With traditional instrumentation, stomatal conductance is difficult to measure. Traditional meters, such as a porometer, are slow and laborious. Typically, a single measurement can take several minutes and measuring many samples is very time consuming. Moreover, an instrument to measure stomatal conductance needs regular calibration and maintenance which just adds to the frustration of taking these very important physiological measurements.
Edaphic Scientific provides an easy-to-use and low maintenance solution for continuous stomatal conductance measurements. Stomatal conductance can be measured at a minimum of five-minute intervals but a single leaf can be measured continuously for several days to weeks.
As the sensor is connected to a data logger, measurements can be performed at any time of the day without the need for a researcher or technician to be present. With a modem connected to the data logger, stomatal conductance of a leaf can be viewed and monitored anywhere in the world with an internet connection.
A continuous data set of stomatal conductance over several days to weeks will provide important insights into the physiology of plants in response to experimental and environmental treatments.
Mounts of leaf and stem epidermises or bare cuticles, useful in both general anatomical and specialized phylogenetic studies, can be prepared by a maceration process using Jeffrey’s solution (equal volumes of 10% aqueous CrO3 and 10% HNO3).
Leaves, including those of conifers, and stems with cuticles thick enough to maintain integrity when isolated are amenable to this process. Dried specimens are hydrated by boiling in water; fluid-preserved specimens are washed thoroughly in water; fresh specimens need no pretreatment. Specimens are cut to a convenient mount size and trimmed so as to allow adequate and even penetration of the macerating fluid. Laminar leaf segments are left with one edge untrimmed so that upper and lower epidermises remain contiguous.
Cylindrical leaf and stem segments are slit lengthwise through about half their thickness. Specimens are macerated in Jeffrey’s solution for 1 to 4 days until unwanted tissues are loosened and easily freed from the epidermis (or bare cuticle, if that is desired).
The macerating fluid is then washed out completely with changes of water and specimens are stained in a 0.5% aqueous solution of safranin. Dehydration is accomplished with several changes of tertiary butanol.
All unwanted tissues not removed by agitation during previous steps of the process are removed by teasing prior to mounting. Specimens are then mounted on slides in Canada balsam and dried in the usual manner.
The use of transparent replicas is a convenient method for the microscopic study of surfaces. Perfectly dry structures can be replicated with plastic substances such as ‘Collodion’, ‘Formvar’ or methacrylate, but a serious disadvantage to the general use of these materials is that they ‘mist’ when allowed to harden in a moist atmosphere.
Accordingly, they cannot be used satisfactorily with specimens that either become distorted when dry or, like many biological specimens, have a naturally wet surface.
The following method, which overcomes this difficulty, consists essentially of taking a primary impression of the surface with a silicone rubber plastic and using this to form secondary transparent replicas.
A fast image processing method is proposed for detecting stomata and measuring stomatal aperture size in individual images. The accuracy of aperture measurements is 97%. A prototype mobile application is developed to assist field measurements.
A technique for enzymatically cleaning leaf epidermal strips of Nicotiana glauca so that guard cells remain the only intact and viable cells is described.
The method relies on differential sensitivities of epidermal, mesophyll, and guard cell walls to digestion with a mixture of Cellulysin and hemicellulase.
Guard cells isolated by this procedure remain joined at their ends as duplexes, retain continuous cell walls, and are free of epidermal and mesophyll cell wall fragments and cytoplasmic debris. Yields range from 90–95% of original cells. All recovered cells concentrate neutral red and exclude trypan blue. In addition, duplexes isolated from either adaxial or abaxial surfaces show significant increases in width and aperture over dark controls when illuminated in solutions containing 30 mM KCl. Yields approach those necessary for standard methods of biochemical analysis. A brief comparison with existing techniques for guard cell isolation is included.
Epidermal strips of leaves of the Gramineae can be prepared using the following technique: The mature leaf is dipped in boiling water to kill the cells, and decolorized in boiling 70% alcohol. It is cleared and softened in 88% lactic acid. Epidermal, mesophyll and vascular tissue is removed from a selected constant area of the leaf leaving an epidermal strip 1-3 cm in length. This is inverted on a slide, stained in lactopheno-cotton blue, and destained in 88% lactic acid. Transverse and longitudinal sections of the strip are obtained at this stage. The epidermal strip is finally mounted on a slide in 88% lactic acid. The preparation is photographed with a 35 mm camera using transmitted light, and a yellow filter in the microscope lamp. Photomicrographs of known enlargement are then prepared from which accurate measurements can be recorded. The technique is applicable to both fresh and herbarium material.