High efficiency transformation of cultured tobacco cells

Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants.     

Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD₆₀₀, 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of ‘bacoside A’ than the untransformed roots. Maximum biomass accumulation (6.8 g l^?1) and ‘bacoside A’ content (10.02 mg g^?1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.     

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD₆₀₀) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.     

Expression of GroES TB antigen in tobacco and potato

As the world races towards a plant-based bioeconomy, plants known to be ideal and economical bioreactors are being harnessed for the production of recombinant proteins. The major immunodominant 10 kDa GroES TB antigen (Chaperonin 10) gene from Mycobacterium tuberculosis was selected for expression in plants as a putative tuberculosis (TB) subunit vaccine candidate. Two crops, tobacco and potato, were engineered by stable plant transformation for expression of the 10 kDa GroES TB antigen using non-viral binary vectors. The integration of the GroES TB gene into the genomes of tobacco and potato was confirmed by PCR and Southern blotting. The expression of the GroES TB antigen in tobacco was 0.04–1.2 % of the total soluble protein (TSP). However, the expression of the same TB antigen in the Indian potato cv. Kufri bahar was comparatively low (0.033 % of TSP). The recombinant GroES plant derived protein was characterised and confirmed by MALDI-TOF–TOF and ELISA. This is the first report of the expression of the 10 kDa chaperonin in tobacco and potato.     

Production of hairy root cultures of lettuce (Lactuca sativa L.)

Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.     

A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant

In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2 %. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66 %, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.     

Isolation and characterization of Histone1 gene and its promoter from tea plant (Camellia sinensis)

One 1.2 kbp long sequence was cloned by using PCR with primers that were designed from cDNA sequence of CsH1 gene (Genbank: EU716314) from tea plant (Camellia sinensis). According to the 1.2 kbp sequence, a 0.6 kbp sequence was isolated from tea plant genomic DNA using DNA Walking Method. Sequence analysis revealed that the 1.2 kbp sequence is a CsH1 gene consisting of 1 exon and 2 introns, the border of exton and intron sequences conforming to the GT–AG rule, and the 0.6 kbp sequence was found to be the promoter of CsH1 gene which contains basic promoter elements, TATA-box and CAAT-box. Abscisic acid responsiveness cis-acting element, elictor-responsive element, GA response element, light response cis-acting element and TC-rich repeats were also represented. To further study the activity of this promoter, the sequence was used to drive a GUS fusion gene in Agrobacterium-mediated transformation of tea plant somatic embryos, leaf discs and calli of tobacco (Nicotiana tabacum L.) where a high level of GUS expression was both observed in the tobacco calli and tea plant somatic embryos. These results suggest that the CsH1 gene promoter isolated is capable of conferring nuclear gene expression.     

Agrobacterium tumefaciens-mediated genetic transformation and production of stable transgenic pearl millet (Pennisetum glaucum [L.] R. Br.)

A synthetic gene encoding the antimicrobial peptide magainin has been designed, cloned, and engineered for regulation by the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (nos) terminator. The plant expression cassette was introduced into the vector pSB11-bar (with the glyphosate [Basta®] resistance gene, bar), and the recombinant plasmid was mobilized into Agrobacterium tumefaciens strain LBA4404 for the generation of a super-binary vector pSB111-bar-mag. Magainins, positively charged amphipathic antimicrobial peptides of 21–26 amino acid residues, are potential candidates for the development of disease resistant transgenic plants. Six-wk-old pearl millet (Pennisetum glaucum [L.] R. Br.) calli and A. tumefaciens harboring pSB111-bar-mag were cocultivated in a medium supplemented with 400 μM acetosyringone and 3.3 mM l-cysteine. Out of 3,000 infected calli subjected to selection on phosphinothricin medium, 82 calli showed sectors of healthy growth, resulting in a transformation frequency of 2.73%. Among 13 Basta-tolerant putative transformed plants, eight were fertile and their transgenic nature and expression of the transgene was characterized by Southern and Northern blot analyses, respectively. Subsequent T₁ progenies co-segregated for bar and magainin genes in a 3:1 ratio. Bioassays that challenged the eight transgenic T₁ plant progenies against three highly virulent strains of Sclerospora graminicola, viz., Sg 384, Sg 445, and Sg 492 failed to show resistance. The failure of synthetic magainin gene to confer resistance against downy mildew in pearl millet may be attributed to the complexity of the cell wall and cell membrane of the pathogen.     

An efficient <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation protocol of <Emphasis Type="Italic">Withania somnifera</Emphasis>

This is the first report on Agrobacterium rhizogenes-mediated transformation of Withania somnifera for expression of a foreign gene in hairy roots. We transformed leaf and shoot tip explants using binary vector having gusA as a reporter gene and nptII as a selectable marker gene. To improve the transformation efficiency, acetosyringone (AS) was added in three stages, Agrobacterium liquid culture, Agrobacterium infection and co-culture of explants with Agrobacterium. The addition of 75 μM AS to Agrobacterium liquid culture was found to be optimum for induction of vir genes. Moreover, the gusA gene expression in hairy roots was found to be best when the leaves and shoot tips were sonicated for 10 and 20s, respectively. Based on transformation efficiency, the Agrobacterium infection for 60 and 120 min was found to be suitable for leaves and shoot tips, respectively. Amongst the various culture media tested, MS basal medium was found to be best in hairy roots. The transformation efficiency of the improved protocol was recorded 66.5 and 59.5?% in the case of leaf and shoot tip explants, respectively. When compared with other protocols the transformation efficiency of this improved protocol was found to be 2.5 fold higher for leaves and 3.7 fold more for shoot tips. Southern blot analyses confirmed 1–2 copies of the gusA transgene in the lines W1-W4, while 1–4 transgene copies were detected in the line W5 generated by the improved protocol. Thus, we have established a robust and efficient A. rhizogenes mediated expression of transgene (s) in hairy roots of W. somnifera.     

A comparison study of Agrobacterium-mediated transformation methods for root-specific promoter analysis in soybean

Key message

Both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.

Abstract

An efficient genetic transformation system is crucial for promoter analysis in plants. Agrobacterium-mediated transformation is the most popular method to produce transgenic hairy roots or plants. In the present study, first, we compared the two different Agrobacterium rhizogenes-mediated hairy root transformation methods using either constitutive CaMV35S or the promoters of root-preferential genes, GmEXPB2 and GmPAP21, in soybean, and found the efficiency of in vitro hairy root transformation was significantly higher than that of in vivo transformation. We compared Agrobacterium rhizogenes-mediated hairy root and Agrobacterium tumefaciens-mediated whole plant transformation systems. The results showed that low-phosphorous (P) inducible GmEXPB2 and GmPAP21 promoters could not induce the increased expression of the GUS reporter gene under low P stress in both in vivo and in vitro transgenic hairy roots. Conversely, GUS activity of GmPAP21 promoter was significantly higher at low P than high P in whole plant transformation. Therefore, both in vitro and in vivo hairy root transformation systems could not replace whole plant transformation for promoter analysis of root-specific and low-P induced genes in soybean.     

Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation

For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes. Here we report a significant variation in different tissues of Indica rice (Oryza sativa L. cv. Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium. An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction. Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands. We observed that the physical presence of preincubated scutella is required for vir gene induction. Conditioned medium from which preincubated scutella were removed did not induce the vir genes. Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level. These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.     

A reliable and efficient protocol for induction of hairy roots in Agastache foeniculum

Hairy root culture system is a valuable tool to study the characteristics of gene expression, gene function, root biology, biochemical properties and biosynthesis pathways of secondary metabolites. In the present study, hairy roots were established in Anise hyssop (Agastache foeniculum) via Agrobacterium rhizogenes. Three strains of Agrobacterium rhizogenes (A4, A7 and 9435), were used for induction of hairy roots in four various explants (hypocotyl, cotyledon, one-month-old leaf and five-month-old leaf) of Anise hyssop. The highest frequency of transformation was achieved using A4 strain in one-month-old leaves (51.1%). The transgenic states of hairy root lines were confirmed by PCR (Polymerase chain reaction) method. High performance liquid chromatography analysis revealed that the production of rosmarinic acid (RA) in transformed roots of A. foeniculum was almost 4-fold higher than that of the non-transformed roots. In a separate experiment, hairy roots obtained from one-month-old leaves inoculated with A4 strain, were grown in liquid medium and the effects of different concentrations of salicylic acid (0.0, 0.01, 0.1 and 1 mM) and chitosan (0, 50, 100 and 150 mg L^?1) (as elicitor) and sucrose (20, 30, 40 and 50 g L^?1) on the growth of hairy roots were evaluated. The results showed that, 30 g L^?1 sucrose and 100 mg L^?1 chitosan increased the biomass of hairy root cultures and application of salicylic acid reduced the growth of hairy roots compared with control roots.     

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae)

Key message

Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species.

Abstract

Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.     

Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp

The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.     

Lead phytoavailability change driven by its speciation transformation after the addition of tea polyphenols (TPs): Combined selective sequential extraction (SSE) and XANES analysis

Background and aims

Metal species in rhizosphere soil profoundly influence their mobility and phytoavailability. Clarifying the speciation transformation of heavy metals helps understand their translocation and accumulation in plants.

Methods

Single extraction, selective sequential extraction (SSE) and X-ray absorption near-edge structure (XANES) spectroscopy were employed to investigate the speciation transformation of lead (Pb) and its influence on metal accumulation in tea plants after the addition of tea polyphenols (TPs).

Results

Pb content was decreased in young leaves and stems, whereas increased in roots, after TPs were amended to soil. Both SSE and XANES analysis suggested bioavailable Pb was transformed to organically bound Pb after the addition of TPs. The increased percentage of organically bound Pb might be fixed in the cell wall of plant root through a ternary complex formed between the Pb-organic matter complex and cell wall components. Therefore, Pb translocation from roots to young tissues was decreased.

Conclusions

Pb phytoavailability change was driven by its speciation transformation after the addition of TPs. Combined SSE and XANES spectroscopy represent powerful tools to study metal speciation transformation in plant and soil systems.     

Development of efficient miniprep transformation methods for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium rhizogenes

Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes.     

Indole-3-acetic acid and indole-3-butyric acid in tissues of carrot inoculated withAgrobacterium rhizogenes

The role of auxins in induction of roots byAgrobacterium rhizogenes was studied in carrot root disks. Transformed roots were produced on root disks by inoculation withA. rhizogenes, A4. Measurement of indole-3-acetic acid (IAA) by gas chromatography-mass spectrometry (GC-MS) indicated that there was a significant increase in the concentration of IAA in transformed callus and induced roots compared with initial IAA concentrations in carrot disks. Indole-3-butyric acid (IBA) was found to occur naturally in carrot roots. The presence of IBA, a potent root inducer, must be taken into account when assessing the role of auxin during transformation and induction of roots byA. rhizogenes.     

Spacer length-dependent protection of specific activity of pollen and/or embryo promoters from influence of CaMV 35S promoter/enhancer in transgenic plants

The influence of the CaMV 35S promoter/enhancer on expression profiles of four Arabidopsis thaliana pollen- and/or embryo-specific promoters, APRS, ESL, MXL, and DLL, was tested in transgenic tobacco plants. Individual promoters were fused to the gus reporter gene and cloned in head-to-head orientation with the CaMV 35S:hpt expression unit within the same T-DNA. With the exception of the TATA-less promoter DLL, all other combinations generated interactions between the promoter under investigation and 35S promoter/enhancer resulting in ectopic β-glucuronidase (GUS) expression in vegetative organs and tissues, the most susceptible being the stem, followed by callus, leaf, and root. To eliminate this crosstalk, DNA spacers of length 1, 2 and 5 kb were cloned between the interacting sequences. Ectopic GUS staining was dependent on the affected promoter as well as the distance between the 5′-end of the CaMV 35S promoter and the reporter gene translation start site. When this distance was less than 1 kb strong ectopic GUS staining was observed in all vegetative tissues, similar to the CaMV35S:gus expression profile in transgenic tobacco plants. Insertion of spacer DNA sequences of increasing length resulted in gradual reduction of ectopic GUS staining in tested plants. Of the tissues and organs related to plant reproduction, only anthers and seed coats in the early stages of seed development showed ectopic GUS staining. Developing pollen and embryos showed a pattern of GUS activity consistent with the predicted role of a developmental stage-specific promoter in transgenic tobacco plants.