Conventional plant breeding methods using chemical- and radiation-mutagenesis are time-consuming and laborious, and require expensive screens on large populations to identify rare plants with the desired mutation. Here, we focus on the use of engineered sequence-specific nuclease and gene targeting (GT) technology to allow highly efficient, precise modification of plant genomes. Plant genome modifications with engineered sequence - specific nuclease [e.g., zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs) and CRISPR/Cas based RNA-guided DNA endonucleases] occur through imprecise repair, via the non-homologous end joining pathway, of DNA double strand breaks introduced by the specific nuclease used. GT is an extremely rare event in which exogenous DNA harboring the desired mutation integrates into the endogenous homologous sequence via homologous recombination. To isolate rare clones that have undergone targeted gene replacement by GT, direct gene-specific selection and positive-negative selection have been used in higher plants. However, the positive selectable marker gene should be completely eliminated from the GT locus to leave only the desired mutations in the target gene. Thus, we also describe precise marker excision using piggyBac transposon and intrachromosomal recombination. These technologies represent powerful tools, not only for studying basic biology and applied biotechnology but also for enhancing plant breeding.

Grafting is a cultivation method that exploits a cooperative relationship between partner plants possessing different genomes. It is most commonly used for the propagation and cultivation of trees, shrubs, and fruit vegetables. In addition, as represented by florigen (flowering hormone) experiments, grafting has been utilized in the field of plant physiology to clarify the mechanism of long-distance transport by which signals arising in organs that perceive an environmental change are transmitted to response organs. Recent analytical technology has revealed that some specific RNA molecules are also transported through phloem tissue as genetic information to execute coordinated organ growth and development. Therefore, it is anticipated that the RNA transport system could be applied for the improvement of cultivars of various horticultural crops, if the mechanism were controllable by artificial means. We reported here that the mobile siRNAs produced from the hairpin-structure transgene controlled by a companion cell specific promoter can induce the transmissible gene silencing in our grafting system. The epigenetic mutation was maintained through tissue culture and then inherited to the progenies. Our results suggest the potential application using the mobile promoter-targeting siRNAs to improve horticulture plants cultivars by grafting.

One of the goals of plant biotechnology is the production of genetically engineered crops, such as rice, wheat, and maize, with improved resistance to diseases. For this objective, finding viral molecules critical for virus replication seems to be important for specifying molecular targets for the application of this technique. Thus, biological, biochemical, structural and cytopatholgical studies of viral particles, viral proteins and virus infected cells were carried out to clarify functions and possible roles of each of 12 viral proteins of Rice dwarf virus, a double-stranded RNA virus. Among 12 proteins of the virus, Pns12 viroplasm protein, first appeared in infected cells, is considered to play an important role in virus replication, thus, chosen as a target for RNA interference. As expected, transgenic rice plants that knocked down the expression of the gene for the Pns12 showed strong resistance to virus infection. This strategy was successfully applied to develop rice plants resistant to viruses in the same group, Rice gall dwarf virus and Rice black - streaked dwarf virus. On the other hand, in Rice stripe virus, a negative stranded tenuivirus, most effective targets for RNA interference were nucleocapsid and movement protein, thus showed that molecular targets for RNA interference for obtaining resistant plants differ in viruses with different replication strategies.

Recent advances in the technologies for developing genetically modified crops have highlighted the importance of epigenetic controls in plants. Several studies have suggested that double-stranded RNA (dsRNA) can induce sequence-specific DNA methylation and silence expression of target genes. Moreover, DNA methylation-mediated gene silencing can be stably transmitted to the next generation without dsRNA trigger constructs. By exploring and elucidating the molecular mechanisms behind these phenomena, we have achieved a greater understanding of epigenetic molecular mechanisms and this knowledge has provided the foundation for new techniques, the so-called New Plant Breeding Techniques (NBT). Further advances in our understanding of the molecular mechanisms of RNA-directed DNA methylation (RdDM) will enable improvements to these new techniques.

Strigolactones (SLs) were initially characterized as root-derived signals for parasitic and symbiotic interactions with root parasitic plants and arbuscular mycorrhizal fungi. In 2008, SLs or the downstream metabolites were shown to act as endogenous phytohormones that regulate shoot branching. After the discovery, many researchers demonstrated that SLs play important biological roles in plant development. Moreover, biochemical functions of SL-biosynthetic enzymes have been uncovered. In this review, we summarize recent advances in SL researches regarding diverse biological roles and biosynthesis.

Strigolactones (SLs) are plant secondary metabolites which function as germination stimulants of root parasitic plants and hyphal branching factors of symbiotic arbuscular mycorrhizal (AM) fungi in the rhizosphere. In planta, SLs also work as plant hormone regulating shoot and root architecture, photomorphogenesis, secondary growth, etc. To date, more than 20 SLs have been characterized from root exudates of various plant species. In this review, we will discuss on diverse stereochemical structures of natural SLs and distribution of SLs in the plant kingdom.

Biochemical composition of bulk plant extracts provides limited and averaged information. Homogenization procedure destroys spatiotemporal information of cells included in the tissue. It is essential to understand specific metabolic functions assigned to individual cell types. So, we have developed and applied in-situ Live Single-cell MS which can identify the molecules of a single cell directly with simultaneous observation by a video-microscope. In this technical note, we overviewed its methodology and potentials to apply for plant biology.

In recent decades, laser optics has been strenuously adapted to biological and biotechnological applications. Laser has high monochromaticity, directivity, and coherency and these properties are considered to be suitable for manipulating and/or observing intact cells with high special resolution. In this technical note, several laser-based microsurgery and non-linear optical microscopy techniques for plant metabolite analysis was discussed.