Single T-DNA Research Articles

Knockout mutation in TaD27 enhances number of productive tillers in hexaploid wheat.

Recent advances allow the deployment of cluster regularly interspaced short palindromic repeats (CRISPR)-associated endonucleases (Cas) system for the targeted mutagenesis in the genome with accuracy and precision for trait improvement in crops. CRISPR-Cas systems have been extensively utilized to induce knockout or frameshift mutations in the targeted sequence of mostly negative regulating genes for wheat improvement. However, most of the reported work has been done in non-commercial varieties of wheat and introgression of edited alleles into breeding population comes with the penalty of unwanted linkage-drag. Wheat yield is controlled by various genes such as positive and negative regulators. The TaD27 gene is described as a negative regulator of shoot branching or tillering and involved in the biosynthesis of strigolactones. In this study, we developed Tad27 knockout mutant lines of an elite wheat cultivar that showed a twofold increase in the number of tillers and 1.8-fold increase in the number of grains per plant. Subsequently, enhancing the grain yield without any morphological penalty in the architecture of the plants. The co-transformation of regeneration enhancing growth regulator, Growth Regulating Factor 4 (GRF4) and its cofactor GRF-Interacting Factor 1 (GIF1), under single T-DNA cassette improved the regeneration efficiency up to 6% of transgenic events from mature embryos of wheat. Our results indicate that the CRISPR-mediated targeted mutagenesis confers the potential to knockout yield-related negative regulators in elite cultivars of wheat that can substantially enhance grain yield per plant and this strategy can be harnessed for the improvement of future wheat.

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology.

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership.

Upregulation of LmHxt1 gene is associated with reduced virulence of Leptosphaeria maculans on Brassica napus

AbstractLeptosphaeria maculans is a fungal pathogen that causes heavy losses of Brassica napus crops. To develop novel means of plant protection it is necessary to understand the virulence mechanisms of pathogens. In this study we investigated a T-DNA insertion mutant of L. maculans that exhibited a hypovirulent phenotype (pHxt1ins). The mutant contains a single T-DNA insertion that affects expression of a single gene identified as a transmembrane hexose transporter (LmHxt1). This gene exhibited enhanced expression in the pHxt1ins mutant during in vitro cultivation. The excess of glucose in the cultivation medium lowered expression of LmHxt1 in the mutant line, suggesting that the gene might function as a sugar sensor. Lack of virulence of the pHxt1ins mutant in planta was observed using fluorescence microscopy in GFP labeled lines ruling out asymptomatic growth and showing inability to successfully colonize the apoplastic space of B. napus. On the other hand, in vitro growth did not differ between pHxt1ins and control lines. Interestingly, the mutant showed altered mycelium morphology and a change in conidial germination. Overall, our results suggest that the LmHxt1 gene is a novel important virulence factor for L. maculans. We also propose pHxt1ins mutant as a tool to study the role of sugar transporters in fungal metabolism and pathogenesis.

Obtaining of transgenic barrelclover plants (Medicago truncatula) producing chicken interferon gamma for veterinary use

At the Laboratory of Plant Genetic and Cellular Engineering, Department of Genetics and Biotechnology, St. Petersburg State University, five transgenic Medicago truncatula plants were obtained through Agrobacterium-mediated transformation, carrying one of the variants of the heterologous chicken interferon-gamma gene under the control of the constitutive 35S CaMV promoter. Among these, one plant harbored an unmodified gene insertion, while four had a modified gene with a deletion at the protease recognition site, providing resistance to proteolytic degradation.
 We demonstrate the application of the SWPOP-PCR “genome walking” method to determine the integration sites of T-DNA into the plant genome, identify the number of insertion copies and their orientation. Analysis of the obtained sequences revealed that only one plant exhibited a single T-DNA insertion, which represents the most optimal structure for stable expression.
 Upon self-pollination of T0 plants, 39 offspring were obtained and subjected to testing for the presence and expression of the transgene. Among them, six homozygous plants were identified using molecular methods. Quantitative assessment of transgene expression levels showed significant differences among representatives of different lines and among the offspring derived from a single transformed plant. Among the T1 and T2 progeny, the presence of the heterologous interferon protein in plant tissues was confirmed through Western blot analysis.
 The engineered barrelclover plants hold potential as bioreactors for the production of chicken interferon-gamma for veterinary applications. The use of an edible plant allows eliminating protein extraction and purification procedures, thereby resulting in a noteworthy reduction in production expenses of up to 80%.

Pyramiding stacking of multigenes (PSM): a simple, flexible and efficient multigene stacking system based on Gibson assembly and gateway cloning.

Genetic engineering of complex metabolic pathways and multiple traits often requires the introduction of multiple genes. The construction of plasmids carrying multiple DNA fragments plays a vital role in these processes. In this study, the Gibson assembly and Gateway cloning combined Pyramiding Stacking of Multigenes (PSM) system was developed to assemble multiple transgenes into a single T-DNA. Combining the advantages of Gibson assembly and Gateway cloning, the PSM system uses an inverted pyramid stacking route and allows fast, flexible and efficient stacking of multiple genes into a binary vector. The PSM system contains two modular designed entry vectors (each containing two different attL sites and two selectable markers) and one Gateway-compatible destination vector (containing four attR sites and two negative selection markers). The target genes are primarily assembled into the entry vectors via two parallel rounds of Gibson assembly reactions. Then, the cargos in the entry constructs are integrated into the destination vector via a single tube Gateway LR reaction. To demonstrate PSM's capabilities, four and nine gene expression cassettes were respectively assembled into the destination vector to generate two binary expression vectors. The transgenic analysis of these constructs in Arabidopsis demonstrated the reliability of the constructs generated by PSM. Due to its flexibility, simplicity and versatility, PSM has great potential for genetic engineering, synthetic biology and the improvement of multiple traits.

An accurate, reliable, and universal qPCR method to identify homozygous single insert T-DNA with the example of transgenic rice.

Early determination of transgenic plants that are homozygous for a single locus T-DNA insert is highly desirable in most fundamental and applied transgenic research. This study aimed to build on an accurate, rapid, and reliable quantitative real-time PCR (qPCR) method to fast-track the development of multiple homozygous transgenic rice lines in the T1 generation, with low copy number to single T-DNA insert for further analyses. Here, a well-established qPCR protocol, based on the OsSBE4 reference gene and the nos terminator, was optimized in the transgenic Japonica rice cultivar Nipponbare, to distinguish homozygous single-insert plants with 100% accuracy. This method was successfully adapted to transgenic Indica rice plants carrying three different T-DNAs, without any modifications to quickly develop homozygous rice plants in the T1 generation. The accuracy of this qPCR method when applied to transgenic Indica rice approached 100% in 12 putative transgenic lines. Moreover, this protocol also successfully detected homozygous single-locus T-DNA transgenic rice plants with two-transgene T-DNAs, a feature likely to become more popular in future transgenic research. The assay was developed utilizing universal primers targeting common sequence elements of gene cassettes (the nos terminator). This assay could therefore be applied to other transgenic plants carrying the nos terminator. All procedures described here use standardized qPCR reaction conditions and relatively inexpensive dyes, such as SYBR Green, thus the qPCR method could be cost-effective and suitable for lower budget laboratories that are involved in rice transgenic research.

Identification of T-DNA structure and insertion site in transgenic crops using targeted capture sequencing.

The commercialization of GE crops requires a rigorous safety assessment, which includes a precise DNA level characterization of inserted T-DNA. In the past, several strategies have been developed for identifying T-DNA insertion sites including, Southern blot and different PCR-based methods. However, these methods are often challenging to scale up for screening of dozens of transgenic events and for crops with complex genomes, like potato. Here, we report using target capture sequencing (TCS) to characterize the T-DNA structure and insertion sites of 34 transgenic events in potato. This T-DNA is an 18 kb fragment between left and right borders and carries three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1 genes) that result in complete resistance to late blight disease. Using TCS, we obtained a high sequence read coverage within the T-DNA and junction regions. We identified the T-DNA breakpoints on either ends for 85% of the transgenic events. About 74% of the transgenic events had their T-DNA with 3R gene sequences intact. The flanking sequences of the T-DNA were from the potato genome for half of the transgenic events, and about a third (11) of the transgenic events have a single T-DNA insertion mapped into the potato genome, of which five events do not interrupt an existing potato gene. The TCS results were confirmed using PCR and Sanger sequencing for 6 of the best transgenic events representing 20% of the transgenic events suitable for regulatory approval. These results demonstrate the wide applicability of TCS for the precise T-DNA insertion characterization in transgenic crops.

Molecular Characterization and Efficacy Evaluation of Transgenic Maize Harboring cry2Ab-vip3A-cp4epsps for Insect Resistance and Herbicide Tolerance.

Insect infestation and weed interference have a seriously negative impact on the growth, yield, and grain quality of maize. In this study, transgenic maize plants harboring three exogenous genes, cry2Ab, vip3A, and cp4epsps, that were constructed into a single T-DNA were developed for protection against insects and weeds. The transgene integration sites on the chromosomes in two transgenic maize events, CVC-1 and CVC-2, were determined using whole genome sequencing and specific PCR detection. As revealed by laboratory insect bioassays, these two transgenic events exhibited strong insecticidal toxicity against three major species of Lepidoptera insects, including Mythimna separata, Helicoverpa armigera, and Spodoptera frugiperda, with mortality rates exceeding 96%, 100%, and 100%, respectively, after six days of infestation. In addition, CVC-1 exhibited a high tolerance to glyphosate under field conditions. The successful expressions of cry2Ab, vip3A, and cp4epsps in various tissues at different developmental stages of CVC-1 were validated at the transcriptional and translational levels using quantitative real-time reverse transcription PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. These findings demonstrated that the transgenic maize CVC-1 developed using this triple gene construct has excellent insect resistance and herbicide tolerance, which may provide a valuable germplasm resource and data support for future maize breeding of insect and weed control.

Cmcrf1, a Putative Zn2Cys6 Fungal Transcription Factor, Is Involved in Conidiation, Carotenoid Production, and Fruiting Body Development in Cordyceps militaris.

Cordyceps militaris is a high-value medicinal and edible fungus that produces many bioactive compounds, including carotenoid, and thus, improving the carotenoid productivity of C. militaris will increase its commercial value. However, little is known about the genetic regulatory mechanism of carotenoid biosynthesis in C. militaris. To further understanding the regulatory mechanism of carotenoid biosynthesis, we performed a large-scale screen of T-DNA insertional mutant library and identified a defective mutant, denoted T111, whose colonies did not change color from white to yellow upon exposure to light. Mutation analysis confirmed that a single T-DNA insertion occurred in the gene encoding a 695-amino-acid putative fungal-specific transcription factor with a predicted Zn2Cys6 binuclear cluster DNA-binding domain found uniquely in fungi. Targeted deletion of this gene, denoted C. militaris carotenogenesis regulatory factor 1 (Cmcrf1), generated the ΔCmcrf1 mutant that exhibited drastically reduced carotenoid biosynthesis and failed to generate fruiting bodies. In addition, the ΔCmcrf1 mutant showed significantly increased conidiation and increased hypersensitivity to cell-wall-perturbing agents compared with the wild-type strain. However, the Cmcrf1 gene did not have an impact on the mycelia growth of C. militaris. These results show that Cmcrf1 is involved in carotenoid biosynthesis and is required for conidiation and fruiting body formation in C. militaris.

MoBiFC: development of a modular bimolecular fluorescence complementation toolkit for the analysis of chloroplast protein–protein interactions

BackgroundThe bimolecular fluorescence complementation (BiFC) assay has emerged as one of the most popular methods for analysing protein–protein interactions (PPIs) in plant biology. This includes its increasing use as a tool for dissecting the molecular mechanisms of chloroplast function. However, the construction of chloroplast fusion proteins for BiFC can be difficult, and the availability and selection of appropriate controls is not trivial. Furthermore, the challenges of performing BiFC in restricted cellular compartments has not been specifically addressed.ResultsHere we describe the development of a flexible modular cloning-based toolkit for BiFC (MoBiFC) and proximity labelling in the chloroplast and other cellular compartments using synthetic biology principles. We used pairs of chloroplast proteins previously shown to interact (HSP21/HSP21 and HSP21/PTAC5) and a negative control (HSP21/ΔPTAC5) to develop standardised Goldengate-compatible modules for the assembly of protein fusions with fluorescent protein (FP) fragments for BiFC expressed from a single multigenic T-DNA. Using synthetic biology principles and transient expression in Nicotiana benthamiana, we iteratively improved the approach by testing different FP fragments, promoters, reference FPs for ratiometric quantification, and cell types. A generic negative control (mCHERRY) was also tested, and modules for the identification of proximal proteins by Turbo-ID labelling were developed and validated.ConclusionsMoBiFC facilitates the cloning process for organelle-targeted proteins, allows robust ratiometric quantification, and makes available model positive and negative controls. Development of MoBiFC underlines how Goldengate cloning approaches accelerate the development and enrichment of new toolsets, and highlights several potential pitfalls in designing BiFC experiments including the choice of FP split, negative controls, cell type, and reference FP. We discuss how MoBiFC could be further improved and extended to other compartments of the plant cell and to high throughput cloning approaches.

Molecular identification and efficacy assessment of a glufosinate-tolerant and brown planthopper-resistant transgenic rice line

Insect pests and weeds are the two major biotic factors affecting crop yield in the modern agricultural system. In this study, a brown planthopper (BPH) resistance gene (BPH9) and glufosinate tolerance gene (bar) were stacked into a single T-DNA cassette and transformed into an indica rice (Oryza sativa L.) line Guangzhan 63-4S. A stable transgenic line H23 with a single T-DNA insert was generated, with the T-DNA cassette located on chromosome 3. Field resistance trial using H23 revealed high tolerance to glufosinate and excellent resistance to BPH. These results propose H23 as valuable germplasm for BPH-resistance and glufosinate-tolerance breeding in rice.

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.

Large scale genomic rearrangements in selected Arabidopsis thaliana T-DNA lines are caused by T-DNA insertion mutagenesis

BackgroundExperimental proof of gene function assignments in plants is based on mutant analyses. T-DNA insertion lines provided an invaluable resource of mutants and enabled systematic reverse genetics-based investigation of the functions of Arabidopsis thaliana genes during the last decades.ResultsWe sequenced the genomes of 14 A. thaliana GABI-Kat T-DNA insertion lines, which eluded flanking sequence tag-based attempts to characterize their insertion loci, with Oxford Nanopore Technologies (ONT) long reads. Complex T-DNA insertions were resolved and 11 previously unknown T-DNA loci identified, resulting in about 2 T-DNA insertions per line and suggesting that this number was previously underestimated. T-DNA mutagenesis caused fusions of chromosomes along with compensating translocations to keep the gene set complete throughout meiosis. Also, an inverted duplication of 800 kbp was detected. About 10 % of GABI-Kat lines might be affected by chromosomal rearrangements, some of which do not involve T-DNA. Local assembly of selected reads was shown to be a computationally effective method to resolve the structure of T-DNA insertion loci. We developed an automated workflow to support investigation of long read data from T-DNA insertion lines. All steps from DNA extraction to assembly of T-DNA loci can be completed within days.ConclusionsLong read sequencing was demonstrated to be an effective way to resolve complex T-DNA insertions and chromosome fusions. Many T-DNA insertions comprise not just a single T-DNA, but complex arrays of multiple T-DNAs. It is becoming obvious that T-DNA insertion alleles must be characterized by exact identification of both T-DNA::genome junctions to generate clear genotype-to-phenotype relations.

Knockdown of Mitochondrial Uncoupling Proteins 1 and 2 (AtUCP1 and 2) in Arabidopsis thaliana Impacts Vegetative Development and Fertility.

Mitochondrial uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that dissipate the proton electrochemical gradient generated by the respiratory chain complexes. In plants, these proteins are crucial for maintaining mitochondrial reactive oxygen species (ROS) homeostasis. In this study, single T-DNA insertion mutants for two (AtUCP1 and AtUCP2) out of the three UCP genes present in Arabidopsis thaliana were employed to elucidate their potential roles in planta. Our data revealed a significant increase in the Adenosine triphosphate (ATP)/Adenosine diphosphate (ADP) ratios of both mutants, indicating clear alterations in energy metabolism, and a reduced respiratory rate in atucp2. Phenotypic characterization revealed that atucp1 and atucp2 plants displayed reduced primary root growth under normal and stressed conditions. Moreover, a reduced fertility phenotype was observed in both mutants, which exhibited an increased number of sterile siliques and a lower seed yield compared with wild-type plants. Reciprocal crosses demonstrated that both male fertility and female fertility were compromised in atucp1, while such effect was exclusively observed in the male counterpart in atucp2. Most strikingly, a pronounced accumulation of hydrogen peroxide in the reproductive organs was observed in all mutant lines, indicating a disturbance in ROS homeostasis of mutant flowers. Accordingly, the atucp1 and atucp2 mutants exhibited higher levels of ROS in pollen grains. Further, alternative oxidase 1a was highly induced in mutant flowers, while the expression profiles of transcription factors implicated in gene regulation during female and male reproductive organ/tissue development were perturbed. Overall, these data support the important role for AtUCP1 and AtUCP2 in flower oxidative homeostasis and overall plant fertility.

Highly efficient generation of bacterial leaf blight-resistant and transgene-free rice using a genome editing and multiplexed selection system

BackgroundRice leaf blight, which is a devastating disease worldwide, is caused by the bacterium Xanthomonas oryzae pv. oryzae (Xoo). The upregulated by transcription activator-like 1 (UPT) effector box in the promoter region of the rice Xa13 gene plays a key role in Xoo pathogenicity. Mutation of a key bacterial protein-binding site in the UPT box of Xa13 to abolish PXO99-induced Xa13 expression is a way to improve rice resistance to bacteria.Highly efficient generation and selection of transgene-free edited plants are helpful to shorten and simplify the gene editing-based breeding process. Selective elimination of transgenic pollen of T0 plants can enrich the proportion of T1 transgene-free offspring, and expression of a color marker gene in seeds makes the selection of T2 plants very convenient and efficient. In this study, a genome editing and multiplexed selection system was used to generate bacterial leaf blight-resistant and transgene-free rice plants.ResultsWe introduced site-specific mutations into the UPT box using CRISPR/Cas12a technology to hamper with transcription-activator-like effector (TAL) protein binding and gene activation and generated genome-edited rice with improved bacterial blight resistance. Transgenic pollen of T0 plants was eliminated by pollen-specific expression of the α-amylase gene Zmaa1, and the proportion of transgene-free plants increased from 25 to 50% among single T-DNA insertion events in the T1 generation. Transgenic seeds were visually identified and discarded by specific aleuronic expression of DsRed, which reduced the cost by 50% and led to up to 98.64% accuracy for the selection of transgene-free edited plants.ConclusionWe demonstrated that core nucleotide deletion in the UPT box of the Xa13 promoter conferred resistance to rice blight, and selection of transgene-free plants was boosted by introducing multiplexed selection. The combination of genome editing and transgene-free selection is an efficient strategy to accelerate functional genomic research and plant breeding.

Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus.

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

Direct Germline Transformation of Cotton Meristem Explants With No Selection.

Regeneration of transgenic plants without selectable markers can facilitate the development and commercialization of trait stacking products. A wide range of strategies have been developed to eliminate selectable markers to produce marker-free transgenic plants. The most widely used marker free approach is probably the Agrobacterium-based 2 T-DNA strategy where the gene-of-interest (GOI) and selectable marker gene are delivered from independent T-DNAs (Darbani et al., 2007). The selectable marker gene is segregated away from the GOI in subsequent generations. However, the efficiency of this 2 T-DNA system is much less than the traditional 1 T-DNA system due to the inefficiency of T-DNA co-transformation and high rate of con-integration between the GOI and selectable marker gene T-DNAs. In contrast, no selection transformation utilizes a single T-DNA carrying the GOI and thus eliminates the need to remove the selectable marker insert and potentially provides a viable alternative marker-free system. In this study, we reported the successful regeneration of transgenic cotton plants through Agrobacterium inoculation of seed meristem explants without the use of selective agents. Regeneration of putative transgenic plants were identified by GUS histo-chemical assay. The germline transmission of transgene to progeny was determined by segregation of pollen grains, immature embryos and T1 plants by GUS expression. The results were further confirmed by Southern analyses. The marker-free transformation frequency in this no selection system was similar to current meristem transformation system with selection (0.2%–0.7%). The strategy for further improvement of this system and its implication in improving cotton transformation pipeline and in developing transgene-free genome editing technology is discussed.

The Salt Sensitivity Induced by Disruption of Cell Wall-Associated Kinase 1 (SlWAK1) Tomato Gene Is Linked to Altered Osmotic and Metabolic Homeostasis

Tomato cell wall-associated kinase 1 (SlWAK1) has only been studied in biotic stress response and hence its function in abiotic stress remains unknown. In a screening under salinity of an insertional mutant collection of tomato (Solanum lycopersicum L.), a mutant exhibiting lower degree of leaf chlorosis than wild type (WT) together with reduced leaf Na+ accumulation was selected. Genetic analysis of the mutation revealed that a single T-DNA insertion in the SlWAK1 gene was responsible of the mutant phenotype. Slwak1 null mutant reduced its shoot growth compared with WT, despite its improved Na+ homeostasis. SlWAK1 disruption affected osmotic homeostasis, as leaf water content was lower in mutant than in WT under salt stress. In addition, Slwak1 altered the source-sink balance under salinity, by increasing sucrose content in roots. Finally, a significant fruit yield reduction was found in Slwak1 vs. WT under long-term salt stress, mainly due to lower fruit weight. Our results show that disruption of SlWAK1 induces a higher sucrose transport from source leaf to sink root, negatively affecting fruit, the main sink at adult stage.

Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants

BackgroundGeneration of marker-free transgenic plants is very important to the regulatory permission and commercial release of transgenic crops. Co-transformation methods that enable the removal of selectable marker genes have been extensively used because they are simple and clean. Few comparisons are currently available between different strain/plasmid co-transformation systems, and also data are related to variation in co-transformation frequencies caused by other details of the vector design.ResultsIn this study, we constructed three vector systems for the co-transformation of allotetraploid Brassica napus (B. napus) mediated by Agrobacterium tumefaciens and compared these co-transformation methods. We tested a mixed-strain system, in which a single T-DNA is harbored in two plasmids, as well as two “double T-DNA” vector systems, in which two independent T-DNAs are harbored in one plasmid in a tandem orientation or in an inverted orientation. As confirmed by the use of PCR analysis, test strips, and Southern blot, the average co-transformation frequencies from these systems ranged from 24 to 81% in T0 plants, with the highest frequency of 81% for 1:1 treatment of the mixed-strain system. These vector systems are valuable for generating marker-free transgenic B. napus plants, and marker-free plants were successfully obtained in the T1 generation from 50 to 77% of T0 transgenic lines using these systems, with the highest frequency of 77% for “double T-DNA” vector systems of pBID RT Enhanced. We further found that marker-free B. napus plants were more frequently encountered in the progeny of transgenic lines which has only one or two marker gene copies in the T0 generation. Two types of herbicide resistant transgenic B. napus plants, Bar+ with phosphinothricin resistance and Bar+EPSPS+GOX+ with phosphinothricin and glyphosate resistance, were obtained.ConclusionWe were successful in removing selectable marker genes in transgenic B. napus plants using all three co-transformation systems developed in this study. It was proved that if a appropriate mole ratio was designed for the specific length ratio of the twin T-DNAs for the mixed-strain method, high unlinked co-insertion frequency and overall success frequency could be achieved. Our study provides useful information for the construction of efficient co-transformation system for marker-free transgenic crop production and developed transgenic B. napus with various types of herbicide resistance.

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.