a master review paper for tissue culture about Tissue culture and influence of salt stress on growth and nutrient acquisition

1. Tissue culture and influence of salt stress
on growth and nutrient acquisition
Abstract
Salinity or salt stress is a serious problem around the world because it affects on crops
production. Because of the differences among plants in their ability to grow under salt
stress, there are many studies to determine the salt tolerant plants, in other words, the
upper level of salt stress that can tolerate and how we can positively influence plant's
growth under salinity conditions. These studies are stand on based tests which interest in
either morphological and physiological changes. These studies clarify that the change in
the chemical parameters of the culture medium could lead to undesirable effects on plant
growth and nutrient elements. Some of these studies were applied in two ways; by
exposing the studied plants to direct salinity shock or gradual salinity shock. Using these
two ways revealed differences in the results among these two ways, and showed that the
gradual shock was more effective in the study of plant tolerance to imposed salinity
stress.
Introduction
Salinity is a serious environmental restriction to crop production around the world
(Flowers and Yeo, 1995) which is the total amount of soluble salt in soil (Marschner
and Termaat, 1995 as cited by Deniz GÖL, 2006) that generally defined as one in
which the electrical conductivity (EC) of the saturation extract in the root zone exceeds 4
dSm−1 at 25°C and has an exchangeable sodium percentage of 15 (Ghassemi et al.,
1995; Szabolcs, 1992 as cited by Foolad, 2004). This problem is widespread in irrigated
Afnan Zuiter.

2. agriculture –where causes from 25% to 50% damage- and in marginal lands associated
with poor drainage or high water tables (Postel, 1989 as cited by Shannon, 1993).
Regional distribution of salt-affected soils (in million hectares) (Dajic, 2006)
Why we face soil salinity?
Soil salinity is founded by many factors: high surface evaporation of water leaving
salts and other substances behind, weathering of native rocks, low precipitation, and poor
cultural practices (Foolad, 2004). In addition to use recycled water, drainage water, or
poor quality water on crops to preserve water quality and the occurrence of cyclic drought
conditions (Shannon, 1993). Furthermore, human-induced processes resulting in the
accumulation of dissolved salts in the soil water (Dajic, 2006).
How soil salinity can affect on crop production?
By causing ion toxicity, water deficit, and/or nutritional imbalance. Also the high
salinity in the root zone severely delay normal plant growth and development,
furthermore, humidity, temperature, light and soil fertility will interact with soil salinity
and affect the crop production (Deniz GÖL, 2006; Gomaa and Gaballa, 2004).
High salinity causes hyperosmotic stress and ion disequilibrium that produce
secondary effects or pathologies (Hasegawa et al., 2000b; Zhu, 2001 as cited by Yokio

3. et al, 2002) also, cytotoxic ions (Na+ and Cl-) are compartmentalized into the vacuole
and used as osmotic solutes (Blumwald et al., 2000; Niu et al., 1995 as cited by Yokio
et al, 2002).
How we can solve this problem?
Elimination of salt from the soil is time consuming and very expensive (Deniz
GÖL, 2006) so the effective way to fix this problem is to produce salt tolerant crops
(Shannon, 1993 ; Deniz GÖL, 2006) by apply conventional breeding programmes, the
use of in vitro selection, pooling physiological traits, interspecific hybridization, using
halophytes as alternative crops (Flowers, 2004) or by using both transgenic applications
and molecular marker technology (Deniz GÖL, 2006) in addition to agricultural
management (Shannon, 1993).
What is the salt tolerant plants?
Salt tolerance is a character that is determined by a complex array of genes and
genetic mechanisms which are influenced in their expression by other environmental
interactions (Shannon, 1993).
How we can distinguish the crop tolerant plants?
Genetic variation in salt tolerance among lines and cultivars has been reported for many
plant species (Shannon et al, 1987 as cited by Shibli et al, 2000) and this offer a method
for selection and breeding programs for salt tolerant crops (Shannon, 1990) so can test
for plant tolerance to salinity by measuring growth responses and tissue mineral content
(Feigin et al, 1987) like; chlorosis, necrosis, and stunting as an indicators for non salt
tolerant plants (Shibli et al, 2000).

4. Basic strategies for the development of salt tolerant plants:
There are many ways to develop a salt tolerant plants, that could by using
conventional breeding and selection between the existing cultivars, by the introgression of
salt tolerant genes from wild progenitors into crops that have retained many of their salt
tolerance traits, also by developing new crops from some of the wild species that
currently inhabit saline environments (halophytes) by breeding and selection for
agronomic characteristics, moreover the using of tissue cultures to select single salt
tolerant cells for plant regeneration or to produce salt tolerance through somaclonal
variation, finally the individual genes for salt tolerance can be identified, isolated and
manipulated across conventional genetic barriers through molecular biological techniques
(Shannon and Noble, 1990 as cited by Shannon, 1993).
But both breeding and screening germplasm for salt tolerance facing several
constraints; different phenotypic responses of plants at different growth stages, different
physiological mechanisms, complicated genotype × environment interactions, and
variability of the salt-affected field in its chemical and physical soil composition (Arzani,
2008).
Briefly, mechanisms of salt tolerance are divided into two main types: those
minimizing the entry of salt into the plant (or at least their accumulation in photosynthetic
tissues) and those minimizing the concentration of salt in the cytoplasm. The regulation of
Na+ uptake and transport across the plasma membranes and tonoplast will be a key factor
determining the plant cell response to salinity stress (Dajic, 2006).

5. Examples of strategies for the selection, breeding and development of
salt tolerant plants (Shannon, 1993).
Some examples of genetic transformation resulting in enhanced salt tolerance (Dajic,
2006)
Why we using cell and tissue culture?
Cell and tissue culture has been used to assess plant reaction to salinity drought
and other stresses (Shatnawi et al, 2009; Arzani, 2008) and it is amenable for screening

6. plant genotypes for salinity tolerance (Shibli et al, 2003) in many plant species because it
offers greater control than in vivo growth conditions (Shilbi and Al-Juboory, 2002) and
provides a consistent plant response to the imposed treatments (Shibli et al, 2003).
Beside that, In vitro cultures have the advantage of clear visibility for monitoring shoot
and root responses in the presence of the imposed stress (Shibli et al, 2001). Moreover,
consistent subculturing to saline nutrient medium permits the identification of the most
tolerant cell lines, ultimately resulting in the isolation of those few salt tolerant types that
were present in a very large original population (Shiyab et al, 2003), also it acts as an
important tool for studying the physiological effects of salinity at cellular level (Shatnawi
et al, 2009). Also cell culture is an approach to selection of mutant cell lines from
cultured cells and plant regeneration from such cells (somaclones) (Arzani, 2008).
Literature review
All plant can be classified into two major groups based on tolerance to salinity;
halophytes, that can tolerate salinity up to 20% of salts and can successfully grow on 2-
6% of salts, and non-halophytes (glycophytes) that exhibit various degrees of damage and
limited growth in the presence of sodium salts, usually higher than 0.01% (Dajic, 2006).
The tolerance in halophytes depends on its capacity to accommodate extreme
salinity because of very special anatomical and morphological adaptations or avoidance
mechanisms (Flowers et al, 1986 as cited by Yokoi et al, 2002), with more explain, the
ability to compartmentalize ions, which in turn depends on regulation of transpiration, the
tight control of leakage of ions through the root apoplast, the nature of the membranes in
the leaf vacuoles, synthesis of compatible solutes such as glycine, betaine and the ability
to tolerate low K:Na ratios in the cytoplasm of mature cells (Flowers et al, 1997).

7. Salt tolerance of several vegetable species as rated by the salinity threshold and percent
yield decline (Shannon and Grieve, 1999).
Screening Procedures
These procedures can be applied by collecting the available informations on salt
tolerant crops, differences between cultivars and closely related species, and sensitivities
to specific ions and environmental interactions, then determining the precise growth stage
which is limiting to productivity and exploring the economical management techniques to
overcome the limitations and finally concerning average salt concentration and
composition of the soil water during sensitive growth periods and the environmental
conditions. Now a selection criterion related to mean yield response in the field should be
done simply by breeding for improved stands through certain tests, for example, ion
selectivity, ion accumulation, osmotic adjustment, organic solutes, and water-use
efficiency (Shannon, 1993).

8. A possible modular design for a process-based plant growth model that
would include salinity effects (Shannon, 1993).
Salinity syndroms
When the plants are exposed to osmotic stress, they exhibit many common
adaptive reactions at the molecular, cellular and whole-plant level, these include
morphological and anatomical alterations and physiological traits associated with

9. maintaining water relations and photosynthesis, in addition to, various metabolic changes,
such as the maintenance of ion and molecular homeostasis, detoxification of harmful
elements and growth recovery, which depends mainly on various signaling molecules,
occur under exposure to salt/drought stress (Dajic, 2006) but not all salinity effects are
negative; salinity may have some positive effects of yield, quality, and disease resistance
(Shannon and Grieve, 1999).
Salinity in general has different effects on plant growth for example; it affects
growth rate by produce smaller leaves, shorter shoot and sometimes fewer leaves by
reducing growth rate (Jocoby, 1994) while it changes the roots’ structure by reducing
their length and mass, therefore roots may become thinner or thicker (Shannon and
Grieve, 1999) and altered leaf color and changes in developmental characteristics
including root/shoot ratio and maturity rate, also the timing of development (Pasternak
et al, 1979). Moreover, its ionic toxicity are generally seen in leaf and meristem damage
or typical nutritional disorder symptoms (Shannon and Grieve,1999). Salinity also
affects seed germination where in the life cycle of plant germination, seedling and
flowering stages are more critical for salt damage (Gomaa and Gaballa, 2004) Maturity
rate also may be delayed or advanced depending on species (Shannon and Grieve,1999).
Salinity and plant growth
Plant growth responses to salinity can vary with: the degree of stress encountered
(mild, moderate or severe), the plant organ, variety or species which is investigated, the
plant developmental stage and the duration of the stress, while the physiological
mechanisms responsible for the growth inhibition induced by salt stress are: turgor
pressure reductions in the expanding tissues, reductions in the photosystem activity of
leaf cells, and direct effects of accumulated salt on critical metabolic steps in dividing and

10. expanding cells. In addition, the two-phase Munns's hypothesis suggests that any varietal
diversity in plant growth responses to salinity will only appear slowly and will be caused
by genotypic differences in rates of salt accumulation. (Neumann, 1997).
There are many equations needed to calculate dry matter, growth value and salt
tolerance ratio (Bekheet et al, 2006)
-Dry matter (%) = Dry weight x100
Fresh weight
-Growth value = _Final fresh weight – Initial fresh weight_
Initial fresh weight
-Salt tolerance ratio = Fresh weight on salt medium__
Fresh weight on salt-free medium
Salinity and shoot growth
The effect of salinity on plants was expressed as reduced shoot dry weight
because the vegetative growth is the most widely used index in studies on salt tolerance
(Cruz et al, 1990 as cite by Shibli et al, 2001). Also the rate of plant growth in nutrient
solution was a function of duration of applied stress (Cooper and Dumbroff, 1973 as
cited by Shibli et al, 2001).
Increased salinity reduced shoot growth (shoot high and dry weight) of African
violet (Saintpaulia ionantha) significantly (Shilbi et al, 2001), and decrease the most
growth parameters (shoot length, leaf number, shoot number, and dry weight for sour
orange (Citrus aurantium) at 150mM NaCl salinity level (Shiyab et al, 2003), while in
apple (Malus domestica Borkh) the microshoot fresh weight, shoot dry weight, shoot
number and shoot height are significantly increased when salinized with less than 100mM
NaCl, and decreased at 100mM NaCl (Shibli et al, 2000).

11. In other hand the Chrysanthemum morifolium can tolerate salinity till 40mM NaCl
and show high decrease in leaf number, dry weight and fresh weight at 300mM NaCl
salinity level (Shatnawi et al, 2009). However, increasing salinity level decreased most
growth parameters (shoot fresh weight, shoot dry weight, and shoot height) of cucumber
(Cucumis sativus L.) which are more pronounced at 75 and 100mM NaCl (Abed
Alrahman et al, 2005).
Also, shoot growth (shoot height and dry weight) of bitter almond (Amygdalus
comnumis) decreased with increasing salinity stress (Shibli et al, 2003). Furthermore, in
muskmelon (Cucumis melo L.) growth was adversely been affected by increased salt level
in both cultures -shoot height, fresh weight and root fresh weight were decreased-, but the
plants did not die, indicating a high degree of tolerance in the tested cultivar. This growth
reduction is accompanied by an osmotic adjustment in tissue cell sap in response to
increasing salt stress (Ztaimeh et al, 2007).
In onion (Allium cepa), number of proliferated shoot buds, average of shoot bud
length, fresh weight (gm) and growth value depressed and decreased as salinity increased
in culture medium. Some shoots remained viable and proliferated few new buds at 6000
ppm salinity in spite of the lowest growth parameters at this concentration. But best result
of salt tolerance ratio was under 2000 ppm salinity because the shoot buds were healthy
and had dark green color (Bekheet et al, 2006).
Salinity and root growth
Salinity affects on root growth in many ways; on root length, root dry matter, root
number, etc. in addition, this influence of salinity differ from plant specie to another, and
differ with same specie (between cultivars) also differ with different salinity
concentrations.

12. For example, root length of cucumber (Cucumis sativus L.) at 50mM NaCl was
increased significantly, but with the increasement of salinity level, there will be a
reduction in root length, root number (Abed Alrahman et al, 2005), while in 'Nabali'
olive (Olea europeae) all cultures had less than 76% rooting naturally when using stem
cutting but by increasing salinity and water deficit of more than 75mM significantly
reduced root number and root length (Shibli and Al-Juboory, 2002).
In other hand, bitter almond (Amygdalus comnumis) rooting was reduced with
increased salinity, as root length and root number, or as rooting percentage went down
from 72.2% [control] to 48.8% [100 mM NaCl] (Shibli et al, 2003). While sour orange
(Citrus aurantium) microshoots gave 80% rooting when grown in vitro at 0, 50 or
100mM NaCl with an average of three roots per microshoot, and no rooting was occurred
on 150mM NaCl or more (Shiyab et al, 2003).
While the root number and dry weight of both 'Roma' and dwarf cv 'Patio' tomato
(Lycopersicon esculentum Mill.) and root length of 'Patio' were decreased with increased
salinity level in the media (Shibli et al, 2007).
Salt tolerance parameters relating relative yield to increasing salinity in the root zone
(Shannon and Greive, 1999).

13. Salinity and germination
Salt tolerant during seed germination stage is a measure of the seeds' ability to
resist the effects of high salinity in the medium (Foolad, 2004). If we talk about
Orobanche cernua, we can find that as salt concentration increased to 75 and 100 mM,
germination percentage was significantly decreased to 14.3 and 9.2%, respectively. The
lowest seed germination (9.2%) was observed in the 100mM NaCl treatment (Al-
Khateeb et al, 2003).
Excessive salt reduce the external water potential which depress water availability
to the seed, so due to osmotic and/or ionic effects of the saline medium the seed
germination will be slower (Foolad, 2004).
Salinity and fruits
Increasing salinity level (more than 7.6 dSm-1
) with tomato "Lycopersicon esculentum
Mill. c.v. Special Pack" significantly decreased the seasonal marketable, non-marketable,
and total tomato yield. While fruit number (with greater than 7.6 dSm-1
) decreased except
for early and late harvesting and number of seeds per fruit decreased (with 18.0 dSm-1
),
the average fruit weight was slightly increased with increasing salinity to 7.6 and 12.8
dSm-1
during all harvesting periods except the early one due to the decrease in fruit
number. Also the fruit acidity was increased with increasing salinity (Shibli, 1993).
Influence of salinity on carbohydrate and sugar
Although salinity elevation caused significant reduction in cucumber (Cucumis
sativus L.) microshoot carbohydrates content at 50 and 75 mM NaCl, it caused
significant elevation at 100 mM NaCl, and the graduate shock caused less carbohydrate
accumulation than direct shock (Abed Alrahman et al, 2005). This accumulation could

14. be due to the increase of carbohydrate synthesis and a reduction in its catabolism
(Noiraud et al, 2000 as cited by Abed Alrahman et al, 2005).
In spite of the elevation of sucrose and fructose in 'Roma' and dwarf cv 'Patio'
tomato (Lycopersicon esculentum Mill.), which the highest sucrose concentration was at
200 mM NaCl in 'Roma' and the fructose at 100 and 150 mM NaCl, sucrose decreased
with in the dwarf tomato 'Patio' and fructose dropped at 50 mM NaCl and started to
increase at 100 and 150 mM NaCl and then decreased at higher concentrations in 'Patio'
(Shibli et al, 2007). While in cucumber (Cucumis sativus L.) the sugar content was
decreased significantly with increased salination in both direct and gradual salination
(Abed Alrahman et al, 2005).
Changes in carbohydrate in response to salinity (Parvaiz and Satyawati, 2008)
Salinity influence on plant proteins and proline
Enzymatic reactions are multiple and complex in responses to salinity, their
influence is related to the change in cytosolic pH which strongly affects the activity of
enzymes. It is generally accepted that enzymes exhibit slightly increased activity under
low concentrations of ions, whereas they start to be inhibited in the presence of NaCl
concentrations higher than 100mM (Dajic, 2006).
Enzymes of halophytes are -in general- as sensitive as enzymes of glycophytes
(Greenway and Osmond, 1972; Flowers et al., 1977 as cited by Dajic, 2006), but some

15. salt tolerant plants exhibit in vitro tolerance of some enzymes to high concentrations of
salts in, and the cell wall enzymes could be more salt-tolerant than cytoplasmic enzymes
of higher plants (Dajic, 2006).
Iranian wheat (Triticum aestivum L.) also underwent the salinity test with its two
cultivars; Sardari and Avland, whereas, superoxide dismutase (SOD) activity in Sardari
was increased starting from 50 mM NaCl, while in Alvand, SOD activity at 50 mM
salinity increased severely, but its activity decreased in higher levels of NaCl content,
namely 100, 150 and 200 mM. Between these levels, there was not a significant
difference (P<5%) with that of control. The same thing was for catalase in the two
cultivars, its activity was increased at 50 mM and decreased again at 100 mM salinity.
Also glutathione reductase (GR) gave the same results. (Esfandiari et al, 2007).
Muskmelon (Cucumis melo L.) plants exhibited significant reduction in tissue
soluble and crude protein contents as NaCl level increased (Ztaimeh et al, 2007). While
in callus cultures of onion (Allium cepa) the total protein gradually enhanced as salt
mixture increased in culture medium and the maximum value of protein content was
recoreded at 6000 ppm of salt mixture (Bekheet et al, 2006).
Effect of salt level (ppm) on total protein (mg/g F.W) of onion tissue cultures (Bekheet et
al, 2006).

16. Whereas soluble protein content of 'Roma' and dwarf cv 'Patio' tomato
(Lycopersicon esculentum Mill.) in response to salinity stress (Shibli et al, 2007). The
same results were collected with cucumber (Cucumis sativus L.), where the salinity cased
a reduction in microshoot content of crude protein at 75 and 100 mM NaCl, but this
reduction was less in the gradual salt shock (Abed Alrahman et al, 2005).
Changes in soluble protein in response to salinity (Parvaiz and Satyawati, 2008)
Leaf proline content in cucumber (Cucumis sativus L.) elevated in both direct and
gradual shock, where the higher elevation was noticed in the gradual shock (Abed
Alrahman et al, 2005). The same was in Chrysanthemum morifolium, the proline content
increased significantly with salinity elevation, where a maximum value of fresh weight
was 80.02 umg/g at 300 mM NaCl and a minimum value of fresh weight was 15.73
umg/g at control 0.0 mM NaCl (Shatnawi et al, 2009).
This accumulation of proline was referred to enhanced activities of the enzyme
involved in proline biosynthesis (Charest and Pan, 1990 as cited by Abed Alrahman et
al, 2005) and to the inhibition of proline oxidase (proline catabolic enzyme) (Yoshiba et
al, 1997 as cited by Abed Alrahman et al, 2005).

17. Salinity and plant hormones
Abscisic acid is an important stress hormone since its concentration increases
when water deficits occur, with its de novo synthesis beginning in the roots, in response
to sensing an insufficient supply of water (Dajic, 2006). ABA can reduce water
consumption, increase water uptake, and mitigate the negative impacts of water deficit by
many ways; First, closure of guard cell stomatal pores upon drought stress and thus the
transpirational water loss is minimized. Second, activation of an array of stress-
responsive genes. Third, certain developmental changes that may make the plants more
adaptive to drought stress which may occur in root development, phase transition, wax
deposition, guard cell patterning and perhaps leaf morphology (Jenks et al, 2007). ABA
will decrease under salinity conditions in halophytes and will accumulate in glycophytes
(Dajic, 2006).
Benzioni et al, 1974 found that, while kinetin with a final concentration 0.1 mg/L
delayed the appearance of necrosis on tobacco leaves for up to 39 days after the
beginning of salination, and with higher concentration (1.0 mg/L) no lesions on leaves
were noticeable until 6 weeks after salination began, but the addition of kinetin to the
saline growth solution reduced shoot growth more than with NaCl alone, and the
dwarfing effect is more pronounced with 1 mg/L kinetin. Also dry matter was pronounced
with low kinetin concentration more than the higher one (Benzioni et al, 1974).

18. The effect of salination and treatment with kinetin on dry weights (g) of tobacco
plants (Benzioni et al, 1974).
(Benzioni et al, 1974).
After saline treatment, ethylene production increased in pepper, tomato, broccoli
and bean shoots, but salinity decreased shoot ethylene production rate in melon, spinach,
and beetroot, while the general effect of salinity in roots was a decrease in ethylene
production, especially in broccoli and bean, except in tomato root, in which a sharp
increase in ethylene production occurred (Zapata et al, 2007). Also we can see that
ethylene accumulation in the headspace of 'Roma' and dwarf cv. 'Patio' tomato
(Lycopersicon esculentum Mill.) was significantly elevated for both cultivars under
treatments with increased salinity compared to the control, and with the increasement of
ethylene, epinasty was increased significantly (Shibli et al, 2007).

19. Ethylene production rate of shoot and roots of control and saline-treate plants 13 days
after transplanting, when plants had been subject to 6 days of 100 mM saline treatment.
Results are the mean ± SE of three replicates (n = 3). Number on bars show fold-increase
or decrease in saline treated plant with respect to control ones (Zapata et al, 2007)
Salinity and plant ions
The quantity of ions that reached the leaves depending upon the ability of the root
system to control the flux of ions to the xylem and the rate of transpiration, while ion
supply to the shoots depends on the ion concentration in the xylem sap and its volume
flux to the shoot which reflects cytosolic ion concentrations (a more sensitive cultivar
having a higher sodium concentration in its cytoplasm than a more resistant variety)
(Flowers and Hajibagheri, 2001).
Concentrations of minerals in muskmelon (Cucumis melo L.) were significantly
affected by elevated salinity levels, where K, Ca, Mg, N and P decreased, with Na
increasing , whereas Na concentration inside tissues was 14 in the in vitro culture and 3.2
times in hydroponic culture at 125 mM NaCl. Furthermore Mg decreased to be 71.4 in
the in vitro culture, and 66.7% in the hydroponic culture at 125 mM NaCl (Ztaimeh et al,
2007).

20. In other hand, sour orange (Citrus aurantium) shoot P, K, and Fe were decreased
with elevated NaCl leveling the direct shock, and this reduction was less in the gradually
shocked culture. Inspite of the reduction in P, K and Fe, Cu content was not significantly
affected by NaCl increasing, and Zn and Na was increased at NaCl level of 200 mM or
higher, also Mn was increased with salinity increasement (Shiyab et al, 2003).
Furthermore, bitter almond (Amygdalus comnumis) mineral acquisition was affected by
the NaCl elevation; whereas Fe, N, P, Ca, and K were decreased with the increasement of
salinity level which more pronounced at 75 to 100 mM NaCl, while Na, Zn, Mn and Cu
were increased with increased salinity level (Shilbi et al, 2003).
Moreover, cucumber (Cucumis sativus L.) microshoot N, Na, K, P, Ca, and Mg
were decreased when NaCl increased, whereas, K and Ca content was significantly
decreased at 75 and 100 mM NaCl in direct shock and had no effect in gradual shock
salinity. Also, P content was decreased significantly at 100mM NaCl in direct shock,
while P content was increased at 50 mM NaCl with gradually salinized. Mg was the same
as P; decreased in direct shock, and increase with gradual shock, while there was no
difference between the direct and gradual salinity for N concentration (Alrahman et al,
2005).
In the selected lines of Trifolium alexandrinum and Medicago sativa, leaves and
stems contained less K+ than those of unselected lines, while in Trifolium pratense, all
plant parts in the selected line again retained less K+. The order of accumulation of K+
was T. pratense > M. sativa > T. alexandrinum. The same thing was for Ca; the T.
alexandrinum selected line had less (not statistically significant) Ca in its leaves at 175
mol mM than the unselected line but did not differ at 0 and 100 mol mM NaCI.
Comparing the species, the Ca contents were in the order T. pratense > M. sativa > T.
alexandrinum. While the Mg contents of the leaves and roots of the two T. alexandrinum

21. lines did not differ significantly, but the stems of the unselected line contained more Mg
than the selected line as same as M. sativa and T. pretense (Ashraf, 1986).
Growing barley (sensitive Triumph and resistant Gerbel cultivars) in 200 mol m-3
NaCl for one month yield 1.5 times NaCl in the roots and shoots of Triumph than in
Gerbel which causing a reduction in K transport to the shoot in Triumph greater than in
Gerbel (Flowers and Hajibagheri, 2001) and in 'Roma' and dwarf cv 'Patio' tomato
(Lycopersicon esculentum Mill.) N, P, and K acquisition was reduced with salinity
elevation, where the reduction was about 68% with 'Roma' and 56% with 'Patio' at 200
mM NaCl, and the same was for Mg, Ca, Fe, Cu and sulfer. In spite of that decrease,
there was an elevation in B, Zn and Mn acquisition with elevation of salinity level (Shibli
et al, 2007).
Na/K ratio and salinity
Reductions in growth with increased salinity might be due to the effect of osmotic
potential (Shiyab et al, 2003) which mean water deficit (Abed Alrahman et al, 2005)
and toxicity of the salt (Shiyab et al, 2003) which means ion toxicity that associated with
excessive uptake particularly of Na+ and Cl- (Abed Alrahman et al, 2005) compared
with K+ (Dajic, 2006) and nutritional imbalance as a result of depressed uptake, shoot
transport and impaired internal distribution of minerals especially K+ and Ca+2 (Abed
Alrahman et al, 2005) in addition to disruption in water structure and a decrease in
hydrophobic interactions and hydrostatic forces within proteins (Dajic, 2006).
Furthermore, Na+ affects the activity of enzymes in two ways; by direct binding
to inhibitory sites or by displacing K+ from activation sites. Additionally, K+ is needed
for protein synthesis, as binding of tRNA to ribosomes requires K+ (Dajic, 2006).

22. Salinity and pH
pH decreased with salinity in wild pear (Pyrus syriaca), bitter almond and 'Spunta'
potato (Shibli et al, 1999) which caused acidic soil environment.
Salinity and electric conductivity (EC)
EC of the medium was increased overtime with increasing salinity in wild pear
(Pyrus syriaca), bitter almond and 'Spunta' potato, and it was correlated positively with
time of the growth (Shibli et al, 1999).
Relation between salinity and irrigation method
The tomato fruit yield per unit of water supplied was on average 1/3 better in drip
irrigation than in the furrow irrigation because, drip irrigation has resulted in an 11%
yield advantage over furrow irrigation, maintained ideal water levels in the soil and
reduced salinity in the root zone when compared with the furrow irrigated plots, and the
possibility of using low salinity water with little or no reduction in yield, thus saving fresh
water for domestic and industrial uses and for irrigation of salt-sensitive crops (Malash et
al, 2008).

23. The effect of irrigation systems, water management strategy and salinity on leaf area
index and plant dry weight of tomato (Malash et al, 2008)
Effect of irrigation methods, water management strategies and fresh and saline water
ratios on water use efficiency in 2001 season (Malash et al, 2008)
Does the salinity cause disorders?
Salinity causes some physiological disorders to 'Nabali' olive "Olea europea", and this is
appeared in stressed culture witth 100 mM or above with 5% shoot tip browning, 10%
stem basal–end browning, and 5% chlorosis (Shibli and Al-Juboory, 2002).

24. Influence of salinity on acclimatization
Increasing salinity to 75% was very effective at increasing survival percentages of
'Nabali' olive "Olea europea" under in vivo acclimatization, and with higher salinity (100
mM or more) the percentage of survivals is decreased (Shibli and Al-Juboory, 2002) but
in sour orange (Citrus aurantium L.) the rooted plantlets were successfully acclimatized
with 90% survival and the acclimatized plants were successfully grown in the greenhouse
with no differences in survival percentages among plantlets which came from the
different salinity treatments (Shiyab et al, 2003).
Suspension cell culture and salinity
In 'Nabali' olive "Olea europea" cells were able to stand up to 75 mM without
significant differences in their growth from the control but at higher salinity or water
stress treatments the growth parameters decreased significantly and cells from the
treatment of 100 mM or more showed more cell aggregates, with the compacted cell
clumps (Shibli and Al-Juboory, 2002).
Does the addition of phosphorous mitigate the salinity effects on plants?
Although the effects of salinity on tomato (Lycopersicon esculentum Mill. Cv
Riogrande) that represented by reduction in shoot weight, plant height number of leaves
per plant, and significant increase in leaf osmotic potential and peroxidase activity were
not influenced by addition of phosphorous (Mohammad et al, 1998), phosphorous
mitigated the adverse effects of increasing salinity like increasing shoot height, dry mass,
leaf osmolarity, root umber, root length, nutrient ions (N, Ca, Fe, K and Mg) and reduce
Mn, Zn and Cu uptake in African violet (Saintpaulia ionantha) (Shibli et al, 2001) while
increasing phosphorous level enhanced root growth through increasing both root length

25. and root surface area at all salinity levels in tomato (Lycopersicon esculentum Mill. Cv
Riogrande) (Mohammad et al, 1998).
Physiological adaptations to salinity
Plants can adapt naturally to environmental conditions by many ways and mechanisms;
ion exclusion, osmotic adjustment and ion regulation, glandular ion excretion, and ion
compartmentation with compatible solutes. These are the plant mechanisms to make
adaptation with salinity as mentioned and explained by (Marcum, 2008).
1- Ion Exclusion
While the major causes of plant growth inhibition under salinity stress are osmotic
stress (osmotic inhibition of plant water absorption), and specific ion effects, including
toxicities and imbalances, monocots (including Poaceae) tend to exclusion saline ions
from shoots, thereby minimizing toxic effects.
2- Osmotic Adjustment and Ion Regulation
Maintenance of cell turgor and plant growth requires sufficient increase in sap
osmolality to compensate for external osmotic stress (osmoregulation, or osmotic
adjustment) which may occur in several mechanisms: selectivity for K+ over Na+ may
occur by selective K+ absorption-vacuolar Na+ compartmentation in root cortical cells or
endodermis, or by selective saline ion excretion through specialized salt glands or
bladders. Monocots -including Poaceae- tends to decrease shoot water content and that is
commonly observed in grasses under salinity stress, though slight increase in shoot
succulence under moderate salinity has been noted in some halophyte grasses .

26. 3- Glandular Ion Excretion
Salt glands or bladders which eliminate excess saline ions from shoots by excretion are
characterized by cutinized cell walls, surrounded by papillae, and they are present in a
number of salt-adapted species; several families of dicotyledons, e.g. Frankeniaceae,
Plumbaginaceae, Aviceniaceae, and Tamaricaceae as multicellular epidermal salt glands,
and Poaceae as a basal cell, attached, or imbedded, into the leaf epidermis, and a cap cell.
Excretion is typically highly selective for Na+ and Cl-, though other ions may be excreted
in minute amounts, such as K+, Ca2+, and Mg2+. Increasing media salinity generally
stimulates excretion up to an optimal level, above which excretion rate may decline. Na+
and Cl- excretion rates were negatively correlated to shoot concentrations, but positively
correlated to leaf salt gland density and salinity.
4- Ion Compartmentation and Compatible Solutes
Salt-tolerant plants growing under saline conditions must restrict the level of ions
in the cytoplasm because enzymes of both glycophytes and halophytes have similar
sensitivities to salt, could be inhibited at concentrations above 100-200 mM
(approximately 8-17 dS m-1). Salt tolerant grasses utilize inorganic ions for a large part
of their osmotic adjustment under saline growing conditions, so salt tolerant plants that
successfully accumulate saline ions for osmotic adjustment above concentrations of 100-
200 mM do so by compartmentalizing them within the vacuole, which typically makes up
90 to 95% of mature plant cell volume.

27. Determinants of salt tolerance related to the main adaptive strategies (salt tolerance and
salt avoidance) of plants exposed to salinity (Dajic, 2006)
Conclusion
Salt tolerant plants are existed naturally or by using biotechnology techniques.
Plants could be salt tolerant as it is their habitat, or acquisite it by exposing the plants to
gradual salinity. If we have one plant of each specie can resist salinity, we can have a line
of this new strain by tissue culture propagation, and then we can face the water quality
problem in plant irrigation. Salt stress is a real problem that should be faced and solved
very soon for human survive, because this problem affects on whole plant; at
morphological and physiological levels.

29. Salt tolerance of crops (cereals, forage crops, vegetables and fruit crops) (Dajic, 2006).

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