Sodium hypochlorite solution

Seeds of C. roseus (cultivar ‘Little Bright Eye’; obtained from NESeed, USA) were used in this study. The seeds were surface sterilized using 75% ethanol for 5 min and then 30% sodium hypochlorite solution (Sigma-Aldrich) for 10 min. After rinsing with sterile ddH₂O for 5 times, the seeds were inoculated on half-strength Murashige and Skoog (½ MS) medium. The seeds were kept in the dark at 30 °C for two days and then transferred to an incubator at 26 °C. For VIGS experiments targeting the CrChlH (accession numbers HQ608936) and CrPDS (accession number JQ655739) in C. roseus, the germinated seeds were grown in the dark for another 3 days. However, for VIGS of TIA pathway genes, the germinated seeds were grown under a light regime of 16/8 photoperiod for 3 days.
For VIGS of ChlH genes in G. inflata, and A. annua, the seeds of respective species were germinated on half-strength MS medium, and seedlings were grown in the dark at 26 ℃. Seeds of G. inflata were treated with H₂SO₄ for 30 min [46 (link)], surface sterilized with 30% sodium hypochlorite solution (Sigma-Aldrich) for 10 min, and germinated on half-strength MS medium for 7 days in dark. Seeds of A. annua were surface sterilized as described for C. roseus seeds and germinated on half-strength MS medium for 6 days in dark.

Plant seeds were surface sterilized according to the protocol published by Rothballer et al. (2003 ) with slight modifications. The seeds were washed in 1% Tween80 (2 min) and subsequent in 70% ethanol (2 min) followed by three washing steps in sterile deionized water. After an incubation step in 13% sodium hypochlorite solution (Sigma-Aldrich® Co., St. Louis, USA) (20 min) seeds were again washed three times in sterile deionized water, incubated in sterile deionized water for 4 h followed by a second incubation step in 13% sodium hypochlorite solution for 10 min. Five washing steps in sterile deionized water completed the surface sterilization. Seeds were then placed on NB (Nutrient Broth) agar plates and incubated for 3 days at room temperature in the dark to allow germination and to control the success of surface sterilization. Only germinated seedling showing no visible contamination were used for further experiments.

Coral pieces were grinded by the use of mechanical mill and the obtained particles were classified into different sizes by the use of sequential sieves.
After being sorted, the coral microparticles were disinfected by immersing in sodium hypochlorite solution (Merck, Germany) for 30 h followed by washing with distilled water several times and vacuum drying. It is worth to mention that the disinfection process of the coral was carried out after they were grinded into micro sized particles, to enhance the exposure of their surface with disinfecting solution. After being disinfected, coral particles were sterilized by being exposure to gamma radiation (25 KG) for 2 h.