This study established a rapid method for the gene expression analysis in potato tubers. The use of microtubers would be useful for primary evaluation of tuber-expressed genes. In the development of transgenic potato or of potato with other genome modifications (e.g., genome editing or RNA-directed DNA methylation (RdDM) and so on) to improve tuber traits, analysis of the target gene is often difficult because of the long cultivation cycle (3–4 months), large areas required, numerous materials for plant cultivation, and considerable efforts needed to obtain transgenic tubers. We demonstrate here rapid and convenient analysis of gene expression in potato microtubers. Enough microtubers for expression analysis can be induced over about 4 weeks in a simple liquid medium in an Erlenmeyer flask. High-quality RNA and protein can be easily prepared from microtubers and used for northern blot, qRT-PCR, and western blot analyses without further purification. We investigated the expression of two tuber-expressed genes (GBSS1 and Vinv) in microtubers derived from the wild-type and from lines derived from RdDM-mediated transcriptional gene silencing. As expected, the expression of both genes was similar between microtubers and normal tubers. Furthermore, we demonstrated that microtubers can be used in western blot and confocal immunofluorescent microscopy analyses. These results suggest that expression analysis using microtubers is a convenient tool for the analysis of tuber-expressed genes such as GBSS1 and Vinv in potato.

SummaryTo induce transcriptional gene silencing (TGS) of endogenous genes of rice (Oryza sativa L.), we expressed double‐strand RNA of each promoter region and thus induced RNA‐directed DNA methylation (RdDM). We targeted constitutively expressed genes encoding calnexin (CNX), protein disulphide isomerase (PDIL1‐1) and luminal binding protein (BiP1); an endoplasmic reticulum stress‐inducible gene (OsbZIP50); and genes with seed‐specific expression encoding α‐globulin (Glb‐1) and glutelin‐B4 (GluB4). TGS of four genes was obtained with high efficiency (CNX, 66.7% of regenerated plants; OsBiP1, 67.4%; OsbZIP50, 63.4%; GluB4, 66.1%), whereas the efficiency was lower for PDIL1‐1 (33.3%) and Glb‐1 TGS lines (10.5%). The heredity of TGS, methylation levels of promoter regions and specificity of silencing of the target gene were investigated in some of the TGS lines. In progeny of CNX and OsbZIP50 TGS lines, suppression of the target genes was preserved (except in the endosperm) even after the removal of trigger genes (T‐DNA) by segregation. TGS of CNX was reverted by demethylation treatment, and a significant difference in CG and CHG methylation levels in the −1 to −250 bp region of the CNX promoter was detected between the TGS and revertant lines, suggesting that TGS is closely related to the methylation levels of promoter. TGS exhibited specific suppression towards the target gene compared with post‐transcriptional gene silencing when GluB4 gene from glutelin multigene family was targeted. Based on these results, future perspectives and problems to be solved in the application of RdDM to new plant breeding techniques in rice are discussed.

In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops.

Apple is a fruit crop of significant economic importance, and breeders world wide continue to develop novel cultivars with improved characteristics. The lengthy juvenile period and the large field space required to grow apple populations have imposed major limitations on breeding. Various molecular biological techniques have been employed to make apple breeding easier. Transgenic technology has facilitated the development of apples with resistance to fungal or bacterial diseases, improved fruit quality, or root stocks with better rooting or dwarfing ability. DNA markers for disease resistance (scab, powdery mildew, fire-blight, Alternaria blotch) and fruit skin color have also been developed, and marker-assisted selection (MAS) has been employed in breeding programs. In the last decade, genomic sequences and chromosome maps of various cultivars have become available, allowing the development of large SNP arrays, enabling efficient QTL mapping and genomic selection (GS). In recent years, new technologies for genetic improvement, such as trans-grafting, virus vectors, and genome-editing, have emerged. Using these techniques, no foreign genes are present in the final product, and some of them show considerable promise for application to apple breeding.

KNOTTED1 (KN1)-like homologous box is a homeodomain transcription factor that is transported intercellularly through plasmodesmata and over long distances via phloem transport in plants. In this study, we investigated KN1 long-distance transport and its regulation by the viral movement protein binding protein of ‘Du Li’ (Pyrus betulaefolia, PbMPB2C) in two species of fruit trees, ‘Du Li’ (rootstock) and ‘Ya Li’ (P. bretschneideri, scion), using grafting experiments. The results showed that PbKN1 mRNA was present in the phloem in both transport directions across the graft junctions of tissue-cultured grafted plants and orchard saplings. To investigate the relationship between PbKN1 and PbMPB2C, we conducted interaction assays to demonstrate their physical interaction. An RNA–protein pull-down assay revealed that PbKN1 mRNA interacted with PbMPB2C protein in vitro but not with PbKN1 and that PbKN1 repressed the binding of PbMPB2C to PbKN1 mRNA. Additionally, using p35S:GFP-PbKN1 transgenic tobacco grafted with wild-type plants, we found that the movement of PbKN1 was increased by the silencing of NtMPB2C. Based on the above findings, we first demonstrate evidence of the bidirectional transport of PbKN1 mRNA and propose that PbMPB2C could act as a negative regulator of the phloem-mediated long-distance transport of PbKN1 mRNA.

Previous attempts to develop RNAi-mediated viroid-resistant transgenic plants using nearly full-length Potato spindle tuber viroid (PSTVd) hairpin RNA (hpRNA) were successful; however unusual phenotypes resembling viroid infection occurred. Therefore, in the present work, transgenic Nicotiana benthamiana lines expressing both partial and truncated versions of PSTVd hpRNA were developed. Specifically, seven partial or truncated versions of PSTVd sequences were selected according to the hotspots of both PSTVd-sRNAs and functional domains of the PSTVd. A total of 21 transgenic lines Nicotiana benthamiana were developed under the control of either the CaMV-35S or the CoYMV promoters. All of the transgenic lines established here were monitored for the induction of phenotypic changes, for PSTVd-sRNA expression and for the resistance against PSTVd infection. Additionally, this study demonstrates the use of inverted repeat construct sequences as short as 26- to -49 nucleotides for both the efficient expression of the PSTVd-sRNA and the inhibition of PSTVd infection.

A small interfering RNA (siRNA) can be custom-produced in plant cells when a transgene comprising an inverted-repeat sequence is used to form the corresponding double-stranded (ds)RNA. This siRNA induces methylation of the homologous DNA through the RNA-directed DNA methylation (RdDM) pathway. As methylation of the promoter region causes transcriptional gene silencing (TGS), dsRNA for the promoter sequence of a gene can induce TGS of the gene, accompanied by modification of chromatin. Such epigenetic variation provides a novel technique to induce silencing of a target gene. Furthermore, once epigenetic variation has occurred, the siRNA need not be present to maintain the stable repression of transcription even in the subsequent generation. Thus, the induction of epigenetic changes is a potentially new plant-breeding technology to improve crops.

BACKGROUND:The Gibberellic Acid (GA) signal is governed by the GAI (Gibberellic Acid Insensitive) repressor, which is characterized by a highly conserved N-terminal DELLA domain. Deletion of the DELLA domain results in constitutive suppression of GA signaling. As the GAI transcript is transportable in phloem elements, a Δ-DELLA GAI (gai) transgenic stock plant can reduce the stature of a scion through transport of gai mRNA from the stock. However, little is known about the characteristics of a scion on a gai stock.RESULTS:Arabidopsis Δ-DELLA GAI (gai) was fused with a T7 epitope tag and expressed under the control of a companion cell-specific expression promoter, Commelina yellow mottle virus promoter (CoYMVp), to enhance transport in the phloem. The CoYMVp:Atgai-T7 (CgT) transgenic Nicotiana benthamiana exhibited a dwarf phenotype and lower sensitivity to GA enhancement of shoot stature. A wild-type (WT) scion on a CgT stock contained both Atgai-T7 mRNA and the translated product. Microarray analysis to clarify the effect of the CgT stock on the gene expression pattern in the scion clearly revealed that the WT scions on CgT stocks had fewer genes whose expression was altered in response to GA treatment. An apple rootstock variety, Malus prunifolia, integrating CoYMVp:Atgai moderately reduced the tree height of the apple cultivar scion.CONCLUSIONS:Our results demonstrate that Atgai mRNA can move from companion cells to sieve tubes and that the translated product remains at the sites to which it is transported, resulting in attenuation of GA responses by reducing the expression of many genes. The induction of semi-dwarfism in an apple cultivar on root stock harbouring Atgai suggests that long-distance transport of mRNA from grafts would be applicable to horticulture crops.

Grafting is an important cultivation method and recent research on the mechanism of interactions between rootstock nad scion is focused on the long-distance transport of mRNA and small RNAs in the phloem. Among these transportable molecules, NACP gene coding NAM, ATAF1/2, CUC2 (NAC) domain protein might be involved in apical meristem development. Here, we report the transport of NACP mRNA between Chinese pear (Pyrus bretschneideri) cv. Yali (scion) and the wild Pyrus betulaefolia Bunge (rootstock). Our results indicated that NACP mRNA can be transported in both directions from the 3rd to 10th day after micro-grafting. It can also be transported to the shoot apex 30 to 70 cm away from graft-union in 2-year-old grafted trees. For further investigation, transgenic tobaccos with 35S: P. betulaefolia-NACP construct were grafted on wild-type tobaccos (Nicotiana tabacum L. cv. Samsun). The sustainable transport of Pyrus-NACP mRNA through the graft-union occurred from the 15th day after grafting.

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.

Expression of MdACS3a, one of the ripening-related ACC synthase genes, plays a pivotal role in initiating the burst of ethylene production by MdACS1 in apple fruit. Although previous studies have demonstrated the presence of MdACS3a-null alleles through deficiency of transcription activity or loss of enzyme activity due to amino acid substitution, which may affect the storage properties of certain fruit cultivars, an overall picture of these null alleles in cultivars is still lacking. The present study investigated the distribution of null allelic genes in 103 cultivars and 172 breeding selections by using a simple sequence repeat (SSR) marker linked to them. The results indicated that both allelic genes were widely distributed throughout the examined cultivars and selections, some occurring as the null genotype, either homozygously or heterozygously, with each null allele. The implications of MdACS3a distribution results and the influence of its null allelotypes in fruit characters are discussed.

Grafting is a common method for clonal propagation of fruit trees. Moreover, it serves as a mean to deal with abiotic stress, adjust tree growth vigor, increase yield, and improve other fruit quality traits. Investigations of rootstock and scion graft relationships have originally focused on anatomical and cellular development, nutrient transport, and hormonal movement across graft union. Discovery of long distance transport of mRNA and small RNAs in phloem tissues of rootstock and scion has provided new opportunities for investigation. In this study, we report on the endogenous transport of Gibberellic acid insensitive (GAI) across graft union of a traditional local Chinese pear cultivar, Pyrus bretschneideri cv. Yali (scion), and a wild Pyrus betulaefolia cv. Bunge (rootstock). Cleaved amplified polymorphic sequence analysis RT-PCR indicated that Pyrus-GAI can be transported within 4 and 10 days after micro-grafting, and it can also be transported to a 10–50-cm tall scion of a 2-year-old grafting tree. To further investigate the transport capacity of Pyrus-GAI transcript, a 35S:pear (P. betulaefolia)-GAI transgenic tobacco (Nicotiana tabacum L. cv. Samson.) was prepared and grafted to wild-type tobacco. RT-PCR indicated that sustained transmission of GAI mRNA through the graft union occurred from the 15th day after grafting. The results have laid a foundation for improving rootstock and regulating the properties of scion in fruit trees by transgenic technology.