Floral Dip Method Research Articles

Plant Growth and Agrobacterium-mediated Floral-dip Transformation of the Extremophyte Schrenkiella parvula.

Schrenkiella parvula is an extremophyte adapted to various abiotic stresses, including multiple ion toxicity stresses. Despite high-quality genomic resources available to study how plants adapt to environmental stresses, its value as a functional genomics model and tool has been limited by the lack of a feasible transformation system. In this protocol, we report how to generate stable transgenic S. parvula lines using an Agrobacterium-mediated floral-dip method. We modified the transformation protocol used for A. thaliana to account for unique traits of S. parvula, such as an indeterminate flowering habit and a high epicuticular wax content on leaves. Briefly, S. parvula seeds were stratified at 4 °C for five days before planting. Plants were grown at a photoperiod of a 14 h light and 10 h dark and a 130 µmol m-2s-1 light intensity, at 22 °C to 24 °C. Eight to nine week-old plants with multiple inflorescences were selected for transformation. These inflorescences were dipped in an infiltration solution of Agrobacterium tumefaciens GV3101 carrying the pMP90RK plasmid. We performed two rounds of flower dipping with an interval of three to four weeks to increase the transformation efficiency. The T1 seeds were collected and dried for four weeks in a container with desiccants before germination to screen for candidate transformed lines. Resistance to BASTA was used to screen T1 plants. We sprayed the BASTA solution three times with an interval of three days starting at two week-old plants to reduce false positives. A BASTA drop test was performed on surviving individual plants to identify true positive transformants. The transformation efficiency was 0.033%, yielding 3-4 transgenic plants per 10,000 T1 seeds propagated.

Ta Ta for now: Thlapsi arvense (pennycress), an emerging model for genetic analyses.

Ta Ta for now: Thlapsi arvense (pennycress), an emerging model for genetic analyses.

TransgenicArabidopsisExpressingacdSGene ofPseudomonas veronii-KJ Alleviate the Adverse Effects of Salt and Water-Logging Stress

Several bacterial genes have been identified for the successful transformation of model and crop plants to cope with a variety of stressful conditions. Here, we isolated acdS gene from Pseudomonas veronii- KJ and cloned into Escherichia coli (DE3) for the successful expression of recombinant 1-aminocyclopropane-1-carboxylate (ACC) deaminase. Then, Arabidopsis thaliana was transformed to express the bacterial acdS for the production of ACC deaminase and were compared to control plants in their response to water-logging and salt stress conditions. We used strong and constitutive 35S promoter from cauliflower mosaic virus to over-express the acdS gene. The plasmid (PCB302-3) including the acdS gene was transferred into Agrobacterium tumefaciens (GV3101) and the floral dip method was carried out for successful transformation of A. thaliana. We subsequently, measured the growth and development of transformed and control plants. The transgenic A. thaliana expressing acdS gene revealed increased tolerance to water-logging and salt stress conditions and were less prone to the adverse effects compare to wild type plants.

Expression of a functional recombinant vascular endothelial growth factor 165 (VEGF165) in Arabidopsis thaliana

Abstract Objective Targeting the protein of interest to a particular tissue to achieve high-level expression is an important strategy to increase expression efficiency. The use of the plant seed oil body as a bioreactor can not only increase the amount of target protein, but also reduce the cost of downstream processing. Methods VEGF165 was expressed in Arabidopsis thaliana seeds via oilbody fusion technology. The pKO-VEGF165 vector was construted and transformed into A. thaliana seeds. T3 transgenic seeds was detected by SDS-PAGE and western blot methods. The cell activity was tested by MTT methods. Result The phaseolin promoter was used to drive seed-specific expression of the VEGF 165 gene in transgenic A. thaliana. The coding region of VEGF 165 was fused to the Arabidopsis oleosin sequence to target the protein to the oil bodies in the seeds of transgenic plants. The T-DNA region of recombinant plasmid pKO-VEGF165 was shifted to A. thaliana seeds via the floral-dip method. Protein was analyzed by electrophoresis and protein hybridization analyses. Finally, MTT assays showed that the oleosin-VEGF165 fusion protein played a part in the proliferation of HUVEC cells in vitro. Conclusion Oleosin-VEGF165 was successfully expressed and it had stimulated HUVEC cell proliferation activity.

An assessment of transgenomics as a tool for gene discovery in Populus euphratica Oliv.

Transgenomics for gene discovery in Populus euphratica. Transgenomics, a member of the omics family of methodologies, is characterized as the introduction of DNA from one organism into another on a genome-wide scale followed by the identification of recipients with altered phenotypes. This strategy allows investigators to identify the gene(s) involved in these phenotypic changes. It is particularly promising for woody plants that have a long life cycle and for which molecular tools are limited. In this study, we constructed a large-insert binary bacterial artificial chromosome library of Populus euphratica, a stress-tolerant poplar species, which included 55,296 clones with average insert sizes of about 127kb. To date, 1077 of the clones have been transformed into Arabidopsis thaliana via Agrobacterium by the floral dip method. Of these, 69 transgenic lines showed phenotypic changes represented by diverse aspects of plant form and development, 22 of which were reproducibly associated with the same phenotypic change. One of the clones conferring transgenic plants with increased salt tolerance, 002A1F06, was further analyzed and the 127,284bp insert in this clone harbored eight genes that have been previously reported to be involved in stress resistance. This study demonstrates that transgenomics is useful in the study of functional genomics of woody plants and in the identification of novel gene(s) responsible for economically important traits. Thus, transgenomics can also be used for validation of quantitative trait loci mapped by molecular markers.

Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.

Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.

Expression of Human FGF18 by Fusion with Oleosin in Arabidopsis thaliana Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.

The Status of Setaria viridis Transformation: Agrobacterium-Mediated to Floral Dip.

Setaria viridis has many attributes, including small stature and simple growth requirements, that make it attractive as a model species for monocots. Genetic engineering (transformation) methodology is a key prerequisite for adoption of plant species as models. Various transformation approaches have been reported for S. viridis including tissue culture-based and in planta by Agrobacterium tumefaciens infection of floral organs referred to as the floral dip method. The tissue culture-based method utilizes A. tumefaciens infection of mature seed-derived callus with subsequent recovery of stable transgenic lines. Vectors found to be most effective contain the hygromycin phosphotransferase selectable marker gene driven by either Panicum virgatum or Zea mays ubiquitin promoters. As for the floral dip method, there are two reports based on Agrobacterium infection of young S. viridis inflorescences. Plants were allowed to mature, seeds were collected, and analysis of the progeny verified the presence of transgenes. Each transformation approach, tissue culture-based and floral dip, has advantages and disadvantages depending on the expertise of personnel and resources available. While the tissue culture-based method results in a higher transformation efficiency than floral dip, implementation requires a specific technical skillset that limits availability of experienced personnel to successfully perform transformations. Less technical experience is required for floral dip; however, a lack of high-quality growth chambers or greenhouses that provide the necessary optimum growing conditions would reduce an already low transformation efficiency or would not result in recovery of transgenic lines. An overview of transformation methods reported for S. viridis is presented in this review.

Increasing branch and seed yield through heterologous expression of the novel rice S-acyl transferase gene OsPAT15 in Brassica napus L.

Branching is a predominant element in the plant architecture of Brassica napus L. and represents an important determinant of seed yield. OsPAT15 (OsDHHC1), a novel DHHC-type zinc finger protein gene, was reported to regulate rice plant architecture by altering the tillering. However, whether heterologous overexpression of the OsPAT15 gene from the monocot rice into the dicot B. napus L. would have the same effect on branching or seed yield is unknown. In this study, the DHHC-type zinc finger protein gene OsPAT15 was determined to have sulfur acyl transferase activity in the akr1Δ yeast mutant in a complementation experiment. Heterologously expressing OsPAT15 transgenic B. napus L. plants were obtained using the Agrobacterium-mediated floral-dip transformation method. As anticipated, OsPAT15 transgenic plants exhibited branching and seed yield. Compared with non-transgenic plants, OsPAT15 transgenic plants had increased primary branches (1.58–1.76-fold) and siliques (1.86–1.89-fold), resulting in a significant increase in seed yield (around 2.39–2.51-fold). Therefore, overexpression of the sulfur acyl transferase gene OsPAT15 in B. napus L. could be used to increase seed yield and produce excellent varieties.

High-efficiency production of bioactive oleosin-basic fibroblast growth factor in A. thaliana and evaluation of wound healing

Basic fibroblast growth factor (bFGF) is a member of the fibroblast growth factors family. It is a highly specific mitogenic factor for many cell types, as though it be involved in wound repair, angiogenesis, nerve nutrition and embryonic development etc. Oil bodies have been applied for medicine, foodstuff and industry field. The heterogonous proteins expressed in oil bodies have distinct advantages, such as less purification steps and low costs. In this study, bFGF was expressed in A. thaliana seeds using oleosin fusion technology. The pOTB-bFGF vector contained an oleosin-bFGF fusion gene and a glufosinate resistance gene for selection. Transgenic A. thaliana lines were obtained by the floral dip method and protein expression was identified by SDS-PAGE and western blotting in transgenic A. thaliana lines. Moreover, MTT assays showed that the oil bodies expressed oleosin-bFGF fusion protein had a remarkable proliferation effect on NIH/3T3 cells and animal experiments showed that it could effectively decrease wound size and accelerate granulation tissue maturation. In conclusion, this may be a better method of producing oleosin-bFGF fusion protein to meet the increasing demand in its pharmacological application.

In planta Agrobacterium-Mediated Transformation of Rice

The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog (MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD600 = 0.8–1.0 and the co-cultivation at 25°C for 48h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gusA gene by PCR using the gusA specific primers in the T0 lines, there were 4 transgenic lines from 286 T0 lines (1.4% transformation efficiency). However, histochemical glucuronidase (GUS) assay demonstrated that only three of four transgenic lines exhibited gusA expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future.

Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae

BackgroundVerticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized. Identifying an endogenous resistance gene may be useful for controlling this disease.ResultsWe cloned the ribosomal protein L18 (GaRPL18) gene, which mediates resistance to Verticillium wilt, from a wilt-resistant cotton species (Gossypium arboreum). We then characterized the function of this gene in cotton and Arabidopsis thaliana plants. GaRPL18 encodes a 60S ribosomal protein subunit important for intracellular protein biosynthesis. However, previous studies revealed that some ribosomal proteins are also inhibitory toward oncogenesis and congenital diseases in humans and play a role in plant disease defense. Here, we observed that V. dahliae infections induce GaRPL18 expression. Furthermore, we determined that the GaRPL18 expression pattern is consistent with the disease resistance level of different cotton varieties. GaRPL18 expression is upregulated by salicylic acid (SA) treatments, suggesting the involvement of GaRPL18 in the SA signal transduction pathway. Virus-induced gene silencing technology was used to determine whether the GaRPL18 expression level influences cotton disease resistance. Wilt-resistant cotton species in which GaRPL18 was silenced became more susceptible to V. dahliae than the control plants because of a significant decrease in the abundance of immune-related molecules. We also transformed A. thaliana ecotype Columbia (Col-0) plants with GaRPL18 according to the floral dip method. The plants overexpressing GaRPL18 were more resistant to V. dahliae infections than the wild-type Col-0 plants. The enhanced resistance of transgenic A. thaliana plants to V. dahliae is likely mediated by the SA pathway.ConclusionOur findings provide new insights into the role of GaRPL18, indicating that it plays a crucial role in resistance to cotton “cancer”, also known as Verticillium wilt, mainly regulated by an SA-related signaling pathway mechanism.

The Impacts of TRR14-Overexpression on Arabidopsis thaliana Growth and Photosynthetic Parameters

Background TRR14 protein is a small protein, a member of a multigene family in Arabidopsis which was found as the fi rst protein during screening seedlings for their resistant to the trehalose sugar. Objectives A number of TRR14-overexpressing plants were subjected to the characterization in the present research, among which, the associated morphological features and changes accompany growth pattern and photosynthesis related parameters. Materials and Methods TRR14 gene was isolated from Arabidopsis Thaliana and cloned into the pBin-35S vector. Recombinant vector was transferred to the Arabidopsis (Col-0) via Agrobacterium tumefaciens using the Floral Dipping method. Seeds from the TRR14 overexpressed (TRR14) and the Col-0 wild-type (WT) plants were shown on soil under long day conditions. Several measurements were then performed including determination of the fresh and dry weights, leaf area, chlorophyll a and b (Chl a and Chl b) content, Chl a/b ratio, total chlorophyll and carotenoids content, soluble and insoluble sugars content, total and soluble protein content, the Hill reaction rate, chlorophyll fl uorescence, as well as photorespiration rate. Meanwhile, the chloroplastic proteins were investigated by SDS-PAGE analysis. Results TRR14 plants showed a signifi cant increase in fresh and dry weights, leaf area, and total and soluble protein content along with a signifi cant decrease in the insoluble sugar contents was observed in comparison to the WT plants. Chl a, Chl b, total chlorophyll content, Chl a/b ratio, carotenoids content, Hill reaction rate, and chlorophyll fl uorescence didn’t show a signifi cant diff erence between TRR14 and WT plants. The SDS-PAGE gel electrophoresis of the chloroplastic proteins showed a thick band with a molecular mass of 25 kDa in TRR14-overexpressed plants, compared to the WT plants. Remarkably, photorespiration rate was decreased in TRR14 plants compared to WT plants. Conclusion The increased biomass of TRR14 transformed plants might be due to its ability in reducing photorespiration through concentrating CO2 in the leaf’s intercellular spaces.

Metabolic Engineering of Photorespiration.

The introduction of two alternative glycolate catabolic pathways in the chloroplasts of Arabidopsis thaliana rendered plants with increased biomass. To introduce these synthetic pathways, the selected genes were stepwise integrated in the nuclear genome of wild-type plants. These plants were transformed by Agrobacterium tumefaciens carrying the binary vectors using the floral dip method. Selection of transformants was conducted using different selection agents and the expression of the transgenes was confirmed by PCR and enzyme activity measurements.

Expression Vector Construction and Genetic Transformation of <i>Paeonia lactiflora</i> Gibberellin 20-Oxidase Gene

GA20-oxidase (GA20ox) gene encodes a key enzyme in gibberellins (GAs) biosynthesis pathway. Previously, we have cloned a PlGA20ox gene (GeneBank accession number: KU886552) from Paeonia lactiflora. To further reveal its function, we constructed an expression vector in the present study and then transformed it into Arabidopsis thaliana plants by floral dip method. The transgenic plants exhibited an early bolting, increased height and improved vegetative growth. These results provided efficient vector tool and functional information of PlGA20ox for future gene engineering in peony.

Transgenesis of Agrobacterium rhizogenes K599 orf3 into plant alters plant phenotype to dwarf and branch

Virulent strains of Agrobacterium rhizogenes (A. rhizogenes) contain Ri plasmids responsible for hairy root development on susceptible plants. Ri plasmid possesses different gene segments. T-DNA region of Ri plasmid pRi2659 in A. rhizogenes strain K599 carries 11 functional genes/coding regions. In this study, we cloned a complete orf3-coding region in T-DNA of plasmid pRi2659, which is 1479 bp in length and encodes 492 amino acid residues. Then, we developed an orf3-containing expression vector named pRI101-AN-orf3 and generated the orf3-transgenic tobacco plants via A. tumefaciens-mediated method. The orf3-transgenic tobacco displayed altered morphological characters including shortened internodes, dwarfisms, more branches, shortened leaf blade length, early flowering, and weak roots. Compared to those of untransformed tobacco, the contents of two endogenous plant hormones, auxins (IAA) and abscisic acid, were decreased whereas the cytokinin content was increased but gibberellic acid content was not consistently obviously changed. Besides, we also transferred orf3 into Arabidopsis thaliana using floral-dip method and obtained orf3-transgenic Arabidopsis homozygous offspring. The orf3-transgenic Arabidopsis displayed morphological characters of dwarfism and more branches similar to those of the orf3-transgenic tobacco. Together, these results indicate that K599 orf3 possesses the functions of altering plant phenotypes and promotes plants to become dwarf and branch.

Overexpression of SpCBL6, a calcineurin B-like protein of Stipa purpurea, enhanced cold tolerance and reduced drought tolerance in transgenic Arabidopsis.

The purpose of the present study was to characterize SpCBL6 (GenBank accession number: KT780442) from Stipa purpurea and elucidate the function of this protein in abiotic stress. The full-length cDNA of SpCBL6 was isolated from S. purpurea by rapid amplification of cDNA ends methods. Laser confocal microscopy was used to analyze the subcellular localization of SpCBL6. The constructs of 35S:GFP-SpCBL6 was used to transform wild-type (WT) Arabidopsis plants (ecotype Columbia-0) with the floral dip method. Quantitative reverse-transcription PCR (qRT-PCR), water potential, photosynthetic efficiency (F v/F m), and ion leakage was performed to investigate the role of SpCBL6 in abiotic stress. The open reading frame of SpCBL6 contains 681bp nucleotides and encodes a 227-amino acid polypeptide. Phylogenetic analysis indicated that SpCBL6 showed the highest similarity with rice OsCBL6. SpCBL6 transcripts were induced by freezing and drought treatments. Subcellular localization analysis showed that SpCBL6 was located in membrane of protoplast. Overexpression of SpCBL6 in Arabidopsis thaliana demonstrated that the transgenic plants were more tolerant to cold treatment, but less tolerant to drought, compared with the plants. qRT-PCR analysis showed that the drought stress marker genes were inhibited in transgenic plants, whereas the cold stress marker genes were enhanced. Further analysis showed that SpCBL6-overexpressing plants showed enhanced water potential, photosynthetic efficiency (F v/F m), and reduced ion leakage compared with the wild-type after cold treatment. Collectively, these results indicate that SpCBL6, a new member of the CBL gene family isolated from S. purpurea, enhances cold tolerance and reduces drought tolerance in plants.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

Characterization of root-knot nematode responsive and root-specific promoter containing PIN domain fromArabidopsis thaliana(L.) Heynh

Root-knot nematodes (Meloidogyne incognita) are obligate plant parasites, causing significant economic loss, that alter expression of host genes in order to establish and maintain their feeding sites in the roots of host plants. In the present study, a nematode-responsive-root-specific gene (AT1G26530) was identified which expressed in roots of Arabidopsis thaliana after nematode infection. Quantitative RT-PCR analysis of this gene revealed maximum (~2.58 fold) up-regulation at 21 days post inoculation of nematode. A 1580 bp region upstream of the translational start site of AT1G26530 was isolated and transformed into Arabidopsis through floral dip method. On analysis of Arabidopsis transgenic plants harboring AT1G26530 prm:: GUS fusion, reporter gene expression was seen exclusively in galls after nematode inoculation. Interestingly, strong GUS activity was observed at early stages of nematode infection, starting from 14 days and was sustained up to 30 days post inoculation. Furthermore, 85 to 93% galls exhibited GUS activity in the nematode feeding sites. The specificity of the activity of the AT1G26530 promoter, in terms of nematode-responsiveness and rootspecificity, makes it a suitable candidate to express dsRNA of nematode genes and engineer plants with resistance against root-knot nematodes using HD-RNAi technology.

Cloning and characterization of the LFY homologue from Chinese cabbage (Brassica rapa subsp. pekinensis)

Flowering is critical to the growth and development of plants, and LFY gene homologues play a major role in flowering initiation. To understand the genetic and molecular mechanisms underlying floral initiation and development in Brassica rapa subsp. pekinensis, BrpLFY, a homologue of LFY, was cloned using RT-PCR. Sequence analysis showed that the cDNA sequence of BrpLFY is 1,341 bp in length, with an ORF of 1,245 bp encoding a predicted protein of 415 amino acids. The predicted protein showed a high degree of identity with LFY homologues from other angiosperm species. Real-time PCR analysis showed that BrpLFY mRNA was detected in all tissues during plant development from the vegetative state to fully differentiated flowers, and its expression was highest in the cotyledon and lowest in the root. BrpLFY expression in the shoot apex increased gradually during vegetative growth and increased dramatically at stage 1 of flower bud differentiation. The relative expression peaked at stage 5 and then decreased in later stages. Moreover, the trend in BrpLFY expression level change in the shoot apex was similar regardless of variety or vernalization method. The relative expression of BrpLFY in leaves gradually decreased with leaf development. We overexpressed the gene in Arabidopsis thaliana using the floral dip method, and examined flowering time in wild-type and transgenic plants. Overexpression of BrpLFY specifically caused early flowering; the transgenic plants flowered 10-14 d earlier than did wild-type plants, and leaf number decreased by 0.5-1 when the plants bolted. Real-time PCR analysis showed that the expression of BrpLFY in transgenic Arabidopsis was higher than in wild-type plants. These results indicate that BrpLFY plays a role in promoting flowering in Chinese cabbage.