Photography as a Tool of Research and Documentation in Plant Tissue Culture

Author(s): Victor Gaba, Danny Shavit, Benjamin Steinitz and Krishnan Kathiravan
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 40, No. 5 (Sep. - Oct., 2004), pp.
536-541
Published by: Society for In Vitro Biology
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In Vitro Cell. Dev. Biol.-Plant 40:536-541, September-October 2004 DOI: 10.1079/IVP2004544
? 2004 Society for In Vitro Biology
1054-5476/04 $18.00+0.00

PHOTOGRAPHY AS A TOOL OF RESEARCH AND DOCUMENTATION IN PLANT TISSUE CULTURE

VICTORGABA1*,DANNY SHAVIT2,BENJAMINSTEINITZ3,ANDKRISHNANKATHIRAVANlt

'Department of Virology, Agricultural Research Organization Volcani Center, P.O.B. 6 Bet Dagan 50250, Israel
2Shavit Professional Scientific Photography, PSP Ltd., P.O.B. 83 Bet Dagan 50250, Israel
3Department of Genetics, Agricultural Research Organization Volcani Center, P.O.B. 6 Bet Dagan 50250, Israel

(Received 30 October2003; accepted 10 December2003; editor E. Yeung)

SUMMARY

Scientific photography is an important facet of plant tissue culture. The aim of photography in plant tissue culture
should be to illustrate clearly the developmental stages occurring in vitro. However, the photographic results presented in
publications are often poor, and morphogenetic responses are often not clearly documented. Plant tissue culture is a very
visual science, and the valuable tool of photography is often not used properly. If the morphogenetic responses are not well
documented, an important part of the research is missed, and the report ends up having limited scientific value. Simple
methods for improving the results of photography in plant tissue culture are discussed, along with photographic equipment,
photomacrography, stereophotomicrography, suitable backgrounds for photography, use of a digital scanner, and the
construction of photographic plates.

Key words: documentation; in vitro development; photography.

I many publications require fuller photographic documentation of

NTRO0t)
'tCTION
Reading the reviews of a colleague's manuscript submitted to a
(good' journal stimulated the writing of this paper. The article was
accepted for publication, although one of the reviewers commented
that the quality of the photographs was too poor to (distinguish the
plants properly! This is not an uncommon story. Many of the
photographs submitted to the specialist plant tissue culture
journals, even when reviewed in a glossy print, are difficult to
understand, much less after reproduction in a journal. It would be
invidious to take examples from the literature for illustration, but
many of the papers in current issues of the plant tissue culture
journals suffer from this problem. Reproduction in many journals
further reduces the quality of the submitted photograph, so that
excellent submitted photographs achieve good (but not excellent)
reproduction in the journal. Without adequate care the photographs
published may not be valid scientific evidence of developmental
stages in vitro and in the greenhouse.
The experimenter has to assure that the photography submitted to
the journal is clear and will bear reduction in quality due to
reproduction, and that the features visible in the photograph support
the points made in the text. If the photograph is not clear, the plant
response is not documented properly despite much investment and
effort, and there is little point in publication as the reader
will be unable to understand what has occurred. Moreover,
*Authorto whom correspondenceshould be addressed:Email vpgaba@
volcani.agri.gov.il1
tPermanent address: Entomology Research Institute, Loyola College,
Chenai 600 030, India.
developmental stages occurring in vitro than are actually given, and
without photographs the written description is often too sparse to be
useful. It is notable that professional photography and processing
costs only a very small fraction of the total cost of a project
including salaries, so it is unwise to restrict the quality and the
quantity of use of' photographic services.
Plant tissue culture is an intensely visual science, and a great
deal of the detail is lost in the conversion of a photograph from color
to black andt white. Furthermore, many published photographs are
much too small, and it is difficult to see details: the trend to plates
with a large number of postage stamp-sized photographs of low
(often digitized) quality must be reversed.
There are many cheap and easy-to-implement improvements in
photography possible fiorplant tissue culture, without sophisticated
equipment. Here we offer advice on the general use of photography
in plant tissue culture projects, followed by some guidance on
technical aspects and the use of photographic equipment.
Photography in plant tissue culture is reviewed by Gray (2000),
who gives technical advice on the use iof the camera and related
procedures (foicus, film type, composition, stereophotomicrography,
depth of field, etc.). We will not discuss here the basic photographic
techniques, for which many primers are available (e.g., Eastman
Kodak Co., 2000). Photomacrography is close-up photography with
an enlarging lens, and is important in plant tissue culture, as it
permits photographic enlargement of explants too small to be
photographed except with a stereomicroscope (see discussion
below). Close-up photography is used to take near-life-size
photographs. Textbooks are available on close-up and
photomacrography, discussing both techniques and equipment

536

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 PHOTOGRAPHY
IN TISSUECULTURE 537

(e.g., Bracegridle, 1994; Constant, 2000; Sholik and Eggers, 2000). POINTS FOR IMPROVEMENT OF PHOTOGRAPHY

Photomicrography is a separate subject not covered here (see, e.g.,

Evennett, 1989; Lacey, 1999). While the points below may be
obvious to some readers, it is clear from the current literature that
there are many problems with the implementation of proper
standards for photography in plant tissue culture.
(1) Scientific photography is an essential part of documentation
in plant tissue culture, and therefore photographs should be
taken routinely throughout a project as documentation, not as
an afterthought.
(2) Use a professional photographer with a properly equipped
photographic laboratory, if possible. Nowadays electronic
cameras are available, and make it very easy for anyone to take,
store, and manipulate large numbers of poor photographs with
a computer. A professional photographer generally does a
much better job. It is curious that many scientists are happy
with poor-quality photography that they have produced
(and perhaps digitally manipulated shoddily) themselves.
However, if the institutional photographer had made a similar
pitiable job, the experimenter would have complained bitterly.
(3) Take the ex/plant out of the box, tube, plate, or jar. It is
necessary to sacrifice the best material to obtain good
photographs. The poor optical qualities of tissue culture
containers make quality photography of the contents near
impossible. Put nothing between the camera and the object.
Photographs of the object in the container are the single
largest problem with visual documentation in plant tissue
culture. Additionally, the subject can generally only be
photographed in close up properly once out of the container.
These points are illustrated in Fig. 1.
(4) Take several different photographs, at different exposures, at
different angles. Photograph different explants showing the
same/similar phenomenon - one picture will often come out
rather better than others. Explants are irregularly shaped
objects and it is hard to predict which shots will best show
the features of interest due to the geometry. Additionally, the
relevance of some details may not be understood until the
end of the project when the report/manuscript is being
written. At project summation, a good collection of
photographs can be very helpful, and will vitiate the
necessity to repeat an experiment for a single observation. Do
not be afraid to burn up film to obtain a range of photographs:
this is much cheaper than having to repeat an experiment to
obtain a good photograph. With a variety of photographs in
stock there is the luxury of being able to choose the best and
most appropriate picture at leisure. Often more detail is
visible in photographs viewed on the computer or slides
projected onto a screen: printing fewer photographs will
recoup the cost of the increased number of photographs.

Flc. 1. A range of photographsof in vitro plant material.Regenerating

cotyledon explants of melon (Cucumis melo L.) cv. Revigal, after a month in
culture in Magenta boxes, as described by Curuk et al. (2002). A, Explants
are hard to view inside the box, due to the poor optical quality of the box
wall, condensation on the box wall, the lateral angle of the photograph, and
the inclusion of much empty space at the top of the box. Condensation is
caused by removal of the box from the warm growth room for photography,
and can be avoided by photography in the growth room. B, Close up of the
explants through the wall of the Magenta box, permitting a better view of the
explants. C, Close up of the explants after removal from the Magenta box.
A black arrow marks vitrified explant material, and a white arrow marks
vitrified leaf primordial; C marks callus. The explants are sacrificed, but
optimal photographic conditions are obtained, and many aspects are clearly
visible. A ruler is included for scale.

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538 GABA ET AL.

Fic. 2. Use of differentbackgrounds.A varietyof differentbackgroundcolors were used for photography,and the color photographs
were laterconvertedinto black and white.A-H, In vitro-growntobaccoplants on:A, black;B, white;C, red;D, darkblue;E, light blue; F,
green;G,light (sandy)brown;H, darkbrown(R, roots).I-L, Meloncallus on differentbackgrounds:I, black;J, white;K, red;L, darkblue.
A ruler is included for scale in each photograph,and has not been replaced by a bar, as this plate is an example.

(5) Take close-up photographs. Filling the viewfinder with the
object of interest is a good method to take quality
photographs. General views are often not very helpful.
Do not show a general picture of 10 plants in a box or 15
explants in a Petri dish: this only gives a broad impression,
but little detail. Convey the information best with a good
close-up photograph of a single explant clearly displaying
the response(s) of interest. Single explants of the treatment
placed alongside the control will often best demonstrate a
point.
(6) Try to get good pictures of the whole developmental
sequence - this will help greatly to explain the 'story' of the
biological response that has been developed. Of necessity the
photographs of various developmental stages will have to be

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 PHOTOGRAPHYIN TISSUECULTURE
taken at different times, and the story assembled retro-
actively. When a particularly good example is observed,
especially of a short-lived developmental phase, sacrifice
that sample to obtain a good picture under optimal
photographic conditions. Any time the present authors have
delayed photography of transient phenomena, this has been
cause for later regret.
(7) Show only the detail of interest. If a photograph (or
photomicrograph) contains a small detail of interest, crop the
picture to remove the unwanted material.
(8) Show a good picture of the buds emerging from the explant,
or the place where the shoots emerge. Do not only show a
general picture of the explant covered with leaves with no
other details visible.
(9) If the photograph does not come out well - repeat the
photography, until the photograph is of suitable quality. This
is similar to repeating the experiment or re-running a gel.
Use a new sample with different geometry, and perhaps a
different lighting angle.
(10) The description in the caption and the text has to match what
is visible in the photograph. If the response you see with your
eyes is not visible in the picture, take the photograph again.
(11) Set up experiments with extra material specifically for
photography, and take the best samples for photography.
(12) Have a metric (tpm, mm, cm) ruler/scale bar in each picture
- even if the photographer says it does not look 'nice' - the
ruler can be excised later and replaced with a scale bar.
(13) When taking photomicrographs, record the conditions and
magnification.
(14) Do not use out of date or improperly stored film.
(15) Photographic film should be developed soon after exposure.
(16) Care should be exercised that the original photographs are
organized and stored properly. Originals of digital photo-
graphs should be maintained separately, and manipulations
performed only on copies. Photographic negatives should be
stored in dry conditions (low humidity), using plastic covers
designed for long storage of films.
(17) Set up the lights and camera prior to bringing the plant
material from the culture room, or removal from the test tube
etc., to prevent the material drying unnecessarily.
(18) It is sometimes possible to take photographs with the explant
out of the laminar flow bench and return the material to
axenic conditions. After optimum illumination for photogra-
phy (or stereophotomicrography or photomacrography) has
been set up and the camera focused on the object in the
closed Petri dish or tube, remove the lid, readjust the focus
slightly for the state of free optical pathway, take the picture,
and immediately replace the lid. The returned material can
continue to develop in culture; sometimes contamination will
occur after several days, during which further development
can be observed. Sometimes the material does not become
contaminated. It can be possible to take photographs and
with luck avoid contamination without compromising
conditions of photography.
(19) It is possible to replace explants on culture medium after
photography, and store at 4oC for a few days, giving time to
evaluate the photographic images. Photographs can be
retaken if necessary from material that has therefore not
developed further.
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539
(20) Exceptions for rare material. If the material to be
photographed is for some reason rare, or cannot be removed
from the container (and there is very little material that
cannot be 'sacrificed'), try the following procedure: the
culture vessels have to be kept 'warm' in the culture room
and transferred to the photographic site (already prepared) as
quickly as possible, to prevent condensation on the lid or
walls caused by cold air. Close-up photographs have to be
taken so that walls of the vessels are not within the focal
plane.
(21) Remove the camera lens cap!
MATERIALS AND METHODS FOR USE IN PHOTOGRAPHY
Camera
We recommend the use of a single lens reflex (SLR) camera on
which lenses can be changed. SLR cameras are the best for
scientific purposes, as what is seen through the viewfinder is the
real frame of the camera picture. The camera should have manual
operation. A tripod and a cable release must be used. The camera
needs at least two lenses, a wide-angle to normal-angle zoom lens
(28-70 mm), and a macro lens. Some zoom lenses are also equipped
with a macro lens. We prefer the use of a good digital SLR camera,
for the reasons above. Additionally, with a digital SLR camera there
are no issues with film use, and there is the important possibility of
instant playback. A digital camera of three million pixels resolution
is recommended as minimum for scientific photography. A handheld
camera must operate at least as fast as 1/60 see, otherwise use a
tripod. Color and black and white film of 100 ASA is recommended.
For photography of fluorescence phenomena, slide film of 400 ASA
is recommended. Black and white film is probably not necessary
nowadays, and has to be developed by the photographer.
Photographs shown here (Figs. 1 and 2) were taken with a Nikon
D100 (digital) camera with 6.1 million pixels (high resolution),
using a 55 mm 'Micro Nikor' macro lens when necessary,
illuminated by a pair of 1000W, 3400 K halogen lamps with
reflective umbrellas.
Lighting
For photography outdoors a flash is needed and should be used
as main lighting or as 'fill-in' lighting to reduce shadows. Indoors
it is best to use tungsten-halogen light of 3400 K color
temperature, diffused (indirect) to reduce shadows and reflec-
tions. The minimum photographic installation is a pair of such
light sources, one at each side of the object, so as not to cast a
shadow. Fluorescent lights give problems with the color balance
with film cameras, although the balance can be corrected with a
digital camera.
Computer Equipment and Software
All recent microcomputers are capable of controlling digital
scanners and image processing. Photographs should be scanned
with a minimum of 300dpi prior to processing. Several image-
processing programs are available, and are included with the
Microsoft 'Office' suite. A more professional image-processing
540 GABA ET AL.
program is Photoshop (Adobe). It is very important to balance the
color on the computer screen (in the image-processing program) so
that the image observed on the screen is what appears when printed.
Do not enlarge digital pictures to the point that the grain is visible.
Beware the loss of resolution that can occur from repeated
processing of the same photograph, and is especially obvious in the
.jpeg format.
Stereomicroscopic Photography
Taking photographs with a stereomicroscope is an essential part
of plant tissue culture research and documentation. A good-quality
camera (as discussed above) is essential, and is best fitted to the
stereomicroscope by a trinocular head (so that the sample can be
viewed easily), using the connector supplied by the manufacturer. It
is possible to use a cheaper digital camera, but the photographic
quality is less good, and connection between the camera and
microscope is problematic and expensive. The field of view of the
specimen should be lit evenly if possible (bilateral illumination),
preferably using a fiber-optic device to avoid heating. A cable
release must be used. The major problem is the depth of field, which
is often very small with a stereomicroscope. Therefore care has to be
taken to focus exactly on the object of interest, as otherwise it will
not be in focus. Some stereomicroscopes are equipped with an iris
diaphragm between the eyepieces and the objective lens. Closing
the diaphragm increases the depth of field, improving the picture
greatly (see Gray, 2000), although with some loss of quality.
Photography with the stereomicroscope requires practice. Record
the conditions (and magnifications) carefully when taking
stereophotomicrographs.
Photomacrography as Replacement for Stereomicroscopic
Photography
Photographs taken using a good-quality camera and macro lens
can replace low-power stereomicroscopic photographs. Photoma-
crography can be used to produce high-quality enlargements up
to 50-fold, with an improved depth of•
field compared to
of•
conventional stereophotomicroscopy (see above), at high quality.
This is because when the object is large enough to photograph with
a macro lens and high-quality digital SLR camera, the photograph
can be taken at enough of a distance so that the entire object is in
focus. In consequence, even though the object does not occupy the
entire photographic frame, the object can be cropped out of the
picture and enlarged (with the computer), observing more detail
because of the high resolution. With such a method it is possible to
capture, for instance, the transitory stage where the meristem dome
appears, or late stages of somatic embryo development (depending
on the species).
Use of Digital Scanners
A digital scanner can be used to record the contents of Petri
dishes during an experiment. The lid of the dish can be exchanged
in the laminar flow bench for an unmarked lid, and the dish inverted
on the scanner. A scanner image made from below the agar always
has a reflection from the lid beside each explant. This method
enables rapid permanent recording of many features of in vitro
culture (size, growth rate between successive scans, color, timing of
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developmental processes), with direct input to the computer for
analysis. However, the photographic quality is only adequate for
scientific record keeping, not for publication, as the instrument is
optimized for recording images on the flat bed and not in three
dimensions. The density of recorded images should balance the
conflicting demands of adequate picture quality, and the size of file
to be stored.
Background Color Contrast
Careful selection of the background is important. Experiment
with different backgrounds, especially if the subject will finally be
published in black and white, not color. Smooth black velvet, or
some other black material, is often the best background for white,
yellow, or green organs from tissue culture. The choice of a dark
background may also be good in other circumstances, but it is
necessary to test. This is illustrated in Fig. 2, where the effect of a
range of backgrounds is seen. It is clearly better to use a black
background for a green plant (Fig. 2A-H), where even the details
are observed more clearly (Fig. 2A). The white background (Fig. 2B)
is less effective: fewer details can be seen, and the roots are hard to
observe. Photography of callus with white, green, and brown sectors
was also attempted (Fig. 21-L), and little of the different colors of
callus can be seen in the black and white photograph, although the
best picture is again observed with black, followed by the white,
backgrounds. Do not take pictures of small plants in pots, as the
plant will be indistinguishable against the brown soil background
on conversion of the color picture to black and white for publication
(see Fig. 2G, H). In such cases it might be better to remove a plant
from a pot and take a photograph against a suitable background.
Generally, the blue color of P-glucuronidase (GUS marker gene)
activity (Jefferson et al., 1987) will show not well against green
tissue in a black and white photograph. It is best to fix the plant
material well with ethanol, which will also remove the chlorophyll,
and enable easy photography.
Construction of Photographic Plates
Photographic plates with a multiplicity of images are a common
feature of scientific publications in plant tissue culture. Professional
preparation of such a plate is best; however, certain rules should be
fiollowed in any case. The best possible original images should be
used. The images should be cropped so that extraneous material is
removed. There should be a uniformity of lighting in each
photograph. The color in a set of photographs for a plate should be
balanced. It is best to try to arrange the images in even rows, i.e.,
2 X 2, 2 x 3, 3 x 3, etc. Arrangements with images of different sizes
are harder to assemble successfully. Do not use so many pictures
that the individual image size is too small to see the object properly
(i.e., no postage stamps). Illustrate any feature with the minimal
number of photographs. Create composite plates with the explants
or plants placed in the same orientation for easier understanding.
One photograph can illustrate several points, saving valuable space,
and assisting in setting the context.
It is essential to record the scale of each photograph in a plate
assembly. Prior to cropping, a scale bar of a known size (e.g. 0.5, 1,
5, or 10 mm) can be constructed for each photograph from the ruler
that was in the original photograph, either manually, or by use of the
image-processing program. The scale bar should be placed inside
 PHOTOGRAPHYIN TISSUECULTURE
the area to be retained after cropping. The bar will be maintained in
the photograph during subsequent manipulations, and thereby serve
as an easy permanent record. An intermediate stage in plate
construction is illustrated in Fig. 2, where each picture still has the
original ruler attached (i.e., has not been replaced by a bar), and
each individual photograph still needs to be cropped to remove
unwanted material.
Label points of interest clearly on figures with lettering that will
be clearly visible following reduction in size of the figure for
publication. Labeling of figures is best performed at the end of
processing of the images. Label points of interest with arrows, using
clearly understood contractions, i.e., shoot meristem (sm),
adventitious meristem (am), bud (B), somatic embryo (sm), globular
embryo (ge), root (R), shoot (S), leaf (L), etc. Use label colors (black
or white) that contrast with the background (e.g., Fig. 2). Take care
that the labels maintain the correct positions during further
manipulations of the photograph. Pay close attention to the
'Instruction for Authors' for the journal to which you wish to submit
your manuscript regarding the details of numbering and labeling
photographic plates, and consult recently published photographs in
that journal. Design plates to fit the size of the published journal
page (single column, as Fig. 1, or page width, as Fig. 2) according to
the 'Instructions for Authors'.
Conversion of Color Plates to Black and White
Often color plates are submitted because researchers do not have
the facility for processing black and white photography. Following
conversion of color plates to black and white for printing, the
quality of the resultant plate may be poor. This problem is best met
by the general improvements in photography that have been
discussed above. Additionally, for the conversion to black and white
it is important to have a good range of contrast in the original color
photograph, especially in the details of interest. It is possible to
scan such a picture with a digital scanner, and following the use of
the option (present in Adobe Photoshop, for instance) of 'conversion
of color to grayscale', to observe the color plate after 'conversion' to
black and white, as in Figs. 1 and 2.
Manipulation of photographs by a computer image-processing
program prior to conversion of a color plate to black and white
involves management of the highlight, mid-tone, and shadow to
obtain contrast in the black and white product not observed in
color. As discussed above, the greater the contrast in the color
original, the easier it is to obtain observable details in the black
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541
and white final. A computerized image can also be converted to
grayscale, to observe the quality of the final black and white
product.
CONCLUSIONS
Scientific photography is an important facet of plant tissue
culture, and has to be worked at to achieve good results, as with
any other technique. The aim of photography in plant tissue
culture should be to illustrate clearly the developmental stages
occurring in vitro, and the final plant product. If these are not
well documented, time, effort, and money are being wasted. Plant
tissue culture is a very visual science, and a valuable tool must
be used effectively.
ACKNOWLEDGMENTS
This paper is contributionno. 507/03 from the AgriculturalResearch
Organization,The Volcani Center,Bet Dagan,Israel.K. Kathiravanreceived
a BOYSCASTFellowship from the Ministryof Science and Technology,
Governmentof India.
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