PROTOCOL 1. Surgical exposure of the intact primary vein.
An expanded leaf is selected and the apical part of the leaf placed on a block of agar (1% w/v). An arc-like incision is made through the upper (adaxial) and lateral epidermis of the petiole of the leaf 4 - 8 mm from the center of the rosette. No incision is made on the basal (abaxial) petiole surface. A second similar incision is made up to 4 mm from the first incision in the direction of the leaf tip. Then, perpendicular to the first incisions, a third cut through the adaxial (upper) epidermis is made to join the first incisions. In all cases, care is taken not to damage the vasculature. Then, using small forceps and a scalpel, extravascular tissues are gently removed from the region of the incisions. This leaves an exposed region of vein. These manipulations are summarized in Fig. 1. The procedure typically takes from 2 to 5 min to perform.See figure in Figures section.
Exposing part of a midvein as described in protocol 1.
a, positions of cuts on the petiole of an expanded leaf. 1 and 2; superficial lateral incisions in the upper surface and sides of the petiole made to avoid severing the primary vein. 3, longitudinal superficial incision made prior to removing extravascular tissue. b, the leaf after removal of extravascular tissues. c, detail of an exposed vein, scale bar = 250 μm.
PROTOCOL 2. Isolation of intact primary vein segments.
An expanded leaf is selected and the leaf blade is cut off with scissors just above the petiole/blade junction so as to leave a small length (a few mm) of leaf blade. The apical part of the leaf blade is then discarded. Using a scalpel, gentle incisions are made through upper (adaxial) and lateral epidermis near the base of the petiole. No incision is made on the underside of the petiole. Using a thumb and forefinger, the cut end near the petiole/lamina junction is gently pulled away to expose the midvein. The vein is then quickly excised for immediate use or for rapid freezing in liquid nitrogen. The procedure, which takes approximately 10 s and works in approximately 50% of cases, is illustrated in Fig. 2.See figure in Figures section.
Preparation of vein segments from expanded leaves as described in protocol 2.
a, positions of cuts on the petiole of an expanded leaf. 1, most of the lamina is removed with scissors. 2, superficial incision in the petiole, b, the leaf stub is gently pulled off the vein. c, the vein is cut off for use. d, remnants of the detached leaf, scale bar = 2 mm. e, extracted primary vein, scale bar = 2 mm. f, detail of an extracted vein, arrowheads show positions of secondary vein branch points; scale bar = 1 mm.
PROTOCOL 3. Extraction of living primary veins attached to the leaf base.
After experience has been gained using method 2 it is possible to adapt this method for the study of live, attached midveins. A ring-like incision is made through the upper (adaxial) and lateral epidermis of the petiole of the leaf 4 - 8 mm from the center of the rosette. No incision is made on the basal (abaxial) petiole surface. Using a thumb and forefinger, the leaf is gently pulled away from the center of the plant. This should leave an attached midvein up to approximately 3 cm-long depending on the size of the leaf. The procedure is outlined in Fig. 3. To prevent desiccation of the attached vasculature, a block of 1% (w/v) agar in 0.5 x Murashige Skoog (MS) medium (pH 5.7) is placed on the soil below the exposed vein. A strip of filter paper previously soaked in 0.5 x MS medium can also be placed on the agar block below the vein to prevent desiccation and enhance visibility. Plants are placed in the light in humidified Plexiglas boxes (30 x 20 x 20 cm) for 1 h prior to experiments to allow recovery. The procedure, detailed in Fig. 3, typically takes about 10 s. However, only 10 – 20 % of vein exposure attempts are successful since veins are vulnerable to breakage during extraction.See figure in Figures section.
Removal of the lamina to expose an attached vein as described in protocol 3.
a, the dashed red line shows placement of a superficial lateral incision on the sides and upper (adaxial) petiole surface made without damaging the midvein. b, the leaf is gently pulled off. c, the remaining vein is left attached to the leaf base. d, leaf vein after extraction, scale bar = 2 mm. e, detail of the extracted vein attached to the petiole base, scale bar = 2 mm.