Floral Dip Method Research Articles

Domestication and engineering of pennycress (Thlaspi arvense L.): challenges and opportunities for sustainable bio-based feedstocks.

Pennycress, as an emerging oilseed crop with high oil content, faces challenges but offers potential for sustainable bioproducts; ongoing research aims to enhance its traits and quality. Pennycress (Thlaspi arvense L.) is an emerging oilseed crop with many advantages, such as high seed oil (27-39%) and monounsaturated fatty acid (55.6%) content, making it an attractive candidate to produce sustainable bioproducts. However, several challenges are associated with domesticating pennycress, including high silicle shatter, which reduces seed yield during harvest, non-uniformed germination rate and high contents of erucic acid and glucosinolates, which have adverse health effects on humans and animals. Pennycress, which can be easily and rapidly transformed using the floral dip method under vacuum, can achieve trait improvements. Ongoing research for pennycress domestication using mutation breeding, including ethylmethylsulfonate treatment and genome editing, aims to improve its quality. Pennycress can be used as an excellent platform for producing industrially important fatty acids such as hydroxy and epoxy fatty acids and docosahexaenoic acid. In conclusion, pennycress is a promising oilseed crop with multiple advantages and potential applications. Continuous improvements in quality and engineering for producing high-value bio-based feedstocks in pennycress will establish it as a sustainable and economically valuable crop.

Functional Characterization of the Effects of CsDGAT1 and CsDGAT2 on Fatty Acid Composition in Camelina sativa.

Triacylglycerols (TAGs) are the storage oils of plant seeds, and these lipids provide energy for seed germination and valuable oils for human consumption. Three diacylglycerol acyltransferases (DGAT1, DGAT2, and DGAT3) and phospholipid:diacylglycerol acyltransferases participate in the biosynthesis of TAGs. DGAT1 and DGAT2 participate in the biosynthesis of TAGs through the endoplasmic reticulum (ER) pathway. In this study, we functionally characterized CsDGAT1 and CsDGAT2 from camelina (Camelina sativa). Green fluorescent protein-fused CsDGAT1 and CsDGAT2 localized to the ER when transiently expressed in Nicotiana benthamiana leaves. To generate Csdgat1 and Csdgat2 mutants using the CRISPR/Cas9 system, camelina was transformed with a binary vector carrying Cas9 and the respective guide RNAs targeting CsDGAT1s and CsDGAT2s via the Agrobacterium-mediated floral dip method. The EDD1 lines had missense and nonsense mutations in the CsDGAT1 homoeologs, suggesting that they retained some CsDGAT1 function, and their seeds showed decreased eicosaenoic acid (C20:1) contents and increased C18:3 contents compared to the wild type (WT). The EDD2 lines had a complete knockout of all CsDGAT2 homoeologs and a slightly decreased C18:3 content compared to the WT. In conclusion, CsDGAT1 and CsDGAT2 have a small influence on the seed oil content and have an acyl preference for C20:1 and C18:3, respectively. This finding can be applied to develop oilseed plants containing high omega-3 fatty acids or high oleic acid.

Overexpression of Genes to Enhance Abiotic Stress Tolerance in Arabidopsis.

Abiotic environmental stressors cause various types of damage to plants and cause significant loss in yield. Abiotic stress tolerance in plants refers to the ability to withstand environmental factors and maintain growth, development, and production. Since this tolerance is controlled by a gene or a set of genes, transgenic activating of these genes in plants often enhances tolerance under abiotic stress. Therefore, this methodology chapter describes a strategy and the corresponding protocols needed to induce a gene by an abiotic stressor, clone the corresponding cDNA into plasmids and Agrobacterium cells, and genetic transformation to the Arabidopsis plants using the floral dip method. The chapter also describes standard assays to evaluate the transgene's effect on the plant's tolerance. Finally, the techniques outlined in this chapter for cloning and generating transgenic plants tolerant to abiotic stress are a versatile approach that can be implemented across various plant species and genes.

Drought stress enhances the efficiency of floral dip method of Agrobacterium-mediated transformation in Arabidopsis thaliana.

The Agrobacterium-mediated floral dip protocol is the most extensively used transformation method for a model plant Arabidopsis thaliana. Several useful methods for Agrobacterium tumefaciens-mediated transformations of Arabidopsis are existing, but they are time consuming and with low transformation efficiency. Here, we developed a transgenic Arabidopsis lines TET12p::TET12-RFP in a short period of time and enhanced transformation efficiency by using a modified transformation method by applying drought stress after floral dip. In this protocol, Agrobacterium cells carrying TET12p::TET12-RFP recombinant vectors were resuspended in a solution of 5% sucrose, 0.05% (v/v) silwet L-77 to transform female gametes of developing Arabidopsis inflorescences. Treated Arabidopsis were then applied with different levels of drought stresses to stimulate plants for the utilization of maximum plant energy in seed maturation process. The applied stresses achieved the fast maturation of already treated inflorescences while stopped the growing of newly arising untreated inflorescence, thus decreased the chances of wrong collection of untransformed seeds. Consequently, the collected seeds were mostly transgenic with a transformation frequency of at least 10%, thus the screening for positive transformants selection was more advantageous on a selective medium as compared to a classical floral dip method. Within 2-3 months, two hundred of individual transgenic plants were produced from just 10 infiltrated plants. This study concludes that application of drought stresses in a specific stage of plant is a beneficial strategy for achieving the transgenic Arabidopsis in a short period of time with high transformation efficiency.

Transformation of Rhodococcus Pigment Production Hydroxylase (PPH) gene into Camelina sativa: an alternative marker for the detection of transgenic plants.

Production of transgenic plants with desired agronomic and horticultural traits has gained great importance to fulfill demands of the growing population. Genetic transformation is also a fundamental step to study basics of plant sciences. Different transformation protocols have been developed and used which are reliable and efficient. These protocols used antibiotic or herbicide resistance genes incorporated along with gene of interest to identify transformed plants from non-transformed ones. These marker genes may pose a threat to human and environment. Use of visual markers enables direct and easier observation of transformed plants with more precision. In current study a gene cassette with 'pigment production hydroxylase (PPH) gene under fiber specific promoter (GhSCFP) and downstream Nos-terminator was designed. After checking the structural and functional efficiency of codon optimized gene using bioinformatics tools, the cassette was sent for chemical synthesis from commercial source. The pigment gene cassette (PPH_CEMB), cloned in pCAMBIA-1301, was transformed into Agrobacterium through electroporation. Agrobacterium-mediated floral dip method was used to transform Camelina sativa inflorescence. After seed setting a total of 600 seed were observed for change in color and out of these, 19 seeds developed a reddish-brown coloration, while the remaining 581 seeds remained yellow. The transformation efficiency calculated on basis of color change was 1.0%. PCR analysis of leaves obtained after sowing reddish seeds confirmed the transformation of pigment production gene, while no PCR amplification was observed in leaves of plants from wild type seeds. From the results it is evident that Agrobacterium-mediated transformation of C. sativa inflorescence is very efficient and environment friendly technique not only for detection of transformed plants but also to study basic cellular processes.

Functional analysis of a susceptibility gene (HIPP27) in the Arabidopsis thaliana-Meloidogyne incognita pathosystem by using a genome editing strategy

BackgroundPlant-parasitic root-knot nematodes cause immense yield declines in crop plants that ultimately obviate global food security. They maintain an intimate relationship with their host plants and hijack the host metabolic machinery to their own advantage. The existing resistance breeding strategies utilizing RNAi and resistance (R) genes might not be particularly effective. Alternatively, knocking out the susceptibility (S) genes in crop plants appears to be a feasible approach, as the induced mutations in S genes are likely to be long-lasting and may confer broad-spectrum resistance. This could be facilitated by the use of CRISPR/Cas9-based genome editing technology that precisely edits the gene of interest using customizable guide RNAs (gRNAs) and Cas9 endonuclease.ResultsInitially, we characterized the nematode-responsive S gene HIPP27 from Arabidopsis thaliana by generating HIPP27 overexpression lines, which were inoculated with Meloidogyne incognita. Next, two gRNAs (corresponding to the HIPP27 gene) were artificially synthesized using laboratory protocols, sequentially cloned into a Cas9 editor plasmid, mobilized into Agrobacterium tumefaciens strain GV3101, and transformed into Arabidopsis plants using the floral dip method. Apart from 1–3 bp deletions and 1 bp insertions adjacent to the PAM site, a long deletion of approximately 161 bp was documented in the T0 generation. Phenotypic analysis of homozygous, ‘transgene-free’ T2 plants revealed reduced nematode infection compared to wild-type plants. Additionally, no growth impairment was observed in gene-edited plants.ConclusionOur results suggest that the loss of function of HIPP27 in A. thaliana by CRISPR/Cas9-induced mutagenesis can improve host resistance to M. incognita.

Functional analysis on sucrose transporters in sweet potato

Sweet potato is an important food crop that can also be used as an industrial raw material. Sucrose is the main form of long-distance carbohydrate transport in plants, and sucrose transporter (SUT) regulates the transmembrane transport and distribution of sucrose during plant growth and metabolism. Moreover, SUT plays a key role in phloem mediated source-to-sink sucrose transport and physiological activities, supplying sucrose for the sink tissues. In this study, the full-length cDNA sequences of IbSUT62788 and IbSUT81616 were obtained by rapid amplification of cDNA ends (RACE) cloning according to the transcripts of the two SUT coding genes which were differentially expressed in sweet potato storage roots with different starch properties. Phylogenetic analysis was performed to clarify the classification of IbSUT62788 and IbSUT81616. The subcellular localization of IbSUT62788 and IbSUT81616 was determined by transient expression in Nicotiana benthamiana. The function of IbSUT62788 and IbSUT81616 in sucrose and hexose absorption and transport was identified using yeast functional complementarity system. The expression pattern of IbSUT62788 and IbSUT81616 in sweet potato organs were analyzed by real-time fluorescence quantitative PCR (RT-qPCR). Arabidopsis plants heterologous expressing IbSUT62788 and IbSUT81616 genes were obtained using floral dip method. The differences in starch and sugar contents between transgenic and wild-type Arabidopsis were compared. The results showed IbSUT62788 and IbSUT81616 encoded SUT proteins with a length of 505 and 521 amino acids, respectively, and both proteins belonged to the SUT1 subfamily. IbSUT62788 and IbSUT81616 were located in the cell membrane and were able to transport sucrose, glucose and fructose in the yeast system. In addition, IbSUT62788 was also able to transport mannose. The expression of IbSUT62788 was higher in leaves, lateral branches and main stems, and the expression of IbSUT81616 was higher in lateral branches, stems and storage roots. After IbSUT62788 and IbSUT81616 were heterologously expressed in Arabidopsis, the plants grew normally, but the biomass increased. The heterologous expression of IbSUT62788 increased the soluble sugar content, leaf size and 1 000-seed weight of Arabidopsis plants. Heterologous expression of IbSUT81616 increased starch accumulation in leaves and root tips and 1 000-seed weight of seeds, but decreased soluble sugar content. The results obtained in this study showed that IbSUT62788 and IbSUT81616 might be important genes regulating sucrose and sugar content traits in sweet potato. They might carry out physiological functions on cell membrane, such as transmembrane transport of sucrose, sucrose into and out of sink tissue, as well as transport and unloading of sucrose into phloem. The changes in traits result from their heterologous expression in Arabidopsis indicates their potential in improving the yield of other plants or crops. The results obtained in this study provide important information for revealing the functions of IbSUT62788 and IbSUT81616 in starch and glucose metabolism and formation mechanism of important quality traits in sweet potato.

Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos.In this study, flower buds with sizes of 5–7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension.The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

Construction and functional analysis of nattokinase-producing cucumber obtained by the CRISPR-Cas9 system

AbstractNattokinase (NK) is effective in the prevention and treatment of cardiovascular disease. Cucumber is rich in nutrients with low sugar content and is safe for consumption. The aim of this study was to construct a therapeutic cucumber that can express NK, which can prevent and alleviate cardiovascular diseases by consumption. Because the Bitter fruit (Bt) gene contributes to bitter taste but has no obvious effect on the growth and development of cucumber, so the NK-producing cucumber was constructed by replacing the Bt gene with NK by using CRISPR/Cas9. The pZHY988-Cas9-sgRNA and pX6-LHA-U6-NK-T-RHA vectors were constructed and transformed into Agrobacterium tumefaciens EHA105, which was transformed into cucumber by floral dip method. The crude extract of NK-producing cucumber had significant thrombolytic activity in vitro. In addition, treatment with the crude extract significantly delayed thrombus tail appearance, and the thrombin time of mice was much longer than that of normal mice. The degrees of coagulation and blood viscosity as well as hemorheological properties improved significantly after crude extract treatment. These findings show that NK-producing cucumber can effectively alleviate thrombosis and improve blood biochemical parameters, providing a new direction for diet therapy against cardiovascular diseases.

Exploring the Potential Applications of the Noninvasive Reporter Gene RUBY in Plant Genetic Transformation

Betalains can be conveniently observed and quantified and, accordingly, have the potential as naked-eye visual screening reporters during plant transformation. RUBY is a new reporter system that uses “2A” peptides to fuse three key genes, CYP76AD1, DODA, and glucosyl transferase, for betalain biosynthesis, and has been successfully used for transformation of rice, Arabidopsis, and cotton, but its potential applications in the genetic transformation of various other plant species remain to be verified. In this study, RUBY was transferred into the hairy roots of Plukenetia volubilis and Nicotiana benthamiana, and was transferred into Arabidopsis by the floral-dip method. The expression levels of CYP76AD1, DODA, and glucosyl transferase were detected by RT−PCR and RT−qPCR, the relationship between the expression level of RUBY and red coloration was analyzed, and the genetic stability of RUBY in transgenic Arabidopsis was studied. The results showed that the expression of RUBY could reconstruct the betalain biosynthesis pathway in the hairy roots of P. volubilis, N. benthamiana, and Arabidopsis plants, indicating that it has the potential for versatile use across species. As a reporter, betalain did not affect callus induction, plant regeneration, development, or fertility. However, when used in plant transformation for observation and visual screening, it needed to accumulate to a certain extent to show red coloration, and it was positively correlated with gene expression. In general, RUBY is a convenient reporter for plant transformation, and has no obvious side effects during plant growth and development. However, the potential application of RUBY for visual screening is highly determined by the expression level, and further improvement is needed.

Targeted Activation of Arabidopsis Genes by a Potent CRISPR-Act3.0 System.

The CRISPR/Cas system has emerged as a versatile platform for sequence-specific genome engineering in plants. Beyond genome editing, CRISPR/Cas systems, based on nuclease-deficient Cas9 (dCas9), have been repurposed as an RNA-guided platform for transcriptional regulation. CRISPR activation (CRISPRa) represents a novel gain-of-function (GOF) strategy, conferring robust over-expression of the target gene within its native chromosomal context. The CRISPRa systems enable precise, scalable, and robust RNA-guided transcription activation, holding great potential for a variety of fundamental and translational research. In this chapter, we provide a step-by-step guide for efficient gene activation in Arabidopsis based on a highly robust CRISPRa system, CRISPR-Act3.0. We present detailed procedures onthe sgRNA design, CRISPR-Act3.0 system construction, Agrobacterium-mediated transformation of Arabidopsis using the floral dip method, and identification of desired transgenic plants.

Molecular and phenotypic characteristics of T1 transgenic yellow cosmos (Cosmos sulphureus) carrying neomycin phosphotransferase II gene

Abstract. Irsyadi MB, Sawitri WD, Purwantoro A. 2022. Molecular and phenotypic characteristics of T1 transgenic yellow cosmos (Cosmos sulphureus) carrying neomycin phosphotransferase II gene. Biodiversitas 23: 6097-6105. Yellow cosmos (Cosmos sulphureus) is a tropical ornamental flower that contains secondary metabolites useful fornatural pesticide application. Plant genetic transformation is one genetic engineering method used to increase secondary metabolite accumulation. However, published reports on this issue have yet to be made available. This is the first report of genetic transformation in transgenic yellow cosmos using neomycin phosphotransferase II (nptII) encoding gene. This study aimed to determine the efficiency transformation and phenotypic character of the T1 yellow cosmos transgenic nptII. This genetic transformation was carried out by the floral dip method mediated with Agrobacterium tumefaciens and characterized based on quantitative and qualitative observations, as well as a cluster analysis of transgenic plants. The T1 transgenic yellow cosmos was confirmed using PCR with a transformation efficiency of 73.33% based on the total plants resistant to Kanamycin and 10.57% based on the total seeds transformed. The presence of the nptII encoding gene was shown in transgenic plant samples with a size of 550 bp. In general, the introduction of the nptII gene generated no novel traits except the resistance to the kanamycin antibiotic. However, there was a decrease and increase in the number of stomata and the size of stomata in transgenic plants, respectively. In addition, there was a change in the type of ray floret to a mixture of ligulate and tubular, indicating that the nptII gene affected the phenotype of yellow cosmos. The result revealed that the nptII gene was inserted fairly randomly into the plant genome.

Characteristics of a radish mutant with longer siliques

The silique is an important organ in plant reproduction and maintains biodiversity among species; however, little is known about the regulation of radish (Raphanus sativus L.) silique development. In this study, we conducted research on the radish long silique (ls) mutant and wild-type (WT) radish and compared their morphological and molecular markers. The results showed that the mutant obtained by ethyl methane sulfonate treatment had the following stable characteristics: the lengths of mutant and WT siliques were 20.50 and 9.10 cm, respectively; and the ovule number per silique of the mutant and WT was 9.5 and 4.5, respectively. Polymerase chain reaction (PCR) analysis revealed abundant polymorphisms between the ls mutant and WT in HZ001, SRC9-022, and OIL2F11 simple-sequence repeat molecular markers, and the expression of LS1 and LS2 (RsNAC66). Arabidopsis thaliana-transformed plants with RsNAC66 overexpression were obtained by the floral dip method. Quantitative PCR showed that LS2 (RsNAC66) was more highly expressed in transformed lines than in WT, and the expression of LS2 (RsNAC66) in the transformed lines was higher in siliques than leaves. Phenotypic analysis revealed abnormal ovule numbers and shoots, and altered plant height in the transformed plant. Phenotypic and gene expression analyses showed that LS2 (RsNAC66) plays an important role on silique length and the number of seeds per silique. Together, the results showed that the radish ls mutant is specific and stable, and thus constitutes an excellent research resource for improving the seed yield of radishes.

Simplified floral dip transformation method of Arabidopsis thaliana

Being a model plant, Arabidopsis transformation is a frequent routine in genetics and molecular laboratories. Here, we explain a modified floral dip method routinely used in our laboratory for Arabidopsis transformation by applying drought stress after plant treatment with Agrobacterium. By using this method, hundreds of individual transgenic plants are produced from just 30 infiltrated plants by increasing transformation frequency from 1% to 10% within 2–3 months.

Targeted base editing in the mitochondrial genome of Arabidopsis thaliana

Beyond their well-known role in respiration, mitochondria of land plants contain biologically essential and/or agriculturally important genes whose function and regulation are not fully understood. Until recently, it has been difficult to analyze these genes or, in the case of crops, to improve their functions, due to a lack of methods for stably modifying plant mitochondrial genomes. In rice, rapeseed, and Arabidopsis thaliana, mitochondria-targeting transcription activator-like effector nucleases (mitoTALENs) have recently been used to disrupt targeted genes in an inheritable and stable manner. However, this technique can also induce large deletions around the targeted sites, as well as cause ectopic homologous recombinations, which can change the sequences and gene order of mitochondrial genomes. Here, we used mitochondria-targeting TALEN-based cytidine deaminase to successfully substitute targeted C:G pairs with T:A pairs in the mitochondrial genomes of plantlets of A. thaliana without causing deletions or changes in genome structure. Expression vectors of the base editor genes were stably introduced into the nuclear genome by the easy-to-use floral dipping method. Some T1 plants had apparent homoplasmic substitutions that were stably inherited by seed progenies, independently of the inheritance of nuclear-introduced genes. As a demonstration of the method, we used it to restore the growth of an organelle transcript processing 87 (otp87) mutant that is defective in the editing of RNA transcripts of the mitochondrial atp1 gene and to identify bases in atp1 that affect the efficiency of RNA editing by OTP87.

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

Development and optimization of a Barley stripe mosaic virus-mediated gene editing system to improve Fusarium head blight resistance in wheat.

Developmental and hormonal regulation of Arabidopsis thaliana ornithine-delta-aminotransferase.

Ornithine aminotransferase (OAT) catalyzes transfer of the delta-amino group from L-ornithine to oxo-glutarate. In plants, this reaction biochemically connects urea cycle, proline cycle, and polyamine biosynthesis pathway. OAT activity is shown to be associated with biotic and abiotic stress responses and nitrogen metabolism, but its physiological role is still unclear. In our study, we decided to investigate transcriptional regulation of the OAT gene in Arabidopsis thaliana under normal conditions and in response to various growth regulators. In the present work, the reporter gene construct containing the Escherichia coli β-glucuronidase gene (gus) under control of the A. thaliana OAT gene promoter was introduced into the genome of A. thaliana ecotype Columbia plants using the floral dip method; GUS activity was assayed in different experimental conditions including hormone treatment, low and high nitrogen and salinity. The GUS activity was analyzed histochemically. Plants were incubated with staining solution containing X-Gluc. We show that under standard growth conditions, the promoter is active during germination and in developing floral organs. OAT promoter activity specifically activates in response to different forms of auxin (IAA, NAA, and 2,4D), cytokinin (6- BAP), ethylene precursor (ACC), high nitrogen and salinity. Analysis of the OAT expression by qRT-PCR confirmed the pattern observed using the GUS reporter system. The OAT gene showed a significantly elevated expression in fourday- old seedlings and in plant roots in response to auxins and cytokinins. The analysis of the OAT promoter structure reveals cis-acting regulatory DNA elements associated with auxin regulation and abiotic stresses. The results of the study indicate that the OAT gene is involved in developmental processes and is regulated by auxin and cytokinins.

Cloning and functional analysis of the promoter of purple acid phosphatase gene <italic>GmPAP14 </italic>in soybean

<p id="C3">The expression of <italic>GmPAP14</italic> was induced under low phosphorus condition, and its overexpression could significantly improve the utilization efficiency of organic phosphorus in <italic>Arabidopsis</italic>. In order to further explore its regulatory mechanism, the promoter sequence of <italic>GmPAP14</italic> was cloned according to the sequence of soybean reference genome. The regulatory elements of <italic>GmPAP14</italic> promoter were predicted by the database PLACE and PlantCARE, which showed that it contained enhancer regulatory elements, tissue-specific expression elements, root-specific expression elements, and P1BS elements (transcription factor PHR1 binding sites). <italic>P_GmPAP14-2568</italic>-<italic>GUS</italic>, <italic>P_GmPAP14-2238</italic>-<italic>GUS</italic>, and <italic>P_GmPAP14-1635</italic>-<italic>GUS</italic> were constructed and transferred into <italic>Arabidopsis thaliana</italic> via Floral dip method. The expressional activities of three fragments of <italic>GmPAP14</italic> promoter were analyzed through GUS staining and activity measurement. The results revealed that <italic>GmPAP14</italic> promoter was mainly expressed in root tip under Pi condition, and GUS staining was extended to the elongation area, mature area, and root hair after low phosphorus treatment. Additionally, <italic>Arabidopsis</italic> plants with <italic>P_GmPAP14-2238</italic>-<italic>GUS</italic> had the highest activity among them. These results lay an important foundation for the further study of gene regulation.

Sandwich Ct real-time PCR identifies single-copy T-DNA integration accumulating in backbone-free transgenic T1 Arabidopsis

A sandwich Ct real-time PCR (SC-PCR) was used to detect single-copy T-DNA plants by visualizing Ct patterns of T-DNA and two reference amplicons. Detecting the T-DNA copy number directly by visualizing the Ct pattern eliminates the errors introduced by multistep calculations of relative Ct values. Using SC-PCR, we found that single-copy T-DNA integrations were more frequent in transgenic T1 Arabidopsis without a vector backbone. On the basis of this phenomenon, we combined the negative screen of the vector backbone and SC-PCR to efficiently identify single-copy T-DNA plants. We found that T-DNA copy number detection was underestimated in transgenic plants containing inverted T-DNA repeats due to hairpin structures formed during PCR, indicating that PCR-based methods for detecting T-DNA copy number should be reevaluated. We solved this problem by releasing T-DNA from the complex structures using restriction enzymes before performing SC-PCR. We also demonstrated that latent Agrobacterium contamination in the T1 transgenic Arabidopsis generated by the floral dip method was exceedingly low and may not affect the detection of T-DNA copy number. Overall, our method provides a whole-set procedure for detecting single-copy T-DNA plants more efficiently than other screening methods including Southern blotting.

Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense ) and Camelina (Camelina sativa ).

Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6×106 cells from pennycress and 3×106 cells from camelina per gram of fresh leaf tissues. Using a 7.3kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.