Lab 6.2: Plant histology & microtechnique

Lab 6.2: Plant histology & microtechnique Purpose The purpose of this lab is to introduce cytology students to basic methods of plant histology. Succe...
Author: Mervyn Todd
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Lab 6.2: Plant histology & microtechnique Purpose The purpose of this lab is to introduce cytology students to basic methods of plant histology. Successful completion of this lab should produce high quality microscope slides containing sectioned and stained plant tissue samples

Background & introduction Meristem histology Plant tissue growth involves the cell division (hyperplasia) of undifferentiated cells (parenchyma) and the growth (hypertrophy) of more differentiated cells. The location of proliferative undifferentiated or dedifferentiated cells in so-called meristematic tissue is of great interest to the horticulturist engaged in plant propagation for commercial or research purposes. Meristematic tissue is most often associated with shoot and root tips (apical meristems). Longitudinal sections allow for the characterization of vasrious cell layers relative to the plant surface (see Figure 1). In some plants meristematic tissue may also be found near nodes (intercalary meristem).

Figure 1. Longitudinal section of a Maize (Zea mays) root tip. This section was stained with Johansen’s Safranin and Fast Green stain. Image from the botanical slide collection by J. Haseloff, Cambridge University.

Root histology A typical root (See figure 2) is comprised of an outer cortex bounded on the outside by an epidermis and on the inside by an endodermis. The epidermis is usually a thin monolayer, however aerial roots of epiphytes have a thick multi-layered epidermis for absorbing water quickly from rain. Botanists typical recognize two forms of cortex. Solid cortex is composed of compact parenchymal cells with very little intracellular space, while lacunate cotex contains large air spaces sandwiched between layers of more dense cotex. It is thought that a lacunate cortex confers an advantage in dense (often soaked) soil, while a solid cortex is more common in well-drained soils. The endodermis typically contains cells with thicker, more lignified (stains well with Safranin O). The inside of the root typically contains a vascular system bounded by a pericycle containing plain, thin-walled cells. The vascular system of monocot roots normally consists of a ring of about six large xylem vessel elements (tracheids). In dicots, there are usually two, three, or four (in rare cases as many as six) bundles of xylem. The phloem is normally present in a ring sorrounding the zylem elements.

BIO-331 Cell biology – Lab 6.2 Plant histology & microtechnique

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Lab 6.2: Plant histology & microtechnique

Figure 2. Transverse section of an orchid (Phallanopsis) aerial root. This section was stained with Johansen’s Safranin and Fast Green stain and imaged using fluoresence microscopy. Image from the Wisconsin Lutheran College botanical slide collection by R. Balza.

Stem histology The stem (see Figure 3), like roots contains a epidermis and cotex but often lack the distinct endodermis visible in root sections. The vascular bundles are arranged in an ordered ring in dicots and gymnosperms, but monocots may possess multiple rings or the vascular bundles may appear scattered throughout the cortex. The mint family (Lamiaceae) has characteristic square stems. The sedges (Carex genus) are known for triangular stems. The central parenchymal cells may be lignified (stain with safranin O).

Figure 3. Transverse section of hibiscus stem. This section was stained with Johansen’s Safranin and Fast Green stain and imaged using brightfield microscopy. Image from the Wisconsin Lutheran College botanical slide collection by R. Balza.

Leaf histology Leaf tissue architechture shows great variability between species. There is no such thing as a “typical” leaf. For this reason leaf structure and histology may be used for taxological purposes. Only in very general ways do all leaves have common histological features: an epidermis with stomata, chloroplast-rich photosynthetic mesophyll, and vascular tissue.

Assignment summary In this lab you will characterize botanical tissue at the cellular level. The preparation of microscope slides from living plant material for visualizing the cellular and tissue architecture is BIO-331 Cell biology – Lab 6.2 Plant histology & microtechnique

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Lab 6.2: Plant histology & microtechnique known as “botanical microtechnique.” These methods find broad application in plant physiology, development, pathology, and molecular biology.

Materials and methods Fixation Obtain a botanical specimen of interest. Small (1 hour 5 min (wash out excess stain) 10 seconds (differentiation) 10 seconds-1 min (neutralize) 10 seconds (dehydrate)

Fast Green Clearing solution xylene + 4 drops 100% ethanol xylene

10 seconds (progressive) 10 seconds (enhance transparency) 10 seconds (remove residual water) 1 minute (dehydrate)

Mount using xylene-base Permount (allow to dry in hood)

Wacker’s trichromatic W-3A botanical stain (optional): One of the most beautiful stains (see Figure 6) developed for botanical tissue in recent years is the so-called W-3A stain developed by Robin Wacker at the University of Wurzburg1. This staining method uses three dyes to label specific histological features of plant tissue. The astra blue dye stains the cytoplasm and cellulosic primary (growing) cell walls a deep blue-green color. Acridine red labels the lignified cell walls brilliant red. Finally the Acriflavin dye labels the cutinized cell walls light pink to orange.

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Wacker, R. (2006) Mikrokosmos p. 210-212.

BIO-331 Cell biology – Lab 6.2 Plant histology & microtechnique

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Lab 6.2: Plant histology & microtechnique

Figure 6. Longitudinal section of a pine (Pinus nigra austiaca) needle. This section was stained with Acridine red, Acriflavine, and Astra blue (WA-3 stain). Image from microscopy-uk.net by Rolf Muller. Required solutions for the W-3A botanical stain: Astra blue solution: 2g safranin dissolved in 100mL dH2O with 2mL glacial acetic acid. Acridine red solution: 1g acridine red 3B dissolved in 100mL 50% ethanol with 2mL glacial acetic acid. Acriflavine solution: 1g acriflavin in 100mL dH2O with 2mL glacial acetic acid. First dry the slides on a heat block set 58oC for 10-15’ as shown above. Deparaffinize the sections using the following series: xylene xylene 100% ethanol 95% ethanol 90% ethanol 70% ethanol 50% ethanol

5 min 5 min 2 min 1 min 1 min 1 min 1 min

Stain the tissue as follows: Acridine red solution dH20 wash Acriflavine solution dH20 wash Astra blue solution Gentle running dH20 wash

5-15 min 15 sec (wash out excess stain) 5-15 sec 15 sec (wash out excess stain) 30 sec – 3 min until no dye remains on slide

Gently blot the excess water off the slide using Kimwipes. Use caution to not touch the tissue. Dehydrate in Triethylphosphate (TEP) Clear in xylene

3x 10 seconds 3x 1 minute

Mount using xylene-base Permount (allow to dry in hood)

BIO-331 Cell biology – Lab 6.2 Plant histology & microtechnique

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