The Cell Membrane Stability (
A
major impact of plant environmental stress is cellular membrane modification,
which results in its perturbed function or total dysfunction. The exact
structural and functional modification caused by stress is not fully resolved.
However, the cellular membrane dysfunction due to stress is well expressed in
increased permeability and leakage of ions out, which can be readily measured
by the efflux of electrolytes. Hence the estimation of membrane dysfunction
under stress by measuring cellular electrolyte leakage from affected leaf
tissue into an aqueous medium is finding a growing use as a measure of
The protocol
The general protocol involves the application
of stress to the leaf after it has been subjected to hardening, followed by the
measurement of electrolyte leakage using the conductometric
method. The most common application is for heat tolerance and therefore
the initial detail is given for heat stress (see schematic outline).
1. The
plant must be exposed to moderate heat stress for at least 24h before the test
in order to allow for hardening (acclimation). The capacity for hardening is a
major component of the capacity for tolerance. Hardening can be achieved in the
natural field environment, if heat stress occurs, or in the greenhouse or a
programmed growth chamber. Exposure of intact plants for 24h to 320C
(cool season plants) to 360C (warm season plants) is sufficient,
even at low light.
2.
Leaf discs or pieces of leaf tissue cut with scissors or even whole small
leaves are detached and placed in standard glass vials that can accommodate a
conductivity electrode (see below). The total area of leaf material per vial is
about 15 to 25 cm2. The exact area is not important and it does not
have to be the same for all vials. The sample is then washed for 2-3 times with
de-ionized water. The water is drained off but samples remain wet so that they
would not desiccate. In the case of screening, at least 10 vials (samples) are
prepared for each genotype. In that case 5 pairs are taken from five different
plants (replicates). For each pair, one vial is designated as treatment (T in
Figure) and the other as control (C in Figure).
3. The
treatment vials are subjected to the heat stress treatment in vitro. (Treatment
can also constitute of low temperature in the case of chilling tolerance). They
are placed in racks and covered (not stoppered) with ‘Saran’ wrap so as to
avoid drying the samples. Racks are placed in thermostated
water bath so that the leaf samples will be completely below the water surface
level. Temperature is set to a predetermined stress (treatment) temperature and
the samples remain in the bath for 1h. The control vials are placed in a rack,
covered with Saran wrap and placed at room temperature. The treatment
temperature should be such that it will result in average population
4.
After treatment 20cc of deionized water is added to each vial making certain
that all leaf materials are submerged. All vials are then placed for incubation
at about 100C (typically, on the lowest refrigerator shelf) for 24h.
After incubation the samples are equilibrated for 1h at room temperature and
the conductivity of the medium is measured by inserting a conductivity
electrode into each vial.
5. All
vials covered with Saran wrap or plastic sheet are placed in an autoclave for
15 min to kill all tissues. Conductivity of all samples is measured after
samples are equilibrated to room temperature.
6.
Calculation: see Figure - where T1 and T2 are treatment conductivities before
and after autoclaving and C1 and C2 are the respective control conductivities.
Calculated results are often better when each T value is calculated against the
average of all C values for the given accession.
CMS
for dehydration tolerance
Two
methods are used: by applying drought stress to samples either in vivo
or in vitro.
In vivo stress: plants
of all materials must be stressed to the same relative water content (RWC) of
about 60% to 70% (depending on crop species) before being sampled (T1 samples)
into stoppered vials and brought to the lab. Samples should not allowed to desiccate between sampling and washing. Control
samples are taken from similar leaves of well-watered plants at RWC close to
98%. Once washed, 20 cc of deionized water is added to all samples and they are
directly placed for 24h incubation period (#4 above). The remaining procedure
is as described above. An example of
such work is given by Tripathy et al., 2000. The method requires the
monitoring of all entries for leaf RWC when subjected to drought stress, in
order to determine time of leaf sampling at the standard RWC.
In vitro stress: This
is a simpler procedure. CMS test under drought stress is devised by subjecting
the stress samples (T1) to desiccation in vitro in a solution of polyethylene
glycol (PEG) (MW 6000 or 8000). Examples are presented by Blum
and Ebercon, 1981 and by Premachandra and
Shimada, 1987. This treatment replaces the heat stress treatment as
described above and it is performed at room temperature. After 24h (or less) of
submergence in PEG the samples are quickly washed to remove PEG from the
surface of the samples and then incubated in deionized water as described
above. PEG concentration and duration of submergence varies with the
experiment, species and PEG. Plants should be subjected to some hardening by
drought stress prior to sampling for the stress treatment.
While there is ample evidence to show
that heat tolerance in terms of plant production can be reasonably predicted by
heat CMS, there is only limited evidence that plant production under drought
stress is related to desiccation CMS, partly because the latter association was
not evaluated as extensively as for heat.