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The Effect of NaCl on the Mineral Nutrient and Photosynthesis

Pigments Content in Pineapple (Ananas comosus) In Vitro Plantlets
B.A. Aziza A. Nur Suraya and H. Sayed M. Zain
Department of Biological Sciences Department of Agrotechnology
Faculty of Science and Technology Faculty of Agrotechnology and Food Science
University Malaysia Terengganu (UMT) University Malaysia Terengganu (UMT)
21030 Kuala Terengganu, Terengganu 21030 Kuala Terengganu, Terengganu
Malaysia Malaysia

Keywords: chlorophyll, carotenoids, tissue cultures, salinity stress

An experiment was conducted to determine the contents of K, Ca, P, Mg and
Na, chlorophyll a, b and carotenoids in pineapple in vitro plantlets under salinity
stress. The cultivars ‘N36’, ‘Morris’ and ‘Sarawak’ were cultured on MS medium
containing 0, 34, 68, 103, 137, 171, 205 or 240 mM NaCl for four weeks. Increasing
amounts of NaCl reduced the K, Ca, P and Mg content while Na increased in all
pineapple cultivars. Levels of chlorophylls and carotenoids generally showed no
change or increased with increasing NaCl to 68 mM and then decreased as NaCl
levels continued to increase. Levels of chlorophylls and carotenoids were much
higher in ‘N36’ than in the other two cultivars.

Salinity is one of the most important environmental stresses limiting plant growth
and productivity. High salt concentration decreases the osmotic potential of the soil
solution creating water stress and causes ion imbalances, toxicity, nutrient deficiencies
and oxidative damage in plants. NaCl in the concentration range 50 to 250 mM inhibits
the growth of most crop plants. However, the response to salinity varies with the species
(Parida and Das, 2005).
Pineapple (Ananas comosus), one of the important horticultural crops in tropical
and sub-tropical regions of the world possesses CAM photosynthesis (Chen et al., 2002).
While the effects of salinity on growth and metabolic processes are well documented in
many plants, knowledge about NaCl induced changes in pineapple is still limited. The
study to determine the level of salt concentrations influencing the mineral nutrient and
pigment contents in pineapple may improve the understanding of tolerance mechanisms,
to the extent they exist, in this species under salinity stress. This could play a role in
improving management practices where pineapple grown in saline areas such as the ‘bris’
area in the east coast of Peninsular Malaysia. Therefore, a study was conducted with the
aim of assessing the effect of NaCl concentrations on K, Ca, Mg, P and Na and
chlorophyll and carotenoid contents in pineapple under tissue culture conditions. Under in
vitro conditions, the cultures are free from deleterious effects of uncontrollable external
environments, technically easy to handle and require minimal management cost as
compared with those under field conditions. Moreover, the salt tolerance assessment
under in vitro conditions using physiological traits could be more effective than the
assessment performed in the field using yield components (Housmand et al., 2005). The
results could also provide an alternative means to formulate growth medium for
preservation of pineapple germplasm under in vitro conditions.


Planting materials of ‘N36’, a hybrid of ‘Gandul’ (‘Singapore Spanish’) and
‘Sarawak’ (‘Smooth Cayenne’), ‘Morris’ (‘Queen’) and ‘Sarawak’ (‘Smooth Cayenne’)
were obtained from the Department of Agriculture (DOA), Pontian, Johor. Explants were

Proc. 7th International Pineapple Symposium 245

Eds.: H. Abdullah et al.
Acta Hort. 902, ISHS 2011

excised from the shoot tips and axillary stem buds of six-month-old pineapple plants for
the establishment of in vitro plantlets. The explants were surface sterilized according to
the standard tissue culture procedure prior to culturing in MS (Murashige and Skoog,
1962) basal medium with 1 mg/L of 6-benzylamino-purine (BAP) (Kiss et al., 1995).
Young shoots were subcultured on MS medium with 0.5 mg/L NAA and 1 mg/L BAP
(Firoozabady and Gutterson, 2003) for shoot proliferation. When axillary shoot tips were
approximately 10-15 mm long they were excised from the three-month-old in vitro
plantlet and transferred into salinity treatment medium. The treatment medium consisted
of MS basal medium with 0, 34, 68, 103, 137, 171, 205 or 240 mM of NaCl added. All
cultures were incubated 28°C under white fluorescent lamps with a 16-h photoperiod
provided for four weeks. Three replications were used for each treatment.
At harvest the plant height and leaf number was determined. Tissues were dried
and measured prior to dry ashing (SIRIM, 1980), the dry ash was dissolved in 0.1 N HCl
and K, Na, Ca and Mg were determined using Inductively Coupled Plasma-Optical
Emission Spectroscopy (ICP-OES). Multi-element Atomic Spectroscopy Standard
Solution 1 (Fluka, Switzerland) was used as the standard. P was determined using yellow
vanado molybdate methods (SIRIM, 1980). Three ml of the solution containing the dry
ash were added with 25 ml of ammonium reagent (0.05 g of ammonium vanadate in
100 ml 1.5 N HNO3 with 1.5 g of ammonium molybdate in 100 ml distilled water) and
shaken for one min. After 30 min, absorbance was read at 410 nm with a UV
spectrophotometer (Bio-Rad, USA). P concentration was calculated based on a standard
Chlorophyll and carotenoids were determined according to method of Bajracharya
(2003). Fresh leaves were homogenized in 80% (v/v) acetone, filtered and made to 50 ml
with acetone prior to measuring the absorbance at 663, 645 and 440 nm. Chlorophyll a, b
and carotenoids were calculated as follows:
 Chlorophyll a = 9.78A663 - 0.99A645
 Chlorophyll b = 21.4A645 - 4.65A663
 Carotenoids = 4.69A440 - 0.268[(20.2A645) + (8.02 A663)


The growth of pineapple in various levels of salinity stress (Table 1) varied with
the cultivar. NaCl up to 205 mM resulted in an increase in the biomass of all cultivars.
The highest dry weight was observed in 68 mM NaCl for ‘N36’ and ‘Morris’, and
103 mM NaCl for ‘Sarawak’. Meanwhile, NaCl higher than 103 mM significantly
inhibited the plant height and leaf number of all cultivars except ‘Morris’. Leaf number of
‘Morris’ was only reduced at levels greater than 171 mM NaCl (Table 1). Hasan and
Abdullah (2007) showed that NaCl below 135 mM did not inhibit the growth of pineapple
plantlets grown in tissue culture. The slower growth rate of crops under salinity stress
might prevent the dilution effect of nutrient elements (Hu and Schmidhalter, 2005).
NaCl at 34 mM and above resulted in less accumulation of K, Ca, Mg and P
compared with control explants with P being somewhat less affected than the cations. In
‘Morris’, P concentrations were gradually reduced until 240 mM NaCl. After a sharp
reduction, P increased in ‘N36’ and ‘Sarawak’ as NaCl increased from 137 to 205 mM.
This increment was significantly lower compared with the control (P>0.05). In most
cases, salinity decreases the concentration of P in plant tissue (Sharpley et al., 1992),
however, some studies indicate salinity either increased or had no effect on P uptake
(Grattan and Grieve, 1999). According to Grattan and Grieve (1994) plant-growing
conditions, plant type and cultivar play a large role in P accumulation. For NaCl at 37 mM
the mineral concentrations in pineapple were not less then 50% of the control (Fig. 1).
Tissue levels of Na increased as the NaCl level in the culture medium increased
(Fig. 1B). Elevated Na concentrations in the media were accompanied by decreases in K,
Ca, and Mg in the tissue (Fig. 1A, C and D) and the results for K and Ca are similar to
result reported previously (Hamed and Ali, 2007). Under elevated salinity the ratios
K+/Na+ and Ca2+/Na+ were much lower than those in the unamended media (Fig. 2).


These ratios varied with the cultivar, with the ratio dropping more rapidly with the
addition of 34 mM sodium in the media for ‘N36’ (70%) and ‘Sarawak’ (62%) than for
‘Morris’ (35%). However, in normal conditions, ‘N36’ and ‘Sarawak’ had higher K+/Na+
and Ca2+/Na+ ratios than ‘Morris’. However, at higher levels of salinity, ‘Morris’ had
higher K+/Na+ ratios than the other cultivars. These decreases could be due to the
antagonism of Na and K at uptake sites or inhibition of uptake processes (Hu and
Schmidhalter, 2001). The K+/Na+ ratio (0.7 to 0.8) seems to indicate the high dry weight
in pineapple (Table 1). A reasonable amount of both K+ and Ca2+ are required to maintain
the integrity and functioning of cell membranes (Wei et al., 2003). Low Ca2+/Na+ ratio
was reported to play a significant role in growth inhibition in addition to causing
significant changes in morphology and anatomy of plants (Ashraf, 2009). In the present
study, the highest growth was exhibited in Ca2+/Na+ ratio lower than the control plant,
which 0.21, 0.08 and 0.09 in ‘N36’, ‘Morris’ and ‘Sarawak’, respectively. In most cases,
high salinity would increase the cytosolic Ca (Hasegawa et al., 2000). There was
relatively little variation in the Mg2+/K+ ratios, which ranged from 0.03 to 0.07 (data not
shown) and the ratios were similar for the three cultivars. The reduction in Mg
concentration might be due to strong competition with Ca (Marschner, 1995).
The contents of chlorophyll a, and b varied significantly when different NaCl
concentrations were applied (Fig. 3). Interestingly, the highest concentrations of
chlorophyll a and b in all cultivars were measured for the treatment with 68 mM of NaCl
(Fig. 3A and B). Among the cultivars, ‘Sarawak’ and ‘N36’ were least affected by
salinity. For ‘N36’, NaCl higher than 137 mM significantly reduced chlorophyll below
that of the control (P>0.05). The reduction in the chlorophyll concentrations might be due
to the decrease in absorption of ions such as Mg (Fig. 1D), which is involved in
chlorophyll formation. Salinity disrupts chloroplasts, possibly by changing the lipid-
protein ratio of pigment-protein complexes or increasing chlorophyllase activity, all of
which could reduce the chlorophyll content (Parida and Das, 2005).
Hypersalinity could also contribute to photoprotection across plant species.
Pineapple cultures produce high amounts of carotenoids in response to salinity stress
(Fig. 3C). Three well-characterized mechanisms, xanthophyll cycle, antioxidant activity
and external foliar waxes are involved. Two carotenoids, zeaxanthin and antheraxanthin,
are epoxidized to violaxanthin, resulting in the harmless dissipation of excess light energy
(Close and Beadle, 2003). In addition, carotenoids are non-enzymatic antioxidants that
scavenge or detoxify reactive oxygen species (ROS) produced by plants under
environmental stress (Ashraf, 2009). Among the cultivars, ‘N36’ shows the highest
carotenoids content and seemed to be the cultivar most tolerant of high, 171 mM, NaCl.
All cultivars in 68 mM NaCl produced the highest carotenoids content as well as growth
and mineral nutrient in tissues. This might be the turning point for salinity tolerance or
toxicity in pineapple. This finding also suggested that carotenoids might be a suitable
biomarker for salinity tolerance in pineapples.

‘N36’, ‘Morris’ and ‘Sarawak’ in vitro plantlets are tolerant to low levels of
salinity (<68 mM). Salinity induced by NaCl altered the mineral nutrient content and
pigment biosynthesis in pineapple tissues. Salinity lower than the toxicity levels increased
the accumulation of minerals in tissues and pigment biosynthesis leading to increased
pineapple growth. Cultivars that showed special characteristics in vitro, such as high
carotenoids content, can be useful as a screen for salinity tolerance, which saves on costly
field experiments. Selected clones should be planted on bris soil and a good irrigation
system may reduce the salinity level soil to one suitable for pineapple growth.

We would like to express our gratitude to the Ministry of Higher Education,
Malaysia for the funding under Fundamental Research Grant Scheme and Universiti
Malaysia Terengganu for the facilities.


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Table 1. The effect of NaCl on the growth pineapple plantlets grown in tissue culture after
four weeks of treatment.

Dry weight Plant height Leaf number

(mg/plantlet) (cm/plantlet) (No./plantlet)
N36 Morris Sarawak N36 Morris Sarawak N36 Morris Sarawak
0 25.3a1 34.0b 82.1b 4.6c 2.1a 2.8bc 8.6d 13.6b 18.0d
34 37.0b 44.4c 87.8b 4.7c 3.0c 3.1c 7.3d 10.6b 17.6d
68 55.0c 55.2d 131.0bc 5.0c 3.6d 3.7d 7.6d 13.6b 20.3d
103 54.6c 47.3c 198.0d 3.7b 3.1c 3.0c 7.3d 11.3b 17.6d
137 47.3bc 44.7c 154.6cd 3.3b 2.5b 2.7bc 5.3bc 11.3b 14.3c
171 46.0bc 43.1c 127.0bc 2.5a 2.0a 2.4ab 5.0b 11.6b 8.0b
205 44.0bc 39.5c 86.4b 2.4a 1.9a 2.3ab 4.3b 6.6a 5.6ab
240 21.6a 23.6a 24.0a 2.3a 1.9a 2.0a 1.6a 5.0a 3.3a
Means in a column followed by the same small letter were not significantly different at p≤0.05 (DMRT).


Potassium (m g/g DW)

0 34 68 103 137 171 205 240
NaCl Treatment (mM)

B 90
Natrium (mg/g DW)

60 N36
50 Morris
40 Sarawak
0 34 68 103 137 171 205 240
NaCl Treatment (mM)

Fig. 1. Effect of NaCl on mineral content in pineapple plantlet cultivar ‘N36’, ‘Morris’
and ‘Sarawak’ after four weeks of treatment. (A) potassium, (B) sodium. The
bar indicates the standard deviation of the mean of the samples. There were
three replicates for each treatment.


Calcium (m g/g DW)
6 Morris

0 34 68 103 137 171 205 240
NaCl Treatment (mM)


D 4
M agnesium (m g/g DW)

0 34 68 103 137 171 205 240
NaCl Treatment (mM)

E 0.035

P hosphorus (m g/g DW)


0.02 N36
0.015 Sarawak


0 34 68 103 137 171 205 240
NaCl Treatment (mM)

Fig. 1. (Continued). Effect of NaCl on mineral content in pineapple plantlet cultivar

‘N36’, ‘Morris’ and ‘Sarawak’ after four weeks of treatment. (C) calcium, (D)
magnesium and (E) phosphorus. The bar indicates the standard deviation of the
mean of the samples. There were three replicates for each treatment.


Fig. 2. The K+/Na+ and Ca2+/Na+ ratios in pineapple plantlets grown in tissue culture after
four weeks of treatment.



Chl a (m g/g FW) 5

4 N36

0 34 68 103 137 171 205 240
NaCI Treatment (mM)

Chl b (mg/g FW)

1.5 Morris


0 34 68 103 137 171 205 240
NaCl Treatment (mM)

Carotenoids (m g/g FW )

1.2 N36
1 Morris
0.8 Sarawak
0 34 68 103 137 171 205 240
NaCl Treatment (mM)

Fig. 3. Effect of NaCl on pigment content in pineapple plantlets cultivar ‘N36’, ‘Morris’
and ‘Sarawak’ after four weeks of treatment. (A) chlorophyll a, (B) chlorophyll b
and (C) carotenoid. The bar indicates the standard deviation of the mean of the
samples. There were three replicates for each treatment.