Article Type: Research Article Article Citation: Abrahám A. Embí BS MBA. (2020). FACILE IN VITRO GLASS SLIDE LIGHT MICROSCOPY METHOD USING TETRACYCLINE
TO VISUALIZE A REPETITIVE PATTERN IN AERIAL PLANT ROOT TIPS FILAMENTOUS NETWORK.
International Journal of Research -GRANTHAALAYAH, 8(12), 155-166. https://doi.org/10.29121/granthaalayah.v8.i12.2020.2699 Received Date: 07 November 2020
Accepted Date: 31 December 2020
Keywords: Tetracycline Adhesion
Cellulose Orchid Air Roots Plant Cell
Apoptosis Plant Cell Wall Filaments
Repetitive Pattern The main purpose of this manuscript is to introduce a facile light microscopy methodology to visualize plant roots filaments. In a previous manuscript in vitro experiments on freshly plucked human hair roots documented the commonly used antibiotic Tetracycline (TE) deleterious effect on soft tissue, severe enough to allow for visualization of an underlying filamentous skeleton. In this manuscript, TE was also evaluated in a similar fashion of in vitro experiments, this time aerial plant roots were immersed in liquid Tetracycline. Images and video recordings are presented where plant aerial root tissue cells appeared to interact with Tetracycline, thus allowing for exposure of an underlying filamentous network. These filaments were documented undergoing biosorption of Tetracycline, thus indicating a probable cellulose base. It is emphasized that a literature search showed similar, albeit visually different displays of roots filaments obtained by using a Scanning Electron Microscopy. The method herein introduced could be an adjunct to existing established methodology in root function research. Two salient advantages are identified, firstly that the essential minimal material and equipment is limited to a light microscope, glass slides, chosen biological material, water and powder Tetracycline. Secondly, the speed in obtaining results would offer researchers a preliminary or perhaps a final correct conclusion.
1. INTRODUCTIONThe deleterious effect of Tetracycline on
the human hair follicle soft tissue was recently demonstrated in vitro [1]. In that paper, in toto human
hairs were immersed in drops of diluted powder of a commonly used antibiotic,
namely Tetracycline (TE). The adhesive property of TE was proposed as one
factor in damaging the human follicle’s metabolism, as well as a myriad of soft
tissue cells. Images of the follicle’s keratin architecture were displayed (Fig
1). The present manuscript introduces a
deleterious effect of liquid TE to aerial plant root tip tissue. Fresh harvested orchid hanging aerial root
tips were exposed to liquid TE by via a previously describe technique dubbed a
Single Slide Preparation (SSP) [2]. The images obtained show a damaging effect of TE on external root
epidermal and internal trichoblats cells, also
documented are detailed images of a continuous filamentous veil seen covering
the interior tissue. The outer epidermal cells failed to exhibit a filamentous
covering network. 2.
MATERIALS AND METHODS
Materials 1) Freshly harvested orchids aerial root tips. 2) Tetracycline 500 mg capsules. 3)
Medicine Dropper. 4)
Microscope glass slides: 25x75x1mm thickness. Pearl Cat. No. 7101 5)
Digital Video Microscope Celestron II
model # 44341, California, USA. 6)
Images downloaded to an Apple Computer MacBook Pro Photo Application. Methods Tetracycline
capsules (500 mg) were pierced at one end and the golden yellow powder
transferred to a clean white glass plate. The power was divided by a double
edge razor blade into 10 piles each estimated to contain 100 mg/pile. The
subdivision progressed until piles of estimated 5 mgs each were reached
(Exhibit I below). EXHBIT I 3. THE LIQUID TETRACYCLINEOn a separate glass
slide approximate a very small amount of TE powder, such as the minimal amount
adhering to a toothpick was placed on the slide. Using a medicine dropper three
small demineralized drinking bottle water drops were delivered and the powder
stirred for a few seconds. For the purpose of the experiments, this aqueous
solution was dubbed “liquid Tetracycline” (LT). 4. THE ROOT TISSUE IMMERSION IN LIQUID TETRACYCLINEFreshly harvested
orchids areal root tips were placed on a glass slide. A sharp blade was used to
perform a longitudinal thin cut of the distal end as shown in Figure 1 below. Figure 1: Freshly
harvested Orchid aerial root tip. Black Arrow: Harvest cut to plant. Red Arrow:
Distal aerial end thin segment for experiment. Orange Line: Delineating
approximate longitudinal thin cut by sharp razor blade. Using small tweezers,
the previously thin distal aerial root cut sample was placed in the LT. The LT
drops were gently dispersed on the slide, this in order to stabilize root
tissue drift towards the drop’s edges.
Once stabilized, the preparation was placed on the optical
video-microscope platform, and allowed to dry. Control images and video
recordings after LT evaporation recorded and saved for downloading into a McBrook Apple Computer photo application. 5. RESULTSTo show the results of
the effect of LT on plant root tissue, we need to refer the reader to an image
showing the effect of LT in human soft tissue tissue
of the human hair follicle a.k.a. root (1) and Figure 2 below. 5.1. HUMAN HAIR FOLLICLE TISSUE IMBEDDED IN LIQUID TETRACYCLINEFigure
2: in present manuscript. Liquid showing stained golden yellow Tetracycline
adhered to hair follicle filaments skeleton. Notice damage to soft tissue of
Dermal Papilla (DP). Also, as primary starting point for hair follicle inner
keratinized filaments. Orange arrow showing: 1 (right upper corner) Keratin
adhesion to keratin-based hair shaft cuticles, Orange Double Arrow: Denoting
amplified area of typical complex filamentous nature. Original image reproduced from: Embi AA. (2020) THE
HUMAN HAIR FOLLICLE AS SENTINEL FOR DRUGS EVALUATION: DEMONSTRATION
OF TETRACYCLINE ADHESION TO HAIR FOLLICLE AS PROPOSED MECHANISM IN
DYSFUNCTIONAL HAIR LOSS. In Print IJRG December 2020. 5.2. PLANT
AERIAL ROOTS TISSUE CONTROL
In the case of distal aerial plant tissue, the
results obtained are comparatively close to its animal counterpart. Control
images of root tip cells immersed in plain water drops show a thick cell wall
and different filament orientation within each trichoblast
cell (Figure 2) Figure
2: Control orchid aerial root thin cut sample
immersed in plain demineralized water showing: Orange Arrows: Different
individual patterns of intracellular filaments orientation in trichoblast cells. Figure 3:
Same orchid aerial root tip cells shown side by side in liquid tetracycline,
after evaporation, showing: Orange Arrows: Root tissue filament network, as
microscope depth of field changes additional filaments appeared; demonstrating
a layering effect between cells. The cell walls appeared thinner. 5.3. PLANT
AERIAL TISSUE IMBEDDED IN LIQUID TETRACYCLINE
Figure
4: Epidermal
cell post three minutes immersion in Tetracycline. Black Arrow: Pointing at
epidermal cell wall already showing TE biosorption. Notice the absence of a filamentous network,
since these are external root cells. For further details documenting tissue cells
layers please link to URL https://youtu.be/XHsGmWoFpLc Or Scan QR Code in upper left corner of image. Figure
5: Same epidermal as in Figure 4 above, now 38
minutes post immersion in liquid Tetracycline showing: Black Arrow: Extensive
cell wall damage indicating apoptosis. 5.4. INTRODUCING
REPETITIVE PATTERN FILAMENTOUS NETWORK OF AERIAL ROOTS
Figure
6: Orchid Aerial root cut end amplified. Orange
Arrows supporting a filamentous repetitive pattern. Figure
7: Image showing complex filaments displayed by
aerial orchid root tip cells. X: Unidentified filamentous structure. RB: Raphide bundle. Double Head Black Arrow: Pointing at
laterally communicating filament structures. Figure
8: Same area as Figure 7 above. Using landmarks
as the raphide bundle (RB) and the unidentified
filamentous structure (x), the observer could appreciate a filamentous
repetitive pattern as demonstrated in Figure 6. Figure
9: Black Arrow: Showing filaments also covering
structures such as xylem vessels 6.
DISCUSSION
In this manuscript, in vitro experiments done on aerial plant root tissue introduces a
facile method utilizing a commonly used antibiotic (Tetracycline) in aqueous
form in direct contact with aerial plant root tissue able to uncover details of
an intercellular filamentous veil. Of relevancy,
also documented for the first time repetitive growth
pattern of filamentous network in aerial plant roots (Figures 3,6,7,8,9). A literature search failed to show the
aforementioned repetitive filamentous pattern in plant roots as shown in these
experiments; instead, a paper was found and it behooves corroboration by
institutional affiliated researchers with a greater access to published data. The Two Faces of Repetition Periodicity and the described phenomena of
repetitive motion of DNA parts in cell division could induce or stunt growth of
cellular structures, and could occur at many levels
during tissue growth formation in both plants and animal tissue [3], [4]. On the other hand, a repetitive protein is essential for
filament structure and function in parasites [5]. Since the antibiotic Tetracycline and its derivatives have
also been reported to impact on the physiology and biochemistry in tissues of
plants and animals [6], it could be stated that it has a deleterious impact on
plant and animal cells, to the point of sort of eliminating barriers in light
microscopy fields, thus allowing for the visualization of the repetitive
patterns herein introduced (Figs 4,5 plus video recording) supporting this
principle. 7.
SUMMARY AND CONCLUSSIONS
I leave you with words from a renowned
biologist “Orchids of many kinds have also adopted this
high life. They lack the ponds that sustain the bromeliads, so they must
collect their nourishment in other ways. Some dangle their roots in the air,
absorbing moisture from the humid atmosphere and rely on the tiny number of
nutriments it might have dissolved on its descent through the forest
vegetation. Others spread their roots over the surface of the branches and
collect the water that has trickled through the leaves and dripped from branch
to branch, gathering a little nutriment on the way.” [7], [8] Keratin as part of
Aerial Root Filaments Regardless of the physics behind light
microscopy imaging, the fact remains that the antibiotic Tetracycline when in
contact with aerial plant roots appears to adhere albeit sparing root filaments
from damage (Figure 10). This finding is in support of an additional substance
such as keratin as part of the root tip filament base [9], plus LT has been previously documented adhering to keratin
in human hair follicle filaments
(Figure 2); furthermore in vitro experiments
have documented “the
explicit features of plant keratin intermediate filaments is a 24–25 nm
periodic structural repeat alone the axis of both the 10 nm filaments and protofilarnents” [10]. The function of the
repetitive filamentous network is theorized to be of physical support to roots
inner tissue. Plant Cell Walls and Liquid Tetracycline Aerial plant root tip epidermal cells walls are
dissolved, thus pointing towards a cellulose base. The facile method herein introduced could
facilitate root function researchers in obtaining a rapid preliminary display
of supporting root tissue filaments. The novel facile rapid method herein
introduced could be used to obtain preliminary; or perhaps final images of
plants and human hair roots filamentous anatomy. Tetracycline Adhesion to Root Filaments Figure 10: Orchid aerial root post immersion in liquid Tetracycline. Black Arrow: Pointing at yellow golden Tetracycline seen on filament supporting a keratin base. Please compare with Figure 2 showing Tetracycline adhesion to keratin in human hair filaments. SOURCES OF FUNDINGThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. CONFLICT OF INTERESTThe author have declared that no competing interests exist. ACKNOWLEDGMENTNone. REFERENCES
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