Genetic modification in plants helps us to understand molecular mechanisms underlying on plant fitness and to improve profitable crops. However, in transgenic plants, the value of gene expression often varies among plant populations of distinct lines and among generations of identical individuals. This variation is caused by several reasons, such as differences in the chromosome position, repeated sequences, and copy number of the inserted transgene. Developing a state-of-art technology to avoid the variation of gene expression levels including gene silencing has been awaited. Here, we developed a novel binary plasmid (pTACAtg1) that is based on a transformation-competent artificial chromosome (TAC) vector, harboring long genomic DNA fragments on both sides of the cloning sites. As a case study, we cloned the cauliflower mosaic virus 35S promoter:β-glucuronidase (35S:GUS) gene cassettes into the pTACAtg1, and introduced it with long flanking sequences on the pTACAtg1 into the plants. In isolated transgenic plants, the copy number was reduced and the GUS expressions were detected more stably than those in the control plants carrying the insert without flanking regions. In our result, the reduced copy number of a transgene suppressed variation and silencing of its gene expression. The pTACAtg1 vector will be suitable for the production of stable transformants and for expression analyses of a transgene.

Potato (Solanum tuberosum L.) is a major global food crop. Contemporary potato production largely utilizes micropropagation to produce healthy seed potatoes. The micropropagation of potatoes is widely achieved through nodal explants using the conventional Murashige and Skoog (MS) medium. Currently, effective culture media that can facilitate rapid propagation are increasingly required for new cultivars that have been developed to possess improved traits. In this study, we evaluated the effect of enhanced meso nutrients (CaCl₂.2H₂O, MgSO₄, and KH₂PO₄) in MS medium on the growth of S. tuberosum. The cultivars used in this study were representative of Japanese, European, and Peruvian lines. Enhanced meso nutrients improved the overall quality of all cultivars, as indicated by longer shoots and larger leaves with dark color, compared with MS medium only. Shoots grown on enhanced mesos were approximately 1.5 times longer than on MS medium. Quantitative ion analysis revealed that plantlets with improved shoot length and leaf quality in most cultivars had increased calcium, magnesium, potassium, and phosphorus uptake than plantlets on MS medium. The results suggest that the reduced iron uptake on 3.0×MS, compared with 2.0× or 2.5×MS mesos, reduced plant growth. This study revealed for the first time that mesos concentrations higher than MS medium concentrations, complemented by enhanced calcium, magnesium, potassium, phosphorus, and iron uptake, play a significant role in improving the in vitro growth of potato.

Dalbergia congestiflora Pittier is a woody plant species grown in Mexico and Central America and widely used as timber wood and medicinal material. Since D. congestiflora is an endangered species, an in-vitro micropropagation technique is needed for mass propagation of D. congestiflora plantlets. Nodal segments of D. congestiflora stem cuttings grown in greenhouse conditions were disinfected with an appropriate protocol and in vitro established on Murashige and Skoog medium (MS) supplemented with 0.05 mg l⁻¹ benzylaminopurine (BA). The explants showed 10% contamination with 90% survival, and the initial shoot was regenerated in 90% of them. Axillary buds of 45-day-old initial shoots were cultured on MS containing BA (0, 0.05, 0.1, 0.5, 1, 1.5 and 2 mg l⁻¹) singly or in combination with α-naphthaleneacetic acid (NAA) (0, 0.1, 0.5 and 1 mg l⁻¹). A higher shoot number (9.6 shoots/explant) was obtained on MS with 1 mg l⁻¹ BA and 0.1 mg l⁻¹ NAA. Rooting was investigated using half-strength MS, 2% sucrose and different concentrations of indole butyric acid (IBA) (0, 0.1, 0.5 and 1 mg l⁻¹). After 30 days of culture, developing shoots were elongated and rooted in culture medium without IBA, with production of 3.2 roots/shoot. Micropropagated plantlets of D. congestiflora were successfully transplanted and acclimatized to a mixture of peat moss and perlite (2 : 1) with 100% relative humidity in greenhouse conditions with 80% survival at 30 days of culture. This micropropagation protocol will contribute to the conservation of D. congestiflora, and assure the mass propagation for sustainable usage of this species.

A NAC domain transcription factor, VND-INTERACTING2 (VNI2) is originally isolated as an interacting protein with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel element differentiation. VND7 directly or indirectly induces expression of a number of genes associated with xylem vessel element differentiation, while VNI2 inhibits the transcriptional activation activities of VND7 by forming a protein complex. VNI2 is expressed at an earlier stage of xylem vessel element differentiation than VND7. Here, to investigate whether VND7 also affects VNI2, a transient expression assay was performed. We demonstrated that VND7 downregulated VNI2 expression. Other transcription factors involved in xylem vessel formation did not show the negative regulation of VNI2 expression. Rather, MYB83, a downstream target of VND7, upregulated VNI2 expression. By using the deletion series of the VNI2 promoter, a 400 bp region was identified as being responsible for downregulation by VND7. These data suggested that VND7 and VNI2 mutually regulate each other, and VNI2 expression is both positively and negatively regulated in the transcriptional cascade.

The commercial use of genetically modified (GM) crops requires prior assessment of the risks to the environment when these crops are grown in the field or distributed. Assessments protocols vary across countries and GM crop events, but there is a common need to assess environmental biosafety. In this study, we conducted an environmental risk assessment in a confined field of GM tomato plants that can produce miraculin, a taste-altering protein that causes sour tastes to be perceived as sweet, for practical use in Japan. The evaluation was conducted for 1) competitiveness (the ability to compete with wild plants for nutrients, sunlight, and growing areas and prevent their growth) and 2) the production of toxic substances (the ability to produce substances that interfere with the habitat and growth of wild plants, animals, and microorganisms). Investigations of plant morphology and growth characteristics as well as tolerance to low temperature during early growth and overwintering for assessment endpoints related to competitiveness showed no biologically meaningful difference between GM tomato and non-GM tomato. In addition, harmful substances in plant residues and root secretions were assessed by the plow-in method, succeeding crop test and soil microflora tests, and it was determined that GM tomato does not exhibit an increase in harmful substances. Based on these results, it was concluded that GM miraculin-accumulating tomato is comparable to conventional tomato and is unlikely to have unintended adverse effects in the natural environment of Japan.

Rice is an important staple crop and fungal blast disease destroys about 10–30% of its global produce, annually. Although genetic manipulation has largely been employed in crop-improvement programmes and agricultural biotechnology, the ease of transformation of several recalcitrant indica cultivars continues to be a challenge. HR-12 and CO-39 are two indica cultivars that are commonly used in breeding programmes, but are susceptible to biotic threats like fungal blast and sheath blight disease. Here in this study, we have optimised a rapid and reproducible transformation protocol for the said cultivars, having compared both the tissue-culture and in-planta methods of transformation. Murashige & Skoog basal media supplemented with maltose and 2.5 mg l⁻¹ 2,4-D induced efficient callogenesis in HR-12, while maltose with 3 mg l⁻¹ 2,4-D gave optimum results in case of CO-39. The media containing 0.5 mg l⁻¹ NAA, 3 mg l⁻¹ BAP, and 1 mg l⁻¹ kinetin yielded a maximum regeneration efficiency of 62% and 65% in HR-12 and CO-39, respectively. The studies with Agrobacterium tumefaciens, LBA4404 strain harbouring pCAMBIA1303 suggested that although these cultivars demonstrated successful gene-transfer, they failed to regenerate efficiently, post-transformation. Alternatively, our modified in-planta piercing and vacuum infiltration-based protocol resulted in 33–35% transformation efficiency in less than half the time required for tissue-culture based transformation method. As per our knowledge, it is among the highest obtained from existing piercing-based direct transformation protocols in rice, and can also be implemented in genetically manipulating other recalcitrant varieties of rice.

To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1— but not SKOR, AKT1, and SAG14—were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

Ongoing research has generated many important lines of the model liverwort Marchantia polymorpha, including mutants and transgenic lines. To maintain these lines, researchers typically spend a lot of time and effort periodically replanting thalli (e.g., every month). To avoid this routine maintenance, researchers have developed methods for cryopreservation of dried and frozen gemmae. In this study, we developed a culture-based method for preserving gemmalings and thalli without encapsulation, drying, or freezing. The method requires only tissue culture on agar medium supplemented with sucrose in the dark at regular temperature (22°C). These culture conditions severely inhibit growth of gemmalings and thalli; however, these tissues remained alive after more than 1 year of storage. Survival rate of tissues using this method was 100% in all tests. This method thus enables preservation of gemmaling and thallus cultures on medium under regular temperature conditions, thereby relieving researchers of labor-intensive routine maintenance.

Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith belongs to the Alpinia genus in the Zingiberaceae family. In East Asia, Alpinia zerumbet has been widely used as food and traditional medicine. Previously, we identified proanthocyanidins (PACs), an anti-plant-virus molecule in A. zerumbet, using Nicotiana benthamiana and tomato mosaic virus (ToMV). Here, we found that PACs from A. zerumbet, apple, and green tea effectively inhibited ToMV infection. Additionally, the PACs from A. zerumbet exhibited greater antiviral activity than those from apple and green tea. The PACs from A. zerumbet also effectively inactivated influenza A virus and porcine epidemic diarrhea virus (PEDV), which acts as a surrogate for human coronaviruses, in a dose-dependent manner. The results from the cytopathic effect assays indicated that 0.1 mg/ml PACs from A. zerumbet decreased the titer of influenza A virus and PEDV by >3 log. These findings suggested that the direct treatment of viruses with PACs from A. zerumbet before inoculation reduced viral activity; thus, PACs might inhibit infections by an influenza virus, coronaviruses, and plant viruses.

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T₁ plants from fertile T₀ plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.

N-Glycosylation is essential for protein stability, activity and characteristics, and is often needed to deliver pharmaceutical glycoproteins to target cells. A paucimannosidic structure, Man₃GlcNAc₂ (M3), has been reported to enable cellular uptake of glycoproteins through the mannose receptor (MR) in humans, and such uptake has been exploited for the treatment of certain diseases. However, M3 is generally produced at a very low level in plants. In this study, a cell culture was established from an Arabidopsis alg3 mutant plant lacking asparagine-linked glycosylation 3 (ALG3) enzyme activity. Arabidopsis alg3 cell culture produced glycoproteins with predominantly M3 and GlcNAc-terminal structures, while the amount of plant-specific N-glycans was very low. Pharmaceutical glycoproteins with these characteristics would be valuable for cellular delivery through the MR, and safe for human therapy.