Acclimatization, survival and growth of in vitro grown
Arabidopsis thaliana seedlings after transplanting to soil.
R.L.M. van Marlen
Supervisor: A. Czerednik and A.J.M. Peeters
Plant Ecophysiology, Utrecht University
Abstract
Plants grown in vitro have a different morphology, physiology and anatomy than
plants grown in soil. In vitro grown plants usually have non-functional stomata, a
weak root system with poor conductivity, a poorly developed cuticle with less
epicuticular waxes and are often hyperhydric due to special conditions in vitro.
These aberrations prevent proper acclimatization from in vitro to ex vitro
conditions. Arabidopsis thaliana displays the same problems when cultivated in
vitro and transferred to soil. Due to natural variation in genotype the different
ecotypes Kashmir-1 (KAS-1) and Shahdara (Shah) display differences in ability
to acclimatize dependent on the time spent in vitro. Growth parameters in Shah
decreased compared to the control group, while growth parameters of KAS-1 did
not decrease compared to the control group. Growth parameters of both
ecotypes correlating well with shoot development, chlorophyll fluorescence and
stomata development.
Introduction
People have been multiplying organisms for commercial gain for centuries. The last
few decades tissue culture, or micropropagation, has proved to be an efficient way to
multiply plants and has been extensively used for rapid production of many plant
species (Pospisilova et al., 1999; Cha-um et al., 2010). Micropropagation is a more
efficient method than traditional breeding, because it makes possible the rapid
production of high quality plants, which are uniform, disease free and can be
produced in every season in a laboratory (Chandra et al., 2010). When transplantation
ex vitro is successful, increase in plant growth can be enormous (Pospisilova et al.,
1999; Chandra et al., 2010). This makes micropropagation an important strategy for
multiplying plants, providing large profits for the companies, which use it (Cha-um et
al., 2010).
However, micropropagation has one disadvantage. When plants are transferred to soil
and are exposed to abiotic stress, many of them die, because their phenotypes are
anatomically and physiologically affected by in vitro conditions (Huylenbroeck et al.,
1998; Pospisilova et al., 1999; Chandra et al., 2010). In recent literature solutions for
this problem can be split into two categories. The first category of solutions is
optimizing the conditions in vitro, for example by changing the quantity of sucrose in
the growth medium or the quantity of light (Haisel et al., 2001; Kadlecek et al., 2001).
This approach does increase growth and survival rate positively, but still a large
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percentage of the plants die during ex vitro transfer (Haisel et al., 2001; Kadlecek et
al., 2001). The second category of solutions consists of gradually changing
environmental conditions during the acclimatization period (Haisel et al., 2001;
Kadlecek et al., 2001). This category includes spraying antitranspirants on plants and
elevating CO2 concentration in the air after transplantation. Slow transition from in
vitro to ex vitro conditions has also been recommended. However, mixed results have
been found about the effectiveness of these methods of plant hardening. Also the costs
of these methods are high, because treating all plants is a labor-intensive job.
(Pospisilova et al., 1999; Chandra et al., 2010; Hazarika, 2010) The aim of this study
is to make a small step towards improving plants acclimatization to ex vitro
conditions by investigating plants with different acclimatization ability.
The first step to the solution of this problem is analysis of the differences between in
vitro cultivated plants and plants grown in soil.
Conditions in vitro are different from conditions in soil in several points. Plants are
grown in vitro in closed containers where the humidity is nearly 100% and airflow is
reduced. The light intensity is often lower partially due to the water vapor blocking
the light. Last, a small amount of carbohydrates is usually mixed in the growth
medium, which could influence ex vitro acclimatization negatively (Pospisilova et al.,
1999; Chandra et al., 2010)
In vitro cultivated plants have an altered morphology, anatomy and physiology
(Pospisilova et al., 1999; Cassels and Curry, 2001). The plants have non-functional
stomata, a weak root system with poor conductivity, a poorly developed cuticle with
less epicuticular waxes and often hyperhydric (Pospisilova et al., 1999; Chandra et
al., 2010).
The retarded stomata and poorly developed cuticle cause high stomatal and cuticular
transpiration when plants are transferred from in vitro to ex vitro conditions (Chandra
et al., 2010). This unrestricted water loss might cause plants to wilt (Pospisilova et al.,
1999; Chandra et al., 2010). Furthermore, absorption of water and nutrients by the
roots might be temporarily impaired, due to damaging of the roots during transfer to
soil, which might also cause wilting (Pospisilova et al., 1999; Kadlecek et al., 2001;
Chandra et al., 2010). The decrease in water supply and the increase in transpiration
might lead to drought stress. Further, the addition of sugar to the growth medium can
cause plants to become heterotrophic and influences acclimatization, when plants
need to switch back to autotrophic (Chandra et al., 2010).
As mentioned before, many plants die after being transferred to soil, but some plants
manage to acclimatize to the new conditions. In many species in vitro cultivated
leaves are not able to develop further after transfer to soil and are replaced by new
leaves, thought not in all plants (Chandra et al., 2010). In both in vitro and newly
formed leaves stomatal density decreases, stomata become elliptical and functional
again, the cuticle and epicuticular waxes develop and leaf thickness increases
(Pospisilova et al., 1999; Chandra et al., 2010). These changes decrease the
transpiration rate and stabilize the water potential (Pospisilova et al., 1999; Chandra et
al., 2010).
A study about the effects of in vitro propagation on Arabidopsis thaliana has not been
done yet, although Arabidopsis is widely used as a model plant. Arabidopsis is a good
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model plant, because its genome is known and plants are small and have a short life
cycle. But is Arabidopsis anatomically and physiologically affected and experience
the same stress as many other plants? When Arabidopsis does not respond to transfer
from in vitro conditions to soil in a manner comparable to other plant species it would
not be wise to use this plant in search of the solution to the problems of in vitro
propagation. The expectation is that Arabidopsis is a good model plant, because it has
proved to be a good model plant and is widely used in other studies (van der Weele et
al., 2000; Koornneef et al., 2004; Millenaar et al., 2005; Verslues et al., 2006;
Lefebvre et al., 2009)
To find out why some plants die after transfer from in vitro to ex vitro conditions and
others survive, the differences in acclimatization of different ecotypes of Arabidopsis
were studied in this research. The approach was to find the differences in
acclimatization ability of Arabidopsis ecotypes Kashmir-1 (KAS-1) and Shahdara
(Shah), making use of the natural variation between Arabidopsis accessions and their
difference in ability to adapt to abiotic stress (Koornneef et al., 2004; Lefebvre et al.,
2009).
We analysed differences in acclimatization ability after plants spent some period in
vitro conditions and then were transferred to soil. We looked at parameters such as the
growth, chlorophyll fluorescence, number and shape of stomata and the appearance of
the plants and compare these parameters in ecotypes Shah and Kas-1.
We found that the two ecotypes KAS-1 and Shah showed different adaptation ability
during stress due to transfer from sterile in vitro conditions to soil. Growth parameters
in Shah decreased compared to the control group, while growth parameters of KAS-1
did not decrease compared to the control group. Growth parameters of both ecotypes
correlating well with shoot development, chlorophyll fluorescence and stomata
development.
Material and methods
Plant material and growth conditions
The seeds of Arabidopsis thaliana Kashmir-1 and Shahdara were sterilized in a flow
cabinet at room temperature. First seeds were soaked for ten minutes in a solution of
bleach and ethanol (ratio 8:2). After that the seeds were rinsed in 70% ethanol three
times. Then they were rinsed another three times in sterile water and dried on sterile
filter paper. With a sterile toothpick the seeds were sown in a sterile medium in a
plastic vessel. The standard growth medium was used: 1/2 MS, sucrose 1,5 % and 0,7
% gel. The vessels were kept in dark in a cold chamber (4°C) for 4 days for
vernalization and then placed in a climate chamber with the following conditions: 8 h
light/16 h darkness, quantum flux density 200 µmol m-2 s-1 temperature 20±1 0C,
relative humidity 65-75 %. A non-sterile control group was sowed directly in soil and
placed in the same conditions. The soil components: potting soil and perlite, ratio 1:2
with added osmocote and Mg 17% both 2 grams per liter perlite and soil. Then 1 liter
of nutrients (0,5 x Hoagland) was added per 6 liters of the mixture.
Experimental design.
After 12, 13, 15 or 20 days after germination (DAG) the sterile seedlings were planted
in pots. The pots were filled with soil mix described above. With tweezers the plants
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were carefully taken out of the vessels, the roots were brushed off in water with a
small brush to get all of the growth medium off. Then the plants were places in a hole
in the soil of the pots and the hole was closed carefully. After potting the plants were
put back in the climate chamber without cover. Harvests were carried out at day 0, 1,
5, 10 and 20 after transferring into the soil; for the control plants harvests were carried
out 5 times beginning from the 12 DAG and then 5, 10, 20 and 30 days after that first
point.
Growth parameters
Each day of harvest 8 plants were randomly selected. Fresh weight (FW), leaf area,
diameter of rosettes, number of leaves and plant height were analysed. Leaf area was
measured with Li-Cor Leaf Area Meter 3100. Plants were dried at 5-6 days at 800C in
paper envelopes and stored in exicator before DW measurement. The dry weight was
analysed with a microbalance. These data were used to calculate the relative growth
rate (RGR) and the net assimilation rate (NAR). The following formulas are used to
calculate RGR and NAR.
RGR = LAR x NAR
(1)
Where LAR is the leaf area ratio, which is the amount of leaf area per unit total plant
mass. NAR is the net assimilation rate, which is the increase in plant dry mass per unit
leaf area. RGR is the rate of increase in plant mass per unit of already present plant
mass (Lambers et al., 2008).
LAR = SLA x LMR
(2)
LAR itself consists of two components. SLA, specific leaf area, which is the amount
of leaf area per unit leaf mass and LMR, leaf mass ratio, which is the fraction of the
total biomass of the plant, which is allocated to the leaves (Lambers et al., 2008).
Chlorophyll fluorescence
For Fv/Fm measurements 10 plants were used per accession. These plants were dark
adapted for at least 20 minutes before measurement Fv/Fm. Per plant one leaf was
selected for measurement on day 0 and marked to be able to follow up on that same
leaf on day each time. On day 10 and 20 after potting newly formed leaves were also
measured to see the difference between in and ex vitro formed leaves. For this
research the PAM-2000 fluorometer was used with the following tuning: measuring
light intensity 6, gain 2, damping 5, saturating pulse 8 seconds 6000 µmol. A small
tube was placed over the sensor so it measures only the leaf directly below it and the
distance to the sensor is the same in every measurement.
Imprints
For analysis of cuticle and stomata imprints were made. 2 plants per accession per
time were taken each day of harvest. To make a mold 2-component dental paste was
mixed and the fresh leaf pushed onto the paste with the topside down. When the
molds had dried leaves were removed and a thin layer of clear nail polish could be
4
applied, while using binoculars to see clearly whether the nail polish is applied well.
When the nail polish had dried it could be peeled off with tweezers and placed upside
down on a slide. Then the imprints were placed under the microscope (objective used
is x20 and x40) and pictures were taken.
Results
Plants of varieties KAS-1 and Shah were growing on a sterile medium in controlled
conditions in the phytotron on a short day regime (8 hours of light and 16 hours of
darkness). After 12, 13, 15 and 20 days after germination (DAG) the plantlets were
transferred to soil. The control group was sown in soil (ex vitro) and was growing in
the same conditions as the in vitro grown plantlets. For in vitro growing plants
observations and measurements were made at the day of transfer to soil (day X0, X is
the number of days after transfer), the day after (day X1) and 5, 10 and 20 (X5, X10
and X20) DAG. We made observations of the control group first day 121, than 125,
1210, 1220 and 1230. The plantlets on the day of transfer (day X0) and the control
plantlets on the first day of measuring (121) are presented in fig. 1.
We observed that shah 13, 15 and 20 days in vitro grown plantlets looked
hyperhydric, had perforations in their cuticles, had long roots compared to the control
plantlets and some wilting at the ends of the leaves. We also observed that Shah,
grown in vitro 13, 15 and 20 days, was forming flower buds. KAS-1 plantlets growing
in vitro were well developed compared to the control plants (fig. 1). Also in KAS-1,
grown 20 days in vitro, we observed some aberrations in cuticle formation. The
cuticle seemed to be thinner in the plants, which have spent 20 days in vitro and the
plants looked hyperhydric. In both KAS-1 and Shah growing in vitro we observed
longer roots and shorter shoots compared to the control plantlets (fig.1).
20 days of after potting we observed that Shah 1220, 1320, 1520 and 2020 were
flowering, changed rosette formation and had a reduced number of leaves compared
to the control plants. Also leaves from in vitro grown plants had smooth edges, while
the control plan had serrated edges (fig. 2). The leaf area (LA) and rosette diameter of
Shah 1220 1520 were smaller than the leaf area and rosette diameter of the control
group (fig 3). KAS-1 plants were well developed and only 2020 formed flower buds
(fig 2). The LA and rosette diameter of 1220, 1520 and 2020 of KAS-1 were comparable
to the LA and rosette diameter of the control group. The LA and rosette diameter of
KAS-1 1320 were bigger than the. LA and rosette diameter of the control group (fig.
3).
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Figure 1. Arabidopsis from varieties KAS-1 and Shah in vitro and the control grown in soil. (A) KAS-1
plantlet from the control group 12 days after germination (DAG), (B) KAS-1 cultured in vitro 12 DAG,
(C) 13 DAG, (D) 15 DAG and (E) 20 DAG. (F) Shah plantlet from the control group 12 DAG, (G) Shah
cultured in vitro 12 DAG, (H) 13 DAG, (I) 15 DAG and (J) 20 DAG.
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Figure 2. Plants of KAS-1 and Shah 20 days after transfer to soil compared to the control
plants. (A) KAS-1 control plantlet, grown ex vitro, 32 days old, (B) KAS-1 plantlet 20 days
after transfer to soil 12 DAG (1220), (C) 13 DAG (1320), (D) 15 DAG (1520 ) and (E) 20 DAG
(2020). (F) Shah control plantlet, grown ex vitro, 32 days old, (G) Shah plantlet 20 days
after transfer to soil 12 DAG (1220), (H) 13 DAG (1320), (I) 15 DAG (1520) and (J) 20 DAG
(2020)
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Figure 3. Leaf area (LA) and rosette diameter of KAS-1 and Shah. (A) Leaf area of KAS-1. (B) Leaf area
of shah. (C) Rosette diameter of KAS-1. (D) Rosette diameter of shah. Error bars represent SE.
*, P<0,05. **, P<0,005.
To analyse the stomata of the leaves we made imprints of the adaxial part of the
lamina. The data are represented in fig. 4 and fig. 5. We observed that stomata formed
on leaves in vitro were clearly bigger, roundly shaped and open widely compared to
stomata formed on control plants. Additionally, we observed that there were more
stomata formed on leaves of in vitro growing plants, compared to the control group.
However, these differences in the number of stomata for KAS-1 after 12 and 15 days
in vitro are not significantly different from the control group. The significant
differences were observed in groups growing longer in vitro.
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Figure 4. (A) Stomata of KAS-1 control plants (con) 12, 17 and 22 days old and in vitro (TC) grown
plants 12, 17 and 20 days old. (B) Stomata of Shah control plants (con)12, 17 and 22 days old and in
vitro grown plants (TC) 12, 17 and 20 days old. The stomata are marked red in Photoshop.
A
B
Fig. 5. The number of stomata per mm2 of KAS-1 (A) and Shah (B) growing in vitro and of control
plants. The controls are plants 12 DAG (121) and 17 DAG (125) days and the in vitro cultured plants
aged 12, 15, and 20 days. Error bars represent SE. **, P<0,005.
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We analysed cross sections of plants grown in vitro at the day of transfer to soil and
compared them with the control plants of the appropriate day. This experiment was
performed with plants 12 DAG and 20 DAG (fig. 4).
The leaves of the Shah plants grown in vitro (for 12 and 20 days) had bigger cells and
bigger intercellular spaces. Moreover, the cells were disorganized and the difference
between palisade cells and mesophyll cells were not clear in in vitro grown plants. On
the contrary, the cells of KAS-1 plants grown in vitro for 12 days were not different
from cells of leaves of the control group. After 20 days in vitro the cells in the leaves
of KAS-1 plants seemed to be bigger and had bigger intercellular spaces. However,
these differences of KAS-1 between control and in vitro grown plants 20 DAG were
less severe than in Shah.
Figure 6. Morphology of KAS-1 and Shah of control group plants 12 and 22 DAG (121 and 1210) and of
in vitro grown plants 12 and 20 DAG (120 and 200)
We analysed the efficiency of photosynthesis using chlorophyll fluorescence as a
parameter of stress and damage of photosystem II. We measured the fluorescence of
the plants on the day of transfer (day 0), the day after (day 1) and 5, 10 and 20 days
after transfer (day 5, day 10 and day 20). Each time we measured the fluorescence of
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the same leaf. On day 10 and 20 after germination we also measured newly formed
leaves (day 10 new and day 20 new). These data are presented in figure 7.
We observed that all plantlets formed in vitro at the day of transfer had an Fv/Fm
value under 0,77, which is a sign of stress. However, in KAS-1 the leaves recovered
later, with the exception of plantlets grown in vitro until 20 DAG. At day 20 more
stress was observed. In case of Shah we observed a diminished ability of in vitro
formed leaves to recover. In both varieties leaves formed de novo were well
developed and did not show any sign of stress.
Figure 7. Chlorophyll fluorescence of KAS-1 and Shah grown in vitro and control (ex vitro). (A)
Fluorescence of KAS-1 control plants and (B) of in vitro grown plants. (C) Fluorescence of Shah control
plants and (D) in vitro grown plants. Error bars represent standard error. Red lines represent stress
threshold at 0,77.
To analyse several parameters of growth analysis we collected plants for fresh weight
and dry weight estimation at the days of observation. Using dry weight and leaf area
values we calculated the relative growth rate (RGR) and the net assimilation rate
(NAR) of Arabidopsis for the whole period of the experiment. The averages of these
parameters are presented in figure 8.
We observed that KAS-1 plants transferred to soil 12, 13 and 15 DAG show a higher
RGR compared to control plants. A decrease of RGR 20 DAG was observed.
In case of Shah all analysed groups showed lower RGR compared to the control
group. The NAR of KAS-1 groups transferred to soil 12, 13 and 15 after germination
were comparable to the control group. NAR of KAS-1 plants transferred to soil 20
DAG was lower than the control group. In case of Shah the NAR of all analysed
groups of in vitro grown plants were lower compared to the control group.
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Figure 8. Parameters of growth analysis. Relative Growth Rate (RGR) of (A) KAS-1 and (B) Shah of
plants cultured in vitro for 12, 13, 15 and 20 days and a control group cultured ex vitro. Net
Assimilation Rate (NAR) of (C) KAS-1 and (D) Shah of plants cultured in vitro for 12, 13, 15 and 20 days
and a control group cultured ex vitro. The RGR and NAR are averages calculated with data of four time
points; day 1, 5, 10 and 20 after transfer to soil. We made measurements of the control group first
day 121 than 5, 10 and 20 days after that.
Discussion
Plants that are transferred from in vitro to ex vitro have stress due to differences in
development in vitro (Pospisilova et al., 1999; Harazaki, 2003; Chandra et al., 2010;
Cha-um et al., 2010).
Tissue culture
In this study it was seen that micropropagated plants had altered anatomical
characteristics as it was reported in different studies (Pospisilova et al., 1999;
Harazaki, 2003; Chandra et al., 2010; Cha-um et al., 2010). These alterations cause a
high transpiration rate in the leaves of the plants due to poorly developed cuticula and
changes in stomata aperture (Pospisilova et al., 1999). This indicates that the plants
are experiencing drought stress. When plants are cultured in a medium they also
become heterotrophic (Pospisilova et al., 1999). Chandra (2010) adds that the sugar in
the medium can cause this and that this influences acclimatization ex vitro. When in
vitro grown plants are transferred to soil they need to become autotrophic, because
they have no energy source any more (Chandra, 2010). In vitro formed leaves are
often unable to develop further and are replaced by new ones (Pospisilova et al.,
1999; Chandra, 2010). This was also seen in Shah in this study.
Growth analysis
In KAS-1 an increase in both RGR and NAR for plants, which had spent up to 17
days in vitro was seen with the most increase in RGR and NAR around 13 days.
12
KAS-1, which had spent more than 17 days in vitro, had both a decreased RGR and
NAR. In Shah we saw a decrease in RGR and NAR in plants, which had spent 12
days in vitro and a further decrease in plants, which had spent more days in vitro.
Reductions in growth under suboptimal conditions, especially water deficit, are seen
more often (Lambers et al., 2008; Harb, 2010). In this case the reduction in RGR and
NAR are the result of a smaller DW and some LA increase. In Shah a decrease in LA
and rosette diameter was also measured. Decreases in leaf area and leaf growth in
leaves formed during drought stress have been documented (Hsiao and Xu, 2000;
Tardieu, 2010) as well as reduction of leaf area in Shah during drought stress
(Bouchabke 2008; Harb, 2010). A decreasing dry weight of the shoot specifically has
been documented as well (van der Weele, 2000). Stress is also a known cause of
reduction in dry weight accumulation (Harb, 2010). Roots are less affected by drought
stress than shoots (Hsiao and Xu, 2000; Lambers et al., 2008) so the assimilates of the
leaves can be invested in root growth (Hsiao and Xu, 2000). In this manner root
growth is favored over shoot growth and roots can explore the ground for water. Also
the distribution of roots can be shifted to wetter soil. The root growth and distribution
can lead to more water uptake for both root and shoot (Hsiao and Xu, 2000). Lambers
et al. (2008) also conclude that these reductions in growth rate (and allocation)
minimize the limitation of growth by a single factor (water deficit). In drought stress
for example, greater allocation to the roots and growth reduction are expected to
minimize the damage (Lambers et al., 2008). Bouchabke (2008) argues that this is a
coping mechanism called dehydration avoidance, which is a coping strategy that
involves the minimizing of water loss and the maximizing of water uptake.
Chlorophyll fluorescence
Chlorophyll fluorescence is a well-known parameter used as an indicator for different
kind of stress, also drought stress (Maxwell and Johnson, 2000; Willits and Peet,
2001; Wright et al., 2009). During stress non-photochemical quenching increases and
often there is photoinactivation of the PS II reaction centers (Maxwell and Johnson,
2000). This causes more non-photochemical quenching and less photochemical
quenching, which influences the efficiency of the photosynthetic apparatus (Maxwell
and Johnson, 2000). PS II is the first system affected by stress in a leaf, because it is
the most susceptible to damage (Maxwell and Johnson, 2000). So the parameter Fv/Fm
is an estimate of the maximum quantum efficiency of photo system II (or the quantum
efficiency is all PSII centers are open)(Genty et al., 1989; Maxwell and Johnson,
2000). Formula 1 describes how to calculate Fv/Fm:
Fv/Fm = (Fm-F0)/Fm
(1)
(Where Fm is the maximum fluorescence and F0 is the yield of fluorescence in
absence of actinic light) (Maxwell and Johnson, 2000). The optimal value is around
0,83 (Maxwell and Johnson, 2000), this value is only seen under optimal conditions
and is lower under normal conditions. When plants are stressed an even lower Fv/Fm is
seen (Maxwell and Johnson, 2000; Willits and Peet, 2001). A threshold value of Fv/Fm
of 0,78 or lower has been chosen as an indicator of stress, although van Huylenbroeck
et al. (1998) used 0,75-0,85 as a typical range of Fv/Fm for non-stressed plants.
Fluorescence indicating stress in leaves of recently transplanted plants was seen with
recovery in KAS-1, but not in Shah. Furthermore we saw that de novo formed leaves
were not stressed in KAS-1 and Shah. Pospisilova, et al. (1999) found that when
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plants are transplanted from a medium to soil, Fv/Fm sometimes decreases the first few
days and increases afterwards. Pospisilova, et al. (1999) and Kadlecek et al. (2001)
argue that photoinhibition was the cause of the decrease in Fv/Fm, which might be
caused by high irradiance.
Stomata aperture
It was also found that in vitro grown Shah had more stomata than the control group
grown in soil. On the other hand there was no significant difference in the amount of
stomata between the in vitro grown group and the control group. Also many in vitro
formed stomata in both ecotypes were big, open and round. Large stomata with
unusual shapes are documented for in vitro grown plants and these stomata usually
demonstrate different stomatal behavior, leading to higher stomatal conductance (van
Huylenbroeck et al., 1998; Pospisilova et al., 1999). The non-functional stomata
together with the aberrations in the cuticle cause high cuticular and stomatal
transpiration, which can lead to wilting of leaves (Chandra, 2010). This is another
indication that the plants experience drought stress.
Roots
The ability of the roots to take up water and nutrients influences growth parameters of
the shoot of a plant. When the plants were transferred it was seen that both ecotypes
had a different shoot/root ratio in comparison to the control plants, sown in soil. Soil
is hard compared to in vitro medium. It has been documented that in in vitro grown
plants water supply is limited by poor hydraulic conductivity of the roots. The root
hairs could be damaged during transfer to soil, which leads to a transient decrease in
water and nutrient uptake (Kadlecek et al., 2001). Root tips can harden in response to
physical hardness of soil (Yamamoto, 2008). This process costs extra energy. This
limited water supply can cause drought stress until the roots have hardened or new
roots are formed.
Flowering
We also noticed that in vitro grown Shah flowers up to 10 days earlier than Shah
control plants. This early flowering could be an escape mechanism for drought stress
(Tardieu and Tuberosa, 2010). Drought escape is accomplished by shortening the
plants life cycle, which allows the plants to reproduce before it becomes to dry in the
environment to survive (Bouchabke et al., 2008).
Conclusion
Two ecotypes KAS-1 and Shah showed different adaptation ability during stress due
to transfer from sterile in vitro conditions to soil. Growth parameters in Shah
decreased compared to the control group, correlating well with shoot development,
chlorophyll fluorescence and stomata development. KAS-1 seems to acclimatize
better and did not show a decrease in growth parameters compared to the control
group until 20 DAG in vitro, which again correlated well with shoot development,
chlorophyll fluorescence and stomata development. It seems that KAS-1 and Shah
have developed different strategies to cope with stress. KAS-1 seems to have
developed drought tolerance, which refers to the ability to survive stress (Bouchabke
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et al., 2008). Shah on the other hand seems to rely on dehydration avoidance and
dehydration escape. Dehydrations avoidance refers to minimizing water loss and
maximizing water uptake. Dehydration escape refers to reproducing before the
environment becomes to dry. Using Arabidopsis as a model plant helps to analyse the
acclimatization ability of a range of different commercially grown plants, which are
cultivated in vitro and transferred to soil.
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