Cover Page Gallery

2021 Vol.38 No.3

“Tsukuba system” as a transient protein expression system in plants

The Tsukuba system is a powerful tool for transient protein expression in plants. The expression level of GFP with this system in Nicotiana benthamiana reached a yield of approximately 4 mg/g fresh mass within 3 days. No other expression systems reached this amount in 3 days. Thus, using our expression system, recombinant proteins can be obtained more rapidly and in large quantities. Furthermore, the Tsukuba system also enhances expression levels of recombinant proteins in several crops, such as tomatoes, eggplants, hot peppers, melons, orchids, soybeans, common beans, and radishes. (Nosaki et al., pp. 297–304)

Photographed by Kenji Miura (Univ. Tsukuba) in Univ. Tsukuba, Japan, 2018. Blue LED light was provided for GFP emission, which was observed with an ultraviolet-absorbing filter, Fujifilm SC-52.
2021 Vol.38 No.2

Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and generates white petals including a pale-yellow colored star-shaped center (left photo in the top). Transgenic I. obscura plants were constructed by introducing the crtW and crtZ genes for producing a red carotenoid astaxanthin. The transgenic plants generated bronze (reddish green) leaves (right pictures in the middle and bottom) and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part (right photo in the top). (See Otani et al., pp. 219–226)
2021 Vol.38 No.1

HIGS method controlled the gene expression of a parasitic plant, Cuscuta campestris

Stem parasitic plant, Cuscuta campestris, develops an attachment structure called “holdfast” on the surface of its stem contacting to the host plant. Epidermal cells of holdfasts showed outgrowth, in which epidermal cell-patterning genes of C. campestris were hypothesized to be involved. To prove this hypothesis, Host-Induced Gene Silencing (HIGS) method was employed (Sultana et al., pp. 47-56). Aritificial miRNA targeting one of the C. campestris epidermal cell-patterning genes was produced in the first host, Nicotiana tabacum (left), loaded to parasitizing C. campestris (center), and then the effect of HIGS was evaluated by letting C. campestris parasitize to the second host, Arabidopsis thaliana (right). This approach successfully silenced the target gene of C. campestris, and repressed the outgrowth of the holdfast epidermal cells.

Photographed by Daiki Fujiwara (Osaka Pref. Univ.). (PENTAX Optio WG-1)
2020 Vol.37 No.4

Strategies of mechanical optimization in plants

This special issue contains articles dealing with new approaches and aspects in plant biomechanics that are developed in the MEXT KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas “Plant-Structure Optimization Strategy”. (Top Left) Four-dimensional observation of Arabidopsis cotyledon pavement cells (Higaki and Mizuno, pp. 429–435). (Top Right) Secondary cell wall deposition in Arabidopsis cotyledon pavement cells induced by the VND7-VP16-GR system (Kunieda et al., pp. 465–469). (Middle Left) Static stress simulation image for a cantilever beam of a low density polyethylene rod (Watanabe et al., pp. 485–488). (Middle Right) Image of passage of sorghum root (yellow) through metal mesh (cyan) buried in Toyoura sand (Yoshida et al., pp. 481–484). (Bottom) Maximum intensity projection of time-lapse images during gravitropism of the pea sprout (Nakaso et al., pp. 475–480).
2020 Vol.37 No.3

Propagation of Polygonatum macranthum using a new sterilization procedure

Polygonatum macranthum is an economically important species owing to its nutritive and medicinal properties. However, the natural propagation of P. macranthum is very difficult to achieve since the species require a long seed germination time, and characterizes an extremely slow rhizome propagation. We developed an efficient protocol for the propagation of P. macranthum from immature seeds with seed coat. The surface sterilization procedure used a new surface sterilization protocol that utilized a low concentration of hypochlorite. Subsequent multiplication of microrizomes was efficiently achieved. (See D. Lekamge et al., pp. 353–357)

Photographed by Shinya Maki (Nagaoka University of Technology, Niigata Agricultural Research Institute Mountain Agricultural Technology Center) in Nagaoka, Niigata, Japan, (Olympus TG-5)
2020 Vol.37 No.2

Technology in tissue culture toward horizon of plant biotechnology

1. Stable transformants of Japanese cedar (Sugi)

Japanese cedar (Sugi, Cryptomeria japonica D Don.) is the most important afforestation coniferous tree in Japan. Since coniferous trees normally have a long juvenile period, the improvement of Sugi by a transgenic approach is effective in terms of shortening the breeding period. We describe a protocol for Agrobacterium-mediated transformation of Sugi using embryogenic tissues as explants isolated from immature seeds (pp. 147-156). The upper image shows fluorescent image of somatic embryos regenerated from the embryogenic tissue introduced with green fluorescent protein gene. The lower image shows transgenic Sugi plants grown in a semi-closed greenhouse. This picture is taken by Ken-ichi Konagaya using MZ FLIII stereo fluorescence microscope (Leica Microsystems) and D60 digital camera (Nikon) at Forestry and Forest Products Research Institute (Ibaraki, Japan).

2. Regeneration of tobacco plastid transformants

This picture shows regeneration shoots of tobacco under spectinomycin selection after bombardment of the plastid transformation vector including the GFP expression cassette. Regenerated shoots with green fluorescence contain transformed plastids, and with red fluorescence still contain wild type plastids. In shoots obtained on first round selection, several mixture patterns of wild type and transformed cells are seen (pp. 223-232). This picture was taken used a fluorescence microscope (Leica MZ 16FA, Leica Microsystems, Germany) with a GFP2 filter (480-nm excitation filter/510-nm barrier filter) at NIAS, Ibaraki, Japan.

3. The culture room used for the production of microtubers

Potato (Solanum tuberosum L.) crops are vegetatively propagated using tubers and facing many risks caused by pathogens. The production of microtubers under sterile conditions is a good way to produce disease-free materials for crop production. Bioreactors have been used for this purpose, but bioreactors are expensive and difficult to handle. We have developed a system using plastic culture bags with forced aeration system for both liquid medium and gaseous phase to produce microtubers of potato. Each bag was 300 mm X 490 mm (total volume 8 l). We were able to place 12 culture bags in each shelf space with dimensions of 120 cm width, 60 cm depth, and 65 cm height, and produced about 250,000 microtubers per year in a 66 m2 tissue culture room using the culture bag system (pp. 233-238). Photographed by Noboru Ohnishi and Kanji Mamiya. This picture was taken using a digital camera (RICOH Caplio R7) at Tochigi, Japan.

4. Genome editing in wheat

Due to the complex allohexaploid genome nature, genome editing is a promising approach in wheat, where developing a mutant is more challenging compared with diploid crops, especially for genetically recessive traits. Using Agrobacterium-delivered CRISPR/Cas9 system, wheat lines with loss-of-function of Qsd1, which controls seed dormancy in barley, was efficiently developed. The edited triple-recessive mutant of TaQsd1 showed longer seed dormancy and is expected to contribute to reducing pre-harvest sprouting of wheat in rain-prone areas. This result was achieved based on the improvement of wheat tissue culture (pp. 177-184) and the development of a method for detection of mutation (pp. 247-251). Photographed by Fumitaka Abe, This picture was taken using a digital camera (Pentax K-7) at Institute of Crop Science, NARO (Ibaraki Japan).

5. Plant regeneration and rooting in transgenic maize

Maize (Zea mays L.) is one of the three most important crops, and a tremendous effort for production of transformed maize has been made since the 1990s. We reviewed progress in maize tissue culture technology, which is a core component of gene transfer and editing processes, and presented typical protocols for creation of transgenic plants for both particle bombardment and Agrobacterium-mediated methods (pp. 121-128). Pictures are of regenerated plants of maize inbred A188, which is a model variety for experiments of tissue culture and transformation. Immature embryos of A188 were inoculated by Agrobacterium tumefaciens that had a bar gene and a GUS gene in its T-DNA. Transgenic cells grown from the immature embryos were selected and plants were regenerated on the media that contained phosphinothricin. Photographed by Yuji Ishida. Both pictures were taken using a digital camera (Nikon Coolpix P310) at JT Plant Innovation Center (Shizuoka, Japan).

6. Genome edited potatoes showing SGA-reduced phenotype.

Potato is one of the most important crops in the world. Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in potato that cause food poisoning issues. Using Agrobacterium-mediated transformation, transgenic potato harboring the TALEN expression vector targeting SSR2 gene, which is encoding a key enzyme for SGA biosynthesis with reduced SGA levels have been generated in previous research (Sawai et al., Plant Cell, 2014). In this issue, we report the transgene-free genome editing in potato through transient TALEN expression by Agrobacterium infection (pp. 205-211). Photographed by Shuhei Yasumoto and Jekson Robertlee (Osaka University) in Osaka, Japan (Nikon D90).

7. Regenerated plants of Tricyrtis ‘Shinonome’ at the flowering stage

The family Liliaceae (Cronquist system) contains various important ornamental plants such as Agapanthus, Hemerocallis, Hosta, Lilium, Muscari, Tricyrtis and Tulipa species. We have been examining the establishment of plant regeneration and genetic transformation systems in liliaceous ornamental plants for their biotechnological breeding and elucidation of the molecular mechanisms determining ornamental traits. Among liliaceous ornamental plants used, Tricyrtis spp. has several advantages for regeneration and transformation studies: efficient regeneration from embryogenic calli, higher transformation efficiency by an Agrobacterium-mediated method, relatively small plant size, ease of cultivation, and taking only 1 year from in vitro regeneration to flowering. We are now investigating the molecular mechanisms for determining plant form, flower color and flower form by using Tricyrtis spp. as liliaceous model plants (pp. 129-140). Photographed by Masaru Nakano, Experimental greenhouse at Faculty of Horticulture, Niigata University (Niigata, Japan). Fujifilm FinePix S9000

8. The concept of in planta particle bombardment (iPB)

The L2 layer cells within the SAM potentially develop into pollen and embryo sac. If genome editing occurs in these cells, the mutation can be inherited to the next generation. Gold particles can accommodate not only DNA but also RNA and protein including Cas9 and TALENs (pp. 171-176).

9. A GFP-expressing transgenic fruit of C. moschata

Cucumber (Cucumis sativus L.) and Cucurbita species (squashes, pumpkins, and gourds), belonging to the Cucurbitaceae family, are among the major vegetable crops in the world. These pictures show GFP-positive fruits of C. moschata appears green under blue light owing to GFP fluorescence (Nanasato and Tabei, pp. 141-146). Photographed by Yoshihiko Nanasato using a digital camera (Sony DSC-HX5V) and an epifluorescence stereomicroscope (Leica MZ16FA) at NIAS (Ibaraki, Japan).

10. Somatic embryogenesis of tea plant (Camellia sinensis) for transformation.

Various kinds of tea beverage are produced from leaves of tea plant (Camellia sinensis). SHIZUOKA prefecture is as famous as a major tea plantation in Japan. Green tea is traditionally the most popular in Japan and it is a part of traditional culture and cuisine. Somatic embryos of tea plant are induced from cotyledons of immature seeds, and secondary embryogenesis occurs by transferring the medium containing auxin to hormone-free medium. Somatic embryos can develop to plantlets in vitro (pp. 195-203). Photographed by Kazumi Furukawa using a digital camera (Olympus tough TG-5). Tea plantation area is at Fuji, Shizuoka, Japan. Somatic embryos and plantlets are cultured at KOSEN (Shizuoka, Japan)

11. Precocious flowering of transgenic apple (JM2)

Apple MdTFL1 (an orthologue of TERMINAL FLOWER 1 from Arabidopsis) is essential to reproductive/vegetative growth and flower initiation in apple. The suppression of MdTFL1 expression caused strong induction to reproductive growth and set flowers on their shoot apexes. The picture represents flowers of MdTFL1 silencing transgenic JM2 of five years old, which bloomed at 12 month after transformation with Agrobacterium method (pp. 163-170). Photographed by Masato Wada using a digital camera (Olympus SP-350) in NIFTS, NARO, Iwate, Japan.

12. Agrobacterium-mediated genetic transformation of ‘Shine Muscat’ grape

The cultivation area and consumption of grape cultivar ‘Shine Muscat’ (Vitis labruscana), which was released by NARO, have rapidly increased. Based on our efficient method for embryogenic callus induction, we established Agrobacterium-mediated genetic transformation of ‘Shine Muscat’ (pp. 185-194). The picture shows the regenerated grape plants in vitro. Photographed by Ikuko Nakajima (Institute of Fruit Tree and Tea Science, NARO) in Tsukuba, Ibaraki, Japan, and (SONY Cyber-shot).
2019 Vol.36 No.4

Soybean transformation system and effect of SYNC1 gene overexpression

The application of useful genes from model plants to crops is an important step to verify its agricultural usefulness. SYNC1, an asparaginyl-tRNA synthetase gene, was previously found through the FOX gene-hunting system of Arabidopsis cDNA by using super-growing root (SR) culture of Lotus corniculatus. In this study, we identified that SYNC1 gene has effects on plant morphology and amino acid contents of seed in soybean (Pages 233–240). The cover shows the Agrobacterium transformation system with GFP expression and plant morphology of SYNC1 overexpressed transgenic plant in soybean.

Photographed and designed by Takahiro Gondo. These pictures are taken using digital camera (Canon EOS Kiss X3) and fluorescence microscope (Olympus SZX12) at University of Miyazaki (Miyazaki, Japan).
2019 Vol.36 No.3

The Lemma-like Sterile Lemmas in Rice (Oryza sativa L.)

Spikelet is a typical inflorescence structure of gramineous plants. The spikelet of rice consists of a top floret, a pair of rudimentary glumes and a pair of sterile lemmas. The floret includes a lemma, a palea, two lodicules, six stamens, and one pistil. The rudimentary glumes are generally regarded as severely reduced bract organs, but the origin of sterile lemmas has been widely debated. Recent studies suggested that the sterile lemmas are the vestigial lemmas of two lateral florets. In the research, the sterile lemma of nsg2 transformed into lemma-like organ provides evidence to support the “three-florets spikelet” hypothesis, which indicate that it is possible to cultivated a “three-florets spikelet” rice. If succeed, the number of grains per panicle would be dramatically increased and affect rice yield. (See Li et al., pp. 125–134)

Photographed by Yun-Feng Li (Southwest University) in Beibei, Chongqing, China, in summer and autum, 2016 (Hitachi SU3500, NIKON SMZ1500, Nikon E600).
2019 Vol.36 No.2

Monitoring Autophagy in Rice Tapetum

Autophagy is crucial for post meiotic anther development in rice; it is required for programmed cell death-mediated degradation of the tapetum and pollen maturation. In this issue, Hanamata et al. (pp. 99–105 and the Supplementary Movie S1) introduces an in vivo imaging technique to analyze the dynamics of autophagy in rice tapetum by expressing GFPATG8, a marker for autophagosomes under the control of tapetum-specific promoters.The cover shows 3-dimensional visualization of autophagosomes/autophagy-related structures in rice tapetum at stage 10 during pollen maturation.This monitoring system offers a powerful tool to analyze the regulation of autophagy in rice tapetum.

Photographed by Shigeru Hanamata and Jumpei Sawada using the LSM5 EXCITER confocal fluorescence microscope (Carl Zeiss, Germany) at Tokyo University of Science. 3D-image processing was carried out using the 3D viewer of ImageJ software.
2019 Vol.36 No.1

Plant Shades

In this issue, Kinoshita et al (Pages 7 to 11) introduces a simplified technique to extract fluorescence signals in freely moving plant leaves. The cover shows fluorescent signals emitted from yellow fluorescent protein and autofluorescence from chlorophyll during a time lapse experiment. This protocol enables seamless image capturing to quantify fluorescent signals using standard microscope software. Photo and design by Natsuko Kinoshita and Aki Sugita, University of Tsukuba, using Leica LasX.

Photograph and design by Natsuko Kinoshita and Aki Sugita, University of Tsukuba, using DFC7000T color CCD camera and LasX (both from Leica Microsystems).
2018 Vol.35 No.4

Albino cla1 mutant with normal tapetum and pollen coat

Plastidial isoprenoid defective cla1 mutants grown in soil show albino and a seedling lethal phenotype. However, when the seedlings were grown in a large agripot with MS agar supplemented with sucrose, only some that grew to the flower stage with albino phenotype were obtained. In cla1-1 mutant, the elaioplast, which is the tapetum cell specific organelle derived from plastid, showed normal phenotype (upper-left photograph), though the chloroplast in cla1-1 has been reported to be the abnormal phenotype previously. The pollen coat derived from tapetum cell also showed normal phenotype in cla1-1 (upper-right photograph). In this issue, Kobayashi et al. report that the plastidial isoprenoid biosynthesis pathway is not critical for the pollen coat formation (pp. 381–385).

Photographed by Rumi Aoyama using the digital camera and the JEM-1200 EX transmission electron microscope (Jeol, Tokyo, Japan) at Japan Woman’s University (Tokyo).
2018 Vol.35 No.3

Botryococcus braunii showa colonies producing hydrocarbon oil

The precursors of triterpene hydrocarbons are synthesized in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. The light-dependent enzyme 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is considered to function as a key-regulatory enzyme in this pathway. In this issue, a docking model analysis suggesting the possible association of HDR with a photosynthetic ferredoxin in this organism is discussed (pp. 297–301).
The cover picture presents an image of algal colonies stained with BODIPY493/503 and DAPI. Hydrocarbon oil (stained in green) is observed around cells (red autofluorescence) including nucleus and organelle nucleoids (stained in blue).

This picture was taken by Hidenobu Uchida using Olympus fluorescence microscope BX51, which was equipped with ×20 objective lens, according to a previous report (Kuroiwa et al. 2012, Cytologia 77: 289–299). Photographing was technically assisted by Haruko Kuroiwa from Japan Women’s University.