Rose is a major ornamental plant, and a lot of cultivars with attractive morphology, color and scent have been generated by classical breeding. Recent progress of genetic modification produces a novel cultivar with attractive features. In both cases, a major problem is the gene-flow from cultivated or genetically modified (GM) plants to wild species, causing reduction of natural population. To investigate whether gene-flow occurs in wild species, molecular analysis with DNA markers with higher efficient technique is useful. Here we investigated the gene-flow from cultivated roses (Rosa×hybrida) to wild rose species planted in close distance in the field. The overlapping flowering periods and visiting insects suggest that pollens were transported by insects between wild and cultivated roses. We examined the germination ratio of seeds from wild species, and extracted DNA and checked with KSN and APETALA2 (AP2) DNA markers to detect transposon insertions. Using two markers, we successfully detected the outcross between wild and cultivated roses. For higher efficiency, we established a bulking method, where DNA, leaves or embryos were pooled, enabling us to that check the outcross of many plants. Our results suggest that wild species and garden cultivars can cross in close distance, so that they should be planted in distance, and checked the outcross with multiple DNA markers.
The plastid is a promising target for the production of valuable biomolecules via genetic engineering. We recently developed a plastid-specific gene delivery system for leaves or seedlings using KH-AtOEP34, a functional peptide composed of the polycationic DNA-binding peptide KH and the Arabidopsis thaliana plastid-targeting peptide OEP34. Here, we established a liquid culture system for inducing multiple shoots in the model plants A. thaliana and Nicotiana benthamiana and the crop plant strawberry (Fragaria×ananassa) and tested the use of these plant materials for peptide-mediated gene delivery to plastids. Our liquid culture system efficiently induced multiple shoots that were enriched in meristems. Using these meristems, we performed KH-AtOEP34-mediated gene delivery to plastids and tested the delivery and integration of a cassette composed of the spectinomycin resistance gene aadA, the GFP reporter gene, and sequences homologous to plastid DNA. Genotyping PCR revealed the integration of the cassette DNA into plastid DNA several days after delivery in all three plants. Confocal laser scanning microscopy and immunoblotting confirmed the presence of plasmid-derived GFP in the plastids of meristems, indicating that the plasmid DNA was successfully integrated into plastid DNA and that the cassette was expressed. These results suggest the meristems developed in our liquid culture system are applicable to peptide-mediated delivery of exogeneous DNA into plastids. The multiple shoots generated in our liquid novel culture system represent promising materials for in planta peptide-mediated plastid transformation in combination with spectinomycin selection.
Salicylic acid (SA) is known to be involved in the immunity against Clavibacter michiganensis ssp. michiganensis (Cmm) that causes bacterial canker in tomato. To identify the candidate genes associated with SA-inducible Cmm resistance, transcriptome analysis was conducted via RNA sequencing in tomato plants treated with SA. SA treatment upregulated various defense-associated genes, such as PR and GST genes, in tomato cotyledons. A comparison of SA- and Cmm-responsive genes revealed that both SA treatment and Cmm infection commonly upregulated a large number of genes. Gene Ontology (GO) analysis indicated that the GO terms associated with plant immunity were over-represented in both SA- and Cmm-induced genes. The genes commonly downregulated by both SA treatment and Cmm infection were associated with the cell cycle and may be involved in growth and immunity trade-off through cell division. After SA treatment, several proteins that were predicted to play a role in immune signaling, such as resistance gene analogs, Ca2+ sensors, and WRKY transcription factors, were transcriptionally upregulated. The W-box element, which was targeted by WRKYs, was over-represented in the promoter regions of genes upregulated by both SA treatment and Cmm infection, supporting the speculation that WRKYs are important for the SA-mediated immunity against Cmm. Prediction of protein–protein interactions suggested that genes encoding receptor-like kinases and EF-hand proteins play an important role in immune signaling. Thus, various candidate genes involved in SA-inducible Cmm resistance were identified.
Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The piggyBac transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because piggyBac has the advantage that it can transpose without leaving a footprint at the excised site. Applying the piggyBac transposon to precise genome editing in plants, we have demonstrated efficient and precise piggyBac transposon excision from a transgene locus integrated into the rice genome. Furthermore, introduction of only desired point mutations into the target gene can be achieved by a combination of precise gene modification via homologous recombination-mediated gene targeting with subsequent marker excision from target loci using piggyBac transposition in rice. In addition, we have designed a piggyBac-mediated transgenesis system for the temporary expression of sequence-specific nucleases to eliminate the transgene from the host genome without leaving unnecessary sequences after the successful induction of targeted mutagenesis via sequence-specific nucleases for use in vegetatively propagated plants. In this review, we summarize our previous works and the future prospects of genetic engineering with piggyBac transposon.
Although it is well known that hierarchical transcriptional networks are essential for various aspects of plant development and environmental response, little has been investigated about whether and how they also regulate the plant cell cycle. Recent studies on cell cycle regulation in Arabidopsis thaliana identified SCARECROW-LIKE28 (SCL28), a GRAS-type transcription factor, that constitutes a hierarchical transcriptional pathway comprised of MYB3R, SCL28 and SIAMESE-RELATED (SMR). In this pathway, MYB3R family proteins regulate the G2/M-specific transcription of the SCL28 gene, of which products, in turn, positively regulate the transcription of SMR genes encoding a group of plant-specific inhibitor proteins of cyclin-dependent kinases. However, this pathway with a role in cell cycle inhibition is solely demonstrated in A. thaliana, thus leaving open the question of whether and to what extent this pathway is evolutionarily conserved in plants. In this study, we conducted differential display RT-PCR on synchronized Nicotiana tabacum (tobacco) BY-2 cells and identified several M-phase-specific cDNA clones, one of which turned out to be a tobacco ortholog of SCL28 and was designated NtSCL28. We showed that NtSCL28 is expressed specifically during G2/M and early G1 in the synchronized cultures of BY-2 cells. NtSCL28 contains MYB3R-binding promoter elements, so-called mitosis-specific activator elements, and is upregulated by a hyperactive form of NtmybA2, one of the MYB3R proteins from tobacco. Our study indicated that a part of the hierarchical pathway identified in A. thaliana is equally operating in tobacco cells, suggesting the conservation of this pathway across different families in evolution of angiosperm.