To analyze root length, five-day-old seedlings grown on agar (for details see Plant Material) were transferred on agar plates containing 1/8 MS, pH 4 supplemented with 200 μM AlCl3. After 9 day incubation, plates were scanned (Canon CanoScan 8800F) and the root growth was measured using the JMicroVision 1.2.7 software.
To measure pollen tubes length, tobacco pollen was transiently transformed with AtNPC4:YFP by particle bombardment. After 6 h of germination in dark, pollen tubes were incubated in liquid simple sucrose medium (pH 5) with or without 50 μM AlCl3 for additional 2 h. Mean growth rate of pollen tubes expressing AtNPC4:YFP and vector-only control was evaluated using the fluorescence microscope Olympus BX-51.
To determine survival rate, 7-day-old seedlings grown on agar (for details see Plant Material) were transferred to 6-well plates with liquid 1/8 Hoagland’s (Kocourková et al., 2011 (link)) solution (pH 4) with 100 μM AlCl3 for 22 days. Plates pictures were taken by Nikon SMZ 1500 zoom stereoscopic microscope coupled to a Nikon DS-5M digital camera. The survival rate was calculated as a number of viable true leaves.
To measure pollen tubes length, tobacco pollen was transiently transformed with AtNPC4:YFP by particle bombardment. After 6 h of germination in dark, pollen tubes were incubated in liquid simple sucrose medium (pH 5) with or without 50 μM AlCl3 for additional 2 h. Mean growth rate of pollen tubes expressing AtNPC4:YFP and vector-only control was evaluated using the fluorescence microscope Olympus BX-51.
To determine survival rate, 7-day-old seedlings grown on agar (for details see Plant Material) were transferred to 6-well plates with liquid 1/8 Hoagland’s (Kocourková et al., 2011 (link)) solution (pH 4) with 100 μM AlCl3 for 22 days. Plates pictures were taken by Nikon SMZ 1500 zoom stereoscopic microscope coupled to a Nikon DS-5M digital camera. The survival rate was calculated as a number of viable true leaves.
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