNA and 50 μg/shot gold. Unexpectedly, the reduction of plasmid DNA from 700 to 70 ng/shot produced transgenic lines with significantly increased numbers of transgene copies. To determine the transgene copy number during plantlet regeneration, relative quantitative real-time polymerase chain reaction (qPCR) was established using fluorescently labeled universal library probes. A correlation of 95% was found for estimation of copy number between Southern blot and qPCR data. Given its speed and high-throughput nature, qPCR can be employed as an effective screening tool to separate high copy number events from low copy number events as early as the shoot formation stage of regeneration. [ABSTRACT FROM AUTHOR]

An efficient protocol was developed for Agrobacterium tumefaciens-mediated transformation of tomato ( Solanum lycopersicum) cultivars using cotyledon explants. The transformation frequency was assessed in response to several different factors, including seed germination medium, seedling age, pre-culture duration, pre-culture and co-cultivation media, inoculation medium, medium pH, washing medium, and kanamycin concentration in initial selection medium. Cotyledons excised from 6-d-old seedlings germinated on half-strength Murashige and Skoog's (MS) basal medium containing 8.9 μM benzyladenine (BA) produced the most suitable explant material. Six days of explant pre-culture and 5 min inoculation with Agrobacterium culture in MS medium, containing 8.9 μM BA, 9.3 μM kinetin, and 0.4 mg l thiamine at pH 5.0, significantly improved the transformation frequency. The addition of a tobacco feeder cell layer, however, did not lead to any significant improvement in the transformation rate. Kanamycin at 20 mg l in the selection medium for the initial 10 d resulted in the highest transformation frequency. Combining the best conditions for each parameter resulted in an overall transformation efficiency of 44.3 %. Gene transfer was confirmed through PCR and Southern blot analyses. Mendelian inheritance ratios were found in 71.5 % of the independent transgenic lines from self-fertilized T progeny. The optimized transformation procedure showed high transformation frequencies for all three tomato cultivars tested, namely, Kashi Vishesh (H-86), Hisar Anmol (H-24), and Kashi Amrit (DVRT-1), and is also expected to give reproducible results with other tomato cultivars. [ABSTRACT FROM AUTHOR]

Rosellinia necatrix is a fungus that infects a wide range of host plants and ruins a variety of commercially important crops. DNA fragments can be introduced into R. necatrix using conventional protoplast-PEG transformation and genome-integrating vectors; however, transformation efficiency with this strategy is quite low. Therefore, to establish a more effective transformation system for the studies of R. necatrix, an autonomously replicating vector was constructed using AMA1 sequences derived from Aspergillus nidulans, which is distantly related to R. necatrix. Use of this vector with AMA1 sequences increased transformation efficiency in R. necatrix, and the vector was maintained as a plasmid in the transformants. Transient and multivariate functional analyses in R. necatrix were performed using co-transformation of multiple pAMA-H vectors, which each carried either an expression cassette for eGFP, mOrange2, or a geneticin resistance gene. Furthermore, fluorescent proteins expressed from the autonomously replicating vectors were dispersed throughout fungal colonies even though the vectors themselves were restricted to the center of each colony. This intriguing phenomenon indicated that gene products could move from the center to the margin in a colony of the filamentous fungi via a cell-to-cell transport system. [ABSTRACT FROM AUTHOR]

Pepper is known to be a recalcitrant species to genetic transformation via Agrobacterium tumefaciens. A. rhizogenes-mediated transformation offers an alternative and rapid possibility to study gene functions in roots. In our study, we developed a new and efficient system for A. rhizogenes transformation of the cultivated species Capsicum annuum. Hypocotyls and foliar organs (true leaves and cotyledons) of Yolo Wonder (YW) and Criollo de Morelos 334 (CM334) pepper cultivars were inoculated with the two constructs pBIN- gus and pHKN29- gfp of A. rhizogenes strain A4RS. Foliar explants of both pepper genotypes infected by A4RS-pBIN- gus or A4RS-pHKN29- gfp produced transformed roots. Optimal results were obtained using the combination of the foliar explants with A4RS-pHKN29- gfp. 20.5% of YW foliar explants and 14.6% of CM334 foliar explants inoculated with A4RS-pHKN29- gfp produced at least one root expressing uniform green fluorescent protein. We confirmed by polymerase chain reaction the presence of the rolB and gfp genes in the co-transformed roots ensuring that they integrated both the T-DNA from the Ri plasmid and the reporter gene. We also demonstrated that co-transformed roots of YW and CM334 displayed the same resistance response to Phytophthora capsici than the corresponding untransformed roots. Our novel procedure to produce C. annuum hairy roots will thus support the functional analysis of potential resistance genes involved in pepper P. capsici interaction. [ABSTRACT FROM AUTHOR]

Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In this study, transient and stable transformation assays using different japonica and indica cultivars showed that the lower transformation efficiency in indica rice was mainly due to the low efficiency in T-DNA integration into the plant genome. Analyses of the global gene expression patterns across the transformation process in different varieties revealed major differences in the expression of genes responding to Agrobacterium within the first 6 h after infection and more differentially expressed genes were observed in the indica cultivar Zhenshan 97 (ZS), with a number of genes repressed early during infection. Microarray analysis revealed an important effect of plant defense response on Agrobacterium-mediated transformation. It has been shown that some genes which may be necessary for the transformation process were down-regulated in the indica cultivar ZS. This dataset provided a versatile resource for plant genomic research to understand the regulatory network of transformation process, and showed great promise for improving indica rice transformation using genetic manipulation of the rice genome. [ABSTRACT FROM AUTHOR]

Selectable marker genes (SMGs) are still useful to efficiently obtain transgenic plants, although marker-free techniques are available, but with limitations. The presence of SMGs, especially bacterial antibiotic resistance genes, in transgenic crops is criticized. Fortunately, several genes isolated from plants are available that can serve as SMGs. Here, I review the plant genes reported to have been used as SMGs. Some are wild-type genes that, when overexpressed, confer a selective advantage during in vitro plant regeneration, whereas some are mutated genes encoding enzymes resistant to inhibitory chemicals. Most of the genes have not yet been tested in a significant number of species. The effect of SMGs expression on the phenotype has often been superficially examined and should be better characterized. The sequence conservation of some SMGs could allow derivation of a SMGs from any plant species, if an intragenic or cisgenic approach to genetic engineering is preferred. I conclude that several promising SMGs have been isolated from plants, allowing avoidance of bacterial genes for transformation, transgene stacking, and intragenic or cisgenic engineering approaches. Nonetheless, further testing in more plant species would be useful to fully assess phenotypic neutrality, efficiency, and versatility. Patent rights restrict the immediate use of most plant SMGs for commercial applications, but freely available marker systems do exist. [ABSTRACT FROM AUTHOR]

To better understand the DNA restriction-modification (R-M) systems for more amenable strain development of the alternative industrial ethanologen, Zymomonas mobilis, three gene knockout mutants were constructed. The gene knockout mutants were tested for their DNA restriction activities by the determination of transformation efficiency using methylated and unmethylated foreign plasmid DNAs. Inactivation of a putative mrr gene encoded by ZMO0028 ( zmrr) resulted in a 60-fold increase in the transformation efficiency when unmethylated plasmid DNA was used. This indicated that the putative mrr gene may serve as a type IV restriction-modification system in Z. mobilis ZM4. To assign the function of a putative type I DNA methyltransferase encoded by ZMO1933 (putative S subunit) and ZMO1934 (putative M subunit), the putative S subunit was inactivated. The gene inactivation of ZMO1933 resulted in a 30-fold increase in the transformation efficiency when methylated plasmid DNA was introduced, indicating that the putative S subunit possibly serves as a part of functional type I R-M system(s). Growth studies performed on the mutant strains indicate inactivation of the type I S subunit resulted in a lower maximum specific glucose consumption rate and biomass yield, while inactivation of the type IV Zmrr had the opposite effect, with an increase in the maximum specific growth rate and biomass yield. [ABSTRACT FROM AUTHOR]

We previously established an efficient Agrobacterium-mediated transformation system using primary calli derived from mature seeds of the model japonica rice variety Nipponbare. We expected that the shortened tissue culture period would reduce callus browning-a common problem with the indica transformation system during prolonged tissue culture in the undifferentiated state. In this study, we successfully applied our efficient transformation system to Kasalath-a model variety of indica rice. The Luc reporter system is sensitive enough to allow quantitative analysis of the competency of rice callus for Agrobacterium-mediated transformation. We unexpectedly discovered that primary callus of Kasalath exhibits a remarkably high competency for Agrobacterium-mediated transformation compared to Nipponbare. Southern blot analysis and Luc luminescence showed that independent transformation events in primary callus of Kasalath occurred successfully at ca. tenfold higher frequency than in Nipponbare, and single copy T-DNA integration was observed in ~40% of these events. We also compared the competency of secondary callus of Nipponbare and Kasalath and again found superior competency in Kasalath, although the identification and subsequent observation of independent transformation events in secondary callus is difficult due to the vigorous growth of both transformed and non-transformed cells. An efficient transformation system in Kasalath could facilitate the identification of QTL genes, since many QTL genes are analyzed in a Nipponbare × Kasalath genetic background. The higher transformation competency of Kasalath could be a useful trait in the establishment of highly efficient systems involving new transformation technologies such as gene targeting. [ABSTRACT FROM AUTHOR]

Efficient Agrobacterium tumefaciens-mediated transformation and a higher recovery of transformed plants of cucumber cv. Poinsett76 were achieved via direct organogenesis from cotyledon explants. Stable transformants were obtained by inoculating explants with A. tumefaciens strains EHA105 or LBA4404, both harboring the binary vector pME508, which contains the neomycin phosphotransferase II ( nptII) and phosphinothricin resistance genes ( bar) conferring resistance to kanamycin and PPT, respectively, as selectable markers and the sgfp-tyg gene for the green fluorescent protein (GFP) as a visual marker driven by the constitutive CaMV35S promoter in the presence of acetosyringone (50 μM). Transformed shoots were obtained on MS Murashige and Skoog (Plant Physiol. 15: 473–497, ) medium supplemented with 1 mg L−1 benzyladenine (BA), 20 mg L−1 l-glutamine and 2 mg L−1 phosphinothricin (PPT) or 100 mg L−1 kanamycin. The regenerated shoots were examined in vivo using a hand-held long wave UV lamp for GFP expression. The GFP screening helped identify escapes and chimeric shoots at regular intervals to increase the growth of transformed shoots on cotyledon explants. Elongation and rooting of putative transformants were achieved on PPT (2 mg L−1) containing MS media with 0.5 mg L−1 gibberellic acid (GA3) and 0.6 mg L−1 indole butyric acid (IBA), respectively. PCR and Southern analyses confirmed the integration of the sgfp gene into the genome of T0 and the progenies. T1 segregation of transgenic progeny exhibited Mendelian inheritance of the transgene. The use of EHA105 resulted in 21% transformation efficiency compared to 8.5% when LBA4404 was used. This higher rate was greatly facilitated by PPT selection coupled with effective screening of transformants for GFP expression, thus making the protocol highly useful for the recovery of a higher number of transgenic cucumber plants. [ABSTRACT FROM AUTHOR]

Agrobacterium rhizogenes mediated transformation combined with a visual selection for green fluorescent protein (GFP) has been applied effectively in carrot ( Daucus carota L.) transformation. Carrot root discs were inoculated with A4, A4T, LBA1334 and LBA9402 strains, all bearing gfp gene in pBIN-m-gfp5-ER. The results indicate that transformed adventitious roots can be visually selected solely based on GFP fluorescence with a very high accuracy. The method requires no selection agents like antibiotics or herbicides and enables a reduction of labour and time necessary for tissue culture. Moreover, individual transformants can be easily excised from the host tissue and cultured separately. All of the 12 used carrot cultivars produced transformed adventitious roots and the frequency of discs producing GFP expressing adventitious roots varied from 13 to 85%. The highest transformation rate was found for A4T and LBA1334 strains possessing chromosomal background of A. tumefaciens C58. The results encourage that visual selection of transformed, fluorescing adventitious roots can be highly effective and applied routinely for the production of carrot transgenic plants. [ABSTRACT FROM AUTHOR]

Transformation efficiencies as high as 107 transformants μg-1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 107 or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations. [ABSTRACT FROM AUTHOR]

Although it is possible to transform wheat, broad application of the technology is limited because of the low overall efficiency and the lack of reliability of the technique. In addition there is little published data on transgene integration patterns and inheritance in wheat. We have generated a population of transgenic wheat and tritordeum lines under different auxin regimes and show that, under the conditions described, the presence of picloram results in higher transformation efficiencies than the presence of 2,4-D. Molecular analysis shows low-copy numbers and simple integration patterns to be prevalent in the transgenic lines. Mendelian inheritance of transgenes in T1 progeny was observed for the majority of lines. [ABSTRACT FROM AUTHOR]

Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol. [ABSTRACT FROM AUTHOR]

The replicating plasmid, pDblet, transformed the budding yeast Kluyveromyces marxianus to an efficiency of 104 transformant μg-1 DNA. Transformed cells showed 1% of segregation rate without affecting their growth rate of 0.69 h-1 and glucose consumption. These results were similar or better than the commonly used pE1 plasmid and suggests that pDblet can be used for cloning genes in K. marxianus. [ABSTRACT FROM AUTHOR]