Agrobacterium Tumefaciens-mediated Transformation Protocol Research Articles

Establishment of genome-editing system and assembly of a near-complete genome in broomcorn millet.

The ancient crop broomcorn millet (Panicum miliaceum L.) is an indispensable orphan crop in semi-arid regions due to its short life cycle and excellent abiotic stress tolerance. These advantages make it an important alternative crop to increase food security and achieve the goal of zero hunger, particularly in light of the uncertainty of global climate change. However, functional genomic and biotechnological research in broomcorn millet has been hampered due to a lack of genetic tools such as transformation and genome-editing techniques. Here, we successfully performed genome editing of broomcorn millet. We identified an elite variety, Hongmi, that produces embryogenic callus and has high shoot regeneration ability in in vitro culture. We established an Agrobacterium tumefaciens-mediated genetic transformation protocol and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated genome-editing system for Hongmi. Using these techniques, we produced herbicide-resistant transgenic plants and edited phytoene desaturase (PmPDS), which is involved in chlorophyll biosynthesis. To facilitate the rapid adoption of Hongmi as a model line for broomcorn millet research, we assembled a near-complete genome sequence of Hongmi and comprehensively annotated its genome. Together, our results open the door to improving broomcorn millet using biotechnology.

Agrobacterium tumefaciens-mediated transformation for the genetic modification of the biotechnologically relevant fungus Aspergillus vadensis through synthetic biology

In the last years, many research efforts have been applied for the development of filamentous fungi as hosts for heterologous protein production. Aspergillus vadensis CBS 113365, a close relative of the industrial workhorse Aspergillus niger, has been suggested as a more suitable cell factory as it does not acidify the culture medium and produces very low levels of secreted proteases. Therefore, efficient methods and tools that allow the genetic manipulation and exploitation of this biotechnologically relevant fungus are needed. To date, only protoplast-mediated transformation and classical cloning strategies have been implemented for A. vadensis genetic modification, which decreases the exploitation capacity of this fungus at the industrial level. In this study, we have adapted and implemented an Agrobacterium tumefaciens-mediated transformation protocol for A. vadensis for the first time, and applied the FungalBraid system to genetically modify this species by means of synthetic biology. As proof of concept, we have successfully complemented and fluorescently labelled a uridine auxotrophic A. vadensis pyrA- strain and generated A. vadensis mutants carrying the Penicillium expansum-based expression cassette for the heterologous production of the antifungal protein PeAfpA from P. expansum. Even though we have yet to find the conditions that trigger PeAfpA production in this species, the implementation of the ATMT method reported here, along with the application of the FungalBraid system, will greatly aid in this task and will facilitate the exploitation of A. vadensis as a fungal workhorse for protein production for multiple biotechnological applications.

Towards a Cardoon (Cynara cardunculus var. altilis)-Based Biorefinery: A Case Study of Improved Cell Cultures via Genetic Modulation of the Phenylpropanoid Pathway.

Cultivated cardoon (Cynara cardunculus var. altilis L.) is a promising candidate species for the development of plant cell cultures suitable for large-scale biomass production and recovery of nutraceuticals. We set up a protocol for Agrobacterium tumefaciens-mediated transformation, which can be used for the improvement of cardoon cell cultures in a frame of biorefinery. As high lignin content determines lower saccharification yields for the biomass, we opted for a biotechnological approach, with the purpose of reducing lignin content; we generated transgenic lines overexpressing the Arabidopsis thaliana MYB4 transcription factor, a known repressor of lignin/flavonoid biosynthesis. Here, we report a comprehensive characterization, including metabolic and transcriptomic analyses of AtMYB4 overexpression cardoon lines, in comparison to wild type, underlining favorable traits for their use in biorefinery. Among these, the improved accessibility of the lignocellulosic biomass to degrading enzymes due to depletion of lignin content, the unexpected increased growth rates, and the valuable nutraceutical profiles, in particular for hydroxycinnamic/caffeoylquinic and fatty acids profiles.

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

Agrobacterium tumefaciens-mediated transformation of Coniella granati

Pomegranate fruit rot caused by Coniella granati is among the most devastating diseases threatening pomegranate production. The pathogenic mechanism of this pathogen remains largely unknown due to lack of genetic transformation method. Herein, we developed an approach to the Agrobacterium tumefaciens-mediated transformation (ATMT) of C. granati using a plasmid vector encoding the green fluorescent protein (GFP) and hygromycin resistance (Hyg) genes. This approach yielded C. granati transformants that exhibited uniform, stable green fluorescence. We further optimized this ATMT protocol, enabling us to achieve a transformation efficiency of up to 300 transformants per 0.5 cm2 mycelial plug. Together, we thus provide the first report of the stable transformation of C. granati, laying a foundation for future functional studies characterizing this economically important fungal pathogen.

Introduction of the Nicotiana protein kinase (NPK1) gene by combining Agrobacterium-mediated transformation and recurrent somatic embryogenesis to enhance salt tolerance in cauliflower

Cauliflower is exposed to various biotic and abiotic stresses, including increased salinity due to the intensive irrigation of crops. Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules that play important roles in regulating innate immune responses in plants. Based on involvement of tobacco MAP kinase kinase kinase (NPK1) in stress response, the effect of the expression of NPK1 transgene to NaCl salt stress tolerance in cauliflower KFRM4 lines was studied. The Agrobacterium tumefaciens-mediated transformation protocol, using EHA101(pSHX004) vector harbouring the NPK1 and phosphinothricin N-acetyltransferase (bar) genes, the cyclic somatic embryogenesis regeneration pathway, the application of acetosyringone (AS) during co-cultivation and a delayed phosphinothricine (PPT) selection procedure provided sufficient transformation efficiency of 7.33% without escapes. PCR analysis indicated the integration of both NPK1 and bar transgenes in regenerated cauliflower lines. Transgenic cauliflower lines, exposed to NaCl stress in vitro, showed higher growth rates, greater ability to retain chlorophyll and carotenoids, and increased osmotic regulation capacity compared with non-transformed control plants. The tolerance level of transformed lines correlated with the level of NPK1 gene expression estimated by RT-qPCR, and the L2 line with the highest NPK1 expression displayed the greatest tolerance to NaCl stress. None of the obtained cauliflower transformed lines grown in greenhouses showed any morphological or yield differences compared with non-transformed plants. Furthermore, the expression of the bar gene facilitated the tolerance of transformed lines to the total herbicide PPT, applied at concentrations 2–3 times higher than those routinely used for weed control in the crop field. The results underlined that constitutively expressing NPK1 can significantly contribute to enhanced salt stress tolerance in cauliflower, suggesting that this could be a promising basis for the creation of new stress tolerance cruciferous vegetable lines.

Establishment of an Agrobacterium mediated transformation protocol for the detection of cytokinin in the heterophyllous plant Hygrophila difformis (Acanthaceae).

This is the first report of a highly efficient Agrobacterium tumefaciens-mediated transformation protocol for Acanthaceae and its utilization in revealing important roles of cytokinin in regulating heterophylly in Hygrophila difformis. Plants show amazing morphological differences in leaf form in response to changes in the surrounding environment, which is a phenomenon called heterophylly. Previous studies have shown that the aquatic plant Hygrophila difformis (Acanthaceae) is an ideal model for heterophylly study. However, low efficiency and poor reproducibility of genetic transformation restricted H. difformis as a model plant. In this study, we reported successful induction of callus, shoots and the establishment of an efficient stable transformation protocol as mediated by Agrobacterium tumefaciens LBA4404. We found that the highest callus induction efficiency was achieved with 1mg/L 1-Naphthaleneacetic acid (NAA) and 2mg/L 6-benzyladenine (6-BA), that efficient shoot induction required 0.1mg/L NAA and 0.1mg/L 6-BA and that high transformation efficiency required 100µM acetosyringone. Due to the importance of phytohormones in the regulation of heterophylly and the inadequate knowledge about the function of cytokinin (CK) in this process, we analyzed the function of CK in the regulation of heterophylly by exogenous CK applicationand endogenous CK detection. By using our newly developed transformation system to detect CK signals, contents and distribution in H. difformis, we revealed an important role of CK in environmental mediated heterophylly.

Thyrostroma carpophilum insertional mutagenesis: A step towards understanding its pathogenicity mechanism

Thyrostroma carpophilum, a causal agent of shot hole disease of stone fruits, cause severe loss in economically important fruit crops of Kashmir. Understanding its pathogenesis at molecular level will aid in devising a better management strategy. In this study, we optimized Agrobacterium tumefaciens mediated transformation (ATMT) conditions for T. carpophilum using PBIF2-EGFP construct. Using this protocol, we obtained 328 positive transformants per 104 spores and subsequent sub-culturing of transformants on selective and non-selective media resulted in stable T-DNA integration. Southern blot analysis revealed that most of the transformants embodied single T-DNA integration. Using this method, we obtained a small-scale transformant library (2050 transformants). Among this pool, we tested 1005 transformants for their pathogenicity; out of which 185 showed complete pathogenicity loss, 35 displayed reduced virulence and 785 were pathogenically similar to wild type. Out of this experimental stock, three transformants from each category were randomly selected to dissect the infection assay. The findings deciphered that transformants with complete pathogenicity loss failed to penetrate the host tissue and a few transformants failed to sporulate in laboratory. Transformants from reduced category could not form appressorium and occasionally sporulated. Transformants similar to wild type were morphologically and pathogenically similar to wild type because of un-alteration in their modus operandi. Our work provides a new platform to understand the pathogenicity mechanism of T. carpophilum. The optimized ATMT protocol will help in developing large transformant library that can help to identify the virulence arsenals necessary for the pathogen to cause disease.

Advancing Functional Genetics Through Agrobacterium-Mediated Insertional Mutagenesis and CRISPR/Cas9 in the Commensal and Pathogenic Yeast Malassezia.

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens-mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens, to deliver both a CAS9-gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens-mediated transformation to aid in the identification of gene functions in M. furfur, through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion.

Transformation of Corynespora cassiicola by Agrobacterium tumefaciens

The fungus causing target spot disease, Corynespora cassiicola (Berk. & M. A. Curtis) C. T. Wei, poses an increasing threat to watermelon (Citrullus lanatus), muskmelon (Cucumis melo), and cucumber (Cucumis sativus); the most economically important cucurbit crops grown in China. An understanding of the molecular mechanisms underlying the pathogenicity of C. cassiicola is essential for the development of new strategies to control this disease-causing fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) might be useful to obtain transformants of C. cassiicola, for the ultimate identification of genes involved in pathogenicity. In the present work, we established and optimized an ATMT protocol using A. tumefaciens strain AGL-1 carrying the vector pATMT1 for C. cassiicola. Efficiency of ATMT was 102–148 transformants per 106 conidia and successive subculturing of transformants on non-selective and selective media demonstrated that the integrated transfer (T)-DNA was stably inherited in C. cassiicola transformants. The integration of the hygromycin B phosphotransferase (hph) gene into C. cassiicola was validated by PCR and Southern blot analyses, which revealed that nearly 90 % of the transformants contained single-copy T-DNA. The transformants with altered phenotypes were characterized. Three of these transformants completely lost pathogenicity and other three showed strongly impaired pathogenicity relative to the Cc-GX strain on muskmelon leaves. These results strongly suggest that ATMT may be used as a molecular tool for identifying genes relevant to pathogenicity in the fungus C. cassiicola, an emerging threat to several agronomically important plants in China.

Agrobacterium-mediated transformation of Ceratocystis albifundus

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium’s virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.

Embryogenic Callus as Target for Efficient Transformation of Cyclamen persicum Enabling Gene Function Studies.

Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus.

Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ∼ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (∼ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

CRISPR/Cas9-Mediated Mutagenesis of Four Putative Symbiosis Genes of the Tropical Tree Parasponia andersonii Reveals Novel Phenotypes.

Parasponia represents five fast-growing tropical tree species in the Cannabaceae and is the only plant lineage besides legumes that can establish nitrogen-fixing nodules with rhizobium. Comparative analyses between legumes and Parasponia allows identification of conserved genetic networks controlling this symbiosis. However, such studies are hampered due to the absence of powerful reverse genetic tools for Parasponia. Here, we present a fast and efficient protocol for Agrobacterium tumefaciens-mediated transformation and CRISPR/Cas9 mutagenesis of Parasponia andersonii. Using this protocol, knockout mutants are obtained within 3 months. Due to efficient micro-propagation, bi-allelic mutants can be studied in the T0 generation, allowing phenotypic evaluation within 6 months after transformation. We mutated four genes – PanHK4, PanEIN2, PanNSP1, and PanNSP2 – that control cytokinin, ethylene, or strigolactone hormonal networks and that in legumes commit essential symbiotic functions. Knockout mutants in Panhk4 and Panein2 displayed developmental phenotypes, namely reduced procambium activity in Panhk4 and disturbed sex differentiation in Panein2 mutants. The symbiotic phenotypes of Panhk4 and Panein2 mutant lines differ from those in legumes. In contrast, PanNSP1 and PanNSP2 are essential for nodule formation, a phenotype similar as reported for legumes. This indicates a conserved role for these GRAS-type transcriptional regulators in rhizobium symbiosis, illustrating the value of Parasponia trees as a research model for reverse genetic studies.

Improved Genetic Transformation of Sugarcane (Saccharum spp.) Embryogenic Callus Mediated by Agrobacterium tumefaciens.

Sugarcane (Saccharum spp.) is a monocotyledonous semi-perennial C4 grass of the Poaceae family. Its capacity to accumulate high content of sucrose and biomass makes it one of the most important crops for sugar and biofuel production. Conventional methods of sugarcane breeding have shown several limitations due to its complex polyploid and aneuploid genome. However, improvement by biotechnological engineering is currently the most promising alternative to introduce economically important traits. In this work, we present an improved protocol for Agrobacterium tumefaciens-mediated transformation of commercial sugarcane hybrids using immature top stalk-derived embryogenic callus cultures. The callus cultures are transformed with preconditioned A. tumefaciens carrying a binary vector that encodes expression cassettes for a gene of interest and the bialaphos resistance gene (bar confers resistance to glufosinate-ammonium herbicide). This protocol has been used to successfully transform a commercial sugarcane cultivar, SP80-3280, highlighting: (i) reduced recalcitrance and oxidation; (ii) high yield of embryogenic callus; (iii) improved selection; and (iv) shoot regeneration and rooting of the transformed plants. Altogether, these improvements generated a transformation efficiency of 2.2%. This protocol provides a reliable tool for a routine procedure for sugarcane improvement by genetic engineering. © 2017 by John Wiley & Sons, Inc.

Optimization of a Bacopa monnieri-based genetic transformation model for testing the expression efficiency of pathway gene constructs of medicinal crops

A fast regenerating Agrobacterium tumefaciens-mediated transformation protocol for Bacopa monnieri (L.) Wettst. was developed as a model system for heterologous expression of terpenoid indole alkaloid pathway genes from Catharanthus roseus (L.) G. Don. The direct regeneration of shoots from leaf explants co-cultured with A. tumefaciens resulted in the integration of a tryptophan decarboxylase (tdc) and strictosidine synthase (str) cassette ( ) in the regenerated progeny. The highest transformation efficiency (83.88%) was achieved when leaf explants were infected on the adaxial laminar surface by manual pricking with 48- to 72-h-old suspensions (OD600 = 0.5–0.6) of A. tumefaciens strain LBA1119 (carrying the binary vector pMOG22). The heterologous expression of tryptophan decarboxylase and strictosidine synthase genes that are otherwise not present in B. monnieri plants was confirmed through semi-quantitative PCR and metabolite quantification assays. The entire protocol duration from co-cultivation through regeneration of transgenic plants to their establishment in the glass house took 40–45 d. The developed B. monnieri model can be used to test expression cassettes carrying genes for plant secondary metabolic pathway engineering, especially those genes that are expressed in differentiated cell, tissue, or organs.

Standardization of Agrobacterium tumefaciens-mediated genetic transformation protocol in Punica granatum L. cv. Kandhari Kabuli

A protocol for Agrobacterium tumefaciens-mediated genetic transformation in Punica granatum cv. Kandhari Kabuli has been developed using cotyledon and hypocotyl explants via both direct and indirect organogenesis pathways. Agrobacterium tumefaciens strain LBA 4404 harboring gus and npt-II genes in a binary vector pBI 121 had been used for genetic transformation studies. Pre-culturing of explants for 48 h (2 days) on regeneration medium before agrobacterial infection significantly increased the transformation frequency. Co-cultivation period of 48 h (2 days) was found to be optimum for genetic transformation. Cefotaxime in a concentration of 500 mg/l was found optimal for controlling the excessive bacterial growth and transformed shoots were selected using 50 mg/l kanamycin. Only the transformed cells were able to grow on the selective regeneration medium. Transgenic shoots were rooted on half-strength MS medium supplemented with 0.10 mg/l NAA, 0.02% activated charcoal, 50 mg/l kanamycin and 500 mg/l cefotaxime and subsequently acclimatized to the culture room conditions. Stable transformation and expression were confirmed on the basis of GUS histochemical and spectrophotometric assays. This genetic transformation protocol will provide a platform for genetic manipulation of pomegranate for incorporation of genes governing various horticulturally important traits in future.

Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea.

Botryosphaeria dothidea is a severe causal agent of die-back and cankers of many woody plants and causes great losses in many regions. The pathogenic mechanism of this pathogen has not been well explored due to lack of mutants and genetic information. In this study, we developed an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for B. dothidea protoplasts using vector pBHt2 containing the hph gene as a selection marker under the control of trp C promoter. Using this protocol we successfully generated the B. dothidea transformants with efficiency about 23 transformants per 10(5) protoplasts. This is the first report of genetic transformation of B. dothidea via ATMT and this protocol provides an effective tool for B. dothidea genome manipulation, gene identification and functional analysis.

Agrobacterium tumefaciens - Mediated transformation of Woodfordia fruticosa (L.) Kurz.

In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

Efficient genetic transformation of Jatropha curcas L. by means of vacuum infiltration combined with filter-paper wicks

Jatropha curcas L. (Jatropha) is a promising plant for the production of biodiesel in arid regions. To improve its productivity, it is important to understand the molecular functions of key genes in Jatropha, and to seek ways to modify important agronomic traits of Jatropha via molecular breeding. However, Jatropha is notorious for its recalcitrance to transformation. Thus, an improved protocol for efficient and reproducible Agrobacterium tumefaciens-mediated transformation is essential for molecular biology research and breeding in this species. Because resistance of Jatropha to A. tumefaciens infection is one of the key factors limiting transformation efficiency, we attempted to improve infection efficiency by comparing various co-culture conditions with the use of a β-glucuronidase (GUS) assay. The LBA4404 strain of A. tumefaciens was more efficient than the EHA105 strain for transformation of Jatropha. Vacuum infiltration of an A. tumefaciens suspension with Jatropha explants, combined with co-cultivation on filter-paper wicks moistened with co-culture medium instead of on solid medium, significantly improved transformation efficiency. In these conditions, GUS-positive sectors were observed in 93.0 ± 23.6% of infected explants. Moreover, a variant of the yellow fluorescent protein gene, Venus, was used as a visible marker, which proved effective for discrimination of candidate transgenic shoots from escape shoots. Transgene integration was confirmed by means of polymerase chain reaction and Southern hybridization analyses. Transgenic shoots were regenerated from 23% of explants from the vacuum-filter-paper treatment, which is sufficient for practical use.