Organ xylem vulnerability to cavitation.
The optical vulnerability method (OV) was used to quantify xylem vulnerability (Brodribb et al 2015). This technique is minimally invasive and allows simultaneous monitoring of xylem cavitation throughout the plant. Data produced using the OV technique has been shown to correspond very closely with vulnerability data collected using hydraulic and x-ray methods in Tomato (Skelton, Brodribb, & Choat, 2017) and many other species (Gauthey et al., 2020). A combination of custom-made “cavicams” (opensourceov.org) and a microscope were used simultaneously on each plant to monitor cavitation during dehydration.
Leaves : a cavicam was secured to a single fully expanded leaf per plant to view a region 5x5mm that included secondary and tertiary veins. Tissue was illuminated from underneath by LEDs (white-light emitting diodes). Images were acquired every 2 min for 8 days. Cameras were refocussed as necessary.
Petioles : a region of one petiole on an adjacent leaf per plant was selected close to the main stem. Using a sharp razor blade, a window of around 15 mm x 8 mm was carefully cut parallel to the surface to remove the epidermis and cuticle on one side of the petiole, exposing the xylem. The area was coated in hydrogel (Tensive Gel; Parker Laboratories Inc., Fairfield, NJ, USA) to improve light transmission and reduce evaporation from the surface. The section of petiole was then secured in a cavicam which acquired images as described above using reflected light.
Peduncles: a region of one peduncle supporting several open flowers, a variable number of buds and no fruit was selected per plant. A region of around 15 mm was carefully scraped with a fingernail to remove the epidermis and cuticle, exposing the xylem. No razor blade was used as the epidermal tissue was softer than the petiole equivalent and razor blades easily cut too deep into the vascular tissue. The area was coated in hydrogel then secured in a cavicam which acquired images using reflected light.
Petals: Images of one petal per plant from an intact apical inflorescence were captured with a Leica DFC450 digital camera, mounted on a Leica M205A stereomicroscope (Leica Microsystems, Wetzlar, Germany). The petals of a newly opened flower were taped to a glass plate to prevent movement/shrinkage during image capture. Light was transmitted through the petal illuminating the midrib xylem bundle and images captured as described above.