ESTIMATING STABILITY PARAMETERS AND STRESS INDICES USING ELITE SALT TOLERANT RICE GENOTYPES

ACHARYA N.G RANGAAGRICULTURAL UNIVERSITY,
AGRICULTURAL COLLEGE,BAPATLA.
MASTER'S SEMINAR
TOPIC : ESTIMATING STABILITY PARAMETERS AND STRESS INDICES USING
ELITE SALT TOLERANT RICE GENOTYPES
COURSE NO : GP-591
DEPARTMENT : Genetics and Plant Breeding
SUBMITTED TO : SUBMITTED BY:
Dr.T.Srinivas, D.Sri Tejaswi
Professor & Head, BAM-20-25,
Department of GPBR . Department of GPBR.
AGRICULTURAL COLLEGE, BAPATLA
DEPARTMENT OF GENETICS AND PLANT BREEDING 1

Contents:
• Introduction
• Salinity
• Distribution of saline soils in Andhra Pradesh.
• Statistics
• Stability
• Standard Evaluation Score
• Case Studies.

Scientific classification:
• Kingdom : Plantae
• Division : Angiosperms
• Class : Monocotyledonae
• Order : Poales
• Family : Poaceae or Graminae
• Genus : Oryza
• Species : sativa

Introduction:
• Rice is one of the important staple food crops of the world and demand for rice increases with the
increase in the population worldwide.
• Rice is a self - pollinated crop, which consists of diploid chromosome number of 24 (2n = 24).
• It is spectacularly diverse, both in the way that is grown and how it is used by humans.
• But its productivity is decreasing due to various stresses i.e., both biotic and abiotic stresses.
• Various stresses limits productivity of rice worldwide, among abiotic stresses, salinity adversely
affects about 50% of the agricultural land and 20% of the total land area worldwide (Hussain et al.,
2018).

Salinity
• Salinity is the most common abiotic problem which results in huge yield loss.
• Salinity occurs due to sea water intrusion, indistricimate use of fertilizers, poor quality of
irrigation water, increased aquaculture, poor irrigation practice or insufficient irrigation water in
seasons/years with low rainfall.
• Salinity is associated with alkaline soils in inland areas where evaporation is greater than the
precipitation. Increased level of saline ground water caused by over exploitation of natural
resources.
• Salinity affects the respiration and photosynthesis of plants. It decreases biological nitrogen
fixation and soil nitrogen mineralization.

• Salinity is one of the most important abiotic stresses can directly affect on plant growth and
development (Arshad et al., 2012).
• Salinity effects rice growth by reducing germination rate, reduction in plant height, tillering, poor
root growth and increased spikelet sterility.
• Many studies have documented that rice is relatively tolerant to salinity at the germination stage
but seedling stage and reproductive stage are two most salinity-sensitive growth stages, which
directly related to crop yield .
• Salt tolerance is a complex traits influenced by intensity of soil stress and adopted management
practices.
• Breeding salt tolerant rice varieties with stable yield under salinity with lodging resistance is
necessary to mitigate adverse affects of climatic change in coastal areas.

• Salinity is caused due to high accumulation of calcium, magnesium as well as sodium and then
anions such as SO4
-2, NO3
-, CO3
-2, HCO3
- and Cl-.
• Saline soil is the soil containing sufficient soluble salt to adversely effect the growth of most of the
with a lower limit of electrical conductivity of saturated extract (Ece) being 4 dSm-1.
• Salinity adversely effects the quality and quantity of crop produce (Gepstein et. al 2006).
• To maximize productivity of rice under saline soils, there is an urgent need to look for discovery of
genes imparting salt tolerance and their introduction in salt sensitive rice cultivars.

Distribution saline soils in Andhra Pradesh:

Statistics:
• In the world, rice crop is cultivated in an area of 162.06 M. ha and the production is 503.17
million metric tones (USDA 2020/2021).
• In India, area under rice is 44 M. ha and the production is 121.46 million tonnes (USDA
2020/2021).
• In Andhra Pradesh, production of rice is 8.6 million tonnes (www.epwrfits.in).
• More than 800 m. ha of world's land area is salt affected which constitutes more than 6% of world
land area.
• In India, salt affected land accounts for 6.73 m. ha which is expected to increase to 16.2 m. ha by
2050.
• In Andhra Pradesh out of 0.274 m. ha salt-effected soils, 0.196 m. ha area is sodic and 0.077 m. ha
area is saline.

Stability:
• Stability : Ability of a genotype to produce a narrow range of phenotypes, that perform well over
a range of environments.
• It is important to identify genotypes with show consistent performance over different
environments.
• Such identification of stable genotypes can be done by carrying out stability analysis of multi
location trails.
• Stability analysis: An analysis to estimate the adaptability of a genotype, helps in accurate
estimation of yield.
• Stability is one of the key objective in the development of plant varieties that have high yield
potential combined with better and wider adaptability over different agro-climatic conditions.

Score Observation Tolerance
1 Normal growth, no leaf symptoms Highly tolerant
3 Nearly normal growth, but leaf tips
or few leaves whitish and rolled
Tolerant
5 Growth severely retarded, most
leaves rolled; only a few are
elongating
Moderately Tolerant
7 Complete cessation of growth; most
leaves dry; some plants drying
Susceptible
9 Almost all plants dead or dying Highly susceptible
Standard Evaluation Score (SES) of Visual Salt Injury at Seedling Stage:

Score Observation Tolerance
1 Normal growth, spikelet sterility at ≤ 5% Highly tolerant
3 Growth slightly stunted, spikelet sterility at > 5% - 20% Tolerant
5 Growth moderately stunted, ¼ of leaves brown, panicles
partially exerted, spikelet sterility at 21% - 40%
Moderately tolerant
7 Growth severely stunted with about ½ of all leaves become
brown, panicles poorly exerted, highly sterility 41% - 70%
Sensitive
9 Growth severely stunted with almost all the leaves become
brown and affected, panicles not exerted, delayed heads or
papery florets/chaffy panicle with very high sterility at >
70%
Highly sensitive
Standard Evaluation Score (SES) of Reproductive Stage:

CASE STUDY - I
Additive main effects and multiplicative interaction analyses of yield performance
in rice genotypes for general and speciﬁc adaptation to salt stress in locations in
India.
Krishnamurthy et al. (2021)
Journal : Euphytica
NASS Score : 7.53

Materials and methods:
• The present investigation was conducted in 13 salt stress locations across India, representing five
saline environments and eight alkaline environments, during the wet seasons of 2014 and 2015
• Thirteen salt-tolerant rice genotypes used in this study were obtained from the International Rice
Research Institute (IRRI), Philippines, National Agricultural Research and Extension System
(NARES) and Indian Council of Agricultural Research (ICAR) institutes of India.
• They were evaluated across 13 locations during the kharif season of 2014 and 2015, along with
three checks, namely CST 7-1 (coastal salinity), CSR 27 (inland salinity) and CSR 36 (alkalinity).
• Seeds were sown from the last week of May to the first week of June, depending on the location.
Trials were laid out in a randomized complete block design (RCBD) with three replications.
Observations on days to 50% flowering, plant height, productive tillers, spikelet fertility and grain
yield were recorded at each location.

• The best AMMI model family is advised to use the FR-test and assess model diagnosis and to
identify signiﬁcant interaction principal components (IPCs) in the AMMI model using
AMMISOFT software
• AMMI constitutes a model family, with AMMI0 having no IPC, AMMI1 having 1 IPC, AMMI2
having 2 IPC, and so on up to AMMIF (residual discarded).
• The AMMI model equation is:

Result:
• Seven AMMI model families were identiﬁed based on the FR-test at p <= 0.01 (since AMMISOFT is limited to 7
IPCs) for grain yield in the different salinity environments

Winner’’ genotypes and numbers of mega-environments for the additive main effects and
multiplicative interaction (AMMI) model family for rice genotypes evaluated under 13 saline and
alkaline soil conditions during 2014 and 2015.

• The genotype CHK3 won in all AMMI model families, and it won in terms of maximum number
of environments, with 26, 21, 13, 10, 8 and 8 in the AMMI0, AMMI1, AMMI2, AMMI3, AMMI4
and AMMI5 model families.
• The AMMI1 model, genotype CHK3 was the winner genotype in 21 environments, followed by
genotype CHK2, which was the winner in 18 environments
• The genotypes with broad and narrow adaptation suggested that genotypes CHK3, CHK2 and
GN05, with an average grain yield of 3487, 3280 and 3086 kg ha-1, were broadly adapted to the
larger mega-environments which had 21 salt stress locations.
• while genotypes GN13, CHK1 and GN11 are genotypes with narrow adaptation, with an average
grain yield of 2498, 2642 and 2273 kg ha-1 over the environments .

A ranking table showing the top 5 genotypes according to AMMI1 and AMMIF model families for 16 rice
genotype.

AMMI1 mega-environment display for 16 rice genotypes evaluated under 26 environments of
saline and alkaline conditions.

• Genotype GN13 exhibited a grain yield of 3487.9 X 1.29 = 4498 kg ha-1, i.e. 4498 - 2498 = 2000
kg ha-1 (80%) yield advantage over genotypes with broad adaptation in the E01 environment and
3487.9 X 1.19 = 4149 kg ha-1, i.e. 4149 - 2498 = 1651 kg ha-1 (66%) yield advantage over broad
adaptation in the E25 environment.
• Genotype CHK1 showed 3487.9 X 1.003 = 3497 kg ha-1 grain yield, i.e. 3497 - 2642 = 855 kg
ha-1 (32%) yield advantage and 3487.9 X 1.009 = 3518 kg ha-1, i.e. 3518 - 2642 = 876 kg ha-1
(33%) yield advantage over genotypes showing broad adaptation E12 and E14 environments.
• GN11 recorded 3487.9 X 1.05 = 3661 kg ha-1 of grain yield, i.e. 3661 - 2273 = 1388 kg ha-1
(61%) yield advantage over genotypes with broad adaptation then E21 environment.

Conclusions:
• Genotype CHK 3 was found to be the most ideal and best performer in the majority of the
environments.
• The genotypes CHK 2 is found to be the most stable, with above average yields and genotype
GN05 (IR 87952-1-1-1-2-3-B) had high mean yield, stability and used as donor for breeding
programs across salt-affected soils.
• The genotypes GN 13 (IR 87938-1-1-2-1-3-B) and GN 11(IR 87938-1-2-2-1-3-B) showed 60–80%
yield advantage showing that these genotypes could be used for speciﬁc environments of salt-
affected soils in India.

CASE STUDY - II
Effect of salinity and use of stress indices of morphological and physiological traits
at the seedling stage in rice
Krishnamurthy et al. (2016)
Journal : Indian Journal of Experimental Biology
NASS Score : 6.93

Materials and Methods:
• Rice 131 indigenous collections (ICs) were obtained from DRR (Directorate of Rice Research,
Hyderabad) and assessed their responses to salinity stress in hydroponic conditions along with a
sensitive check (IR 29) and a tolerant check (FL 478) in non saline and saline conditions
• The experiment was conducted in a controlled glasshouse at Central Soil Salinity Research
Institute (CSSRI), Karnal under two salinity stress situations, namely salinity stress (EC-10 dS m)
and non saline (EC- 1.2 dS/m) hydroponics at seedling stage
• These rice accessions were grown in hydroponics using Yoshida nutrient solution and screened for
salinity tolerance at the seedling stage (14 days after sowing) using IRRI standard protocol.
• Nutrient solution was salinized by adding NaCl to obtain the desired levels of salinity. Salinity (EC
10 dS/m) was induced at the seedling stage and the desired level of salinity was maintained for the
next 14 days.
• The modified standard evaluation system (SES) was used in rating the visual symptoms of salt
toxicity and genotypes scored after two weeks of salinization.

131 rice accessions and vigour score in Non saline (N) and saline (S) conditions

Estimation of Na and K content
• The shoot samples were oven dried (70°C to constant weight), ground and analyzed by flame
photometer (PFP7, Staffordshire United Kingdom) for sodium and potassium.
• Relative root/shoot length, Stress Susceptibility Index (SSI) and Stress Tolerance Index (STI) can
be calculated

Results:
• Salinity stress caused lesser effects on the morphological characters shoot and root length in three
rice accessions IC 545004, IC 545486 and IC 545215.
• The per cent reduction of root and shoot was minimal in IC 545004 (30.77 and 39.05%, vigor 5)
followed by IC 545486 (34.88 and 27.63%, vigor 5) and IC 545215 (35.07 and 30.22 %, vigor 5)
in saline stress over non stress.
• The low values of stress susceptibility index (SSI) indicate those genotypes which perform well
under stress and respond to the potential environment. Accessions ICs 545004, 545486 and 545215
were amongst the bottom 10 candidates for SSI root length, shoot length and Na' content.
• Higher values of STI indicate the superiority of genotypes due to both high yield potential and
stress tolerance.
• Accession 545004 appeared among the top five contenders for STI root length, shoot length and
vigor, accession 545486 had the lowest SSI for shoot length and Na' content and had maximum
STI for root length amongst all the accessions. Hence, accessions 545486 followed by accession
545004 and 545215 performed well under saline stress and were superior over other accessions.

Comparsion of mean, range of 131 rice accessions in saline (EC-10 dS m) and non saline (EC- 1.2 dS/m)

Correlation :
• Significant and positive correlation was found between root and shoot length in non stress and
saline stress conditions .
• Strong and negative correlation between Na content and K uptake was observed in saline stress as
well as in non stress conditions as well.
• Vigor score was positively correlated with Na content under saline stress.
Stress Indices:
• Relative lengths were positively correlated with STI indices for root and shoot lengths.
• STI shoot length was also found to be strongly correlated to STI root length .
• The SSI root length has a positive Correlations SSI of shoot length .

Correlation coefficient of morphological and physiological parameters of 131 rice accessions
in saline and non saline stress at seedling stage

Correlation coefficient of stress indices morphological and physiological parameters of 131 rice
accessions in saline and non saline stress at seedling stage

Conclusion :
• Three rice accessions IC 545004, IC 545486 and IC 545215 were the best performers on the basis
of the morphological, physiological criteria and stress indices.
• These three accessions performed better in saline stress and can be used in genotypic studies and
in future breeding programs.
• It may be concluded that screening at the seedling stage along with other morphological,
physiological parameters and stress indices is useful to know the salt tolerance potential of rice
genotypes.

CASE STUDY - III
Evaluation of rice genotypes under salt stress at the seedling and reproductive
stages using phenotypic and molecular markers.
Rubel et al. (2014)
Journal : Pakistan Journal of Botany
NASS Score : 6.67

Materials and methods:
• A total of 27 traditional and improved rice genotypes were used including one salt tolerant cultivar
BINA dhan8, nine high yielding varieties (HYVs), sixteen advanced lines and one land race
(Kashrail) of Bangladesh.
• BINA dhan-8 is the salt tolerant variety of BINA, is used as one control (tolerant) whereas BINA
dhan7 is used another control (susceptible), developed at Bangladesh Institute of Nuclear
Agriculture (BINA).
• Three markers RM10772, RM7075, RM296 were selected to evaluate 27 rice genotypes for salt
tolerance. The genotypes having similar banding pattern to BINA dhan8 were considered as
tolerant and similar to BINA dhan7 were considered as salt susceptible.

Phenotypic study of salinity tolerance at seedling stage:
• The genotypes were screen for salt tolerance at seedling stage in hydroponic system using IRRI
standard protocol . Salinized and nonsalinized setups with 3 replications were maintained.
• The evaluation was done nutrient solution was salinized by adding crude salt to obtain desired EC
of 12 dS/m. The modified standard evaluation system (SES) was used in rating the visual
symptoms of salt toxicity (IRRI, 1997).
• This scoring discriminated the susceptible from the tolerant and the moderately tolerant genotypes.
Initial and final scoring was done at 13 d and 22 d after salinization.
• For phenotypic observation plant height, root length and total dry matter was recorded at salinized
and non-salinized conditions.

Screening of rice genotypes at the reproductive stage:
• The genotypes were evaluated for their tolerance to salinity under sustained water bath using IRRI
standard protocol . The experimental design was completely randomized design with three
replications.
• Two setups were maintained: normal and salinized. Pregerminated seeds of rice genotypes were
sown in perforated glass fibre pots. The pots were placed in glass fibre trays with tap water.
• After 2 weeks, seedlings were thinned and the water level was raised to about 1 cm. The pots were
salinized at EC 6 dS /m 3 weeks after sowing and EC was monitored in every week.
• Data were recorded for plant height (cm), days to flowering, days to maturity, number of effective
tillers/plant, number of field grains, number of unfilled grains, total dry mater (g), percent fertility
and grain yield (g).

Genotyping of salinity tolerant rice genotypes:
The banding patterns of 27 germplasm were scored compared with tolerant control and susceptible
control variety and similar banding pattern with BINA dhan8 were considered as tolerant and BINA
dhan7 were considered as salt susceptible.

Results and discussion :
Seedlings at early growth stage in salinized (EC 12 dS/m) and non-salinized condition.

Seedlings stage performance of genotypes under salinized (EC 12 dS/m) and non-salinized
condition.

Performance of rice genotypes under salinized condition (EC 12dS/m) grown in
hydroponic system at the seedling stage
• Maximum reduction of plant height was
observed in the variety S-37 SL-
25(59%), minimum plant height
reduction was observed in Kashrail
(22.6%)
• These results indicated that plant height
was reduced due to salinity stress that
salinity might directly or indirectly
inhibit cell division and enlargement
during plant growing period. As a result,
leaves and stems of the affected plants
appeared stunted.
• Reduction of dry biomass increased
with the increased of salinity level.

Performance of rice germplasm under non-salinized and salinized (EC 6 dS/m) condition at the
reproductive stage.
• Under salt stress (EC 6 dS/m) 27 rice genotypes showed wider variation for yield and yield
contributing characters. In salinized condition the genotypes had less vigorous growth whereas in
non-salinized condition they had been showed vigorous growth

Reproductive stage performance of genotypes under salinized (EC 6 dS/m) and non-
salinized condition.

DEP 45
Genotyping evaluation of rice genotypes using SSR markers
• PBRC-37, Kashrail, PBSAL-655, FL-378, FL-478, STL-15 and BRRI dhan47 had similar band
with BINA dhan8 which is salt tolerant
• S-39 L-15, S-37 SL-37, S-37 SL-32, BINA dhan5, AYT SL-1, AYT SL-7, AYT SL-23, AYT SL-
32, PYT SL-20 and BRRI dhan28 had similar band with BINA dhan7 which is salt susceptible.
• There was no common variety found moderately tolerant in all the tested markers.
The genotypes Kashrail, BRRI dhan-47, FL-378, STL-15, PBRC-37 and PBSAL-655 were
identified as tolerant on the basis of phenotypic (agronomic performance) and genotypic
(reaction with markers) studies.

Banding profiles of 27 rice genotypes using primer RM10772
Banding profiles of 27 rice genotypes using primer RM7075
Banding profiles
of 27 rice
genotypes using
primer RM296.

Conclusions:
• Based on Standard Evaluation Score (SES) for visual salt injury at seedling stage, 8 genotypes
were salt tolerant, 4 were moderately tolerant and the 15 were susceptible.
• At the reproductive stage, 6 genotypes were tolerant and 11 of them were susceptible.
• SSR based marker identified 7 genotypes as tolerant and 10 of them were susceptible for all three
markers compared to two checks.
• 6 genotypes were tolerant in both phenotypic and SSR screening.

CASE STUDY - IV
Genetic analysis of grain yield and its associated traits in diverse salt tolerant rice
genotypes under coastal salinity condition.
Manohara et al. (2019)
Journal : Journal of Cereal Science
NASS Score : 8.45

Materials and Methods:
• The study was carried out during 2018 in the experimental site located in Chorao Island, North Goa
district, Goa State.
• The trial comprised of 82 rice genotypes of which 75 were test entries and seven were check varieties
(CSR 10, CSR 27, CSR 36, CST 7-1, Pusa 44, NSICR 222 and local check variety Goa Dhan 2).
• All these genotypes were received as part of the Salinity Tolerant Breeding Network (STBN) component
of the Stress Tolerant Rice for Africa and South Asia (STRASA) project (IRRI-ICAR collaborative
project).
• The trial was laid out in an augmented block design in three blocks . The genotypes were planted at 20
cm x 15 cm spacing in 8 rows of 3-meter length with a plot size of 4.8 m2.
• Recommended practices were followed to raise a good crop. The observations were recorded on five
randomly selected plants per genotype for the traits plant height, number of tillers, number of productive
tillers, panicle length, grains per panicle, per cent fertility, and 1000 grain weight, For days to 50%
flowering, days to maturity, and grain yield observation as per plot basis.
• The data of 82 genotypes was statistically analyzed using Window stat (Genetic variability and
correlation) and SPSS (Principal Component Analysis) software.

Results:
• CV which measures the dispersion of the variable ranged from 4.7% for days to maturity to 33.37% for
grains per panicle. PCV ranged from 4.14% to 30.62% and GCV from 3.66% to 24.36%. High PCV
and GCV was observed only for grains per panicle.
• Broad sense heritability are classified into low (<30%), medium (30% to 60%), and high (>60%). In this
study, heritability varied from 23.19% for plant height to 85.06% for grain yield.
• Genetic advance as percent of mean in this study ranges from 4.32 for plant height to 39.91 for grains
per panicle.
• High heritability coupled with high genetic advance as per cent mean was recorded for grains per
panicle, per cent fertility, and grain yield. This suggests these traits are less influenced by the
environment, governed by additive gene action and therefore selection can be practiced based on
phenotypic performance.
• High heritability coupled with moderate genetic advance in percent of mean was observed for panicle
length indicating the both additive and non-additive gene effects for control of the characters.
• High heritability and low genetic advance shows the non-additive type of gene action and was observed
in traits days to 50 per cent flowering and days to maturity.

Estimates of mean, variability, heritability and genetic advance for ten characters in 82 rice
genotypes

Phenotypic correlation coefficients among ten yield components in 82 rice genotypes under
coastal salinity condition

Correlation :
• Grain yield recorded a significant and positive association with per cent fertility, grains per panicle ,
test weight, and panicle length. This suggested an increase in grain yield is an association of these
characters.
• Grain yield with days to 50 per cent flowering and days to maturity was significant and negative
indicating a decrease in grain yield in the late-maturing genotypes.
Principal component analysis
• PCA identifies traits that contribute to most of the variation within a group of genotypes.
• PCA revealed characters per cent fertility, number of tillers, number of productive tillers, plant height,
and grain yield contributed for most of the variation in the studied genotypes

Three major principal components and factor loadings (eigen vectors) for different
yield attributing traits

Conclusions:
• The results indicate the presence of adequate genetic variability in the studied genotypes.
Characters grains per panicle, per cent fertility, panicle length, and test weight showed a strong
and positive correlation with grain yield.
• PCA revealed characters per cent fertility, number of tillers, number of productive tillers, plant
height, and grain yield contributed for most of the variation in the studied genotypes.
• Therefore, emphasis may be given for selecting such traits under coastal salinity conditions to
bring genetic improvement in rice.

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