Agarose

The Arabidopsis thaliana HTR10mRFP homozygous line, where the sperm cell nuclei were visualized with mRFP [32 (link)], and heterozygous +/gcs1 mutant with a background of HTR10mRFP (+/gcs1^HTR10mRFP) [8 (link)] were used for the transformation experiments in this study. Briefly, seeds were surface-sterilized and germinated on Murashige and Skoog (MS) medium (pH 5.7) supplemented with 1% sucrose (FUJIFILM Wako Pure Chemical Co., Osaka, Japan), 0.5 g/L 2-morpholinoethanesulfonic acid (monohydrate) (NACALAI TESQUE, Inc., Kyoto, Japan), and 0.8% agarose (FUJIFILM Wako Pure Chemical Co., Tokyo, Japan). No antibiotics were used for the germination of HTR10mRFP seeds, while +/gcs1^HTR10mRFP seeds were germinated with 100 mg/L kanamycin for the selection of gcs1 loci. Two- to three-week-old seedlings were transferred to the soil and grown at 22 °C under a 16 h light/8 h dark cycle. Then, three to four weeks after acclimatization, a bacterial solution of Agrobacterium tumefaciens GV3101 harboring each binary vector was spread on the flower buds for infection [33 (link)].

All experiments using animals were performed according to the guide for the care and use of laboratory animals approved by the Animal Use Review Board and Ethical Committee of Osaka Medical College (permit no. 30036) and the Institute for Integrated Radiation and Nuclear Science, Kyoto University (KURNS; Kumatori, Osaka, Japan) (permit no. 2018-9). The animals used for the study were eight-week-old male Fischer rats weighing approximately 200–250 g (Japan SLC; Shizuoka, Japan). Each rat was anesthetized by an intraperitoneal injection of three different anesthetics mixed together: medetomidine (ZENOAQ, Fukushima, Japan) (0.4 mg/kg), midazolam (SANDOZ, Yamagata, Japan) (2.0 mg/kg), and butorphanol (Meiji Seika, Tokyo, Japan) (5.0 mg/kg). The animal’s head was fixed using a stereotactic frame (Model 900; David Kopf Instruments, Tujunga, CA, USA). The F98 tumor cell implantation into the rat brain was conducted using a surgical procedure that was previously adopted by our research group to confirm the efficacy of a novel boron compound [16 (link),17 (link)]. The F98 cells—diluted in a 10 μL solution of DMEM containing 1.4% agarose (Wako Pure Chemical Industries, Osaka, Japan) at a concentration of either 10³ cells for the therapeutic experiments or 10⁵ cells for the biodistribution experiments—were injected at a rate of 20 μL/min by an automatic infuser pump.

We used younger leaves to detect ethylene evolution. Shoot apicals were placed in 50 mL vials containing 35 mL of Murashige Skoog’s medium (M.S.), with a pH of 5.8, 3% sucrose, and 1% agarose (FUJIFILM Wako Pure Chemical Corp., Tokyo, Japan) (Murashige and Skoog, 1962 ) to create a gas space of 15 mL. To observe the ACO activity, 200 µM of ACC (Wako) was added to the M.S. medium and incubated at 30 °C for 24 h under light/dark 16/8 h conditions. M.S. medium with 0 µM of ACC was also prepared as a control. After incubation, 1 mL of the air phase in the vials was subjected to gas chromatography using a stainless-steel column packed with activated alumina, heated to 50°C, and flame-ionization detection at 120°C (GC14-B; Shimadzu Corporation, Kyoto, Japan). The methodology is described in Supplementary Figure S1.