Increased Transformation Efficiency Research Articles

Genetic evidence for a novel competence inhibitor in the industrially important Bacillus licheniformis

Natural genetic competence renders bacteria able to take up and, in case there is sufficient homology to the recipient’s chromosome, integrate exogenously supplied DNA. Well studied in Bacillus subtilis, genetic competence is—in several aspects—known to be differently regulated in Bacillus licheniformis. We now report on the identification of a novel, chromosomally encoded homolog of a competence inhibitor in B. licheniformis (ComI) that has hitherto only been described as a plasmid borne trait in the ancestral B. subtilis NCIB3610. Bioinformatical analysis that included 80 Bacillus strains covering 20 different species revealed a ComI encoding gene in all of the examined B. licheniformis representatives, and was identified in few among the other species investigated. The predicted ComI of B. licheniformis is a highly conserved peptide consisting of 28 amino acids. Since deletion of comI in B. licheniformis DSM13 resulted in twofold increased transformation efficiency by genetic competence and overexpression resulted in threefold decreased transformability, the function as a competence inhibitor became evident.

Anti-Restriction Protein, KlcAHS, Promotes Dissemination of Carbapenem Resistance.

Carbapenemase-producing Klebsiella pneumoniae (KPC) has emerged and spread throughout the world. A retrospective analysis was performed on carbapenem-resistant K. pneumoniae isolated at our teaching hospital during the period 2009–2010, when the initial outbreak occurred. To determine the mechanism(s) that underlies the increased infectivity exhibited by KPC, Multilocus Sequence Typing (MLST) was conducted. A series of plasmids was also extracted, sequenced and analyzed. Concurrently, the complete sequences of blaKPC−2-harboring plasmids deposited in GenBank were summarized and aligned. The blaKPC−2 and KlcAHS genes in the carbapenem-resistant K. pneumoniae isolates were examined. E. coli strains, carrying different Type I Restriction and Modification (RM) systems, were selected to study the interaction between RM systems, anti-RM systems and horizontal gene transfer (HGT). The ST11 clone predominated among 102 carbapenem-resistant K. pneumoniae isolates, all harbored the blaKPC−2 gene; 98% contained the KlcAHS gene. KlcAHS was one of the core genes in the backbone region of most blaKPC−2 carrying plasmids. Type I RM systems in the host bacteria reduced the rate of pHS10842 plasmid transformation by 30- to 40-fold. Presence of the anti-restriction protein, KlcAHS, on the other hand, increased transformation efficiency by 3- to 6-fold. These results indicate that RM systems can significantly restrict HGT. In contrast, KlcAHS can disrupt the RM systems and promote HGT by transformation. These findings suggest that the anti-restriction protein, KlcAHS, represents a novel mechanism that facilitates the increased transfer of blaKPC-2 and KlcAHS-carrying plasmids among K. pneumoniae strains.

Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei

In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude.

Efficiency limit factor analysis for the Francis-99 hydraulic turbine

The energy loss in hydraulic turbine is the most direct factor that affects the efficiency of the hydraulic turbine. Based on the analysis theory of inner energy loss of hydraulic turbine, combining the measurement data of the Francis-99, this paper calculates characteristic parameters of inner energy loss of the hydraulic turbine, and establishes the calculation model of the hydraulic turbine power. Taken the start-up test conditions given by Francis-99 as case, characteristics of the inner energy of the hydraulic turbine in transient and transformation law are researched. Further, analyzing mechanical friction in hydraulic turbine, we think that main ingredients of mechanical friction loss is the rotation friction loss between rotating runner and water body, and defined as the inner mechanical friction loss. The calculation method of the inner mechanical friction loss is given roughly. Our purpose is that explore and research the method and way increasing transformation efficiency of water flow by means of analysis energy losses in hydraulic turbine.

Transformasi Genetik Pisang Ambon dengan Gen Kitinase dari Padi

<p>One of the main constrains on the<br />productivity and quality enhancement of banana is wilt<br />diseases caused by Fusarium oxysporum (Foc). Production<br />decrease by wilt disease was 63.33%. Therefore, an effort to<br />obtain the banana new variety which is tolerant to fusarium<br />was absolutely necessary to be done. Genetic engineering<br />can be used in new variety improvement, especially for<br />production of pest and disease tolerant varieties.<br />Transformation of banana with chi gene which expressed<br />chitinase enzyme have been used in obtaining the plant<br />resistant to Foc. The goals of the research were to obtain:<br />determine lowest higromisin consentration inhibited nodul<br />growth by tested four consentration of higromisin,<br />determine optimum cocultivation time by tested three times<br />cocultivation, tested asetosiringone added on two times<br />cocultivation, and gen chi introduction at banana<br />transforman shoots with PCR. The explants used were<br />nodule induced from pseudostem of banana cv. Ambon<br />kuning. Genetic transformation done by sowing the explants<br />in bacterial suspension 0, 15, 30, and 45 minutes.The effect<br />of asetosiringone (0 and 100 mg/l) on cocultivation medium<br />was observed. The research results showed that the lowest<br />higromisin concentration inhibited nodule growth was 25<br />mg/l for 5 weeks and the best time for inoculation of nodule<br />were 30 minute. Asetosiringone added on bacterial<br />suspension did not increase transformation efficiency.<br />Chitinase gene transformation using Agrobacterium<br />tumefaciens on banana nodules produced 25 noduly ines of<br />putative transformant on selection media and 34 plants<br />transforman identification by PCR.</p>

Regulation of hydrogen peroxide accumulation and death of Agrobacterium-transformed cells in tomato transformation

Our previous studies demonstrate that Agrobacterium causes tissue browning that subsequently reduces transformation efficiency in tomato transformation. In addition application of lipoic acid (LA) can reduce tissue browning and increase transformation efficiency in different crops. A major challenge in Agrobacterium-mediated plant transformation is the death of Agrobacterium-transformed cells (DATC), which restricts transgenic plant production. Hydrogen peroxide (H2O2) plays a critical role in oxidative stress. However, little is known about the biochemical and molecular mechanisms for DATC. Our biological and correlation analyses showed that Agrobacterium mediated H2O2 accumulation (HOA) and HOA elevation led to DATC during Agrobacterium-mediated tomato cv. MicroTom transformation. Agrobacterium significantly (P < 0.05) increased 4.2- and 1.4-fold expression of WRKY75 (a H2O2-responsive transcription factor) and superoxide dismutase (SOD), respectively, while the application of 4.4 M H2O2 significantly increased 19- and 2.7-fold expression of WRKY75 and 2-cys peroxiredoxin (Cys), respectively, and decreased fivefold SOD expression, compared with a control. LA application significantly (P < 0.05) reduced 1.6-fold HOA and DATC while it significantly increased 1.7-fold expression of Cys, and reduced 2.2- and 1.4-fold expression of WRKY75 and SOD, respectively. The reduction of HOA and DATC was accompanied by suppression of WRKY75 and SOD and activation of Cys. Our results indicated that DATC was regulated by H2O2 that was triggered by Agrobacterium and LA application through their gene regulation. In addition, HOA was associated with a biotic generating reactive oxygen species (ROS) mechanism, and HOA and DATC were likely regulated by an enzymatic ROS scavenging mechanism during Agrobacterium-mediated tomato transformation.

Low Agrobacterium tumefaciens inoculum levels and a long co-culture period lead to reduced plant defense responses and increase transgenic shoot production of sunflower (Helianthus annuus L.).

Agrobacterium-mediated plant transformation is typically conducted by inoculating plant tissues with an Agrobacterium suspension containing approximately 108–109 bacteria mL−1, followed by a 2–3-d co-culture period. Use of longer co-culture periods could potentially increase transformation efficiencies by allowing more time for Agrobacterium to interact with plant cells, but bacterial overgrowth is likely to occur, leading to severe tissue browning and reduced transformation and regeneration. Low bacterial inoculum levels were therefore evaluated as a means to reduce the negative outcomes associated with long co-culture. The use of low inoculum bacterial suspensions (approximately 6 × 102 bacteria mL−1) followed by long co-culture (15 d) led to the production of an average of three transformed sunflower shoots per explant while the use of high inoculum (approximately 6 × 108 bacteria mL−1) followed by short co-culture (3 d) led to no transformed shoots. Low inoculum and long co-culture acted synergistically, and both were required for the improvement of sunflower transformation. Gene expression analysis via qRT-PCR showed that genes related to plant defense response were generally expressed at lower levels in the explants treated with low inoculum than those treated with high inoculum during 15 d of co-culture, suggesting that low inoculum reduced the induction of plant defense responses. The use of low inoculum with long co-culture (LI/LC) led to large increases in sunflower transformation efficiency. This method has great potential for improving transformation efficiencies and expanding the types of target tissues amenable for transformation of different plant species.

Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost.

Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides.

Evaluation of EBV transformation of human memory B-cells isolated by FACS and MACS techniques

Several studies have been performed to develop effective neutralizing monoclonal antibodies. The Epstein–Barr virus (EBV) can efficiently immortalize B-cells to establish lymphoblastoid cell lines (LCL) and so it has been used extensively for transformation of B-cells to produce and secrete immunoglobulin. The present study addressed the effect of TLR7/8 agonist (R848), feeder cells layer and fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) cell separation methods on the transformation efficiency of antibody-producing memory B-cells. For these studies, the antigen used for analyses of antibody formation was the tetanus neurotoxin (TeNT) derived from Clostridium tetani. The results here showed that employing an HFFF.PI6 feeder cell layer, R848 agonist and FACS-mediated purification of memory B-cells led to increased transformation efficiency. Altogether, the effects of the R848 and the feeder cells provided an efficient method for EBV transformation of human B-cells. Moreover, there was an advantage in using FACS sorting of B-cells over the MACS method in the context of EBV transformation and immortalization of precursors of antigen-specific B-cells.

Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.

Bacterial competent cells are essential for cloning, construction of DNA libraries, and mutagenesis in every molecular biology laboratory. Among various transformation methods, electroporation is found to own the best transformation efficiency. Previous electroporation methods are based on washing and electroporating the bacterial cells in ice-cold condition that make them fragile and prone to death. Here we present simple temperature shift based methods that improve DNA transformation and recombineering efficiency in E. coli and several other gram-negative bacteria thereby economizing time and cost. Increased transformation efficiency of large DNA molecules is a significant advantage that might facilitate the cloning of large fragments from genomic DNA preparations and metagenomics samples.

A simple and efficient Agrobacterium tumefaciens-mediated plant transformation of Brassica rapa ssp. pekinensis.

The present study aims to investigate the numerous factors influencing Agrobacterium tumefaciens-mediated genetic transformation of Chinese cabbage (Brassica rapa ssp. pekinensis). Factors affecting transformation efficiency, such as age of explants, Agrobacterium concentration, and effect of acetosyringone, pre-cultivation, infection and co-cultivation time of Agrobacterium were examined. The pre-cultured hypocotyls from young seedlings prior to exposure to Agrobacterium showed higher shoot regeneration. The plant transformation with the modest A. tumefaciens concentrations (0.8 OD) and the 3 days co-cultivation periods increased transformation efficiency. Plant growth hormones [1-naphthyl acetic acid (NAA) and 6-benzyl amino purine (BAP)] were essential for callus and shoot formation. Root formation was effective in half strength MS medium without supplementation of root-inducing hormones. To maintain selection pressure, plant subculture was carried out every 2 weeks with selective antibiotics. The putative transgenic plants were acclimatized in the greenhouse. Polymerase chain reaction was performed to confirm the integration of T-DNA into the genome of transgenic plants. A transformation efficiency of 15 % was obtained. This protocol allows effective transformation and indirect regeneration of Brassica rapa.

Dam and Dcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B-598: development of methods for electrotransformation, conjugation, and sonoporation.

BackgroundButanol is currently one of the most discussed biofuels. Its use provides many benefits in comparison to bio-ethanol, but the price of its fermentative production is still high. Genetic improvements could help solve many problems associated with butanol production during ABE fermentation, such as its toxicity, low concentration achievable in the cultivation medium, the need for a relatively expensive substrate, and many more. Clostridium pasteurianum NRRL B-598 is non-type strain producing butanol, acetone, and a negligible amount of ethanol. Its main benefits are high oxygen tolerance, utilization of a wide range of carbon and nitrogen sources, and the availability of its whole genome sequence. However, there is no established method for the transfer of foreign DNA into this strain; this is the next step necessary for progress in its use for butanol production.ResultsWe have described functional protocols for conjugation and transformation of the bio-butanol producer C. pasteurianum NRRL B-598 by foreign plasmid DNA. We show that the use of unmethylated plasmid DNA is necessary for efficient transformation or successful conjugation. Genes encoding DNA methylation and those for restriction-modification systems and antibiotic resistance were searched for in the whole genome sequence and their homologies with other clostridial bacteria were determined. Furthermore, activity of described novel type I restriction system was proved experimentally. The described electrotransformation protocol achieved an efficiency 1.2 × 102 cfu/μg DNA after step-by-step optimization and an efficiency of 1.6 × 102 cfu/μg DNA was achieved by the sonoporation technique using a standard laboratory ultrasound bath. The highest transformation efficiency was achieved using a combination of these approaches; sono/electroporation led to an increase in transformation efficiency, to 5.3 × 102 cfu/μg DNA.ConclusionsBoth Dam and Dcm methylations are detrimental for transformation of C. pasteurianum NRRL B-598. Methods for conjugation, electroporation, sonoporation, and a combined method for sono/electroporation were established for this strain. The methods described could be used for genetic improvement of this strain, which is suitable for bio-butanol production.

Development of a rapid, low-cost protoplast transfection system for switchgrass (Panicum virgatum L.)

Key messageA switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.In recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

I-SceI-mediated double-strand DNA breaks stimulate efficient gene targeting in the industrial fungus Trichoderma reesei.

Targeted integration of expression cassettes for enzyme production in industrial microorganisms is desirable especially when enzyme variants are screened for improved enzymatic properties. However, currently used methods for targeted integration are inefficient and result in low transformation frequencies. In this study, we expressed the Saccharomyces cerevisiae I-SceI meganuclease to generate double-strand breaks at a defined locus in the Trichoderma reesei genome. We showed that the double-strand DNA breaks mediated by I-SceI can be efficiently repaired when an exogenous DNA cassette flanked by regions homologous to the I-SceI landing locus was added during transformation. Transformation efficiencies increased approximately sixfold compared to control transformation. Analysis of the transformants obtained via I-SceI-mediated gene targeting showed that about two thirds of the transformants resulted from a homologous recombination event at the predetermined locus. Counter selection of the transformants for the loss of the pyrG marker upon integration of the DNA cassette showed that almost all of the clones contained the cassette at the predetermined locus. Analysis of independently obtained transformants using targeted integration of a glucoamylase expression cassette demonstrated that glucoamylase production among the transformants was high and showing limited variation. In conclusion, the gene targeting system developed in this study significantly increases transformation efficiency as well as homologous recombination efficiency and omits the use of Δku70 strains. It is also suitable for high-throughput screening of enzyme variants or gene libraries in T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-015-6829-1) contains supplementary material, which is available to authorized users.

TRANSFORMATION OF IAAM GENE INTO ANTHER-DERIVED EMBRYOS OF LOQUAT MEDIATED BY AGROBACTERIUM TUMEFACIENS

The parthenocarpic gene iaaM was transferred into anther-derived embryos, using Agrobacterium-mediated transformation. The results showed that 2-3-day pre-culture, 15-min infection and 3-day co-culture with 10 mg L-1 AS in the co-culture medium led to an increase in transformation efficiency. Cefotaxime at 250 mg L-1 or Ampicillin at 750 mg L-1 were optimal. Sixteen transgenic lines were obtained. The iaaM gene-specific amplification results showed that 9 strains were amplified at the expected target bands, and the PCR positive rate was 56.3%. The PCR-Southern analysis indicated that 8 strains showed hybridization signal, which further proved that the iaaM gene was integrated into the genome of the embryos, and the transformation rate was about 0.5%.

Disruption of bbe02 by Insertion of a Luciferase Gene Increases Transformation Efficiency of Borrelia burgdorferi and Allows Live Imaging in Lyme Disease Susceptible C3H Mice.

Lyme disease is the most prevalent tick-borne disease in North America and Europe. The causative agent, Borrelia burgdorferi persists in the white-footed mouse. Infection with B. burgdorferi can cause acute to persistent multisystemic Lyme disease in humans. Some disease manifestations are also exhibited in the mouse model of Lyme disease. Genetic manipulation of B. burgdorferi remains difficult. First, B. burgdorferi contains a large number of endogenous plasmids with unique sequences encoding unknown functions. The presence of these plasmids needs to be confirmed after each genetic manipulation. Second, the restriction modification defense systems, including that encoded by bbe02 gene lead to low transformation efficiency in B. burgdorferi. Therefore, studying the molecular basis of Lyme pathogenesis is a challenge. Furthermore, investigation of the role of a specific B. burgdorferi protein throughout infection requires a large number of mice, making it labor intensive and expensive. To overcome the problems associated with low transformation efficiency and to reduce the number of mice needed for experiments, we disrupted the bbe02 gene of a highly infectious and pathogenic B. burgdorferi strain, N40 D10/E9 through insertion of a firefly luciferase gene. The bbe02 mutant shows higher transformation efficiency and maintains luciferase activity throughout infection as detected by live imaging of mice. Infectivity and pathogenesis of this mutant were comparable to the wild-type N40 strain. This mutant will serve as an ideal parental strain to examine the roles of various B. burgdorferi proteins in Lyme pathogenesis in the mouse model in the future.

THE IMPORTANCE OF SALICYLIC ACID AND AN IMPROVED PLANT CONDITION IN DETERMINING SUCCESS IN AGROBACTERIUM-MEDIATED TRANSFORMATION

A succesful Agrobacterium-mediated transformation requires a balanced interaction between the plant and Agrobacterium. We hypothesize that blocking salicylic acid (SA) biosynthesis could increase the plant's susceptibility and gene transfer events. In this study, the influence of the SA biosynthesis on transformation efficiency was investigated. We tested the effect of increasing concentrations of SA inhibitors such as 2-amidoindane-2-phosphonic acid (AIP) and paclobutrazol (PBZ) in Petunia hybrida and Nicotiana benthamiana. As a control, the effect of adding SA was examined. In addition, the effect of lipoic acid (LA) was studied. LA, an antioxidant, is considered to act as a general transformation enhancer by improving the plant's condition. Adding SA inhibitors should lead to improved conditions for Agrobacterium and adding LA should improve conditions for the plant. Explants from fresh leaves of P. hybrida and N. benthamiana were inoculated with A. tumefaciens strain AGL1(pBinGlyRed-Asc1) containing dsRed as a reporter gene. Transient dsRed expresion was determined at 14 days after co-cultivation by counting red fluorescing spots and transformation efficiency was determined from the number of explants with calli surviving on selection media at 30 days. In P. hybrida, 10 ?M AIP and PBZ resulted in the highest transient and stable transformation. However in N. benthamiana, only 50 ?M AIP showed a significant increase and only in transient expresion, and no evidence of any stimulatory effect of PBZ was observed. Adding SA always decreased the efficiencies in both plant species. Furthermore, 10 and 1 ?M LA significantly increased transformation efficiency in P. hybrida and N. benthamiana, respectively, with les browning and necrosis. Our preliminary results suggested that blocking SA biosynthesis promoted gene transfer but responses were variable and not equally succesful between the two plant species. However, repeating these experiments providing us with more data might clarify the picture further.

Heat Shock-Enhanced Conjugation Efficiency in Standard Campylobacter jejuni Strains.

Campylobacter jejuni, the leading bacterial cause of human gastroenteritis in the United States, displays significant strain diversity due to horizontal gene transfer. Conjugation is an important horizontal gene transfer mechanism contributing to the evolution of bacterial pathogenesis and antimicrobial resistance. It has been observed that heat shock could increase transformation efficiency in some bacteria. In this study, the effect of heat shock on C. jejuni conjugation efficiency and the underlying mechanisms were examined. With a modified Escherichia coli donor strain, different C. jejuni recipient strains displayed significant variation in conjugation efficiency ranging from 6.2 × 10(-8) to 6.0 × 10(-3) CFU per recipient cell. Despite reduced viability, heat shock of standard C. jejuni NCTC 11168 and 81-176 strains (e.g., 48 to 54°C for 30 to 60 min) could dramatically enhance C. jejuni conjugation efficiency up to 1,000-fold. The phenotype of the heat shock-enhanced conjugation in C. jejuni recipient cells could be sustained for at least 9 h. Filtered supernatant from the heat shock-treated C. jejuni cells could not enhance conjugation efficiency, which suggests that the enhanced conjugation efficiency is independent of secreted substances. Mutagenesis analysis indicated that the clustered regularly interspaced short palindromic repeats system and the selected restriction-modification systems (Cj0030/Cj0031, Cj0139/Cj0140, Cj0690c, and HsdR) were dispensable for heat shock-enhanced conjugation in C. jejuni. Taking all results together, this study demonstrated a heat shock-enhanced conjugation efficiency in standard C. jejuni strains, leading to an optimized conjugation protocol for molecular manipulation of this organism. The findings from this study also represent a significant step toward elucidation of the molecular mechanism of conjugative gene transfer in C. jejuni.

Editorial: "Agrobacterium biology and its application to transgenic plant production".

Editorial: "Agrobacterium biology and its application to transgenic plant production".

Improvements in Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) for large-scale production of transgenic plants

Cassava (Manihot esculenta Crantz) is a major staple food crop of the humid tropics. As a heterozygous, vegetatively propagated crop, robust transformation protocols must be developed for elite cultivars that allow predictable production of large numbers of independent transgenic plant lines. A high throughput Agrobacterium-mediated transformation system was developed for the elite East African farmer-preferred cassava cultivar TME 204 using the GFP visual marker gene. Inclusion of the antibiotic moxalactam in culture medium used to produce embryogenic target tissues prior to inoculation with Agrobacterium increased recovery of independent GFP-expressing transgenic callus lines by up to 113-fold compared to the control. Enhanced transformation was also observed when TME 204 tissues were pretreated with other cephalosporins, namely cefoperazone, cefoxitin, cefmetazole and cefotaxime. Similar but less dramatic increases in transformation efficiencies were seen for the West African cultivars Oko-iyawo and 60444 when pre-treated with moxalactam. Dilution of Agrobacterium suspensions used for co-culture was found to increase transformation efficiencies, resulting in regeneration at an average of 33 GFP-expressing TME 204 plants per cc settled cell volume at OD600 0.05, compared to 15 plants at the more commonly used OD600 0.5. The optimized transformation systems were successfully utilized for the integration of genetic constructs for disease resistance and nutritional enhancement into more than 750 plants of TME 204.