9(1&2): 205-208, 2015 (Supplement on Rice)

N
IN VITRO SCREENING FOR IMPROVING SALINITY TOLERANCE
IN RICE (ORYZA SATIVA) L.
Save Nature to Survive

QUARTERLY

G. ALAGARASAN*, V. MANI AND CHAVAN NARENDRA RAMESHSING

1
Department of Plant Breeding and Genetics, AC&RI- Killikulam, Tuticorin - 628 252
Department of Plant Molecular Biology and Biotechnology, IGKV, Raipur - 492 012
e-mail: alagarasan.ganesh@hotmail.com
ABSTRACT

INTRODUCTION
Salinity, plays a predominant in role decreasing rice yield is widespread soil
problem in rice growing countries. In response to stress plants are producing
many biomolecules (Prashant Kumar Kar et al., 2013) .The ultimate need for the
development of salt tolerant character in rice is well documented (Flower and
Yeo 1995). Breeding programme for salt tolerance in rice is difficult due to the
involvement of several genes and insufficient knowledge about mechanism(s)
controlling the characters (Yeo et al., 1990). A considerable improvement has
already been made by exploiting the natural variation through conventional
breeding.The other important issue that the increasing demand of rice consumers
in the 21st century cannot be meet only by traditional breeding efforts.In order to
ensure the food security, plant cell and tissue culture techniques are being used
for the genetic improvement and developing salt tolerant lines of rice throughout
the world (Raina 1989). Since the germination of rice genotypes affected by
number of toxic compounds in natural environment(Gupta et al.,2014), tissue
culture technique allows the breeders to create additional variations in rice
genotypes(Lutts et al. 1999 and Sathish et al., 1995).The objectives of this work
was to Standardize the protocol(Abhinash Moirangthem et al., 2014.) for rice
callus culture, In vitro screening of callus under different concentration of NaCl
and identification of salt tolerant callus and regeneration of the callus under
different concentration of NaCl and development of somaclonal varients from the
salt tolerant calli.

MATERIALS AND METHODS

In this, the rice varieties with different adaptation to saline include, FL478, ASD16
and ADT39 were used for the development of salt tolerant high yielding
somaclones. As part of the research, the nutrient medium chosen was Murashige
and Skoog (MS) medium (Murashige, and Skoog1962) with 2 mgl-1 2,4-D for
callus induction (Shukla et al., 2014) and MS +Kin (1mgl/L)+ BAP (1mgl/L)+
NAA (0.1mgl/L) for regeneration.
Yet, thereis a problem here because of changing nature of pH between 5.5 -5.8
was maintained by 0.1N NaOH and moreover carbohydrate source was fulfilled
by adding 30 grams of sucrose (3%) per liter of medium and mixed well. In order
to ensure semi-solid condition 8 grams of agar (0.8%) was added per liter of
medium and melted in the microwave oven for uniform mixing of agar in the
nutrient media. The medium was then poured into the test tube (10 mL each) and
plugged with nonabsorbent cotton to avoid contamination. Test tubes were
autoclaved at 1.01kg/cm pressure at 121C for 20 minutes.
Then medium was carefully taken and allowed to cool at room temperature and
further stored at 10C. Well matured embryo was used as a explant. Seeds were
manually dehusked and surface sterilized with 70% alcohol for 30sec followed
205

This experiment was carried out to critically

evaluate the performance of rice genotypes for
salinity tolerance under in vitro condition.
Genotypes of three different rice varieties
include salt tolerant (FL 478), moderately
tolerant (ADT 39) and susceptible (ASD 16)
were used. Callus was initiated in MS medium
+ 2 mg/L 2,4-D and varying concentrations
of NaCl were added with the medium to
induce salt stress. From statistical analysis it
was revealed that all the genotypes and
treatments and their interaction effect were
significantly different from each other.
According to experimental reports,FL 478
showed significant response for callus
induction and development with 78%
followed by ADT 39 (60 %) and ASD 16
(57%). FL 478, not only for callus induction
but also registered the highest level of callus
development even at high level of Nacl stress.
Similarly, when the callus were transferred to
regeneration media in the same level of Nacl
stress highest level of regeneration was realized
in FL 478 followed by ADT 39 and ASD 16.
Key to the resolution of the screening over
salinity in rice has been a big deal in creating
somaclonal variants of FL 478 and ADT 39
which could be deployed in the field condition
to develop a high yielding salt tolerant variety.

KEY WORDS
In vitro
Callus induction
Regeneration
Salinity screening

Received :
Revised :
Accepted :

09.01.2015
11.02.2015
01.04.2015

*Corresponding author

G. ALAGARASAN et al.,

Table 1: Preparation of stock solution for MS media

Ingredients
Major nutrients (10X)
Ammonium nitrate
Calcium chloride
Magnesium sulphate
Potassium phosphate
Potassium nitrate
Minor nutrients(10X)
Boric acid
Manganese sulphate
Zinc sulphate
Micro nutrients (100X)
Cobalt chloride
Cupric sulphate
Sodium molybdate
Iron stock (50X)
Ferrous sulphate
Na2 EDTA 2H2O
Potassium iodide(100X)
Vitamins & organics(100x)
Nicotinic acid
Pyridoxine.Hcl
Thiamine.Hcl
Glycine

Mg/L

Stock solution

1650
440
370
170
1900

16.50g
4.400g
3.7g
1.7g
100mL
19.00g in 500mL

6.2
22.3
8.6

0.62g
2.23g
0.86g in 250mL

2.5mL

0.025
0.025
0.25

2.5mg
2.5mg
25mg in 100mL

1.0mL

27.8
37.2
0.83

1.393g
1.863g(250mL)
0.083g(250mL)

0.5
0.5
0.1
2

50mg
50mg
10mg
200mg

The results obtained clearly informed that the salt tolerant

genotypes like FL478 recorded higher Callus Induction
Frequency (CIF) at increasing levels of NaCl concentration
than the salt susceptible genotypes like ASD16. Out of these
three genotypes FL478 was highly tolerant to salt stress than
the others (Fig. 1, Fig. 2 and Fig. 3). This conformed the results
of Thach and Pant (1999). This finding revealed that this
technique could be one of the easiest technique to screen the
salt tolerance in rice.

Volume

Similarly it was confirmed by Aditya and Baker (2006). The

Regeneration Frequency (RF) was higher in FL478 in all the
three NaCl stress condition followed by ADT39 whereas the
genotype ASD16 showed lowest RF. At 50 mM NaCl
concentration FL478 showed (47.23) per cent of RF followed
by ADT39 recorded (22.56) per cent and the genotype ASD16
showed the lowest RF of (29.72) per cent. In the 100 mM NaCl

5.0mL

Table 2: Callus induction frequency of three rice cultivars with

three different concentration of growth regulators
Treatment Varieties Plant Growth
Callus Induction
Regulators
Frequency(CIF)

2.5mL

1.0mL

M11

Volume of the solution should be taken/L of medium

M12

by 15% of common bleach for 20 minutes then finally rinsed

several times with the sterile distilled water in laminar air flow
chamber soon, before inoculation into the callus induction
media. Different concentrations of NaCl (0mM, 50mM,
100mM, 150 mM) were added with the callus induction
medium for in vitro salt screening. A view based on salt tolerant
screening and development of salt tolerant somaclones were
done by regenerating the callus with MS +KIN (1mgl-1)+
BAP (1mgl-1)+ NAA (0.1mgl-1) along to the different
concentrations of NaCl.

M13

FL 478
ADT39
ASD16
FL 478
ADT39
ASD16
FL 478
ADT39
ASD16

2,4-D@ 1.5 mg/L

65%

2,4-D@ 2 mg/L

83%

2,4-D@ 2.5 mg/L

57%

RESULTS AND DISCUSSION

In every in vitro screening technique, the callus induction was
the first milestone to achieve. The sharpest significant
differences in callus induction frequency under different
concentrations of saline condition among different genotypes
were noticed (Table 2). Similar trend of responses was observed
in earlier studies (Karim and Zapata, 1994). But at the time of
relative increase of NaCl concentration in the callus induction
and subculture media the callus growth was dramatically
reduced. This showed that the NaCl had an inhibitory effect
on the growth of callus.

Figure 1: Callus induction frequency at different salt concentration

Indicating that, the inability of plant cells and tissues to adjust

with increase of salt over sufficient time periods might be due
to osmotic or ionic shock with increasing NaCl concentrations.
This was also supported by Aditya and Baker (2006). The
callus induction frequency (CIF) decreased with increasing
NaCl concentration. At 50mM NaCl concentration FL478
(42.23%) recorded as better performance for CIF than the
other genotypes. At 100 mM concentration FL478 (28.64%)
performed better and at the 150 mM concentration (13.12%)
gave better performance than others. In all the NaCl
concentration ASD16 was the lowest performing genotype
(Table 3).

Figure 2: Callus regeneration frequency at different salt

concentration

206

IN VITRO SCREENING FOR IMPROVING SALINITY TOLERANCE IN RICE

Table 3: Callus Induction Frequency at different salt concentration

Genotypes

0mM
RI

R II

FL 478
ADT 39
ASD 16

78
60
57

76
63
55

CV = 4.35%;
Genotype
Treatment

SED
0.82916
0.95743

Mean

50mM
RI
R II

Mean

100mM
RI
R II

Mean

150mM
RI
R II

Mean

77
61.5
56

57
35
33

59
36.5
33.5

45
30
25

44
30.5
26

35
25
14

36
23.5
20.5

CD(0.05)
1.80659
2.08607

61
38
34

43
31
27

37
22
21

CD(0.01)
2.53281
2.92464

Table 4: Callus Regeneration Frequency at different salt concentration

Genotypes

0mM
RI
R II

Mean

50mM
RI
R II

Mean

100mM
RI
R II

Mean

150mM
RI
R II

Mean

FL 478
ADT 39
ASD 16

67.5
44.2
39.4

63.35
42.6
37.2

47.2
22.5
24.7

44.7
36.3
25.6

33.7
13.4
13.2

35.35
14.35
14.6

20.5
6.3
5.02

21
7.5
7.5

CV = 4.35%;
Genotype
Treatment

63.2
41
35

SED
1.04246
1.04246

CD(0.05)
2.20995
2.20995

42.2
20.1
26.5

37
15.3
16

21.5
9.2
10

CD(0.01)
3.04493
3.04493

Backer 2006). In the present investigation also the embryogenic

callus from salt tolerant genotypes showed the most tolerance
than the susceptible genotypes. In fact, the salt tolerance can
be considered as a characteristic of embryogenic callus of rice
under in vitro culture conditions. This in vitro technique with
different NaCl stress can also be used as a screening technique
for salt tolerance rather than the field screening would take
more duration.
When regenerated plants are transferred to ex vitro condition
under standardized environmental variables, they can exhibit
non-genetic or epigenetic changes as well as heritable and
genetic variation, but reversible (Karp 1991). Theoretically,
salt tolerance of individual plants could be correlated with
that of its isolated cells and tissues under in vitro condition
(Tal 1994) but this correlation is not always absolute (Casas et
al., 1991). Against the above backdrop, in vitro selected
materials are to be assessed at different growth stages by
growing them under glass house with different levels of salt
stresses (Shanthi et al., 2010). This procedure will help in
understanding the inheritance of salt tolerance from tissue
level to complete new plant level.

FL 478(50mM)

ACKNOWLEDGEMENT
ADT39(50mM)

The authors would like to thank Department of Plant Breeding

and Genetics, AC&RI-Killikulam for providing financial support
and research facilities.

ASD16(50mM)

concentration FL478 showed maximum RF of (33.73) per

cent and the ASD16 recorded the minimum of (13.26) per
cent. While comparing the 150 mM Nacl concentration the
genotype FL478 recorded the maximum of (20.50) per cent
and the genotype ASD16was recorded almost nil RF and
ADT39 recorded (6.32) per cent RF (Table 4).

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