P E TA L R E P L I C AT I O N
Single-Step Surface Replication of Flower
Petals for SEM
Gaurav Sharma1, M. L. Sharma 2 , S. K. Sharma 2, and H. P. S. Kang 2
1. Center for Environment and Vocational Studies, 2. SAIF/CIL, Panjab University, Chandigarh, India
BIOGRAPHY
Gaurav Sharma has
completed his masters
degree in environment
sciences at Panjab University and is now looking for a research
assignment. He was the
recipient of the ICON
trophy for best paper presentation in biological sciences of the Electron Microscope
Society of India at the XXVI Annual Conference on Electron Microscopy and Allied
Fields held at Central Potato Research Institute, Shimla, India, April 2003.
ABSTRACT
The surface morphology of a flower petal
can be a significant marker for the characterization of its variety. This paper
describes a new single-step replication
technique for petal surfaces using a cosmetic peel-off facial material. The technique is an easy and quick method to prepare replicas for SEM as compared to conventional and has great potential to study
the surface morphological features of any
flower. The SEM study revealed differences
in cellular morphology and patterning on
the epidermal layers of differently
coloured flower petals.
KEYWORDS
single-step replica, scanning electron
microscopy, flower petals, plant sciences
ACKNOWLEDGEMENTS
I would like to thank Dr Ravinder Mittal of
Cadila/Zydus group for supplying the replication material used in this study.
A U T H O R D E TA I L S
Gaurav Sharma, Center for Environment
and Vocational Studies, Panjab University,
Chandigarh-160014, India.
Email: [email protected]
Microscopy and Analysis, 19 (1): 21-23 (UK),
INTRODUCTION
The perianth of a flower consists of petals collectively known as the corolla. The petal is a
non-reproductive (sterile) part of the flower,
usually brightly coloured, typically showy and
well defined in a mature flower [1]. Each
flower has a different pattern to its petal. The
petal may be separated or fused into one;
dicotyledons usually have 4 or 5 or multiples of
4 or 5, monocotyledons usually have 3 petals
or multiples of 3 petals. These differences are
for several reasons. The design of the petal is
used to increase surface area to improve the
performance of catalyzer to attract insects and
birds for pollination. They serve to attract pollinators for many species. Sometimes they are
extremely fragrant. However the cellular
arrangement and construction on the epidermal layer of the flower petal plays an important role in taxonomy.
The epidermal differences are most apparent when viewed using scanning electron
microscopy (SEM). However, the methodology
of direct methods of processing flower specimens [2-3] is prone to various artifacts and
examination at higher accelerating voltages
[4] is a tedious job.
The replica technique is an indirect method
of examination of those materials which are
otherwise not easy to process by conventional
means for electron microscopy. Replica techniques can be used to study patterning and
development of the plant epidermis [5-6]. In a
conventional single- or double-replica technique a celluloid plate is softened by a drop of
amyl acetate or acetone and pressed against
the specimen in order to form a cast or mould
for examination under SEM [7-9].
The cellular arrangements on a flower petal
are highly fragile and subject to collapse from
the slightest pressure. Here we describe a
material for making moulds with a single-step
replica technique in which the step of applying
pressure is avoided; the technique is very
handy and easy to use in comparison with
available conventional techniques [10]. It replicates the morphological features of the minutest fragile ornamentation on the flower
petal surface giving results comparable to
material prepared by critical point or freeze
drying. The need for a positive replica does not
arise, which is again a long and tedious process
[11] and requires polymerization of some plastics or polymers in an incubator at higher temperature, which may damage the negative
replica and fine detail.
M AT E R I A L S A N D M E T H O D S
The rose-scented varieties of Rosa damascena
and R. gallica of red, yellow, pink and orange
MICROSCOPY
AND
colour were used for this study.
The replication material was ‘Orange peeloff Home Facials’, a product of the Cosmetic
Division, Cedilla Healthcare Ltd., Zydus Group,
Ahmedabad, which is easily available in the
market. The peel-off paste is a combination of
natural orange-peel extract, fruit AHA and
skin vitamins A, C, and E.
A tiny drop of the peel-off paste was diluted
20% with alcohol, so that it filled minute
crevices, and placed on petal surfaces without
detaching the flowers from plants and spread
evenly with a wooden spatula without applying any pressure. This formed a thin layer on
both surfaces of the flower petals. After 20-25
minutes the paste hardened into a very thin
film. The film was peeled off gently with fine
tweezers and discarded as it may contain some
loose debris of the petal surface. A second
coating of peel-off was then applied in the
same way and used for examination under
SEM.
The technique requires patience and practice to make moulds without damaging the
petals. Once set, the replica, which had reasonable tear-resistance and tensile strength,
was peeled off easily. The film was attached to
an SEM stub using double adhesive tape,
keeping the impression side uppermost. The
replicas were coated with a 20-nm conducting
film of gold using an ion-beam sputter coater
(JEOL, JFC 1100). The replicas were examined
at different accelerating voltages in a JEOL,
JSM 6100 scanning electron microscope. Secondary electron images at different magnification were taken in reverse electron polarity
mode with a Pentax K1000 camera using
35mm, B&W, 100ASA, Ilford Pan film. The single-step replica provided negative images of
the epidermis ornamentation but in reverse
electron polarity it gave positive images.
R E S U LT S A N D D I S C U S S I O N
The petal epidermis of a rose is made of many
spherical glandular cells of different shape and
size having vertical striations on the projecting
nipple-like structure in the centre. These cells
were arranged in a precise pattern. Tightly
packed together, the cells were smooth, giving
the petal a silky texture and velvety sheen on
both ventral and dorsal surfaces (Figs 1-4). The
data of the uniform cellular patterns formed
by these cells give specific characteristics to
their variety. The cells with striated tips on the
dorsal side might be responsible for emitting
scented fragrance into the atmosphere. The
ventral epidermis surface of these petals had a
different but uniform pattern of cells of different sizes joined together in a fixed pattern.
The ornamentation on epidermal layers of
A N A LY S I S • J A N U A R Y 2 0 0 5
21
b
a
Figure 1:
Scanning electron micrographs of replicas of dorsal (a) and ventral (b) epidermal surfaces of red rose petals. Scale bar = 10 µm.
flower petals of different coloured roses gave
variation in the cell arrangement, pattern and
look.
The coloured varieties of roses have different morphological features on their dorsal
and ventral petal epidermal surfaces. Figures
1a and 1b show the red variety, which revealed
the precise striated structure on the tips of
glandular cells of different sizes on the dorsal
surface whereas on the ventral side cells were
large and formed a mosaic pattern. Figures 2a
and 2b show the pink variety, which shows
fewer striations and nipple-like features are
missing on the tips of cells whereas on the ventral surface the patterns are smaller. Figures 3a
and 3b show the yellow variety, which has
similar structure on dorsal surface to the red
variety but the ventral side is similar to the
pink variety. Figures 4a and 4b show the
orange coloured variety, which has a different
glandular structure to the cells with assorted
sizes and less striation on the tips of cells
whereas the patterning of cells on ventral surface is entirely different to the other varieties.
CONCLUSIONS
We have demonstrated that this technique is a
quick and easy method of examining flower
petal morphology. The technique is most useful in the characterization of different scented
geraniums. The variation in ornamentation,
size and shape of glandular structure on the
surface of flower petal is helpful in the characterization of different flowers.
a
b
Figure 2:
Scanning electron micrographs of replicas of dorsal (a) and ventral (b) epidermal surfaces of pink rose petals. Scale bar = 10 µm.
22
MICROSCOPY
AND
A N A LY S I S • J A N U A R Y 2 0 0 5
The replicas used in this study fulfilled all the
basic requirements of replica preparation and
examination under SEM. The replication material has the following additional advantages
over conventionally used replicating materials. It yields high resolution replicas with uniform success rate irrespective of the area
under study. It can be used for any type of surface: hard/fragile, thin/thick topography, and
living/non-living. It does not require any pretreatment of the surface under examination.
The replica can be archived for later examination. Different types of information can be
obtained through negative replica in normal
SEM mode and in reverse polarity SEM. The
material has strong potential for the study of
morphological features of any flower and can
P E TA L R E P L I C AT I O N
a
b
Figure 3:
Scanning electron micrographs of replicas of dorsal (a) and ventral (b) epidermal surfaces of yellow rose petals. Scale bar = 10 µm.
be an easy and handy tool for characterization. Finally, the new replication method is
suitable for field studies. This technique is also
useful in other scientific investigations of hard
or soft material with SEM.
Figure 4:
Scanning electron micrographs of replicas of
dorsal (a) and ventral (b) epidermal surfaces
of orange rose petals. Scale bar = 10 µm.
a
REFERENCES
1. Gray, A. Gray's Lessons in Botany, Ivision, Blakeman, Taylor &
Co., New York.1878.
2. Neinhuis, C., Eldelmann, H. G. Methanol as a rapid fixative
for the investigation of plant surfaces by SEM. Journal of
Microscopy 184(1):14-16, 1996.
3. Pameijer, C. H. Replica techniques. In: Hayat MA (ed)
Principles and techniques of scanning electron microscopy,
VI. Biological applications. Van Nostrand-Reinhold, New
York, pp 45-93, 1975.
4. Polowick, P.L., Sawhney, V. K. Scanning electron microscopic
study on the initiation and development of floral organs of
Brassica napus. Americal Journal of Botany 73(2):254-263,
1986.
5. Williams, M. H., Green, P.B. Sequential Scanning Electron
Microscopy of a Growing Plant Meristem. Protoplasma
146:77-79, 1988.
6. Liod, A. M. et al. Epidermal Cell Fate determination in
Arabidopsis; Patterns Defined by a Steroid-Induced
Regulator. Science 266:436-439, 1994.
7. Crankshaw O. S. Introduction of replica techniques for
scanning electron microscopy. Scanning Electron Microscopy
IV:1751-1757, 1984.
8. Fujita, T., Tokunaga, J. Inone, H. Scanning electron
microscopy of skin using celluloid impression. Arch. Histol.
Jap. 30:321-326,1969.
9. Bernstein, E. O., Jones, C.P. Skin replication procedures for
scanning electron microscopy. Science 166:252-253,1969.
10. Sharma, G., Sharma, M. L, Kohli, R. K. A new replicating material for SEM studies, XXVI Annual Conference on Electron
Microscopy and Allied Fields, April 16-18, 2003, CPRI, Shimla.
11. Pameijer, C. H. Replicating techniques with new dental
impression material in combination with different negative
impression material. Scanning Electron Microscopy 2:571574, 1979.
b
©2005 John Wiley & Sons, Ltd
MICROSCOPY
AND
A N A LY S I S • J A N U A R Y 2 0 0 5
23