Comprehensive screening and selection of okra (Abelmoschus esculentus) germplasm for salinity tolerance at the seedling stage and during plant ontogeny

Comprehensive screening and selection of okra ( Abelmoschus esculentus ) germplasm for salinity tolerance at the seedling stage and during plant ontogeny 1 first-author 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:asifpbg@uaf.edu.pkknnike@yahoo.com †E-mail:asifpbg@uaf.edu.pkknnike@yahoo.com asifpbg@uaf.edu.pk 2 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:asifpbg@uaf.edu.pkknnike@yahoo.com †E-mail:asifpbg@uaf.edu.pkknnike@yahoo.com asifpbg@uaf.edu.pk Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan Institute of Horticultural Sciences, University of Agriculture, Faisalabad, Pakistan The okra germplasm was screened for salinity tolerance at the seedling stage and during plant ontogeny. Substantial variation existed in okra for salinity tolerance at the seedling stage. An 80 mmol/L NaCl concentration was suitable for discriminating tolerant and non-tolerant okra genotypes. The pooled ranking of the genotypes, based on individual rankings for each trait (root and shoot length, germination percentage, and relative Na + and K + ) in individual NaCl concentrations, was effective for selecting tolerant genotypes. Genotypes selected at the seedling stage maintained their tolerance to NaCl during plant ontogeny, suggesting that screening of the germplasm entries and advanced breeding materials for salt tolerance at the seedling stage is effective. Among 39 okra genotypes, five were identified as the most tolerant genotypes and showed potential for use in breeding programs that focus on the development of salt-tolerant, high-yield okra cultivars. Salinity tolerance Okra Growth stage Genetic variation Mechanism Potassium to sodium ion ratio (K + /Na + ) Introduction 1 Okra ( Abelmoschus esculentus L.) is a popular vegetable among both the consumers and farmers because it is rich in vitamins and minerals (Oyelade et al., 2003 2003 ). Although the area under okra has progressively increased during last few years, there is a decreasing trend in its yield per hectare (Anonymous, 2008 2008 ). Among identified biotic and abiotic stresses, salinity has been the key factor responsible for yield reduction (Khan et al., 2001 2001 ). Salinity is defined as the amassment of water-soluble salts in the top layer of soil to a level that drastically affects crop production (Rengasamy, 2002 2002 ). Salinization of soils is one of the serious problems for irrigated agriculture, and the situation is most severe in tropical regions (Khan et al., 2003 2003 ). The ion toxicity has negative impacts on plant growth and development due to low water potential within the local root environment (Munns and Tester, 2008 2008 ). Ions of sodium (Na + ) and chlorine (Cl − ) ( > 40 mmol/L) can be toxic to plants at levels that result in an imbalance in plant nutrition due to decreased nutrient uptake and transport to new shoots (Cramer, 2003 2003 ; Tester and Davenport, 2003 2003 ; Munns and Tester, 2008 2008 ). High salinity modifies plant metabolisms, which results in altered plant morphology; cultivar type, duration, and intensity of stress determine the extent of morphological modification (Khan et al., 2003 2003 ; Munns and James, 2003 2003 ). The excess salt can cause decreased seed germination, seedling growth, and dry matter production (Nautiyal et al., 1989 1989 ; Singh et al., 1989 1989 ; Janila et al., 1999 1999 ). Salinity also induces Fe 2+ , K + , and Ca 2+ deficiencies (Singh et al., 2004 2004 ), resulting in yield losses (Hunshal et al., 1991 1991 ). Okra is cultivated in sub-tropical areas, and its cultivation is hampered in saline and sodic (having more Na + ) soil as it is sensitive to such soils. Physiological mechanisms like ion exclusion, ion accumulation, production of compatible solutes, and osmotic adjustment are considered to be associated with genetic variation in salt tolerance. Characteristics of a salt-tolerant variety include Na + exclusion, potassium to sodium ion ratio (K + /Na + ) discrimination, retention of ions in the leaf sheath, tissue tolerance, and ion partitioning into different-aged leaves. Osmotic adjustment, transpiration efficiency, early vigor and early flowering leading to a shorter growing season, and the increased water use efficiency are also the features of typical salt-tolerant varieties (Colmer et al., 2005 2005 ). Salinity affects plant growth most dramatically during developmental stages, and the sensitivity of different crops varies from one growth stage to another (Shannon, 1984 1984 ). Such developmental effects are observed in rice (Akbar and Yabuno, 1974 1974 ), sorghum (Maas, 1986 1986 ), and cowpeas (Maas and Poss, 1989 1989 ). Thus, plant responses to salinity are influenced by its ontogenic stage at the time of stress (Shannon, 1985 1985 ). This suggests that the ability of a plant to respond to salt stress depends upon genes that are functioning at a particular stage of development (Foolad and Jones, 1991 1991 ). For varietal improvement to be effective for salinity tolerance, information about the effect of salinity on all phases of plant growth is essential (Azhar and McNeilly, 1989 1989 ). One strategy to address the problem of salt in soil is identification and exploitation of existing or created genetic variability within plants, specifically, that which gives rise to plant phenotypes capable of sustaining a reasonable yield within salt-affected soils (Ashraf et al., 2003 2003 ). Effective screening protocols depend on an understanding of the response mechanism at different plant growth stages under saline conditions. Knowledge about these mechanisms assists breeders in looking for plants of economic importance with improved salt tolerance (Flowers and Yeo, 1995 1995 ; Khan et al., 2003 2003 ). Okra pod yield is a function of physiological and agronomic characters acting singly or in interaction with each other (Adeniji and Peter, 2005 2005 ). The ultimate yield of a crop depends upon the interaction between its genetic makeup and environmental factors faced during its entire growing period (Humphreys, 1989 1989 ; Ashraf and Fatima, 1994 1994 ). Keeping in view the nutritional and economic importance of okra and deteriorated soil and water quality, a comprehensive sodium chloride (NaCl) screening study was conducted to gain information on the extent of genetic variation in the okra genotypes for salinity tolerance at all the growth and reproductive stages. Materials and methods 1 Assessment of genetic variability for NaCl tolerance in germination and seedling stages of okra 2 The genetically pure selfed seed of 39 okra genotypes (Table 1 1 ) were collected from different sources and subjected to screening for salinity tolerance at the seedling stage. Seeds were surface sterilized in a solution of 2% (0.02 g/ml) hypochlorite for 5 min and washed with distilled water. Individual seeds were planted in triplicate in pots filled with sterilized coarse river sand containing one of six NaCl concentrations as per a randomized complete block design (RCBD). Two pots were used per replication of each genotype. NaCl concentrations of 0, 40, 60, 80, 100, and 120 mmol/L were prepared in half strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950 1950 ). Growth was carried out in a growth chamber adjusted at (28±1) °C, with a relative humidity between 75%–80% and a photoperiod of 16 h. Hoagland’s Solutions were added at 3-d intervals. Data for different seedling traits were recorded from three-week-old seedlings. Seedling traits included percentage of germination, root length, shoot length, Na + and K + concentrations, K + /Na + ratio. The means of all observations were calculated and subjected to analysis of variance (ANOVA) and Pearson’s correlation using a general linear model of SPSS 12 (SPSS, Chicago, IL) and MVSP 3.1 (Kovach Computing Services, Anglesey, Wales), respectively. Box-whisker analysis was performed to assess the level of dissimilarity among accessions using Microsoft Excel (QI Macros). A phenogram was constructed with Euclidian distances based upon the response of okra genotypes to different NaCl concentrations. RL: root length; SL: shoot length; GP: germination percentage; RRL: relative root length; RSL: relative shoot length; RGP: relative germination percentage; RNa + : relative Na + ; RK + : relative K + ; RK + /Na + : relative K + /Na + . The rankings were based on evaluation of each parameter with number 1 being the most tolerant 2 Genotype 14 Rank Overall RL SL GP RRL RSL RGP Na + K + K + /Na + RNa + RK + RK + /Na + Cumulative Acc. No. 019232 1 1 4 5 1 1 2 13 3 8 2 3 2 45 Acc. No. 000010-10237 2 7 3 13 4 8 14 1 13 3 1 5 1 73 Chinese Red 3 3 1 3 5 5 12 8 6 6 13 6 11 79 Ikra III 4 2 11 4 13 10 6 11 2 1 6 8 5 79 Acc. No. 015371 5 4 7 14 9 12 5 7 4 2 12 1 8 85 Punjab Selection 6 8 10 8 6 2 1 5 8 10 8 9 10 85 Parbhani Kranti 7 11 8 2 7 3 7 6 14 13 7 7 7 92 Acc. No. 019231 8 14 6 6 10 14 9 2 12 4 3 10 4 94 Ikra I 9 6 14 7 11 11 11 3 10 11 4 4 3 95 Acc. No. 019223 10 12 5 10 8 6 8 10 5 7 5 13 6 95 Acc. No. 019236 11 13 12 9 3 9 3 14 1 5 14 2 14 99 Acc. No. 019221 12 5 2 11 2 4 13 9 7 12 10 14 13 102 Acc. No. 019217 13 10 13 1 14 13 4 4 11 9 11 12 12 114 IN-1048 14 9 9 12 12 7 10 12 9 14 9 11 9 123 Clemson Spineless 15 19 19 15 20 17 19 24 17 19 21 18 18 226 Acc. No. 019215 16 16 27 22 21 26 28 21 20 20 18 17 16 252 Acc. No. 015373-10811 17 24 20 25 26 16 29 15 22 17 15 26 17 252 University Okra 18 26 18 21 15 31 16 23 25 22 19 22 15 253 Acc. No. 019224 19 20 16 30 17 15 22 19 19 16 34 19 32 259 Acc. No. 019228 20 17 22 19 22 18 17 34 18 26 25 21 21 260 Acc. No. 019216 21 15 17 18 16 21 25 17 28 27 23 32 26 265 Acc. No. 000004 22 25 23 26 25 23 18 25 24 18 26 20 20 273 IN-97 23 23 29 31 18 25 33 20 15 15 30 16 28 283 Green Wonder 24 18 15 17 23 27 20 18 32 30 31 31 34 296 Acc. No. 019211 25 22 24 23 19 19 21 31 27 29 29 24 29 297 Pusa Green 26 21 33 20 30 22 15 26 23 21 35 29 33 308 Ikra IV 27 31 32 32 24 30 24 28 21 24 27 15 23 311 Acc. No. 019218 28 34 21 24 31 29 31 16 31 28 20 27 22 314 Acc. No. 019235 29 29 28 16 32 24 23 27 26 23 33 30 30 321 Sabzpari 2001 30 33 26 34 34 20 26 35 16 25 24 28 24 325 Acc. No. 019234 31 35 35 27 27 34 30 29 30 33 16 25 19 340 Acc. No. 019230 32 32 25 28 28 28 27 33 29 32 22 33 27 344 Acc. No. 000026-10496 33 27 34 35 29 32 34 22 33 31 28 23 31 359 Pusa Sawani 34 30 31 33 33 35 35 30 34 34 17 34 25 371 IN-89 35 28 30 29 35 33 32 32 35 35 32 35 35 391 Acc. No. 019225 36 36 37 36 38 36 36 37 36 36 38 37 38 441 Acc. No. 015380-10934 37 38 39 37 37 39 37 36 37 37 36 36 36 445 Acc. No. 019233 38 39 38 38 39 38 38 38 38 38 37 38 37 456 Acc. No. 015382 39 37 36 39 36 37 39 39 39 39 39 39 39 458 Response of okra genotypes to NaCl salinity during plant ontogeny 2 Based on the overall pooled ranking of the genotypes for salinity tolerance at the seedling stage, ten genotypes from both extremes of salinity tolerance were selected (Table 2 2 ). Selected genotypes were grown to maturity in earthen pots of 30 cm diameter lined with a polyethylene sheet and filled with gravel soil (electrical conductivity of saturation extract (EC e )=1.6 dS/m, saturation=30.65%). Three NaCl concentrations consisting of 0 (control), 60, and 80 mmol/L were created in the growth medium, keeping in view the EC e , pH, and saturation percentage of the soil by adding the appropriate amount of salt in half strength Hoagland’s solution following US Salinity Laboratory Staff ( 1954 1954 ). The experiment was comprised of three replicates for each treatment. Nutrient solutions were applied every 3 d and continued until plant maturation was completed. The experiment was conducted under natural environmental conditions during the summer 2008 and spring 2009. Recommended plant protection practices and cultural practices were adopted until maturity, such as irrigation, weeding, and hoeing. Data were collected at four growth stages viz. seedling stage, bud stage, flowering stage, and maturity for various biochemical/physiological parameters including Na + and K + concentrations, plant height, number of branches, days to first flower, pods per plant, pod weight, pod length, pod girth, and yield per plant. Data for both absolute and relative values were collected from the ten genotypes for Na + and K + concentrations and K + /Na + ratio at all the four growth stages, and the remaining parameters were measured only at the maturity stage. The ANOVA, Pearson’s correlation, and heredity estimates were calculated using SPSS 12, MVSP 3.1, and Microsoft Excel, respectively. Genotype/stage NaCl (mmol/L) Na + (mmol/L) K + (mmol/L) K + /Na + Ikra III 3  Seedling stage 0 3.31 15.73 4.75 60 5.59 10.47 1.88 80 8.52 8.19 0.96 3  Bud stage 0 7.06 40.03 5.67 60 12.60 30.19 2.40 80 18.68 25.75 1.38 3  Flowering stage 0 1.95 30.68 16.36 60 3.57 12.55 3.51 80 10.53 9.93 0.95 3  Maturity stage 0 1.31 22.79 17.37 60 2.99 13.67 4.66 80 4.26 8.13 1.91 2 Acc. No. 019221 3  Seedling stage 0 2.89 32.70 11.35 60 6.46 23.22 3.61 80 14.34 17.21 1.20 3  Bud stage 0 6.87 41.73 6.08 60 12.91 32.34 2.51 80 20.84 22.33 1.07 3  Flowering stage 0 1.90 17.97 9.52 60 4.29 12.53 2.92 80 5.64 11.39 2.02 3  Maturity stage 0 3.00 14.52 4.88 60 4.17 11.34 2.72 80 5.68 8.88 1.57 2 Acc. No. 019236 3  Seedling stage 0 3.72 34.43 9.29 60 7.65 23.16 3.03 80 10.59 12.45 1.18 3  Bud stage 0 4.61 47.68 10.41 60 10.84 25.26 2.33 80 15.04 19.65 1.31 3  Flowering stage 0 2.43 25.22 10.59 60 3.81 15.43 4.07 80 6.01 8.85 1.48 3  Maturity stage 0 2.10 27.67 13.44 60 4.78 18.74 3.93 80 8.33 10.90 1.32 2 Acc. No. 019232 3  Seedling stage 0 2.83 27.25 9.70 60 6.52 21.45 3.29 80 12.83 11.92 0.93 3  Bud stage 0 3.64 52.15 14.45 60 8.13 35.57 4.42 80 12.98 24.24 1.87 3  Flowering stage 0 2.48 29.15 11.79 60 4.11 11.35 2.77 80 7.22 7.53 1.05 3  Maturity stage 0 1.51 13.46 9.00 60 3.64 7.29 2.01 80 6.78 4.95 0.73 2 Acc. No. 015380-10934 3  Seedling stage 0 1.55 45.73 29.55 60 15.46 14.56 0.94 80 29.01 9.03 0.31 3  Bud stage 0 3.69 37.90 10.36 60 16.50 21.49 1.30 80 39.50 16.57 0.42 3  Flowering stage 0 1.02 28.19 28.58 60 6.41 6.25 0.98 80 13.94 4.25 0.31 3  Maturity stage 0 2.82 15.18 5.40 60 7.31 6.09 0.84 80 12.47 6.75 0.54 2 Acc. No. 015371 3  Seedling stage 0 4.44 34.49 7.79 60 6.99 26.08 2.40 80 16.29 17.05 1.05 3  Bud stage 0 2.13 43.35 20.76 60 10.86 32.73 4.72 80 10.90 18.84 1.73 3  Flowering stage 0 1.17 22.26 19.15 60 3.62 12.43 3.44 80 4.48 4.18 0.94 3  Maturity stage 0 1.49 13.49 9.41 60 3.07 10.75 3.54 80 8.32 9.50 1.14 2 Acc. No. 019233 3  Seedling stage 0 2.86 31.41 10.99 60 18.50 12.20 0.66 80 31.83 4.88 0.15 3  Bud stage 0 2.61 27.92 10.83 60 15.03 13.71 0.92 80 35.45 8.74 0.25 3  Flowering stage 0 4.30 36.67 8.54 60 10.45 8.25 0.79 80 15.80 5.31 0.34 3  Maturity stage 0 1.44 18.46 13.01 60 5.08 6.65 1.32 80 7.99 4.06 0.51 2 Acc. No. 000010-10237 3  Seedling stage 0 6.06 30.66 5.07 60 10.12 20.16 1.85 80 18.98 15.38 0.81 3  Bud stage 0 5.51 34.74 6.34 60 10.90 27.23 2.70 80 13.89 20.35 1.46 3  Flowering stage 0 1.49 27.46 19.27 60 6.44 16.86 2.62 80 14.45 6.85 0.47 3  Maturity stage 0 1.75 11.23 6.47 60 2.86 13.90 4.91 80 5.75 9.83 1.72 2 Chinese Red 3  Seedling stage 0 7.45 41.21 5.53 60 13.55 34.16 2.53 80 24.56 26.82 1.09 3  Bud stage 0 10.61 49.40 4.66 60 15.16 38.94 2.58 80 31.94 30.58 0.96 3  Flowering stage 0 2.60 22.39 8.72 60 3.95 9.37 2.39 80 6.05 7.06 1.17 3  Maturity stage 0 1.87 19.99 10.05 60 2.85 8.68 3.06 80 3.69 7.60 2.09 2 Ikra I 3  Seedling stage 0 7.38 39.64 5.37 60 11.27 32.11 2.85 80 18.63 21.91 1.18 3  Bud stage 0 9.21 68.19 7.21 60 16.78 30.01 1.79 80 20.77 22.76 1.10 3  Flowering stage 0 5.06 26.39 5.22 60 7.32 17.11 2.34 80 9.53 12.47 1.31 3  Maturity stage 0 1.90 13.24 7.05 60 4.23 10.78 2.57 80 5.89 7.78 1.33 Results and discussion 1 Genetic variability for NaCl tolerance at the seedling stage in okra 2 The box-whisker analysis provided useful information for the comparison of genotypes and showed differential responses of genotypes. Germination percentage, root and shoot length, and accumulation of Na + and K + , and K + /Na + ratio at the seedling stage under different concentrations of NaCl were considered in the analysis. Increasing salt concentrations significantly affected the germination percentage, root length, and shoot length of genotypes to a varying degree. The ranking of the genotypes was done on the basis of their performance for each trait and under each NaCl concentration, separately. The genotypes were ranked as 1–39 with ‘1’ as the most tolerant and ‘39’ as susceptible based upon their abilities to withstand saline conditions. The ranks under each NaCl concentration were pooled to calculate an overall ranking of the genotypes on the basis of the traits in this study. Ranks depict the performance of genotypes under all salt concentrations. Salinity increases osmotic potential of the soil solution and causes seeds to use more energy to absorb water from the soil, which reduces germination (Kafi and Goldani, 2001 2001 ; Jamil and Rha, 2004 2004 ). Acc. No. 019217 (71.42%), Parbhani Kranti (65.13%), Chinese Red (58.67%) and Acc. No. 015382 (15.50%) were ranked as 1, 2, 3, and 39, respectively, on the basis of germination percentage (Table 1 1 ). Acc. No. 019217 consistently showed the highest germination in increasing NaCl concentrations, and maintained 51.67% germination in 120 mmol/L NaCl, with a mean of 68.28% germination across all of the NaCl concentrations. Similarly the genotypes Parbhani Kranti, Ikra III, Chinese Red, Acc. No. 019231, Ikra I, Acc. No. 019221, Acc. No. 019236, Acc. No. 019223, Punjab Selection, Acc. No. 000010-10237, IN-1048, Acc. No. 015371, and Acc. No. 019232 maintained mean germination percentages ranging from 50.20% to 62.67% in NaCl concentrations. The majority of accessions failed to germinate at 100 and 120 mmol/L NaCl. The differences in the behavior of genotypes could be attributed to the differences in gene frequencies and their interaction with the environment (Maas, 1986 1986 ). A significant negative correlation was observed between percentage of germination and increase in salt concentration (Table 3 3 ). Acc. Nos. 019232 (5.41 cm), 019221 (4.46 cm), and 019236 (3.39 cm) were the top three genotypes on the basis of relative root length. With reference to relative shoot length, Acc. No. 019232 (10.26 cm), Punjab Selection (9.50 cm), and Parbhani Kranti (8.16 cm) secured the top three ranks, respectively. The three-week-old seedlings showed no consistent relationships between root and shoot length in saline or non-saline conditions. The genotypes, five NaCl concentrations and their interactions were significantly different ( P ≤0.01) from each other for germination percentage and root and shoot length (Table 4 4 ). Thus, the different genotypes responded differently under increasing NaCl concentrations. The depressed growth of genotypes may be ascribed to the toxic effect of NaCl and low water potential in the rooting medium (Ayers, 1953 1953 ). GP: germination percentage; RL: root length; SL: shoot length. ‘−’ shows negative correlation Significant correlation ( P < 0.05) Highly significant correlation ( P < 0.01) 2 Parameter 2 NaCl (mmol/L) 6 Correlation coefficient GP RL SL Na + K + K + /Na +  GP 0 1 0.160 0.327 * * 0.327 * * −0.067 −0.486 ** **   40 1 0.641 ** ** 0.688 ** ** −0.756 ** ** 0.643 ** ** 0.704 ** **   60 1 0.788 ** ** 0.875 ** ** −0.829 ** ** 0.736 ** ** 0.820 ** **   80 1 0.793 ** ** 0.845 ** ** −0.843 ** ** 0.739 ** ** 0.836 ** **   100 1 0.924 ** ** 0.932 ** ** 0.453 ** ** 0.885 ** ** 0.918 ** **   120 1 0.941 ** ** 0.930 ** ** 0.676 ** ** 0.907 ** ** 0.926 ** **   Mean 1 0.792 ** ** 0.857 ** ** −0.312 0.714 ** ** −0.647 ** **  RL 0 1 −0.210 0.268 0.234 −0.023   40 1 0.560 ** ** −0.742 ** ** 0.727 ** ** 0.808 ** **   60 1 0.776 ** ** −0.725 ** ** 0.803 ** ** 0.798 ** **   80 1 0.857 ** ** −0.738 ** ** 0.818 ** ** 0.814 ** **   100 1 0.937 ** ** 0.418 ** ** 0.906 ** ** 0.926 ** **   120 1 0.950 ** ** 0.725 ** ** 0.951 ** ** 0.950 ** **   Mean 1 0.792 ** ** −0.106 0.825 ** ** −0.410 ** **  SL 0 1 −0.201 −0.089 0.063   40 1 −0.766 ** ** 0.646 ** ** 0.741 ** **   60 1 −0.866 ** ** 0.786 ** ** 0.842 ** **   80 1 −0.807 ** ** 0.790 ** ** 0.827 ** **   100 1 0.558 ** ** 0.933 ** ** 0.923 ** **   120 1 0.798 ** ** 0.955 ** ** 0.958 ** **   Mean 1 −0.189 0.792 ** ** −0.564 ** **  Na + 0 1 0.067 −0.642 ** **   40 1 −0.751 ** ** −0.894 ** **   60 1 −0.720 ** ** −0.895 ** **   80 1 −0.765 ** ** −0.872 ** **   100 1 0.614 ** ** 0.318 * *   120 1 0.801 ** ** 0.677 ** **   Mean 1 0.153 0.312  K + 0 1 0.254   40 1 0.897 ** **   60 1 0.867 ** **   80 1 0.856 ** **   100 1 0.919 ** **   120 1 0.967 ** **   Mean 1 −0.414 ** ** df : degree of freedom; GP: percentage of germination percentage; RL: root length; SL: shoot length. * Significant at P ≤0.05 Significant at P ≤0.01 Non-significant ( P > 0.05) 2 Source of variation 2 df 6 Mean square for physio-morphological traits GP RL SL Na + K + K + /Na + Genotypes ( G ) 38 14 508.436 ** ** 87.023 ** ** 145.417 ** ** 4 767.340 NS NS 2 058.786 ** ** 40.192 NS NS NaCl concentration ( c ) 5 188 320.735 ** ** 1 302.305 ** ** 10 132.259 ** ** 167 423.024 ** ** 40 750.180 ** ** 3 840.209 ** ** Replications 2 890.363 ** ** 0.162 NS NS 0.416 NS NS 22.544 ** ** 25.075 ** ** 0.272 NS NS G × c 190 892.907 ** ** 3.807 ** ** 20.749 ** ** 5 581.485 ** ** 165.076 ** ** 48.983 ** ** Within+residual 1870 18.232 0.167 0.676 1.815 0.915 0.466 Leaf Na + ion uptake increased with an increase in external NaCl concentrations in all genotypes, but the difference among the genotypes was not significant ( P > 0.05) for Na + accumulation (Table 4 4 ). The interaction between the genotypes and NaCl concentrations was significant, indicating that the genotypes responded differently to increasing NaCl concentrations. A minimum Na + accumulation was observed in Acc. No. 000010-10237 (47.44 mmol/L) and a maximum in Acc. No. 015382 (231.81 mmol/L). On the basis of relative accumulation of Na + , Acc. No. 000010-10237, 019232, and 019231 were classified as tolerant genotypes. The K + concentration was observed to be inversely proportional to the increase in external NaCl concentration. The interaction between the genotypes and NaCl concentrations was significant ( P ≤0.01) (Table 4 4 ), indicating that the accumulation of K + affects genotypes variably in the increasing NaCl concentrations. Acc. No. 019236, Ikra III, and Acc. No. 019232 maintained relatively maximum K + in descending order. The values for K + accumulation ranged from 10.40 to 85.95 mmol/L; Acc. No. 015382 accumulated minimum K + . Under salt stress, plants maintain high concentrations of K + and low concentrations of Na + in the cytosol. Plants regulate the expressions and activities of K + and Na + transporters and H + pumps that generate the driving force for ion transport (Zhu, 2003 2003 ). A greater degree of salt tolerance in plants was found to be associated with a more efficient system for selective uptake of K + over Na + (Neill et al., 2002 2002 ). The selective uptake of K + in contrast to Na + is considered to be one of the most important physiological mechanisms contributing to salt tolerance in many plant species (Poustini and Siosemardeh, 2004 2004 ). An increase in external NaCl concentration significantly affected the leaf K + /Na + uptake ratio within all genotypes, but the difference among the genotypes and replicates was not significant ( P > 0.05) (Table 4 4 ). The interaction between the five genotypes and NaCl concentrations differed significantly ( P ≤0.01) from each other. Increasing salinity adversely affected K + /Na + ratio to a different extent in different genotypes. The K + /Na + uptake ratio varied from 0.05 to 46.07, indicating an inverse proportion with increasing NaCl concentration. On the basis of K + /Na + ratio, Ikra III, Acc. No. 015371, and Acc. No. 000010-10237 were ranked as 1, 2, and 3, respectively (Table 1 1 ). Correlation coefficients among the traits indicated that germination percentage has significant positive correlation with root length, shoot length, K + concentration, and K + /Na + at all concentrations while it has a negative correlation with Na + concentration at 40, 60, and 80 mmol/L and positive correlation at 100 and 120 mmol/L. A similar correlation was observed for root and shoot length. Na + accumulation showed a significant negative correlation with K + concentration and K + /Na + at all NaCl concentrations except at 100 and 120 mmol/L. A significant positive correlation was also found for K + concentration and K + /Na + (Table 3 3 ). A Euclidian distance-based phenogram assigned 39 okra genotypes into two main clusters. All the salt-tolerant genotypes clustered separately from the more susceptible genotypes. Each cluster showed particular morphological characteristics (Fig. 1 1 and Table 5 5 ). GV: genotypic variance; PV: phenotypic variance; GCV: genotypic coefficient of variance; PCV: phenotypic coefficient of variance; H: heterosis; PH: plant height; NB: number of branches; DF: days to 50% flowering; P/Pl: pods per plant; PW: pod width; PL: pod length; PG: pod girth; Y: pod yield 2 Trait 2 Season 2 GV 2 PV 2 GCV 2 PCV 2 H Control 80 mmol/L NaCl Control 80 mmol/L NaCl Control 80 mmol/L NaCl Control 80 mmol/L NaCl Control 80 mmol/L NaCl  PH Spring 123.47 69.10 123.53 69.21 23.88 31.96 23.88 31.99 0.99 0.99   Summer 205.30 195.04 214.67 195.81 15.11 29.78 15.45 29.84 0.95 0.99  NB Spring 0.69 0.41 0.73 0.43 22.12 36.72 22.75 37.64 0.94 0.95   Summer 0.42 0.19 0.54 0.21 17.32 31.29 19.60 32.89 0.78 0.90  DF Spring 16.40 9.63 16.84 9.77 7.92 4.97 8.02 5.00 0.97 0.98   Summer 41.82 49.50 43.85 50.68 14.56 11.50 14.91 11.64 0.95 0.97  P/Pl Spring 9.87 3.95 10.12 3.98 21.92 37.92 22.20 38.06 0.97 0.99   Summer 8.52 11.49 8.95 12.10 14.07 32.80 14.42 33.66 0.95 0.94  PW Spring 0.39 1.16 0.46 1.26 8.12 22.60 8.83 23.54 0.84 0.92   Summer 1.25 1.01 1.46 1.15 20.11 33.15 21.69 35.38 0.86 0.87  PL Spring 0.43 1.58 0.51 1.61 6.99 26.26 7.57 26.45 0.85 0.98   Summer 0.27 0.54 0.66 0.68 6.67 14.30 10.40 16.05 0.41 0.79  PG Spring 0.005 0.01 0.012 0.01 5.37 10.00 8.30 10.29 0.41 0.94   Summer 0.006 0.0019 0.0092 0.017 6.55 3.82 7.60 11.53 0.74 0.10  Y Spring 522.39 164.16 523.04 165.32 21.40 49.82 21.41 50.00 0.99 0.99   Summer 393.84 44.81 395.21 181.05 18.31 21.10 18.34 42.41 0.99 0.24 Based on the overall pooled ranking of the genotypes for salinity tolerance at the seedling stage, Acc. No. 019232, Acc. No. 000010-10237, Chinese Red, Ikra III, and Acc. No. 015371 were selected as the most tolerant. Ikra I, Acc. No. 019236, and Acc. No. 019221 were selected as relatively tolerant, and the two genotypes Acc. No. 015380-10934 and Acc. No. 019233 as non-tolerant (Table 2 2 ). Performance of okra genotypes under saline conditions during plant ontogeny 2 Salinity affects plant growth during all developmental stages. Therefore the crop responses to salinity may vary during the plant ontogeny (Maas and Hoffman, 1977 1977 ; Shannon, 1985 1985 ; Azhar and McNeilly, 1989 1989 ; Maas and Poss, 1989 1989 ). For the selection of salt-tolerant breeding plants, information about the degree of salt tolerance at all plant growth stages in a crop species is important. Without this information, selection at a particular stage may produce individuals that exhibit less tolerance to salinity at the next growth stage. Response to salinity is thought to be controlled by a number of genes that may be stage-specific (Foolad and Jones, 1991 1991 ). The genotypes in this study responded differently in salt concentrations at different growth stages for the accumulation of Na + and K + and K + /Na + ratio in leaves. The concentration of Na + was increased while the K + accumulation and K + /Na + ratio decreased in all the genotypes with increased salinity at all growth stages. However, in the non-tolerant genotypes, Acc. Nos. 019233 and 015380-10934, the Na + concentration was greater than that in the tolerant genotypes at all the growth stages under stress. The tolerant genotypes accumulated low Na + and maintained higher K + and K + /Na + ratio in their leaves. Maximum Na + accumulation was observed at the bud stage in all genotypes at all salt concentrations (Table 2 2 ). The increased uptake may be due to increased diffusion through damaged membranes and restricted outward active exclusion of Na + . This suggests that the okra genotypes used a partial exclusion mechanism for tolerating the toxic ions present in the growth medium (Maas and Nieman, 1978 1978 ; Greenway and Munns, 1980 1980 ; Salisbury and Ross, 1992 1992 ; Shaaban et al., 2004 2004 ; Ahmadi et al., 2009 2009 ). It was observed that the accumulation of Na + in the cell sap was lower in magnitude than the accumulation by the same genotypes in the screening experiment in sand culture. The high K + concentration at higher salinity level is a good criterion for selecting salt-tolerant genotypes (Flowers et al., 1977 1977 ). Maintenance of higher K + /Na + ratio in salt-tolerant genotypes may be one of the reasons for their superior growth under saline conditions (Ashraf and Ahmad, 2000 2000 ). High levels of K + in young leaves are associated with salt tolerance in many plant species (Gorham, 1993 1993 ; Storey et al., 1993 1993 ; Khatun and Flowers, 1995 1995 ). Maintenance of higher K + /Na + ratio in salt-tolerant lines may be attributed to K + /Na + exchange across the plasmalemma of root cortex cells and selective uptake of K + (Jeschke and Wolf, 1988 1988 ; Dashti et al., 2009 2009 ). The sensitive genotypes had K + /Na + ratio less than 1 at higher salinity levels, and the tolerant had K + /Na + ratio more than 1, showing their tolerance at different stages of growth consistently. Plant height and a number of branch lengths were reduced in the okra genotypes with increased NaCl concentration in growth medium. However, the reduction level was low in the tolerant genotypes (Acc. No. 019232, Acc. No. 000010-10237, and Chinese Red) and more in the two sensitive genotypes (Acc. No. 019233 and Ikra I) in both summer and spring plantings (Table 5 5 ). Due to delayed germination, delayed flowering was observed in non-tolerant okra genotypes under saline conditions. The tolerant genotypes started flowering earlier than the sensitive genotypes. Salinity at lower levels is known to delay germination, and at high levels can even reduce germination percentages (Kuhad and Sheoran, 1987 1987 ; Jamil and Rha, 2004 2004 ). The number of pods per plant, pod weight, pod length, and pod girth were significantly reduced by salinity but there was consistency in the tolerance level of the selected genotypes (Table 5 5 ). The effects of salinity and genotypic differences were most apparent for the marketable yield of okra genotypes. There was a significant decrease in tender green pod yield of non-tolerant genotypes, but the tolerant genotypes had relatively less reduction in pod yield (Table 5 5 ). The heritability estimates indicated a high degree of inheritance for all the morphological traits especially during spring (Table 5 5 ); moreover, correlation estimates indicated that fresh green pod yield had significant positive correlation with plant height, pods per plant, pod length, pod width, and pod girth at 80 mmol/L NaCl during both spring and summer (Table 6 6 ). These results suggest that for the improvement of yield-related traits, selections would be more effective at 80 mmol/L NaCl during spring. PH: plant height; NB: number of branches; DF: days to 50% flowering; P/Pl: pods per plant; PW: pod width; PL: pod length; PG: pod girth; Y: pod yield. ‘−’ sign shows negative correlation Significant correlation ( P < 0.05) Highly significant correlation ( P < 0.01) 2 Trait 2 Season 2 NaCl (mmol/L) 8 Correlation coefficient PH NB DF P/Pl PW PL PG Y PH Spring 0 1 −0.240 −0.601 −0.264 0.562 −0.132 0.136 −0.062 60 1 0.278 −0.743 ** ** −0.043 0.891 ** ** 0.447 0.220 0.484 80 1 0.616 * * −0.695 * * 0.480 0.883 ** ** 0.823 ** ** 0.761 ** ** 0.662 * * Summer 0 1 −0.009 0.505 −0.377 0.079 0.263 0.449 −0.228 60 1 0.288 −0.632 * * 0.375 0.521 0.773 ** ** 0.833 ** ** 0.649 * * 80 1 0.414 −0.394 0.457 0.511 0.899 ** ** 0.921 ** ** 0.630 * * NB Spring 0 1 −0.219 0.284 −0.458 0.088 0.238 0.104 60 1 −0.360 0.252 0.330 0.669 * * 0.785 ** ** 0.369 80 1 −0.284 0.481 0.571 0.525 0.733 * * 0.478 Summer 0 1 0.220 0.724 * * −0.230 0.554 0.496 0.261 60 1 −0.322 0.769 ** ** −0.156 0.289 0.034 0.236 80 1 −0.309 0.774 ** ** −0.045 0.578 0.403 0.468 DF Spring 0 1 0.097 −0.370 −0.049 −0.093 −0.034 60 1 0.447 −0.526 −0.224 −0.085 0.012 80 1 −0.184 −0.704 * * −0.823 ** ** −0.605 * * −0.377 Summer 0 1 −0.145 0.021 −0.089 0.134 −0.059 60 1 −0.412 −0.432 −0.323 −0.411 −0.551 80 1 −0.673 * * −0.428 −0.547 −0.682 * * −0.728 * * P/Pl Spring 0 1 −0.284 −0.104 −0.397 0.932 ** ** 60 1 0.195 0.501 0.507 0.823 ** ** 80 1 0.663 * * 0.219 0.723 * * 0.947 ** ** Summer 0 1 −0.581 0.567 0.174 0.097 60 1 −0.147 0.307 0.049 0.388 80 1 0.047 0.665 * * 0.566 0.716 * * PW Spring 0 1 0.089 −0.357 0.079 60 1 0.613 * * 0.449 0.714 * * 80 1 0.850 ** ** 0.841 ** ** 0.843 ** ** Summer 0 1 −0.325 0.385 0.747 ** ** 60 1 0.289 0.841 ** ** 0.838 ** ** 80 1 0.383 0.614 * * 0.712 * * PL Spring 0 1 0.283 −0.042 60 1 0.915 ** ** 0.714 * * 80 1 0.654 * * 0.469 Summer 0 1 0.639 * * 0.026 60 1 0.606 * * 0.394 80 1 0.891 ** ** 0.665 * * PG Spring 0 1 −0.529 60 1 0.615 * * 80 1 0.755 ** ** Summer 0 1 0.556 60 1 0.773 ** ** 80 1 0.766 ** ** The assessments of the tolerant and non-tolerant genotypes for physiological parameters at four growth stages and eight morphological traits at maturity suggest that there was consistency in the salinity tolerance of the selected genotypes until maturity. 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