Optimization of root induction conditions.

<p>A–D. Bright-field (A, C) and green fluorescent (B, D) images of a transformed shoot before (A, B) and after (C, D) 14 days of cultivation in irrigated vermiculite. Scale bar, 5 mm. E. Comparison of root induction conditions (n = 27). A statistical analysis was conducted using the frequencies at 14 days of cultivation. Significant differences (P<0.05 by Fisher's exact test with Holm's P-value adjustment) were observed between two groups that do not share the same lowercase letter. 1/2 MS, half-strength MS salts; SV, 2% sucrose and 1× Gamborg's vitamins.</p

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant <i>Mimosa pudica</i>

<div><p>The sensitive plant <i>Mimosa pudica</i> has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for <i>M. pudica</i> mediated by <i>Agrobacterium tumefaciens</i> (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(<i>N</i>-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (<i>Glycine max</i>) and pea (<i>Pisum sativum</i>). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in <i>M. pudica</i> movements.</p></div

Establishment of transgenic plants.

<p>A. Time course of establishment of transgenic T₀ plants from cotyledonary node explants (n = 160). Each line indicates the frequency of Agrobacterium-infected explants that reached the process indicated in (B). B. Schematic representation of the entire transformation procedure. C. Green fluorescent images of T₁ seedlings of a single T-DNA insertion line (#1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088611#pone-0088611-g006" target="_blank">Figure 6</a>). The zygosity of T₁ progeny [non-transformant (−/−), hemizygote (Tg/−) or homozygote (Tg/Tg)] produced by self-crossing of a T₀ plant could be determined based on fluorescence intensity of the plantlet. D. Green fluorescent image of homozygous T₂ seedling produced by self-crossing of a homozygous T₁ plant. Scale bar, 2 mm.</p

Shoot formation from <i>M. pudica</i> explants.

<p>A–E. Preparation of explants. A 2-day-old seedling cultured in the dark (A) was divided into the root, the cotyledons with petiole (B), and the remaining part (C). The epicotyl containing the shoot apex was then removed from the remaining part (C) to prepare the cotyledonary node explant (D) as illustrated in (E). Dashed lines in (A), (C), and (E) indicate the cutting positions. The circle in (D) indicates the position of the cotyledonary node. SA, shoot apex; Hc, hypocotyl. F, G. Shoot formation from the cotyledonary node (F) and petiolate cotyledon (G) explants after 4 and 6 weeks of cultivation in the presence of 0.5 µg/ml BAP, respectively. H. Comparison of the frequency of explants forming shoots after 4 weeks of cultivation with 0.5 µg/ml of BAP (n = 32). I, J. Effects of BAP and NAA on shoot formation from cotyledonary node (I) and petiolate cotyledon (J) explants after 4 and 6 weeks of cultivation, respectively. The distribution of the number of shoots formed per explant is shown as box-and-whisker plots (n = 32). Lower and upper whiskers indicate the range of values within 1.5 times the interquartile range from the box and circles indicate outliers. Significant differences were observed between two groups that do not share the same lowercase letter [P<0.05 by Fisher's exact test with Holm's P-value adjustment (H) or Steel-Dwass test (I, J)]. Scale bars, 1 cm (A, F, G), 1 mm (B–D).</p

Genomic Southern blot analysis of T₀ plants.

<p>Genomic DNAs of 13 independent T₀ plants (lines #1-13) and a non-transformed plant (C) were analyzed by <i>Eco</i>RI digestion and detection of the <i>sGFP</i> sequence. The size of several bands is shorter than the minimal length expected from the intact T-DNA sequence (3.0 kb; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088611#pone-0088611-g002" target="_blank">Figure 2A</a>), suggesting that the T-DNA sequence had undergone truncation and/or rearrangement.</p

Changes in medium pH during co-cultivation.

<p>A. Effects of Agrobacterium strain LBA4404 harboring pSB111-GFP (LBA), cotyledonary node explants (Exp), and MES buffer (MES) on medium pH (n = 4). The co-cultivation media were initially adjusted to pH 5.8 and sterilized by filtration to circumvent the pH decrease caused by autoclaving. In the absence of MES buffer, pH values had already declined in the time it took to prepare the Agrobacterium suspension in co-cultivation medium (∼30 minutes). B. Changes in pH of MES-buffered medium initially adjusted to pH 6.1 (n = 5). The medium was sterilized by autoclaving and then used for co-cultivation of Agrobacterium and explants. The effect of Silwet L-77 treatment prior to co-cultivation was also examined. Data are the means ± SD. The pH of the medium was measured at 0, 3, 6, 9, 12, 18, 24, 48, and 72 hours after the initiation of cultivation.</p

Optical Property Analyses of Plant Cells for Adaptive Optics Microscopy

<div><p>In astronomy, adaptive optics (AO) can be used to cancel aberrations caused by atmospheric turbulence and to perform diffraction-limited observation of astronomical objects from the ground. AO can also be applied to microscopy, to cancel aberrations caused by cellular structures and to perform high-resolution live imaging. As a step toward the application of AO to microscopy, here we analyzed the optical properties of plant cells. We used leaves of the moss Physcomitrella patens, which have a single layer of cells and are thus suitable for optical analysis. Observation of the cells with bright field and phase contrast microscopy, and image degradation analysis using fluorescent beads demonstrated that chloroplasts provide the main source of optical degradations. Unexpectedly, the cell wall, which was thought to be a major obstacle, has only a minor effect. Such information provides the basis for the application of AO to microscopy for the observation of plant cells.</p> </div

Workflow for 5′-DGE library preparation.

<p>(a) 1st strand cDNA is synthesized from mRNA. (b) At the 5′ end of the mRNA, cDNA synthesis continues onto the DNA/RNA chimeric oligonucleotide. (c) Three-cycle PCR is performed to produce the double-stranded cDNA. (d) the double-strand cDNA fragments are digested by EcoP15I. (e) Digested P2-attached 5′ tag fragments are captured by streptavidin-magnet beads and ligated with P1 adapter. (f) 5′-DGE library is amplified, and 97 bp fragments are purified after PAGE.</p

Reprogramming from leaf cells to chloronema apical cells.

<p>(<b>A</b>) Upper parts of gametophores. (<b>B–D</b>, <b>F–P</b>) Dissected leaves after indicated time. (<b>E</b>) Intact leaf. Bars in <b>A</b>: 1 mm; <b>B</b>: 1 mm for <b>B–D</b> and <b>I–L</b>; <b>E</b>: 0.2 mm for <b>E–H</b> and <b>M–P</b>.</p

Expression patterns of transcription factors, epigenetic-related and reprogramming-related genes.

<p>(<b>A</b>) Heat map representation of expression patterns during reprogramming for reprogramming-related genes. Numbers in parentheses indicate JGI protein identifiers. (<b>B</b>) Expression patterns of transcription factor genes sorted according to gene expression pattern tree. Four distinct clusters are identified to the left. (<b>C</b>) Expression patterns of epigenetic-related genes.</p