OBJECTIVE
 To stain lignin of the plant section and observe under the microscope.
 To described the arrangement of Lignin and the surrounding tissues as
observed under microscope
HYPOTHESIS:
The lignin cannot be stained by phloroglucinol and are evenly distributed within
the stem of dicotyledonous plants
PRINCIPLE
Lignin is a chemical compound derived from wood and is found in the secondary
cell walls of plants. It is a polymer of aromatic subunits derived from phenylalanine.
Lignin is found in the spaces in the cell wall between cellulose, hemicelluloses and
pectin components. It is covalently linked to hemicelluloses. It is cross linked to
different polysaccharides and thus provides mechanical strength to the cell wall and
also to the whole plant. Lignin polymers are hydrophobic and hence impermeable to
water whereas the polysaccharides are permeable to water.
It constitutes about 30% of dry weight in woody plants and is the most abundant
organic compound after cellulose. The lignin content varies considerably even
within plants of the same species.
MATERIALS REQUIRED
1. Potato
2. Double edged razor blade
3. Dropper
4. Brush
5. Glass slide and coverslip
6. Needle
7. Phloroglucinol stain
8. Petridish
9. Watch glass
10. Stem of a Commelina plant.
PROCEDURE
1. Took a potato and cut it into a block. Made a slit in the middle of the potato
block using a razor blade.
2. Placed the stem of the plant in the middle of the slit.
3. Cut thin sections of the potato block along with the stem into a petridish
containing water.
4. Transferred the only the cut sections of the stem using a brush into a petridish
containing water.
5. Added three drops of phloroglucinol stain to a watch glass using a dropper.
6. Transferred the potato sections using a brush into the watch glass containing the
stain.
7. Took a clean glass slide and add two drops of water onto the slide and
transferred the sections onto the slide using a wet brush.
8. Placed a cover slip onto the slide with the help of a needle or forceps.
9. Removed the solution from the edges of the cover slip using a blotting paper and
observed under the microscope.
10. Drew and described the arrangement of Lignin and the surrounding tissues as
observed under microscope.
RESULTS
Lignified walls become red.
A DRAWING SHOWING THE SECTIONS OF POTATO BLOCK WITH THE
COMMELINA STEM AS VIEWED FROM THE MICROSCOPE:
DISCUSSION OF RESULTS:
The red colour seen in the section indicate lignin staining
Lignin staining is heavy in both xylem and inter-fascular fibres while other regions
had no coloration due to absence of lignin.
The principle behind the action of the stain is that the cinnamaldehyde end groups
of lignin appear to react with phloroglucinol-HCl to give a red-violet color (Gahan,
1974). Although the reaction is not very sensitive, because of the ease of staining,
this procedure is still often used as one of the tests for the presence of lignin in
plant cell wall
Description of lignin
Lignin was a cross linked phenolic polymer arranged in a hyper branched
topology with no regular repeating structure.
It was observed that the lignin were rarely distributed in the G-layer of
phloem fibres and more in the S1 layers and very low in the G- layers of the
xylem fibres.
Some fibres cells showed multi-layered structure; not every cell contained
lignin; and only a few cells had lignin and in higher concentrations; the cells
which did not have lignin were widely distributed around those with lignin.
Lignin was abundant in the secondary cell walls and xylem vessels and the
fibres that strengthens plants providing structural support for the upward
growth of plants and enabling the long distance water transport.
The highest lignin levels were found in the compound middle lamella and
the cells corners due to possession of sclerenchyma cells.
Lignin was found out to be closely following the cellulose microfibrils
orientation in the secondary cell walls and therefore polymerization of
monolignols was affected by the arrangement of polysaccharide which
constituted the cell wall.
It was also observed that the layering of fibres generally alternated thick and
the layers with different fibrillar orientation.
According to the tissues, some tissues especially the xylem tissue is very
highly lignified to increase support and minimize side way movement of
water out of the vessel, yet meristematic tissue lack lignin.
Conclusion:
Lignin is found in the secondary cell walls of plants and the highest amount
is found in the xylem vessels. The major role of lignin is to provide rigidity
of walls and provide extra strength. The presence of lignin is determined by
the distribution of stained areas using phloroglucinol stain. The distribution
of lignin depends on several factors and even differs in plants of the same
species.
RECCOMENDATIONS
 Before starting the experiment sterilize the laminar air flow chamber using
spirit.
 Ensure that while placing the cover slip over the section, Avoid the presence
of air bubbles in between the cover slip and section.
 Always disinfect your work area when you are finished.
REFERENCE
1. David T. Dennis & David H. Turpin (1990): Plant Physiology, Biochemistry
and Molecular Biology, Longman Scientific & Technical group UK.
2. Irene Ridge (1996): Plant Physiology, CBS Publishers, New Delhi, India
3. C. Jeffrey (1982): An introduction to Plant Taxonomy. 2nd Edition,
Cambridge University Press.
4. C. Dutta (1956). A class Book of Botany, 10th Edition, Oxford University
Press, USA
5. Clive A. Stace (1989): Plant taxonomy and Biosystematics, 2nd Edition,
Chapman & Hall Inc. USA.
6. Neil A. Campbell & Jane B. Reece (2005): Biology. 7th Edition, Pearson
Education, Inc. Benjamin Cummings Publishing Company.
7. Yeung, E. 1998. A beginner’s guide to the study of plant structure. Pages 125142. Retrieved from http://www.ableweb.org/volumes/vol-19/9-yeung.pdf