Evaluation of Resistance Improvement of Soybean (Glycine Max (L)

Merr.) against Salinity using Mass Selection and Gene Expression of

Salinity Tolerant

Nini Rahmawati

, Rosmayati

, Delvian

, Mohammad Basyuni

and Hirosuke Oku

Faculty of Agriculture, Universitas Sumatera Utara, Medan 20155, Sumatera Utara, Indonesia

Faculty of Forestry, Universitas Sumatera Utara, Medan20155, Sumatera Utara, Indonesia

Centers of Molecular Biosciences, University of the Ryukyus, Okinawa, Japan

okuhiros@comb.u-ryukyu.ac.jp

Keywords: Soybean, Saline Tolerant Gene, Mass Selection.

Abstract: Saline soils are land that has not been utilized widely for the cultivation of soybean due to the toxic effects

that interfere with the growth of the plant. This research aims to get the soybean genotypes that have salinity

resistant through mass selection methods and gene expression testing of third generation of saline tolerant of

the soybean genotypes that salinity resistant (F3). The research of mass selection conducted on research’s

land with salinity of land 5-6 mmhos/cm and molecular analysis carried out at the Center of Molecular

Biosciences University of the Ryukyus, Japan. Molecular analysis of saline tolerant gene at the root of

soybean selection results F3 and soybean grobogan varieties showed mRNA expression gene DREB5,

GPRP3, P5CS, bZIP, ERF and NHX1 higher in selected soybean that salinity resistant F3 that was treated by

salinity compared to the controls, while the level of gene expression GmCLC1 and PAP3 lower than the

control. Comparison of gene expression levels in soybeans that given salinity stress show there has been an

increased of expression genes that associated with the ability of adaptation of plants to salinity stress.

1 INTRODUCTION

Salinity is one of the important abiotic factors that

limiting the production of soybean in the

world. Reclamation of soil is not an economical

option to increase soybean production that

experiencing in salinity stress. Therefore, genetic

improvement for salt tolerance is a more cost

effective option. Conventional breeding has

contributed significantly to enhancement of soybean

production in the last 50 years. Through conventional

breeding, it is easy to manipulate the inheritance of

qualitative properties that are less sensitive to

environment changes, but quantitative properties like

yield or tolerance to abiotic stress were significantly

influenced by the environment (Pathan et al., 2007).

Some plants develop mechanisms to cope with

these stresses, in addition there are also being

adapted. The majority of the cultivation of plants are

vulnerable and could not survive in high salinity

condition, or even survive, but with yields reduced. A

study of the response plant to salinity is important in

efforts to achieve effective plant filtering technique.

Soybean varieties showed broad spectrum in its

ability to tolerate salt. Filtering of soybean genotypes

have been conducted to identify genetic properties

that show a high tolerance to salt stress. Currently, the

breeding is the main strategy to improve salt tolerance

in soybean (Phang et al., 2009).

Plant breeding in the future will be further lead to

the use of techniques and methodologies of molecular

breeding using genetic markers. The use of

"molecular breeding" has been promising simplicity

of the constraints and challenges in plant breeding

complexity. Selection indirectly using molecular

markers that are bound to the desired properties has

allowed individual studies on the related to the

selection of the double properties and inaccuracy of

measurement due to the expression of properties that

caused by external factors of double genetic locus

(Sudarmi, 2013).

Genes that induced by abiotic stresses such as

high salinity have been found and provide an

important opportunity to improve the tolerant

Rahmawati, N., Rosmayati, ., Delvian, ., Basyuni, M. and Oku, H.

Evaluation of Resistance Improvement of Soybean (Glycine Max (L) Merr.) against Salinity using Mass Selection and Gene Expression of Salinity Tolerant.

DOI: 10.5220/0008387500300037

In Proceedings of the International Conference on Natural Resources and Technology (ICONART 2019), pages 30-37

ISBN: 978-989-758-404-6

properties to high salinity through genetic

engineering approach. Target genes include genes

that encode the enzymes that necessary for the

biosynthesis of various osmoprotectant, enzymes that

eliminated reactive oxygen species, end

embryogenesis proteins (LEA), enzymes to detoxify

and transcription factors (Santoso et al., 2012).

Molecular approach through screening is done at

the plant to determine the genes that are play a role in

the resistance mechanism (Turan et al., 2012;

Ermawati, 2011).

The main mechanism for salt tolerance is to

minimize salt that was taken up by the roots, split

them at the level of cells and tissues so as do not reach

concentrations that contain of toxic on the cytosol in

the leaves. Candidate genes, among others for ion

transport, osmoprotectant, and make plants grow

faster in saline soil. A study of gene expression in the

roots and leaves has been widely reviewed and

various suggestions have been submitted to increase

plant resistance in saline soils (Munns, 2005). Some

researchers have reported several genes that

associated with tolerance soybean against salinity

stress, among others P5CS, GmCLC1, GmSOS1,

GmNHX1, GmbZIP, GmDREB, GPRP3 (Phang et

al., 2008; Peng et al., 2012; Lan et al., 2011; Celik

and Atak, 2011; Gao et al., 2011; Zhang et al., 2009;

Li et al., 2006; Liao et al., 2003; Sun et al., 2013).

2 MATERIALS AND METHODS

2.1 Study Area

Mass selection starts from elders to the fourth

generation (F4) was conducted in the Paluh Merbau

village, Percut Sei Tuan, Deli Serdang district with a

height of ± 1.5 m above sea level and salinity levels

5-6 mmhos/cm that was conducted in May 2011 to

the May 2013.

2.2 Procedures

Molecular analysis carried out in the greenhouse and

laboratory of Center of Molecular Biosciences

(COMB) University of the Ryukyus, Okinawa, Japan,

from October to December 2012. The limit of

selection that used was 10% of the plant

population. And the heritability was analyzed using

the following formula:

Criteria of heritability:

> 0.5 : High

0,2 - 0,5 : Moderate

< 0.2 : Low

(Stansfield, 1991).

Soybeans that will be used for molecular analysis

planted in greenhouse in a tub of plastic for 14 days

and were given the control treatment (without salinity

stress) and the treatment of salinity stress given for 72

hours after 14 HST by using a commercial salt

powder (Red Sea Salt, Houston, TX, USA) by DHL

5-6 mmhos/cm. Media salinity level is done every day

by using a salinity refractometer S/Mill-E (Atago Co.

Ltd., Tokyo, Japan). RNA isolation is done by using

RNA easy Plant Mini Kit (Qiagen). Total RNA

quantitative test using nanodrop, while the qualitative

test using electrophoresis and agarose. The next stage

is the synthesis of cDNA with Hexamer Random

Method using High Capacity RNA-to-cDNA

(Applied Biosystems). Primary will be used for the

analysis of gene expression and housekeeping genes

(for normalization of target genes) were 8 primers

that designed using data from NCBI and software

Genetyx (Table 1).

Table 1: Primary that Designed and Used for Gene

Expressions Analysis.

Gene

Forward primer

sequence [5’-3’]

Reverse primer

sequence [5’-3’]

DREB5

GTGAGCGATGAC

CAGGTTCATG

CATCCAAATC

ATCCCACATGGG

GPRP3

CTGCTGCTGCTT

ATGGTGCTCA

ATGCTTTCCA

TGCTTGCCAAAC

P5CS

GGAAGTGCACAT

ACTGATTCCG

TGGACCCCGA

GCATGAATCCTG

BZIP

CACCAGTTGGTG

ACTGTTCAGA

CCTTTACCAG

CTTTCCCAGTTG

ERF3

GCTCCTGAGATC

TCATCCATGC

GTCACCAGAT

AGCAAGGATTCC

CLC1

TTACATGGGTGG

AGCTGGCTGA

GCGAAGTCCT

ACTTGTCTGAAG

NHX1

GCATCACGATAA

CCACAGATCC

CATCAAACTT

ACGCCACAAGCG

PAP3

GTCGGAGATGGT

GGAAATCAAG

GCCATCATCA

TTGCGATTCCAG

ACTI I

ATTTTGACTGAG

CGTGGTTATTCC

GCTGTCTCCA

GTTCTTGCTCGT

2.3 Data Analysis

Analysis of genes expression were using Real Timer-

PCR that using Fast SYBR®GreenMaster Mix. RT-

PCR result data were analyzed to determine the level

of expression of each variety and treatment.

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Evaluation of Resistance Improvement of Soybean (Glycine Max (L) Merr.) against Salinity using Mass Selection and Gene Expression of

Salinity Tolerant

3 RESULT AND DISCUSSIONS

3.1 Heritability, Selection Limits and

Selection Advancement of Salinity

Resistant Soybean Selection

Selection of soybeans to obtain plants that have the

potential to be developed as saline tolerant varieties

showed the progress of selection is not yet stable, but

observation variable of production character has high

heritability values. Soybean production is determined

by genetic factors, besides that, it also strongly

influenced by environmental conditions, especially

changes in soil salinity levels. Selection of salinity

resistant soybean ranging from elders to the fourth

generation showed the average of each observation

variable of growth and production showed diverse

results (Table 2 and Table 3). Growth and production

of best soybean is reached on the second generation

of selection. Soybean production per plant in the

second generation reached 12.00 g. While on the third

generation of plant production (weight of seeds per

plant) reached its low point of 1.6 g per plant. The

growth and production of soybean increased again in

the fourth generation of selection, where production

per plant was 10.55 g.

The highest soybean production was obtained in

the second generation, namely 12.0 g/plant (Table 2).

This is supported by the environmental conditions

with the level of soil salinity is relatively

stable. While in the third generation, there was a

decrease of the production average due to changes in

soil salinity is very high, reaching 10.36 mmhos/cm.

Ghassemi-Golezani and Taifeh-Noori (2011)

reported the production of soybeans that grown in soil

with DHL 9 dS/m decreased by 354.55% compared

to the DHL 0 dS/m.

Heritability value of the first generation to fourth

generation showed a change (Table 4 and Table

5). Observation variables of production character

such as harvest age, number of pods, number of

containing pods, the number of empty pods, and the

weight of seeds/plants that are tend to have high

heritability values in each generation. Heritability is

one of the most important considerations in plants

evaluating, selection method and crossbreeding

system. More specifically, heritability is part of the

total variation in properties that caused by genetic

differences among observed plants. Heritability is the

ratio between the genetic variance to the phenotypic

variance. Phenotypic variance is influenced by

genetic and environmental factors. Heritability value

of production characters at every stage of selection is

likely to be stable and included in high or moderate

criteria. This shows that genetic factors are more

dominant to controlling the characters. Roy (2000)

stated that the success of selection is determined by

the existing of diversity that controlled by genetic

factors, while Ceccarelli et al. (2007) suggested that

selection on the stress environment conducted in the

target environment so as to maximize the expression

of genes that control the yield capability and plant

adaptability. Marquez-Ortiz et al., (1999) stated a low

heritability value means that environmental factors

have greater influence than genetic factor.

Table 2: The Average of Agronomic Observation Variable

from Elders to Second Generation.

Variable Observation

Generation

Elders

First

Second

Plant Height (cm)

20.30

20.86

34.60

Number of

Branches(branch)

1.60

1.80

5.40

Flowering Age (day)

29.80

29.12

30.50

Harvest Age (day)

68.00

86.53

84.90

Number of Pods (pod)

10.10

3.83

44.40

Containing Pod (pod)

9.80

3.64

42.00

Empty Pod (pod)

0.30

2.40

Seed/Plant Weight (g)

2.90

0.66

12.00

Table 3: The Average of Agronomic Observation Variable

from Third to Fourth Generation and Optimal Condition.

Variable Observation

Generation

Optimal

Condition

Third

Fourth

Plant Height (cm)

29.20

34.10

61.24

Number of

Branches (branch)

3.90

4.00

2.90

Flowering Age (day)

29.00

29.20

35.00

Harvest Age (day)

72.90

73.15

76.12

Number of Pods (pod)

9.80

32.15

60.50

Containing Pod (pod)

8.60

27.7

53.15

Empty Pod (pod)

1.30

4.45

6.35

Seed/Plant Weight (g)

1.60

10.55

14.04

Selection limit and selection advancement of each

stage of selection can be seen in Table 6. The highest

selection limit was on the second and fourth

generation, namely 10.16 g/plant, while the lowest

selection limit was in the third generation, namely

0.21 g/plant. Selection progress also showed diverse

results. Selection regress occurred from the second

generation to the third generation, there was 2.38.

Selection progress rebound in the second and fourth-

generation, namely 3.57 and 3.93.

Changes in saline environments also causes the

selection boundary to the character of production is

not yet stable. The highest selection limit is achieved

by the second generation, namely 10.16 and

decreased to 0.21 in the third generation due to

ICONART 2019 - International Conference on Natural Resources and Technology

increased soil salinity. Selection boundary on the

fourth generation increased again to 10.16 and 1.69

for the selection progress. Selection progress in this

research showed an increase in the third and fourth

generation. Selection progress is a value which is a

parameter of the success of the selection that we

done. Sketchily, the value of the selection progress is

the difference of the initial population and the further

population that has experiencing selection (Idris et al.,

2011). The high value of selection progress is a

manifestation of the value of diversity additive in a

population. The diversity of additive itself is a

necessary component for recurrent selection (Sutoro,

2006).

Table 4: Heritability Value of the First Generation and

Second Generation.

Variable Observation

First

Generation

Second

Generation

Criteria

Plant Height (cm)

0.89

0.45

Number of Branches

(branch)

0.00

Flowering Age (day)

0.00

0.05

Harvest Age (day)

0.39

0.00

Number of Pods (pod)

0.98

0.50

Containing Pod (pod)

0.97

0.50

Empty Pod (pod)

0.88

0.41

Seed/Plant Weight (g)

0.98

0.50

H = High, M = Medium, L = Low

Table 5: Heritability Value of the Third Generation and

Fourth Generation.

Variable Observation

Third

Generation

Fourth

Generation

Criteria

Plant Height (cm)

0.76

0.95

Number of Branches

(branch)

0.00

0.20

Flowering Age (day)

0.26

0.13

Harvest Age (day)

0.81

0.52

Number of Pods (pod)

0.67

0.91

Containing Pod (pod)

0.68

0.93

Empty Pod (pod)

0.00

0.83

Seed/Plant Weight (g)

0.75

0.82

H = High, M = Medium, L = Low

Uncontrolled environmental conditions are one of

the obstacles to assemble soybean that salinity

resistant that has high yield capability. Ashraf (2004)

stated that the direct selection in the field about

quantitative properties which is tolerant to high

salinity still difficult because of uncontrolled

environmental factors. One approach to improve the

efficiency of the breeding program is to adopt a new

selection criterion based on the knowledge of

physiological processes, which is the delimiter from

production plants at the time of exposure of high

salinity.

Table 6: Selection Limit and Selection Progress every

Generation.

Generation

Selection limit Of

Products/Plants

Selection Progress

Elders

2.37

First Generation

0.48

4.68

Second

Generation

10.16

2.38

Third

Generation

0.21

3.57

Fourth

Generation

10.16

3.93

3.2 Molecular Analysis of Saline

Tolerant Genes in Soybean Roots

Resistant to Salinity

Strong interactions between agronomic properties

which are morphological markers with environmental

factors encourage the use of molecular breeding

methodology using genetic markers to support the

selection of soybean that salinity resistant. Salinity

stress caused some changes in physiological

processes, metabolism, and the expression of several

genes that allegedly played an important role in the

adaption response of plants to salinity stress. Santoso

et al., (2012) describes the genes that was induced by

abiotic stresses such as high salinity have been found

and provide an important opportunity to improve the

properties of tolerant to high salinity through genetic

engineering approach. Target genes include genes

that encode the enzymes that necessary for the

biosynthesis of various osmoprotectant, enzymes that

eliminated the reactive oxygen species, late

embryogenesis proteins (LEA), enzyme for

detoxification and transcription factors. This research

examined the expression of several genes that are

responsive to salinity stress, namelyDehydration

Responsive Element Binding Protein 5 (DREB5),

Glycine and Proline Rich Proteins 3 (GPRP3), Δ1-

Pyrroline-5-carboxylate synthetase (P5CS), bZIP

Transcription Factor (ZIP), EREBP/AP2

Transcription Factor (ERF), Gm Chloride Channel 1

(GmCLC1), Gm putative Na+/ H+ antiporter

(NHX1), and Purple Acid Phosphatases 3 (PAP3).

Molecular test of saline tolerant gene at the root

of soybean selection result F3, soybean grobongan

varieties and Burangrang varieties can be seen in

Table 7, Figure 1 and Figure 2. In Table 7 and Figure

Evaluation of Resistance Improvement of Soybean (Glycine Max (L) Merr.) against Salinity using Mass Selection and Gene Expression of

Salinity Tolerant

1, it can be seen that the mRNA expression of

Dehydration Responsive Element Binding Protein 5

( DREB5) genes, Glycine and Proline Rich Proteins 3

(GPRP3), Δ1-Pyrroline-5-carboxylate synthetase

(P5CS), bZIP Transcription Factor (ZIP),

EREBP/AP2 Transcription Factor (ERF) and Gm

putative Na+/H+ Antiporter (NHX1 ) higher (up-

regulated) in selected soybean that salinity resistant

F3that was given salinity treatment compared with

the control treatment, while the level of gene

expression of Gm Chloride Channel 1 (GmCLC1)

and Purple Acid phosphatases 3 (PAP3) lower (down-

regulated) than control treatment. P5CS genes

showed the highest level of expression enhancement

that is 1.75 fold in soybean that treated salinity.

Table 7: Level of mRNA Expression of Salinity Tolerant

Genes (fold) on soybean genotypes that salinity resistant

and Grobogan varieties on Control Treatment and Salinity

Stress 5-6 mmhos/cm.

Gene

Soybean Result of

Selection Salinity

Resistant F3

Grobogan Varieties

Soybean

Control

Salinity Stress

Control

Salinity

Stress

DREB5

0.61

0.94

0.54

0.71

GPRP3

0.40

0.56

0.46

0.49

P5CS

0.76

1.75

1.09

1.30

BZIP

0.35

0.51

0.35

0.25

ERF3

0.59

0.92

0.60

0.68

CLC1

0.63

0.28

0.60

0.38

NHX1

0.44

0.54

0.60

0.38

PAP3

0.43

0.31

0.50

0.34

Shinozaki and Yamaguchi-Shinozaki (1997) also

mentions that some of the genes that responsive to

drought stress, high salinity, and cold temperatures at

the level of transcription (mRNA) have been widely

reported. The amount of mRNA of the gene that is

responsive to salinity decreases if the stress on the

plant is stopped. This is an evidence that these genes

induced by salinity on plant growth environment.

Gene expression that induced by environmental

stress will produce proteins that function as a signal

conductor from the surface of plant cells into the cell,

enzymes that involved in the biosynthesis of

molecules that influence the defense mechanisms

(such as proline, some types of carbohydrates and

polyamine), or a transcription factor that activates the

expression of genes that play a role in plant defense

mechanisms against the stress. Cis and elements trans

involved in gene expression that induced by stress has

been widely analyzed in detail and carefully to ravel

the mechanisms of plants in defend themselves

against environmental stress. Dehydration-responsive

element (DRE) is cis elements that contained in a

promoter and co-regulate the expression of genes in

times of drought stress, high salinity and cold

temperatures. This element has a motive sequences

A/GCCGAC that detected by DRE-binding protein

(DREB) transcription factor (Hardiarto, 2010). One

of the DREB gene subfamily is GmDREB5. This

gene plays an important role in the soybean plant

resistance to drought stress by recognizing the

response of dehydration (Lan et al., 2011). The results

of research showed the level of gene expression

DREB 5 and GPRP3 gene (Glycine and Proline Rich

Proteins 3) in third generation of selected soybean

salinity increases with salinity stress (Figure

1). Penget al. (2012) also reported an increase in gene

expression, particularly in soybean roots that induced

by salinity stress. DREB5 and GPRP3 allegedly play

an important role in mediating independent pathways

of ABA (abscisic acid) of the salinity stress (Phang et

al., 2008; Peng et al., 2012). Decrease in leaf water

potential to stimulate the synthesis of ABA. ABA

concentration in the crown would affect the

expression of genes that determine the synthesis of

proteins (including protein function and protein

regulator). Functional protein that referred is among

LEA proteins, proteinase, detoxifying enzymes, and

synthesis regulator and osmotic controller enzymes

osmotic, namelyproline, betaine and sugar. It appears

that the rapid response of plants is closing of

stomata. While the density of stomata allegedly

changed after the plant experienced continual stress

in a relatively long time (weekly to monthly) other

changes in the crown, leaf senescence, changes in

root growth, vernalization changes, when flowering

and seed filling. There appears to be the link between

a decrease of relative water content of leaves that

followed by an increase in ABA with synthesis of

proline as osmotic control compound (Shinozaki and

Yamaguchi-Shinozaki, 1997; Passiora, 1996;

Swasono, 2012).

MRNA expression of several genes in unselected

soybean Grobongan varieties that treated salinity also

showed an enhancement that is DREB5, P5CS and

ERF3 compared with controls (Table 5 and Figure

2). P5CS genes showed the highest level of

expression of 1.30 fold. While the level of gene

expression GPRP3, ZIP, CLC1, NHX1 and PAP3 in

soybean Grobongan varieties that are subjected lower

salinity treatment than the control.

ICONART 2019 - International Conference on Natural Resources and Technology

Figure 1: Level of Gene Expression in Selection Soybean

that Salinity Resistant F3, Grobogan varieties (**t <0.001,

*t> 0.05 Compared Control Using t test).

Figure 2: Level of Gene Expression in Soybean Grobogan

Varieties (** t <0.001, * t> 0.05 Compared Control Using t

test).

Proline is the main compounds that can protect the

cells through stabilization of proteins and cell

membranes. On a variety of plant species, found

accumulation of proline in salinity stress. Proline

accumulation is regulated by a balance between the

synthesis and catabolism. Δ1-Pyrroline-5-

Carboxylate Synthetase (P5CS) is a key enzyme in

the mechanism of proline. , P5CS is primarily

responsible genes in the biosynthesis of proline and

proline decomposition by the proline dehydrogenase

enzymes that is an enzyme in mitochondria and the

other mechanisms that can increase the concentration

of proline. The level of gene expression is very

important to understand the mechanism of proline

accumulation (Ashraf and Foolad 2007; Lutts et al.,

1999). The results of research showed the expression

of gene P5CS in selected soybean salinity F3

increases with salinity stress, as well as soybean

Grobogan varieties despite an increase in that gene

expression was lower than selected soybean salinity

(Figure 1 and Figure 2). Celik and Atak’s research

(2011) also showed an increase in gene expression

P5CS by increasing the salt concentration in the

growing media, especially in soybeans that salinity

tolerant.

BZIP gene expression Transcription Factor (ZIP)

in selected soybean that salinity resistant also showed

an increase in the presence of salinity stress, while in

soybean Grobongan varieties and Burangrang

decreased due to salinity stress. Gao et al. (2011)

explained that over-expression of GmbZIP1increases

the response of transgenic plant to forming of an

independent ABA and triggering stomatal closure

under stress conditions, thereby potentially increasing

the tolerance to multiple abiotic stresses including

high salt stress. So far, the relationship between salt

stress and stomata is still largely unknown. In this

research, it is known that GmbZIP protein play a

positive role in stomatal closure as effect of salt

induction. The results showed that GmbZIP can act as

a positive regulator of the salinity response by

controlling stomatal closure. Thus, under salinity

stress, excess GmbZIP in transgenic plants may be

able to prevent the entry of Na+ and Cl-, stomatal

closure, and reduce membrane cell damage that

caused by ions and so that increases tolerance to salt

stress.

The results also showed gene expression

EREBP/AP2 Transcription Factor 3 (ERF3) increases

in selected soybean that salinity resistant that

experiencing salinity, in soybean Grobogan varieties

increased expression of the gene is not as high in

selected soybean (Figure 1 and Figure 2). The same

result has also been reported to Zhang et al., (2009) in

the treatment of salt stress, mRNA GmERF3

accumulates after 5 hours of initiation and reached a

peak after 10 hours of initiation. GmERF3 transgenic

tobacco than given 200 mM NaCl stress is able to

maintain the green leaves and roots that can grow

well. Allegedly biotic and abiotic stresses, including

salinity stress can regulate the expression of GmERF3

and abundance of transcriptional activator GmERF3

and most likely regulate transcription, up-regulate

several genes that induced by stress, and their protein

products contribute to increase resistance to stress

conditions. In addition, the accumulation of soluble

sugars and proline as osmolite, plays an important

role in plants that exposed to stress.

In addition to increased osmolite, maintenance of

ion homeostasis is an important strategy in plants to

survive in salinity stress. This mechanism will

prevent the toxicity effect that repercussions of ion

poisoning which causes cytoplasmic organelle

membrane damage. GmNHX1 and GmCLC1 are

genes that regulate ion homeostasis in soybean (Li et

al., 2006). The results of research showed that the

salinity stress also increases the gene expression of

Evaluation of Resistance Improvement of Soybean (Glycine Max (L) Merr.) against Salinity using Mass Selection and Gene Expression of

Salinity Tolerant

Gm Putative Na+/H+ antiporter (NHX1) in selected

soybean that salinity resistant F3, while in soybean

Grobogan varieties and Burangrang, gene expression

is decreased in the presence of salt stress. It shows

gene expression of NHX1 in selected soybean that

salinity resistant F3 more resistant to salinity stress

than soybean Grobogan varieties. Staal et al. research

results (1991) also showed the activities of NHX

tonoplast higher on Plantogomaritima NaCl tolerant

compared to Plantogomaritima NaCl

sensitive. Research Li et al., (2006) showed the

transgenic cell vacuole of GmNHX1-YFPcontained

Na+ accumulation, whereas in control YFD vacuoles

is no accumulation of Na+. Allegedly ion transporter

that located in the plasma membrane and tonoplast

can help the release of ion from cells and ions

compartment within the cell, thereby reducing the

effects of ion poisoning.

GmCLC1 gene encodes a protein of Cl- to the

vacuole, namely by transferring ions from the

cytoplasm into the vacuole to reduce the toxic effects

of salts (Li et al., 2006). GmCLC1 genes also play an

important role to increase plant tolerance to salinity,

reducing damage to the structure of the membrane,

increasing osmotic adjustment and regulation of

antioxidant enzymes in salinity stress conditions (Sun

et al., 2013). Gene expression Gm Chloride Channel

1 (GmCLC1) in selected soybean that salinity

resistant F3, soybean Grobogan varieties on NaCl

stress conditions lower than those in the control

treatment. Instead, the research of Sun et al. (2006)

showed increased expression of GmCLC1 which

increases the tolerance of transgenic plants

Populusdeltoides × P. euramericana 'Nanlin 895'

against salinity stress. This difference is expected

because Cl compartment not only increased because

GmCLC1 expression and the presence of other

mechanism of Cl released that reduce toxic effects on

plants.

Salinity stress not only induces the accumulation

of proline, sugars and other osmolite, but it can also

lead to increased production of reactive oxygen

species (ROS) that can cause damage to lipid

membranes, proteins and nucleic acids that can cause

cell death. Plants develop enzymatic protection

mechanisms that can be scavenged ROS and prevent

the damaging effects of free radicals. Liao et

al. (2003) stated that the GmPAP3 genes alleged

related to soybean adaptation to NaCl stress through

its involvement in the depuration of reactive oxygen

species (ROS). The results showed GmPAP3

expression in selected soybean that salinity resistant

lower than the treatment without salt stress, so it is

necessary to put other efforts to help plants cope with

oxidative stress that caused by ROS. Exogenous

antioxidants applications such as ascorbic acid,

salicylic acid, glutathione, tocoferol, ubiquinone,

ubiquinol, and cysteine are expected to help soybean

plants to overcome the problems of the free

radicals. Research of Sitinjak et al. (2012)

demonstrated the application of ascorbic acid at a

dose of 500 ppm on soybean Grobogan varieties in

saline soil with DHL 5-6 mmhos/cm that produce the

highest production. Based on these results, in the

third stage studies of ascorbic acid is applied to

increase soybean resistance to salinity stress.

4 CONCLUSIONS

Salinity-resistant soybean selection shows success

which is indicated by high and moderate heritability

in production characters and increased selection

progress. Molecular test of the third generation

salinity tolerant gene shows the expression of

DREB5, GPRP3, P5CS genes, bZIP Transcription

Factor (ZIP), EREBP/AP2 Transcription Factor

(ERF3) and Gm Putative Na

Antiporter (NHX1)

were higher in selected F3 salinity-resistant soybeans

that received salinity stress compared to control

treatment, whereas GmCLC1 and PAP3 genes is

lower than the control treatment. Increasing the

heritability and expression of several salinity tolerant

genes in salinity-resistant soybean related to their role

in plant defense mechanisms against salinity stress

shows the potential for selected soybeans to be

developed as a salinity tolerant variety.

ACKNOWLEDGEMENTS

The authors wish to express sincere thankfulness to

Directorate General of Higher Education, Ministry of

Research and Higher Education, Indonesia for

financial support on Sandwichlike Program and Prof.

Hirosuke Oku (Centers of Molecular Biosciences,

University of the Ryukyus, Okinawa, Japan) as

supervisor and facility support in this study.

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Evaluation of Resistance Improvement of Soybean (Glycine Max (L) Merr.) against Salinity using Mass Selection and Gene Expression of

Salinity Tolerant