BIOLOGIA (PAKISTAN) 2013, 59 (2), 341-344
PK ISSN 0006 - 3096
Soluble protein contents from in-vivo and in-vitro sources of Brassica juncea, var.
poorbiraya, under salt stress
ATHAR HUSSAIN
Department of Botany, Govt. College University Lahore
ABSTRACT
Soluble protein contents, produced under salt stress, from in vivo and in vitro sources of Brassica juncea
were evaluated. Salt stress was created by NaCl alone and by combination of NaCl + CaCl2. 2H2O + MgSO4.7H2O
ranging from (control) 0 to 200mM equimolar strength. Results indicated a gradual increase in soluble protein
contents in in vitro samples while in vivo samples showed gradual decrease in protein contents under increasing salt
stress conditions. In vitro studies might establish possible co-relation, between salt stress, water stress, proteins and
antioxidant.
Key words., Brassica juncea var. poorbiraya, soluble proteins, salt stress. water stress, tissue culture, antioxidant.
________________________________________________________________________________________________
INTRODUCTION
Salinity and drought are the twin
environmental stresses which are badly affecting
plant life (Kavi-Kishor et. al., 1995, Skirver & Mundy
1990). Up to 20% of irrigated land in arid and
semiarid regions is affected by salt; the area is
expanding with every passing day (Mühling &
Läuchli, 2003). Of the two factors salinity is the most
important factor which is badly hampering plant
productivity and thus survival of the plants (Eker et
al., 2006). Furthermore the salt tolerance studies, in
plants had shown a close relationship in salt and
water stress phenomena (Munir & Aftab, 2009).
Under salt stress plants had to manage the stress
imposed by low external water potential and high
ion toxicity, due to accumulation of ions, inside the
plants (Romero-Aranda et al., 2006). It has been
observed that salt stress leads to oxidative stress
resulting in the accumulation of Reactive Oxygen
Species (ROS) and free radicals (Azevedo-Neto et.
al.,, 2006; Ashraf 2009), not only increasing the
antioxidant contents but also its activity (Frary et al.,
2010).
High salt concentration either causes an
increase in the N-contents and high protein content
in some glycophytic plants (Abed El- Baki, 1996) or
an increase in soluble proteins (Shaddad et al.,
2005). It has also been reported by Kuznetsov et al.,
(2007) that under environmental stress number of
N-containing compounds accumulates in plants,
amino acids like proline, asparagine and amino
butyric are produced which can play important roles
in osmotic adjustment of plant under saline
conditions (Gilbert et al., 1998).
*Corresponding author:[email protected]
Brassica juncea var. poorbiraya, is a major
oil seed crop of Indo-Pak subcontinent. In vitro
studies for salt tolerance of Brassica juncea has
been reported by Jains et al., (1991 a, b). The salt
tolerance mechanisms exhibited by cells towards
salinity have been shown associated with a number
of factors, which includes ion exclusion, ion
compartments, favourable ion balance, proline
accumulation (Daines & Gould, 1985, Shah et al.,
1990, Yang et al., 1990) production of certain
polyanimes and rapid release of stress ethylene
(McCue & Hanson, 1990) by changing the hormonal
balance (Nilsen & Orcutt, 1996) as growth in saline
soils is controlled by hormonal signals rather than
water relations (Munns, 2002). The levels of several
polypeptides especially some basic proteins had
been reported to be stimulated by addition of NaCl
to culture medium (Yen et al., 1997).
Study was an attempt to develop cell lines
of Brassica juncea var poorbiraya, which can
endure the stress of salt for cultivation in the salt
claimed soils of Punjab.
MATERIALS AND METHODS
Seeds of Brassica juncea var. poorbiraya
were collected from NARC (National Agriculture
Research Council) Islamabad and were germinated
aseptically in Petri plates. The plumule, hypocotyls,
root and leaf were obtained from fifteen days old
seedling. Each ex-plant was incubated on MS
(Murashige & Skoog 1962) medium supplemented
with 3 mg/L NAA + 1 mg/L BAP in the presence of
various concentrations (0, 10, 25, 50, 100, 150, 200
mM) of NaCl alone or a combination of NaCl +
CaCl2. 2H2O + MgSO4.7H2O in equimolar strength.
Physical conditions of cultures were
maintained at 26 ± 1°C under 16 hours photoperiod
342
A. HUSSAIN
at 3K lux of cool white light provided by florescent
tubes. Explants and calli were sub-cultured after
regular interval of 4 to 6 weeks. Protein estimation
was carried out on 12 weeks old callus after
Roenson & Johnstone (1961).
RESULTS AND DISCUSSION
In-vivo soluble protein contents of leaf
decreased gradually with increasing salt stress
(Table 1) while in-vitro results showed gradual
increase in soluble proteins. Salt stress created by
combination of salts were more stress inducing as
compared to when single salt was used in spite of
the fact that both were used in equal strength. Same
trend was observed in plumule, hypocotyl and root.
In all cases in-vivo reading showed reduction in
soluble protein formation while in vitro results
showed gradual increase in the soluble protein
contents.
At the molecular level one of the most
extensively characterized stress response in higher
plants is the synthesis of stress shock proteins
BIOLOGIA (PAKISTAN)
(SSP) as was also observed in this study. The
proteins, being capable of creating more stability in
+
the presence of high concentration of Na in the
cytoplasm, are reported to be synthesized under a
variety of stresses such as high temperature,
desiccation, heavy metals, chilling, anoxia and
salinity (Uma et. al., 1995).
Various plant species, even the different
parts of the same plant are reported to differ in
adopting different strategies for response to different
levels of salt stress in relation to protein
accumulation (Yen et. al., 1997). Thus metabolic
changes make plants respond differently to the
stress. It was observed in Bruguiera parviflora, that
total protein contents of leaf gradually decreased
with increasing NaCl concentration (Parida et al.,
2004). In the same fashion, total protein contents of
tomato cultivars decreased by increasing salt stress
(Zeynep et al., 2010). Same trend was also
observed in Phaseolus vulgaris, but in Phaseolus
acutifolius increasing NaCl concentration does not
affect relative water or protein contents (Yurekli et
al., 2004).
Table 1: Quantitative expression of soluble proteins produced under salt stress created by NaCl and
NaCl + CaCl2 + MgSO4, from in vivo and in vitro sources of Brassica juncea
Strength
equimolar
Control
0
25
50
100
150
200
Salts
used
A
B
A
B
A
B
A
B
A
B
A
B
A = NaCl
L = Leaf,
P = Plumule,
H = Hypocotyl
Rc = Root callus
L
0.50
0.50
0.44
0.41
0.37
0.32
0.30
0.25
0.26
0.12
0.15
0.02
Lc
0.10
0.10
0.15
0.13
0.18
0.16
0.21
0.20
0.35
0.33
0.58
0.52
P
0.15
0.15
0.13
0.11
0.10
0.08
0.09
0.07
0.08
0.06
0.07
0.05
Proteins mg/g
Pc
0.10
0.10
0.56
0.19
0.55
0.49
0.60
0.42
0.67
0.46
0.73
0.50
H
0.12
0.12
0.11
0.10
0.11
0.09
0.10
0.07
0.10
0.05
0.06
0.04
Hc
0.34
0.34
0.41
0.52
0.44
0.52
0.45
0.54
0.48
0.56
0.50
0.65
Rc
0.46
0.46
0.18
0.20
0.22
0.30
0.32
0.41
0.46
0.59
0.91
0.89
B = NaCl + CaCl2 + MgSO4
Lc = Leaf callus
Pc = Plumule callus
Hc = Hypocotyl callus
This variation in metabolic response can be
attributed to the kind and distribution of various
endogenous and/or exogenous hormones. A
correlation might exist between stress and hormone
distribution or vice versa. Increasing salt stress was
observed to increase abscisic acid (Sibole et al.,
1998), IAA level in leaves of Lycopersicum pennelli
behaved similarly (Yurekli, 2004). Moreover as it
VOL. 59 (2)
SOLUBLE PROTEIN CONTENTS FROM BRASSICA JUNCEA
was reported by Hare et al., (1997) that application
of cytokinin activated transcription of stress –
inducible genes in plants. It seems that under stress
genetically modified plants (GMPs) with desired
genes could be promising solution for biotic and
abiotic stresses (Mohamed et al., 2010), which are
affecting food production so badly. In vivo and in
vitro studies showed considerable difference in their
response. This might be due to the application of
exogenous hormones and response of individual
cells of callus (in vitro) rather than response of cells
of organs (in vivo). For stress studies biotic and/or
abiotic it seems desirable to use in vitro cell source
instead of in vivo source. This might establish
possible co-relation, between salt stress, water
stress, proteins and antioxidant.
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