Breeding of a practical rice line ‘TJTT8’ for phytoextraction of cadmium contamination in paddy fields

<p>Previously, we showed that <i>qCdp7</i>, an allele identified in the high-Cd-accumulating <i>indica</i> rice variety ‘Jarjan,’ is associated with effective phytoextraction of Cd from paddy soils. However, ‘Jarjan’ may not be practical for phytoextraction because it is susceptible to seed shattering and culm lodging, which are unfavorable traits for mechanical rice harvesting. In this study, to develop a practical rice line for phytoextraction, we introduced the <i>qCdp7</i> allele into ‘Tachisugata,’ a rice variety with a nonshattering habit and lodging resistance, to produce a new high-Cd-accumulating rice line designated ‘TJTT8.’ This line inherited high-Cd accumulation and brown pericarps from ‘Jarjan’ and a nonshattering habit and lodging resistance from ‘Tachisugata;’ all of these traits are necessary for rice intended for Cd phytoextraction in Japan. Backcross inbred lines (BILs) were produced by two backcrosses to ‘Tachisugata’ after a cross between ‘Jarjan’ and ‘Tachisugata.’ ‘TJTT8’ was selected from the BILs by means of marker-assisted selection and phenotypic evaluation. When ‘TJTT8,’ the parents, and ‘Cho-ko-koku’ which is a high-Cd-accumulating <i>indica</i> variety were cultivated in Cd-contaminated paddy fields in four locations in Japan, ‘TJTT8’ exhibited lodging resistance and shattering resistance that were higher than those of ‘Jarjan’ and ‘Cho-ko-koku’ and equivalent to those of ‘Tachisugata.’ ‘TJTT8’ accumulated Cd in the aerial parts of the plants at concentrations ranging from 6.5 to 22.7 mg m⁻²: it showed significantly higher Cd accumulation than ‘Tachisugata’ and was equivalent to ‘Jarjan’ and slightly superior to ‘Cho-ko-koku.’ Soil Cd concentration was estimated to have been reduced by 8.7–33.6% based on the amount of Cd accumulation in the aerial parts of the plants. Thus, we succeeded in using the <i>qCdp7</i> allele to produce a practical rice line for Cd phytoextraction by improving several agronomic traits for compatibility with Japanese cultivation systems.</p

Solubility of PTX and gPTX in different solvents.

<p>* <i>Solubility of H₂O was referred as described previously</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107976#pone.0107976-Mandai1" target="_blank">[19]</a>.</p><p>** <i>CEP consists of Cremophor EL and ethanol in PBS (12∶12∶76 volume %)</i>.</p><p>Solubility of PTX and gPTX in different solvents.</p

Anticancer efficacy of different gPTX formulations with repeated administration in HT-29 cells tumor-bearing mice.

<p>gPTX-IL (open circle with line), gPTX-L (open triangle), gPTX-L with trastuzumab (open square), trastuzumab (closed square), CEP-IL (closed circle), CEP-L (closed triangle), or PBS (cross) was intravenously injected at day 0, 10, and 20. The dose of each administration was 150 mg/kg gPTX. A and B, Changes in tumor volume. C, Changes in body weight. D, Survival curves. Data are presented as the mean ± S.D. The changes of tumor volume and body weight in the mice administered with gPTX-L do not show S.D. after day 10. The <i>P</i> value shown compared with gPTX-IL and gPTX-L with trastuzumab treated group at day 43 (n = 4). *, <i>P</i><0.05.</p

Cytotoxicity of different gPTX formulations by the MTT assay.

<p>A, The IC₅₀ values after drug exposure for 72 h are shown. B, The IT₅₀ values at IC₁₀₀ are shown. The data are presented as the mean ± S.D. for three independent experiments. *, <i>P</i><0.05. **, <i>P</i><0.01. ***, <i>P</i><0.005. ****, <i>P</i><0.001. NSD, no significant difference.</p

Influence of the lipid composition and incubation time for drug encapsulation by the solubility gradient method.

<p>A, The encapsulation efficiency (EE) of gPTX-L with various lipid compositions was evaluated after 30 min of incubation for gPTX encapsulation. B, The drug retention of gPTX-L with different lipid compositions was evaluated in medium supplied with 10% FBS at 37°C. C, The efficiency of drug encapsulation under different durations of incubation was evaluated with a DPPC to Chol ratio of 3∶1.</p

Schematic illustration of remote loading with a solubility gradient.

<p>CEP-encapsulated liposomes (CEP-L) and gPTX dissolved in 40% EG were mixed and incubated. gPTX was encapsulated into liposomes under a solubility gradient.</p

Characterizations of gPTX.

<p>A, The hydrophobicity of PTX and gPTX was evaluated by reverse-phase HPLC using a C₁₈ column at a flow rate of 1 mL/min with 55% (v/v) methanol under an isocratic condition. B, The ability of 3 µM PTX (open circle) and gPTX (open triangle) to stabilize porcine tubulin polymerization was evaluated using a Tubulin Polymerization Assay Kit (Cytoskeleton). The fluorescent reporter was detected with excitation at 360 nm and emission at 420 nm. Each dot represents the mean ± S.D. (n = 3).</p