Phloem
The detailed
structure of sieve elements in the phloem cannot be observed easily without the
use of special staining techniques. Consequently, some of the material used in this
exercise will be fresh. Sections of living material are usually more difficult
to interpret than commercial slides. Therefore certain prepared slides will be
used for orientation, and to demonstrate the arrangement of cells in the
phloem, as well as the associations of phloem & xylem.
Primary Phloem of
squash (Cucurbita) Picture 1
Study prepared
slides of cross and longitudinal sections of Cucurbita
stems.
Locate the xylem
and phloem. Does the phloem occur on one side of the xylem (collateral bundle)
or on both sides (bicollateral)????
Study hand
sections and stain with Toluidine Blue. Compare these
with the commercial slide.
Cucumber Vascular
Bundle: It has Phloem on two sides of the Xylem Picture 2
Cucumber Vascular Bundle
showing the Phloem. Picture 3 The dark cells are Companion
Cells & the largest cells are Sieve Tube Members.
High-power study
shows the three components of phloem tissue: Sieve Elements (here Sieve Tube
Members), Companion Cells (small cells accompanying the sieve elements), and
Phloem Parenchyma cells (intermediate in size between sieve elements and
companion cells).
The end walls of
the sieve elements seen from the surface in cross sections,
bear highly differentiated Sieve Areas. These end walls are called Sieve
Plates. The protoplasts of adjacent sieve tube members form a continuum through
the sieve plates. Picture 4
These connections
are the Connecting Strands. Each is encased in Callose,
a carbohydrate wall substance chemically distinct from the cellulose that lines
the sieve pores.
Staining shows
that sieve elements appear end to end in longitudinal series and thus, form
Sieve Tubes.
The lateral walls
of sieve tubes bear relatively undifferentiated Sieve Areas. The pores in the
sieve areas are much larger than typical pits and resemble those in the sieve
plate.
Top view of a
Sieve Plate from a commercial slide at high magnification Picture 5
Commercial slide of Phloem seen in Longitudinal section: Note the Red-Stained material which
contains Callose. also note
the Sieve Plates Picture 6
In order to
demonstrate Callose in fresh material we will use
free-hand longitudinal sections of Cucurbita stained with Aniline Blue. Aniline Blue
preferentially stains callose. Furthermore, stained callose emits fluorescence under ultra-violet and violet
light.
Staining Procedure
It is sufficient to stain the sections with
Aniline Blue.
Wait 5 min.
Rinse with water and observe.
Detailed protocol for Aniline Blue Staining
Place sections in IKI for 3 minutes,
Rinse with water
Stain 5 minutes with 0.1% aqueous aniline
blue.
Wash briefly with IKI
Mount in water.
Callose will stain blue. However, Aniline Blue will
also stain other materials in the section so you need to locate the xylem which
is auto-fluorescent, then the phloem. Picture 7
Look for
concentrations of the stain in the phloem region, and locate the presence of
sieve plates in the highly stained areas. Callose
accumulates at the Sieve Plates due to the pressure that exists in the Phloem.
Observe these
sections with a fluorescence microscope that clearly shows the sieve plates
because of aniline blue fluorescence. These will appear white or light blue
against a dark background. Picture 8
Plastids will
fluoresce red. Xylem fluorescence will also be blue but you can easily identify
it due to the characteristic secondary wall thickenings.
Overall view of a
longitudinal section of cucumber
stem stained with Aniline Blue and seen with
Violet Fluorescence. The cell walls of the Xylem (left) are auto-fluorescent
while the fluorescence of the Phloem (right) is due to Callose
which has stained with Aniline Blue.
The sieve plates
will be the most fluorescent areas because callose
accumulates there normally and becomes more concentrated after wounding. The
sieve plates vary in their orientation. Some are perpendicular to the long axis
of the stem while others may have 45O angles of inclination. The latter can be
seen in face view in longitudinal sections. This allows you to see the sieve
pores. Picture 9
Cucumber Phloem
stained with Aniline Blue & Viewed with Violet Light Fluorescence.
Longitudinal
sections of Cucumis stems were stained with Aniline
Blue and photographed with a fluorescence microscope after excitation with
Violet Light.
Callose in the Phloem is fluorescent and appears
bright under these conditions. It has a yellow/green color. This is partly due
to the blue color of the dye.
The Sieve Plates
are especially fluorescent because they contain a lot of associated Callose and Callose accumulates
there upon injury.
Lateral Sieve
Pores are also fluorescent.
Xylem is autofluorescent and appears similar to the Phloem. Xylem
can be identified because of its characteristic secondary walls.
Plastids appear a red dots due to the fluorescence of Chlorophyll Picture
10
What else to do:
•
Root
–
Zea, x.s. Locate
phloem, then draw sieve tube elements and companion
cells.
•
Leaf
–
Zea, x.s.. Note the pits on the vessel member walls
•
Stem
–
Cucurbita, x.s. & l.s.
•
First examine x.s. slides, figure out where phloem should be, and then
examine l.s. slides. Note
the sieve tube members and sieve plates.