Arabidopsis thaliana Columbia (Col-0) accession was used as a wild-type control. Transgenic plants possessing HTR10p::HTR10-mRFP or -Clover (Ingouff et al., 2007 (link); Kawashima et al., 2014 (link)), RPS5Ap::H2B-GFP or -tdTomato (Adachi et al., 2011 (link)), and RPS5Ap::tdTomato-LTI6b (Mizuta et al., 2015 (link)) genes were used to visualize the sperm cell nuclei, female gametophytic cell nuclei, and female gametophytic cell membrane, respectively. A tetraspore (tes) mutant, tes-4 (CS9353; Spielman et al., 1997 (link); Yang et al., 2003 (link)) was obtained from the Arabidopsis Biological Resource Center and crossed with the pollen from the HTR10p::HTR10-mRFP line.
A. thaliana seeds were sterilized in a solution containing 2% Plant Preservative MixtureTM (Cosmo Bio, Tokyo, Japan), 50 μg/mL magnesium sulfide, and 0.1% Tween 20 for several days at 4°C. The seeds were sown on Murashige and Skoog (MS) medium [1 × MS salt (Duchefa Biochemie, Haarlem, Netherlands), 2% sucrose, 1 × Gamborg's vitamin solution (Sigma, St. Louis, MO, USA)] solidified with 0.3% Gelrite (Wako, Osaka, Japan) and adjusted to pH 5.7 with KOH. Plants were germinated and grown in a growth chamber at 22°C with continuous light. Two-week-old seedlings were transferred to soil and grown at 22°C in a plant growth room.
A. thaliana seeds were sterilized in a solution containing 2% Plant Preservative MixtureTM (Cosmo Bio, Tokyo, Japan), 50 μg/mL magnesium sulfide, and 0.1% Tween 20 for several days at 4°C. The seeds were sown on Murashige and Skoog (MS) medium [1 × MS salt (Duchefa Biochemie, Haarlem, Netherlands), 2% sucrose, 1 × Gamborg's vitamin solution (Sigma, St. Louis, MO, USA)] solidified with 0.3% Gelrite (Wako, Osaka, Japan) and adjusted to pH 5.7 with KOH. Plants were germinated and grown in a growth chamber at 22°C with continuous light. Two-week-old seedlings were transferred to soil and grown at 22°C in a plant growth room.
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