1. Based on in Vitro Seed Germination of Rice Cultivars.
Bhupinder Singh1, K. Raja Reddy1, and Tim Walker2
1Department of Plant & Soil Sciences, Mississippi State University, Mississippi State, MS
2Horizon Ag. LLC, Memphis, TN
Developing a Screening Tool for Osmotic Stress Tolerance Classification
Based on in Vitro Seed Germination of Rice Cultivars.
Introduction
Figures and Tables
Figures and Tables cont.
The first impact of drought in rice (Oryza sativa) phenology occurs during seed germination and seedling emergence that lead to impaired seedling vigor.
The adoption of dry-seeded, delayed-flood culture with alternate wetting and drying irrigation management options to grow and sustain rice is favored in US Mid-south due to environmental and ground-water depletion concerns.
Selecting cultivars for such a system is challenging and information is needed on how the rice seeds respond not only to soil temperature but also to soil moisture conditions.
Polyethylene glycol (PEG) system to impose low osmotic potential to study seed germination traits is ideal to quantify variability among rice genotypes.
Low drought-tolerant
(CDRI < 3.14)
Moderately drought-tolerant
(CDRI = )
High drought-tolerant
(CDRI = )
Cheniere (2.94)
CL111 (3.23)
Cocodrie (3.35)
CL151 (2.96)
Lakast (3.27)
Rex (3.37)
CLXL729 (2.97)
XL753 (3.27)
RU (3.37)
Mermentau (3.02)
CL152 (3.30)
CLXL745 (3.37)
CL142-AR (3.03)
RU (3.45)
RU (3.52)
Low drought-tolerant(CDRI ≤ minimum CDRI SD)
Moderately drought-tolerant (minimum CDRI SD < CDRI ≤ minimum CDRI SD)
High drought-tolerant (minimum CDRI SD < CDRI ≤minimum CDRI SD)
Very High drought-tolerant (minimum CDRI SD < CDRI minimum ≤ CDRI SD)
Objectives
The objectives of this study was to quantify the effect of osmotic potential on germination rate of rice cultivars grown in the Mississippi rice production systems.
To classify the rice cultivars into different drought tolerant groups based on cumulative drought response indices (CDRI) based on seed germination traits.
Materials and Methodology
Table 2. Classification of 15 rice cultivars into low, moderately, and high drought tolerant groups based on cumulative drought response indices (CDRI), along with individual CDRI scores (in parenthesis). Each CDRI is the sum of individual drought response indices (IDRI) of the selected seed-based parameters.
Seed Material:
Rice seed grown at the Delta Research and Extension Center, Stoneville, MS were evaluated for seed germination response to different levels of osmotic stresses developed using polyethylene glycol commonly called PEG (molecular weight 8000).
Results and Discussion
Fig. 1. Germination time course of four rice cultivars representing different tolerant levels at a range of osmotic potentials (0 to -1.0 MPa). Cumulative germination data is represented using symbols and germination time coarse is represented using lines fitted with a three-parameter Sigmoidal function.
Seed Germination Testing:
Study was conducted in vitro at the Environmental Plant Physiology Laboratory, Mississippi State University, MS.
The germination of rice seeds was performed according to the rules described by the Association of Official Seed Analyst (AOSA).
The design was completely randomized block design with two-factors (15 genotypes × 6 levels of treatments) where each genotype was replicated four times with 100 seed per replication.
The amount of PEG required to develop desired osmotic potential was calculated according to the equation developed by Michel (1983).
Seed were treated with Captan 50-WP [cis-N-{(trichlormethyl)thio-4-cyclohexene-1,2- 2.5g kg-1 seed prior to germination test to avoid any fungal infection during the test.
Seed were then placed in a sterilized plastic plates layered with two layers of paper towels moistened with an appropriate concentration of PEG and then incubated (Fisher Scientific, Suwanee, GA) at constant set temperature at 25 ± 1.0 ºC.
The plates were examined after every 2 to 4-hour interval following incubation.
Data for temperature was recorded using three WatchDog Model 100 data loggers placed at the top, middle, and bottom shelves of the incubator.
Seed with radicle length equal to or longer than half of seed length were considered as germinated seed and were discarded subsequently.
The experiment was terminated when no germination occurred for 3 consecutive days or 15 days after incubation. In addition, 100 seed weight was recorded for each genotype and correlated to maximum seed germination.
Time-series for Seed Germination
Three-parameter Sigmoid functions best described the rice seed germination and time series data for all the cultivars studied with R2 ranging from 0.92 to 0.99 (Fig. 1).
Maximum seed germination ranged from 67% (Cheniere) to 94% (RU ) under control conditions without the addition of PEG (Table 1).
Most rice cultivars did not germinate at or beyond -1.0 MPa osmotic potential, and cultivars Cocodrie, Lakast, Rex, and RU germinated to a limited extent at -0.8% MPa osmotic potential (Fig. 1).
Maximum Seed Germination (MSG)
The estimated maximum seed germination percentages varied among the cultivars and linear and quadratic responses were observed in different cultivars (Fig. 2 & Table 1).
Cultivars differed significantly (P < ) for MSG and cultivar by osmotic potential interactions were also significant (P < ).
Cultivars varied significantly for maximum osmotic potential, estimated when seed germination was zero (MSGOPmax), which ranged from MPa (Cheniere) to MPa (CL142 AR) (Table 1).
Seed Germination Rate (SGR)
Seed germination rate (1/t50) declined linearly with declining osmotic potential (Fig. 2 & Table 1).
Cultivars differed significantly (P < ) for SGR and cultivar by osmotic potential interactions were also significant (P < ).
Cultivars varied significantly for maximum osmotic potential, estimated when seed germination rate was zero (GROPmax), which ranged from MPa (Lakast) to MPa (CL142 AR) (Table 1).
Positive and linear correlation was observed between SGR and maximum seed germination (MSG), R2 = 0.73.
Cumulative Drought Response Indices
Among 15 cultivars, Cheniere was least tolerant and RU most tolerant to osmotic stress based on seed germination traits (Table 2).
The identified tolerance among the rice cultivars and the numerical scores assigned will be helpful for the rice producer to select a variety for seeding based on region-specific yield potential.
Curve Fitting Procedure for Seed Germination-time Course Data:
The cumulative seed germination time course data was fit to a 3-parameter Sigmoidal function (Eq. 1) to generate estimates of maximum seed germination percentage (MSG) at time t, the shape and steepness of the function (Grate), and a time to reach 50% of maximum germination (t50).
Y=MSG / {1 + exp[−(t − t50) / Grate]} (Eq. 1)
The MSG and the rate of development (SGR) derived by the reciprocal of t50 were further analyzed using linear and quadratic model functions, which provided regression constants to estimate maximum osmotic potential when seed germination was zero (MSGOPmax) and maximum osmotic potential when seed germination rate was zero (GROPmax), respectively, for all rice cultivars.
Fig. 2. Osmotic potential effects on maximum seed germination and seed germination rate of four representative rice cultivars with different drought tolerance levels.
Cultivars
Seed weight (mg)
MSG(%)
Equation constants R2
MSGOPmax
(MPa)
GRGOPmax a b c
CHENIERE
23.9
67.2
67.0
73.9 -
0.92
-1.19
0.22
0.23
0.96
-0.95
COCODRIE
24.1
96.3
89.3
-25.6
-98.9
-0.83
0.38
0.30
-1.25
LAKAST
25.0
92.9
87.7
-42.9
-108.6
0.94
-0.72
0.26
0.95
-1.44
MERMENTAU
23.4
84.8
84.6
8.4
-87.0
0.97
-1.03
0.28
-1.22
REX
28.1
92.6
89.6
-46.2
-123.6
0.98
-0.68
0.40
0.35
0.99
-1.17
CL 111
25.8
92.4
91.4
-20.7
-108.4
0.33
-1.07
CL 142 AR
27.9
93.8
87.9
-69.5
-204.9
-0.51
0.36
-1.04
CL 151
91.8
88.2
-46.4
-147.5
-0.63
0.31
-1.10
CL 152
22.0
91.2
89.2
-30.5
-119.8
-0.75
0.37
-1.06
XL 753
21.7
79.9
79.1
-17.1
-99.2
-0.81
-1.13
CLXL 729
21.8
82.5
78.0
-46.9
-122.7
0.29
-1.15
CLXL 745
23.2
87.2
85.7
-25.0
-109.7
0.93
-0.78 RU
24.4
94.2
90.8
-43.8
-119.0
-0.71
0.41
-1.24 RU
23.1
91.6
91.5
-9.8
-115.9
-0.85
-1.12 RU
89.4
-5.7
-0.88
0.34
-1.11
Cultivar effect
***
† The significance levels ***, **, and NS represent P ≤ 0.001, P ≤ 0.01, and P>0.05 respectively.
Cumulative Drought Response Indices (CDRI) for Seed Germination:
Individual drought response index (IDRI) for the linear model function for SGR (a, and b), GROPmax, and for MSGOPmax were calculated by dividing the minimum value (Pmin) among all cultivars by the maximum value of specific cultivar (Ps), as shown in Eq. 2.
IDRI = Pmin / Ps (Eq. 2)
where, Pmin is the minimum values among all cultivars for P parameter and Ps is the observed values of the specific cultivars for P parameter.
The rice cultivars were then classified into drought tolerant groups based on cumulative drought tolerance index (CDRI) generated by summation of the IDRI values (Eq. 3).
CDRI = amin / as + bmin / bs + GROPmaxmin / (GROPmaxs +
(MSGOPmaxmin / (MSGOPmaxs) (Eq. 3)
where a, and b are the cultivar specific linear equation constants for SGR generated using regression functions in SigmaPlot 13.
Conclusions
Maximum seed germination and seed germination rate decreased significantly with decreasing osmotic potential among all the rice cultivars.
Cultivars varied in their response to osmotic potential, and seed-based traits provide promise as a screening tool.
Since seed weight was poorly correlated with MSG, SGR, and CDRI, weight-based traits could not be used in drought response screening.
Of the 15 rice cultivars studied, 5 were classified as low, 4 as intermediate, and 6 were tolerant to drought stress.
Future research is needed to test these cultivars under field conditions at specific growth stages and other stresses that affect seed germination traits.
Reference
Michel, B.E Evaluation of the water potentials of solutions of polyethylene glycol 8000 both in absence and presence of other solutes. Plant Physiol. 72:66–70.
Table 1. Seed weight, maximum seed germination percentage (MSG),quadratic equation constants, regression coefficient (R2) for MSG, maximum osmotic potential when seed germination was zero, linear equation constant, regression coefficient (R2) for germination rate, and maximum osmotic potential when seed germination rate was zero of 15 rice cultivars.
Acknowledgement
Data Analysis
The seed parameters were analyzed using one way ANOVA procedure (PROC GLM, SAS 9.4) to determine the effect of osmotic potential and cultivar effects on seed parameters.
We are very thankful to Mississippi Rice Promotion Board for funding for this study and Environmental Plant Physiology Laboratory staff for technical help.