1. Indol 3- Acidic Acid (IAA) and Kinetin
Influnce on Root Border Cells Production
Zina Al-Saffar; Senin Ermisilia and Robin Vervoorn
AVANS University of Applied Science – ATGM department-Breda
Abstract
Root border cells are metabolically active cells that are released from a plant root tip into the
rhizosphere. Root border cells are recently used as a source to quantify genotype and phenotype.
The aim of this study was to stimulate and enhance root border cells production for cucumber,
lettuce and tomato using kinetin and indole-3 acidic acid (IAA) as stimulus. The seeds were
challenged with 4 different concentrations of kinetin and IAA (0.01 µM, 0.1 µM, 1 µM and 10 µM),
control seeds were germinated using tap water. Following germination, seeds with emerged roots
were examined for root border cells production and border cells were counted using a light-
microscope. DNA from harvested cells was extracted and further used for PCR assay. Results showed
that IAA and kinetin stimulated the three crops under study to increase border cells production. The
cucumber had higher root border cells production at 0.1 µM for both IAA and kinetin hormones, the
tomato seeds showed increase in border cells number at 10 µM kinetin and the lettuce showed
increase at 0.01µM for kinetin and 10 µM for IAA. No single hormone concentration or exact
hormone type that caused the increase was able to be determined as the three crops responded
differently to the different hormone concentrations and for the both two hormones. Adult plant
from seeds germinated in IAA and kinetin showed different root grew and development, plant grew
in IAA showed long, smooth root with branched root were is plant grew in kinetin showed dwarfed,
rough root with no branch root formation. The polymerized chain reaction amplified fragments on
agarose gel were within the expected sizes. Lettuce showed fragments with a size of (+/-250 bp) and
(+/- 350 bp) for tomato.
Introduction
Root border cells are metabolically active cells
that are released from a plant root tip into the
rhizosphere [1] .Root border cells provide a
biotic boundary between the rhizosphere and
the root which is fundamental for the
rhizosphere modification [2]. The function of
the root border cells is to provide
environmental protection for the growing root
tip by regulating the balance of the pathogenic
- and beneficial microorganisms and excessive
minerals in the rhizosphere. [1] [2]
The root tip is essential to the plants
survival. [3] Within the root tip is the root
apical meristem region (RAM). From the RAM

2. region all the primary root tissue and the root
cap are derived. The RAM consists of cells that
are actively engaged in division, growth and
differentiation. [4]. The production of border
cells appear to be regulated, but little is
known about this process. Border cell
production is thought to be a continuous by-
product of constitutive turnover of the root
cap [5], but recent studies reported that
border cell separation is not necessarily
continuous but can be turned on and off by
the plant by controlled endogenous and
environmental signals and can also be induced
experimentally [6] [7]. A research conducted
on pea (Pisum sativum) showed that a high
concentration of carbon dioxide admitted in
the soil atmosphere can override endogenous
signals that regulate the amount of root
border cells that are being released by the
plant into the rhizosphere. [8]
Auxin and cytokinin are both plant
hormones that stimulate plant growth. Auxin
plays an important role in coordinating plant
development signals and root cap [9] [10].
Indole-3 acidic acid (IAA) which is one of the
forms of auxin that is responsible for the root
system architecture and various stage of root
development. Cytokinin stimulates cytokinesis
(cell division). A study revealed that cytokinin
deficiency reduces root meristem size and
activity. Many studies clearly suggest that
cytokinin affects the RAM [11] [12] [13] [14].
Additionally, auxin and cytokinin opposed
interaction plays an important role in
controlling the root meristem development.
This interaction is regulated through certain
integrators that regulate auxin and cytokinin
metabolisms and signaling pathway to
determine a specific developmental output in
the root meristem.
Recently, interests in using border
cells as a tool in plant studies have been
increased because of their low cost and
simplicity of obtaining required materials. In
addition border cells can be obtained from a
single plant and within relatively short time.
Expression of transferred genes in border cells
can also be studied as they are differentiated
cells and they can be easily adapted to a wide
range of biochemical, physiological and
molecular biological assays [15]. The objective
of this study which was carried out at AVANS
university of applied science – Breda and
supported by Rijk Zwaan – Fijnaart, was to find
a method to increase the production of root
border cells either by changing environmental
conditions or by changing endogenous
signalling. Here, IAA and kinetin will be tested
to study their role to influence the root tip
Ram tissue to increase border cells
production. The plants that were challenged
were cucumber (Cucumis sativus), lettuce
(Lactuca sativa) and tomato (Solanum
lycopersicum).
Additionally, auxin and cytokinin opposed
interaction plays an important role in
controlling the root meristem development.
This interaction is regulated through certain
integrators that regulate auxin and cytokinin
metabolisms and signaling pathway to
determine a specific developmental output in
the root meristem.
Materials and methods
Seeds Sterilization and Germination
The three types of seeds used in this
research, cucumber, tomato and lettuce, were
kindly delivered by Rijk Zwaan. The first step
in the research was to germinate the seeds.
The tomato seeds were surface sterilized in
0.5% NaOCl (Sigma Aldrich) for 45 minutes .
Seeds then were rinsed in sterilized distilled
water six times before germination. A stock
solution with a concentration of (10 mM) was
used to prepare the four concentrations of

3. Indole-3-acetic acid (IAA) hormone (Sigma
Aldrich) and Kinetin (Sigma Aldrich). IAA was
dissolved in 1ml of 0.1 % DMSO (Emplura),
kinetin was dissolved with 1ml of 1M of NaOH
(Sigma Aldrich, Netherlands). These solutions
were brought up to final volume of 10ml with
PBS. The following concentrations were
prepared from the previously prepared stock
solutions (0.01; 0.1; 1 and 10 µM) and used for
seeds treatment. The control seeds were
germinated in tap water. All the seeds were
germinated in eco-tubes (Thermo Fisher). The
cucumber seeds were germinated in 100 µl
solution. The lettuce and tomato seeds were
germinated in 30 µl solution. The cucumber
and the tomato seeds were incubated at 26oC
(Ika KS 4000 I). The lettuce seeds were
incubated at room temperature 20°C. Once
root emerged, seeds were checked for border
cells production.
Border Cells Counting
All seeds with root length between
(0.5 - 2 mm) were collected, washed with
100µl PBS, vortexed for 1min and border cells
were harvested by centrifugation for 10min at
1300 rpm (Eppendorf) . The pellet containing
border cells was dissolved with 30µl PBS and
samples from each treatment were pooled
together and border cells were counted with
Bürker-Türk counting chamber slide
(Marienfeld), the number of border cells
counted was calculated by the mean number
of cells per plant by dividing the number of
cells counted by the number of seeds were
the border cells were harvested from. The
solution containing the remains of cells were
then stored at -20o
C to extract DNA.
DNA extraction
The DNA from the previously
harvested border cells was extracted using
Extract-N-Amp kit (Sigma Aldrich). Leaves
from an adult plant of crop under the study
were used as a positive control. An adjustable
volume of the extraction buffer was added to
volume of the border cells solution and the
leaf sample. The sample was then vortexed
briefly for 1min and further incubated at 95°C
in a water bath for 30min. After incubation, an
equal volume of the dilution solution was
added, briefly mixed and samples were then
stored at -20o
C to be used for PCR assay.
DNA Purification
Before the DNA was used for PCR, the
DNA was purified by adding an equal volume
of Phenol-Chloroform-isoamylalcohol (Sigma
Aldrich) to the extracted DNA solutions.
Afterwards, the solutions were vortexed and
centrifuged for 2 minutes at 14000 rpm. A 350
µl of the supernatant was transferred to a
clean 1.5 ml tube and 1 ml of 100% ethanol
was added to the supernatant, vortexed
briefly and incubated at -20°C for 10 minutes.
Then the solution was centrifuged for 20
minutes at 14000 rpm and 500 µl of 70%
ethanol was added to the pellet. The mixture
was vortexed and centrifuged for 5 minutes at
14000 rpm. The pellet was dried by incubating
at 40°C. In the final step, 50 µl of milliQ was
added to the pellet to dissolve the
precipitated DNA.
PCR and Gel Electrophoresis
The PCR reaction was carried out for
the previously extracted DNA of the harvested
border cells. For 20 µl PCR product, 1 µl of
each specific crop primer was added to 4 µl

4. DNA template, 4 µl PCR level water and 10 µl
of Extract-N-Amp PCR Ready Mix (Sigma
Aldrich). The PCR program was set for: Initial
denaturation at 94°C for three minutes,
regular denaturation at 94°C for 1 minute,
annealing at 52°C (lettuce), 58°C ( tomato) and
55°C (cucumber) for 1 minute, extension at
72°C for 1 minute and final extension at 72°C
for 10 minutes. The amplified fragments were
visualized on 1% agarose gel. [16]
Results and Discussion
Effect of Altered IAA and Kinetin
Hormone Concentrations on
Germination, Root Growth and Border
Cell Number
Germination and Root Growth
Cucumber, Tomato and Lettuce
The effects of IAA and Kinetin on root
growth and cells differentiation can be seen in
Fig. 1. In the IAA treatment the germinated
seeds showed long, smooth root grew with
visible lateral branched roots, while in Kinetin
treatment, germinated seeds showed rough,
dwarf roots grew with no lateral roots
formation.
Antagonistic effects of IAA and kinetin
similarly control the growth and the
outgrowth of branches in root. The high
localized Auxin concentration in the quiescent
center of the root tip meristematic cells is
responsible to maintain stem cells population,
cell elongation and outgrowth initiation of the
lateral roots. Whereas; just above the
meristem, in the transition zone, cytokinin
(Kinetin) represses auxin movement from the
tip and auxin action thus represses cell
elongation and lateral roots initiation [17]. In
another word, the distance of the lateral root
initiation from the root tip is regulated by
Cytokinin concentration. The high Cytokinin
concentration at the root cap represses IAA
action and inhibits lateral root initiation in the
surrounding area of the tip, which is crucial for
enabling uninterrupted elongation zone, thus
ensuring the elongation of smooth primary
root tip free from lateral roots. Above the
elongation zone, where concentrations of
Cytokinin decrease, lateral roots can initiate
[18]. These hormonal mechanism roles for
both IAA and Kinetin on root growth can
explain the different root shapes of our
challenged seeds. The germination of seeds in
presence of Kinetin hormone inhibited the
Auxin action which is necessary for root
elongation and lateral branched root
initiation.
Border Cell number
Cucumber
After 24h germination in eco tubes
containing 30µl of the different hormone
concentrations, seeds with root length
between (0.5 -1 mm) were washed and the
harvested border cells were counted. The
highest production of the border cells was
observed when seeds were germinated in 0.1
µM for both IAA and kinetin hormone. The
Figure 1. Effects of IAA and Kinetin hormones on root
growth and development. Cucumber seeds germinated in
0.1 uM Kinetin (upper plant) and 0.1uM IAA (lower plant)
for 7 days at 26
o
C.

5. number of border cells was (32000
cell/ml/plant) and (9800 cell/ml/plant)
respectively, compared with (5750
cell/ml/plant) from seeds grown in tap water
(control), (Figure 2). No border cells were
counted when root length exceeded 2.5 cm.
Tomato
Tomato seeds were germinated for 7
days in eco tubes with 30µl of different kinetin
and IAA concentrations. In each treatment,
border cells were harvested pooled together
and counted. The root length ranged between
(0.5 – 2 mm), the highest number of border
cells (7500 cell/ml/plant) was observed when
seeds were grown in (10 µM) Kinetin
compared with (3500 cell/ml/plant) of seeds
grown in tap water (Figure 3). No border cells
were able to be harvested from seeds
challenged with different IAA concentration as
the roots length were still too short to
produce border cells at the time of cell
counting. Additionally, no difference in seeds
germination was observed, when seeds were
either sterilized or not sterilized (results not
shown). Though, sterilization of the seeds
helped to avoid sample contamination that
usually accompanied with long incubation
time.
Lettuce
When lettuce seeds were grown in
different IAA and Kinetin concentrations until
root is (0.5 - 1 mm) long. Roots were washed
and border cells from each treatment
harvested, pooled together and counted.
Seeds challenged with (1.0 µM) IAA and (0.01
µM) Kinetin showed increase in border cells
production. The counted border cell number
was 13750 cell/ml/plant compared with 7500
cell/ml/plant from control seeds. No border
cells were harvested when seeds grown in 10
µM of IAA and Kinetin hormones, (Figure 4).
Figure 3. Different effects of Kinetin hormone on
Border cells production for Tomato seeds. Tomato
seeds germinated in different Kinetin concentrations
for 7days, Seeds from each treatment with root length
between (0.5-2mm) were washed with PBS pooled
together and counted using counting chamber.
Seeds challenged with IAA and Kinetin
hormones showed increase in border cells
production compared with seeds grown in tap
water. Crops responded differently to the
tested hormones, as each crop influenced by
Figure 2. Effect of different IAA and Kinetin concentration
on border cells production for cucumber seeds. Seeds
germinated in 30µl of different IAA and Kinetin hormones
for 7 days at 26
o
C. Border cells from seeds with root length
of (0.5-1mm) of each treatment were harvested pooled
together and counted using counting chamber.

6. different hormone type and different
hormone concentration, as no single hormone
type or certain hormone concentration caused
the increase in border cells production. Plant
regulates border cells release during growth
and development and in response to different
environmental effects [18]. Auxin and IAA are
shown to increase ethylene production in root
tip [17]. This increase is shown to induce the
release of border cells from the root tip [19].
The release of cells from the root tip into the
surrounding will stimulate the root tip to
produce new set of border cells as their
accumulation on the root tip ceases the
border cells meristem to produce new cells
[20] [16]. The quantitative increase in border
cells number indicate an important role for
IAA and Kinetin phytohormones on border
cells production, a hypothesis that requires
future work to validate it.
Border Cells as Simple Research Tool for
PCR Assay
The harvested cells from the above
experiments were used as resource for
genomic and biotechnological studies. DNA
was extracted from the harvested Border cells
of (tomato, lettuce and cucumber) and further
used for PCR assay. A specific 350-bp fragment
of T26 for tomato crop was polymerase chain
reaction amplified using primer 1 (5’
-
GGCTTCTACTTGTGCAGTTTCG-3
’
) and
primer 2 (3’
- GCTTGACAGGATCAAAATTGG-5’
),
(Figure 5). 250-bp fragment of R17a for
Lettuce was amplified using primer 1 (5’
-
GTCGCAATCAACACAACGG-3’
) and primer 2 (3’
-
GTGAACTTAGGTGCAAATAAGAAAG-5’), (Figure
6). All amplified fragments were within the
expected size. The presence of band in the
negative control for Lettuce indicates the
contamination of the sample with plant
material. The PCR amplified products of the
cucumber border cells showed nonspecific
bands with large size on agarose gel (results
not shown).
Figure 4. Different effects of IAA and Kinetin hormones on
Border cell production for Lettuce seeds. Seeds germinated in
different IAA and kinetin hormones for 4 days. Border cells
from seeds with root length (0.5-1mm) were harvested,
pooled together and counted using counting champers.
Figure 5. Amplified fragment of T26 in tomato. Lane 1:
mole marker III; lane 2: 0.01µM kin; lane 3: 0.1µM kin; lane
4: 1µM kin; lane 5: 10µM kin; lane 6 tab water; lane 7 (+)
control leaf DNA; lane 8: (-) control; lane 9: mole marker
VII.

7. Border cells have multiple biological
functions in enhancing root growth, defending
plant root from all kinds of dangers and
regulating populations of soil borne microflora
in the rizosphere. Hence, genes expressed by
border cells can be used for the controlled
delivery of chemicals into the root tip region
where water and nutrient uptake, gravity
sensing as well as infection by pathogens and
symbionts is initiated [21] [22]. Recent studies
have reported that border cells share similar
functional roles in defense to those of
mammalian white blood cells in which a “trap”
consisting of extracellular DNA (exDNA) and
antimicrobial proteins function in immune
responses to microbial pathogens [20] [21].
Border cells traps bacteria and fungi in a host-
microbe specific manner by producing a
complex of exDNA and antimicrobial proteins,
a defense mechanism that is similar to that
found in neutrophil cells [23] [24]. By other
studies it was proposed that root cap
functions in a manner similar to that of
mammalian brain stem and that of bone
marrow in its capacity to control programmed
movement in response to specific pathogens
environmental or signals [21]. A better
understanding of border cell genes controlling
this process may yield new insights into
factors underlying root- rhizosphere
community structure and its role in crop
production [25].
Moreover, in many plant root studies
determining the number of the released
border cells is required. Using the traditional
quantitative methods, border cells counting
can be expensive and time consuming
especially when there are several numbers of
plants under study or when the cells sample is
contaminated. Thus, looking for alternative
method that is time efficient and less
expensive is required. Using specific primer
design for a gene or sequence that is
expressed constitutively in root border cells
(i.e. Brd13) [26] along with qPCR can be used
as an economic method for quantifying single
species cell number [27].
Statistic
Border cell numbers represent the
mean number of seedlings used. Variation
measurement within the same group was not
calculated because the samples from each
treatment were pool together and counted.
Conclusions
Both IAA and Kinetin increased border
cells production in all the three tested crops.
No specific hormone concentration was able
to be determined as plants responded
differently to different hormone
concentrations and different hormone type.
Figure 6. Amplified fragment of R17a in Lettuce. Lane 1:
Mole Marker III; lane 2: 10µM IAA; lane 3: 1µM IAA; lane 4:
0.1µM IAA; lane 5: 0.01µM IAA; lane 6: 10 µM kinetin; lane
7: 1µM kinetin; lane 8: 0.1µM kinetin; lane 9: 0.01µM
kinetin; lane 10: (+) control; lane 11: tab water; lane 12: (-)
control; lane 13: mole marker VII.

8. Sterilization of the seeds before
germination did not affect root grow or
border cells production, but it helped to
reduce sample contamination.
DNA extracted from border cells can
be used to study genes expressed in root tip
or other biotechnological analysis. In our
study, we were not able to use the thickness
of bands formed on agarose gel to calculate
the number of border cells used to extract
DNA, as the sample contained traces of root
and plant pieces. Thus it was not possible to
make sure that the extracted DNA represent
border cells only.
Recommendation
Differences in germination rate within
the same set of seeds were one of the
problems we encountered. Thus, germinating
more seeds for each experiment can help to
select a group of seeds within a close root
length.
A recent study [26], reported that
brd13 gene is expressed constitutively in
border cells. Thus, using specific primers
sequences for Brd13 along with qPCR assay
can be used as a new method to count border
cells [27].
We also recommend to test the effect
of another interesting enzyme (Pectin Methyl
Esterase PME) on Border cells production as
previous studies showed that it help to release
border cells from the root tip and increase
their production [17] [4] [19].
Acknowledgment
We sincerely thank Dr. Robert Sijbrandi for his
scientific comments and guidance, Dr.
Jojanneke Welzen and Paul de Graag from Rijk
Zwaan for their warming helps.
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