Abraham, A.

Kumar, L. S. S.

Solomon, S.

English

Publications of the IAS Fellows

A Technique for the Anatomical Study of Root Parasitism
BY
L. S. S. KUMAR, A. ABRAHAM
AND
S. SOLOMON
(Botany Department, College of Agriculture, Poona, India)
With Plate V
STEPHENS (1912) was the first to study the structure and developmentof the haustoria of Striga lutea Lour, parasitic on maize roots. Later,
Saunders (1933) carried out more extensive studies on the parasitism of this
plant and found certain differences between resistant and susceptible strains
of Sorghum vulgare in regard to the rapidity and extent of penetration of the
parasite.
In our investigations on the attack of Striga, a root parasite on Sorghum
vulgare and on several other hosts (Kumar and Solomon, 1941), we have
examined three species of the parasite, viz. Striga lutea, S. densiflora, and S.
euphrasioides. Any attempt to study by the usual methods of section cutting
the differences in the type of attack by different species of Striga on different
varieties of Sorghum possessing varying degrees of resistance to parasitism
would be most laborious and time-consuming. It was therefore found
necessary to devise a technique by which the whole material is made nearly
transparent and then any desired tissue, in the present case lignified tissues,
stained with, an almost transparent stain. The method had to be rapid and
easy of manipulation in order to deal with the large amount of material under
study.
Various techniques and their modifications were tried but the results were
not satisfactory. The method suggested by Debenham (1939) for the micro-
scopic examination of the xylem of whole plant organs, besides taking too long,
did not give very transparent preparations. After several trials it was found
that staining by the Feulgen technique after properly clearing the material
gave very satisfactory results. Subsequently, to test the possibility of wider
application of this method, the technique was tried on a variety of other
materials including fern fronds, Selaginella stem, roots of seedlings, flower
buds, leaves, &c. The results showed that if the material is not very thick
and can be well cleared, the lignified elements take on a very characteristic,
bright, reddish-violet colour.
[Annali of Botany, N.S. Vol. VI, No. 21, January, 1943.]
966-11
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178 Kumar, Abraham, and Solomon—A Technique for
MATERIAL
Striga seeds were sown in a Petri dish with moist blotting-paper at the
bottom and one or two grains of Sorghum were placed in the centre and sur-
rounded by a wad of cotton to keep them moist. The Petri dish is covered with
a piece of cardboard, slit on one side to allow the Sorghum leaves to come up,
and the dish is kept in an incubator at 300 C. in the dark. Within two to three
days the Sorghum grains germinate and the main root grows horizontally on
the blotting-paper, which is kept moist by the addition of a little water every
day. The main root gives rise to many lateral roots. The Striga seeds which
lie near the host roots germinate and establish contact with them. The dishes
are examined every day and material collected for fixation. It was found
possible to procure enough material in the different stages in about two
weeks.
FIXATION
As materials are to be mounted whole, they should be of convenient size
and not larger than is necessary for the particular study. Small lengths, about
0-5 to 1 cm., of the host root with the parasite attached are cut with a pair of
sharp scissors and immediately transferred to the fixative. Hillary (1939) has
shown that for successful application of the Feulgen technique a fixative con-
taining chromic acid should be used. We have used, with uniformly good
results, Nawaschin's fluid both for cytological and morphological studies, and
as materials may be left in this fixative for a week or two without any damage,
it was used throughout the investigation.
WASHING AND CLEARING
After keeping the material in the fixative for at least 24 hours it is washed in
running water for one hour and then passed to 70 per cent, alcohol after keeping
in 30 per cent, and 50 per cent, alcohol for one hour each. The material is
left in 70 per cent, alcohol at least overnight; this hardens the material and
prevents maceration during subsequent treatment. Material which cannot be
handled immediately may be stored in 70 per cent, alcohol.
The fixed material is brought down to water after passing through 50 per
cent, and 30 per cent, alcohol and given a few changes of water. The method
then used is that devised by Schultz (1897) for demonstration of bone in
mammalian embryo in which the soft tissues are cleared with potassium
hydroxide. The material is left in 1 per cent, potassium hydroxide solution
for one to two days. A higher concentration of hydroxide tends to macerate
soft tissues. When the material is properly cleared the vascular strands
should be easily visible through the outer tissues, which should be almost
transparent. The cleared material is then washed in running water for about
30 minutes and is ready for staining.
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the Anatomical Study of Root Parasitism 179
STAINING
This is done by a modification of the Feulgen reaction now widely used
for chromosome studies (de Tomasi, 1936). The Feulgen reaction as applied
to chromatin involves a chemical reaction in which as a result of a mild hydro-
lysis of thymonucleic acid contained in the chromosomes an aldehyde is
liberated. This aldehyde when brought into contact with decolorized
fuchsin sulphurous acid restores the natural colour of fuchsin and thus stains
the chromosomes containing the liberated aldehydes. Margolena (1932) has
stated that lignin, suberin, and cutin also give a persistent positive reaction
with the Feulgen test. Relying on the observations of Mehta (1925), she
infers that this positive reaction is due to the presence of aldehydes in the
molecules of these three substances. In the present technique this positive
reaction of lignin to the Feulgen test is applied in staining the lignified ele-
ments of both host and parasite. The most important points on which a suc-
cessful reaction depends are (1) use of an absolutely colourless 'leuco basic
fuchsin', and (2) correct hydrolysis.
Basic fuchsin suitable for this purpose is difficult to obtain. We tried four
samples of basic fuchsin, and of these Grubler's special basic fuchsin gave
the best result, being superior to the certified stain supplied by Coleman &
Bells (certificate No. CF. 17). The stain is prepared in the following way:
pour 100 c.c. of boiling distilled water over 0-5 gm. of powdered basic fuchsin
in a flask; shake vigorously and allow to cool to 500 C.; filter into a bottle with
a ground glass stopper and add 10 c.c. of N/HC1; cool to 250 C. and add 0-5 gm.
of potassium metabisulphite. Leave the bottle always well stoppered and in
a dark place. The liquid becomes colourless within 24 hours and can be used
till it begins to assume a reddish tint due to oxidation.
As shown by Hillary (1939), hydrolysis is a critical part of the procedure
and the optimum period of hydrolysis varies with the fixative used and some-
times also with the material. With Nawaschin's fixative, 15 to 20 minutes
hydrolysis in dilute HC1 (8 c.c. of concentrated HC1 of s.g. 1-19 to 100 c.c. of
distilled water) at a constant temperature of 6o° C. in an electric incubator was
found the best for the materials used in the present study. Material which is
ready for staining is transferred from water to cold HC1 of the same strength
as used for hydrolysis. After 1 or 2 minutes it is transferred to dilute HC1 at
6o° C. and hydrolysed for 15 mins. Then the acid is poured off and some
decolorized fuchsin solution, prepared as detailed above, is added. The
material is left in this solution from 6 hours to overnight. Then it is washed in
running tap-water for about 10 minutes and then given two or three changes
of water made slightly alkaline by the addition of a few drops of 1 per cent.
KOH solution; this treatment intensifies the stain. It is found advantageous
to omit the usual washing in SO2 water, as the retention of a very light stain in
the cellulose walls is found desirable. The material is then rinsed in distilled
water and passed up rapidly through alcohol grades of 30, 50, 70, and 95 per
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180 Kumar, Abraham, and Solomon—A Technique for
cent, strength, allowing about 3 minutes in each. Then it is changed to
absolute alcohol, then to xylol-alcohol, keeping in each for about 5 minutes
and finally taken to pure xylol. After leaving in xylol for 1 hour the material
is mounted in thin Canada balsam. The slide is placed horizontally on a hot
plate at about 400 C. for a few hours with a 5 gm. weight on the coverslip.
This helps to flatten slightly the material and is useful for certain types of
observations. More balsam is added at the edge of the coverslip if necessary.
COMMENTS
Previous studies on the structure and development of haustoria of Striga
give only details of certain stages, as they have been mainly based on serial
sections (cf. illustrations of Stephens (1912) and Saunders (1933)). The
advantages of the present technique are: (1) it is possible to follow all the
stages from germination of the Striga seed to the development of the adult
parasite; (2) a complete and clear idea of the type and degree of haustorial
penetration at any stage of development of the parasite on the host root can be
obtained; and (3) it is easy to make comparative studies under controlled
conditions. However, it is not to be expected that this technique, though
facilitating anatomical examination of unsectioned material, will completely
eliminate the need for section cutting; it reduces considerably, however, the
latter type of work and is distinctly superior to it for the type of observations
mentioned above. Another advantage is that material which has been stained
and brought to the xylol stage can be examined under the microscope, rough
diagrams made of the haustoria, and then, if desired, can be taken to paraffin
for embedding in the usual manner and serial sections made which can be
counterstained with light green or any other suitable stain and examined.
These observations together with those made prior to embedding the material
give a much clearer idea of the anatomy than can be obtained from serial
sections alone.
It is hoped that this technique, with modifications where necessary, will
prove to be of wider application. Plate V shows photomicrographs of prepara-
tions made by this method. Brief notes on the anatomical features are given in
the explanation to the plate. A detailed anatomical account will be published
when the studies now in progress are completed.
SUMMARY
A new technique for the anatomical study of root parasitism is described.
It involves fixation of the material in Nawaschin's fluid, clearing in 1 per cent,
potassium hydroxide solution and then staining by a modification of the Feul-
gen technique.
The advantages of this technique over the usual method of sectioning are
indicated.
This research was carried out under the 'Scheme of Investigations into the
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the Anatomical Study of Root Parasitism 181
attack of Striga on Sorgktm', financed by the Imperial Council of Agricultural
Research, New Delhi.
LITERATURE CITED
DEBBNHAM, E. M., 1939: A Modified Technique for the Microscopic Examination of the
Xylem of whole Plants or Plant Organs. Ann. Bot., N.S., iii 10. 369-73.
DE TOMASI, J. A., 1936: Improving the Technic of the Feulgen Stain. Stain. Tech., xi. 4.
137-44-
HILLARY, B. B., 1939: Use of the Feulgen Reaction in Cytology. I. Effect of Fixatives on the
Reaction. Bot. Gaz., ci. 2. 276-300.
KUMAR, L. S. S., and S. SOLOMON, 1941: A List of Hosts of some Phanerogamic Root Para-
lites attacking Economic Crops in India. Proc. Ind. Acad. Sci. xiii. 3. 151-6.
MARGOLENA, L. A., 1932: Feulgen Reaction and some of its Applications for Botanical
material. Stain. Tech., vii. 1. 9—16.
MEHTA, M. M., 1925: Biochemical and Histological Studies on Lignification. I. The Nature
of Lignin, its Physiological Significance and its Estimation in Timbers. Biochem. Journ.,
xix. 958.
SAUNDERS, A. R., 1933: Studies in Phanerogamic Parasitism, with particular reference to
Striga lutea Lour. Science Bull. No. 128, Dept. of Agri., S. Africa.
SCHULTZ, O., 1897: Uber Herstellung und Conservirung durchsichtiger Embryonen zum
Studium der Skeletbildung. Verhandl der Anat. Gesellsch. xi. 3-5.
STEPHENS, E. L., 1912: The Structure and Development of the Haustorium of Striga lutea.
Ann. Bot., xxvi. 1067-76.
EXPLANATION OF PLATE V
Illustrating the article by L. S. S. Kumar, A. Abraham, and S. Solomon, on 'A Technique
for the Anatomical Study of Root Parasitism'.
The photomicrographs are mounts of portions of Sorghum root with Striga seedlings in
various stages of parasitic development upon it. Figs. 1-6 and 10 are of Striga lutea while
Figs. 7-9 are of S. dentiflora.
Fig. 1. Striga lutea. The seed has germinated and the end of the radicle which forms the
primary haustorium has penetrated the cortex of the host root. The cotyledons are still en-
closed by the seed coat. Some idea of the transparency of the preparation can be obtained from
the fact that though the Striga radicle passes below the Sorghum root it is very clearly visible.
(X 40.)
Fig. 2. The left-hand portion seen in the above figure under higher magnification. Under
this focus it would appear that the haustorium is not in contact with the vascular cylinder of
the host, whereas in another focus (see Fig. 1) it is clearly seen to be in contact with it. ( X 130.)
Fig. 3. The haustorium has enlarged; vascular elements are developed in the parasite and
close contact with the vascular elements of the host is established, though no penetration into
it has taken place. The origin of a branch, which later develops into a secondary haustorium,
is seen as a lateral outgrowth to one side of the primary haustorium. ( X 80.)
Fig. 4. A later stage after the cotyledons and plumule have come out of the seed coat.
Haustorial cells have penetrated into the vascular tissue of the host. The first pair of leaves are
seen as rudiments. ( X 80.)
Fig. 5. Still later stage showing deeper penetration of the host tissue by the haustorial cells.
(X80.)
Fig. 6. Portion of the above under higher magnification to show more clearly the hauatorial
connexion. ( X 160.)
Fig. 7. Striga densiflora. A portion of the host root attacked by two Striga seedlings from
opposite sides showing that penetration of a seedling by one haustorium does not interfere
with the penetration by another. The seedling on the upper side is apparently older than the
one below, in which the cotyledons are still enclosed by the seed coat. ( x 50.)
Fig. 8. The haustorial connexion of the above under higher magnification, showing clearly
the tracheides of the parasite and 'haustorial hyphae', which are connected to the tracheides
and have deeply penetrated the host vascular tissue. (X 160.)
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182 Kumar, Abraham, and Solomon
Fig. 9. Another specimen of S. dentiflora showing the very deep penetration by the haus-
torial cells; note the swollen and branched ends of these cells. From comparison of this with
S. lutea of the same stage (see Fig. 10) it is seen that S. dentiflora shows much deeper penetra-
tion of the vascular tissue; a fact which accounts for the observation that the latter is more
injurious than S. lutea in its attack on the host. ( X 160.)
Fig. 10. S. lutea. Haustorial region magnified, showing clearly the Ugnification of the xylem
elements of both host and parasite. A comparison with Fig. 9 shows that the penetration in
this case extends only as far as the centre of the vascular cylinder of the host. ( X 160I.
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Annals of Botany,
KUMAR, ABRAHAM A SOLOMON - ROOT PARASITISM.
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.V. S. VoL VI. PL V.
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Huth, coll.
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A technique for the anatomical study of root parasitism

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A technique for the anatomical study of root parasitism

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