Journal of Cell and Tissue Research Vol. 16(3) 5901-5910 (2016) (Available online at www. Tcrjournals.com) ISSN: 0973-0028; E-ISSN: 0974-0910
Original Article
IN VITRO SEED GERMINATION AND SEEDLING GROWTH FOR SALT TOLERANCE IN RICE CULTIVARS ? KUMARI, R., KUMAR, P., SHARMA, V. K., AND KUMAR, H. Department of Agricultural Biotechnology and Molecular Biology, Faculty of Basic Sciences and Humanities Dr. Rajendra Prasad Central Agricultural University, Pusa (Samastipur) 848125 (Bihar). E. mail:
[email protected] Received: September 14, 2016; Accepted: October 7, 2016 Abstract: Six selected cultivars of rice namely BPT-5204, MTU-7029, CSR-30, Narendra Usar Dhan-3, Rajendra Bhagwati and Pusa Basmati-1 were screened for salinity tolerance on the basis of in vitro seed germination and seedling growth under different salt stress (02.5%) created by a salt mixture of NaCl, CaCl2, and Na2SO4 in 7:2:1 ratio. At 2% salt stress, germination was only observed in cultivars CSR-30 and Narendra Usar Dhan-3, while at 2.5 % no seed germination was observed. The frequency of seed germination gradually decreased with increasing concentrations of salt stress. As observed with seed germination, seedling growth was also adversely affected with the increase of salt concentrations. The shoot growth was more affected than the root growth. The salt tolerance index (STI) of the rice cultivars was calculated on the basis of seed germination, seedling shoot and root dry weights at different levels of salt stress. Cultivars CSR-30 and Narendra Usar Dhan-3 were found to be salt tolerant, cvs. MTU7029 and BPT-5204 moderately salt tolerant and cvs. Rajendra Bhawati and Pusa Basmati-1 as salt susceptible respectively on the basis of STI. Thus in vitro study can be used for evaluation of salinity tolerance of rice cultivars. Key words: Seed germination, Seedling growth, Salinity, Rice,
INTRODUCTION Rice is one of the most important crops in the world that offers food for more than 50% of the world’s population [1]. It is the first food crop in which complete genome sequence is available. It is an ideal model plant for the study of grass genetics and genome organization due to its relatively small genome size of 430 Mb, compared with other major crops. The productivity of several commercial crops is limited by major abiotic stresses including salinity, drought, water logging and heat. Damages caused by these stresses are responsible for enormous economic loss world-wide. Salinity is a major abiotic stress affecting crops adversely. It is caused by weathering of rocks, heavy irrigation with saline water, the
entry of sea water into freshwater areas, poor water management, high evaporation, and regular use of chemical fertilizers. Salinity is the biggest problem in rice growing areas of many countries [2]. Rice is rated as a salt sensitive crop [3,4]. Salinity affects all stages of the growth and development of rice plant. The response of crop to salinity varies with growth stages, concentration and duration of exposure to salt. In the most commonly cultivated rice cultivars, young seedlings were very sensitive to salinity [5,6]. According to Yoshida [7] rice is more sensitive to salinity during early seedling growth and flowering than other growth stages. Several investigators have stated that salt exerts negative effect on seed germination and seedling growth [8-11]. Inhibition of seed germination and shoot and root was noticed
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J. Cell Tissue Research as a result of sodium chloride treatments in rice [12,13]. Salinity may affect seed germination in two ways, osmotically, by decreasing the ease with which seeds may take up water and ionically, by facilitating the uptake of ions in excess amount to be toxic for the embryonic activity [14,15]. Salt stress is more inhibitor during seed germination than at any other stage of growth in many plants [16]. Seed germination is affected by the increase in salinity [17]. Therefore, this study was conducted to evaluate salt tolerance of six rice cultivars during in vitro seed germination and seedling growth at different levels of salinity. MATERIALS AND METHODS Seeds of six rice cultivars were obtained from the Department of Plant Breeding and Genetics, Rajendra Agricultural University, Pusa, Bihar (Table 1). Seeds were soaked in water overnight. Soaked seeds were treated with 70% ethyl alcohol for 30 sec and then washed with distilled water. Thereafter seeds were treated with 0.1% mercuric chloride (HgCl2) for 10-15 minutes, rinsed with sterile distilled water thrice and placed for germination on filter paper in 10 cm diameter Petri dish containing six different concentrations ranging from 0-2.5% of salt mixture of NaCl, CaCl2, and Na2SO4 in 7:2:1 ratio. The Petri dishes were kept under laboratory conditions. Statistical data analysis: The experiment was laid out in a factorial CRD design [18] with three replications. The data were subjected to analysis of variance and Duncan’s Multiple Range Test (DMRT) for comparing population means. Mean values were compared by two-way ANOVA. The salinity tolerance index, represented as the ratio of the performance of a cultivar at a particular level of salinity to that of control, was calculated on the basis of in vitro germination, and seedling growth based on dry weights of shoot and root following the method of Reddy. and Vaidyanath [19]. RESULTS Seed germination under salt stress: The seeds of six selected rice cultivars were put to germination under different levels of salt concentrations ranging from 0-2.5%. The germination of seeds was initiated within the first week. The first sign of germination was observed by the appearance of the plumule followed by radical. Only in control (0% salt) the seeds of all the cultivars showed germination in the first week. Some seeds of cvs. Narendra Usar Dhan-3 and CSR-30 also germinated in 0.5% salt
solution in the first week. The seeds of all other cultivars germinated in 1.0 and 1.5% salt solutions in the second week. Only seeds of cvs. Narendra Usar Dhan-3 and CSR-30 germinated in 2% salt solution. There was no germination of seeds of all cultivars in 2.5% salt solution. The germination percentage in control (0% salt) in all the cultivars was at par with the mean 93.66%. In 0.5% salt concentration the best seed germination was found in cultivar Narendra Usar Dhan-3 (82.0%) followed by cvs. CSR-30 (81.33%) and MTU7029 (76.66%) which were at par. The minimum germination was found in seeds of cv. Pusa Basmati-1 (63.33%). Similarly in 1% salt, the highest and at par germination was found in cvs. CSR-30 (42.66%), Narendra Usar Dhan-3 (42.66%) and the lowest in cv. Pusa Basmati-1 (22.00%). At 1.5% salt the highest and at par germination was found in cvs. CSR-30 (22.66%), Narendra Usar Dhan-3 (22.00%) and the lowest in cv. Pusa Basmati-1 (2.66%). The germination percentage gradually decreased with increased salt concentrations. Overall the best germination was found in cv. CSR-30 (41.94%) followed by cvs. Narendra Usar Dhan-3 (41.61%), MTU-7029 (36.22%), BPT-5204 (35.44%), Rajendra Bhagwati (30.99%) and Pusa Basmati-1 (30.10%) respectively (Table 2 and Fig. 1). Based on in vitro seed germination in the presence of salt stress, cultivar CSR-30 showed the highest mean salt tolerance index (STI) of 41.45% followed by cv. Narendra Usar Dhan-3 (41.39%) and can be considered as salt tolerant while Pusa Basmati-1 showed the least mean salt tolerance index (STI) of 23.73% followed by Rajendra Bhagwati (25.17%) and can be considered as salt susceptible. The other cvs. MTU-7029 and BPT-5204 with STI of 32.39% and 31.95% respectively, can be considered as moderately tolerant (Table 3 and Fig. 2). Seedling growth under salt stress: Seedling of all six selected cultivars was grown in salt solutions with the salt concentrations ranging from 0-2.5%. The seedling growth was inversely proportional to the salt concentrations. The root growth was more affected than the shoot growth. The fresh and dry weights of seedling were measured to evaluate the seedling growth under different salt stress. Salinity tolerance index (STI) was calculated based on shoot and root dry weights at different levels of salt stresses of all selected six cultivars. STI based on shoot growth was higher than that of root growth at all concentrations of salt indicating the former being less affected than
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Kumari et al.
Germination (%)
50 40 30 20 10 0
BPT-5204
MTU-7029
Narendra Usar Rajendra Dhan-3 Bhagwati Cultivars
CSR-30
BPT-5204
MTU-7029
Narendra Usar Rajendra Dhan-3 Bhagwati Cultivars
CSR-30
Pusa Basmati-1
Narendra Usar Rajendra Dhan-3 Bhagwati Cultivars
CSR-30
Pusa Basmati-1
Narendra Usar Rajendra Dhan-3 Bhagwati Cultivars
CSR-30
Pusa Basmati-1
Mean STI
Fig. 1 45 40 35 30 25 20 15 10 5 0
Fig. 2
Pusa Basmati-1
60
Growth (%)
50 40 30 20 10 0
BPT-5204
MTU-7029
Fig. 3 60 Mean STI
50 40 30 20 10 0
Fig. 4
BPT-5204
MTU-7029
Fig.1: Effect of cultivars on germination (%) of seedling growth. Fig. 2: Salinity tolerance index (STI) of rice cultivars based on germination frequency under different salt concentrations. Fig. 3: Effect of rice cultivars on seedling growth under salinity stress. Fig. 4: Mean STI of rice cultivars based on in vitro seed germination and seedling growth (shoot and root dry weight) under salt stress.
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J. Cell Tissue Research Table 1: Rice cultivars used Sl. No. 1. 2. 3. 4. 5. 6.
Rice cultivars Narendra Usar Dhan-3 CSR-30 Pusa Bsamati-1 Rajendra Bhagwati MTU-7029 BPT-5204
Parentage Leungyai148 x IR 9129-209-2-2-1 xx IR 18272-27-3-1 BR4-10 x Basmati 370 Pusa150 x karnal local IR36 x Type3 Vasistha x Mahsuri GEB24 x TNI x Mahsuri
Developed at NDUAT, Faizabad CSSRI, Karnal IARI, New Delhi RAU, Pusa ANGRAU, Hydrabad ANGRAU, Hydrabad
Table 2: Germination (%) of seeds of rice cultivars on different salt concentrations. Values followed by the same letter in row are not significantly different using Duncan’s Multiple Range Test (DMRT) at 5% Salt (%) 0% 0.5% 1% 1.5% 2% 2.5% Mean CD CV SE (m)
BPT-5204
MTU-7029
93.33±1.76 74.66b±2.90 35.33b±2.90 9.33b±0.66 35.44 5.75 9.02 1.84
94.66±0.66 76.66a±2.66 36.00b±3.05 10.00b±1.15 36.22 5.42 8.33 1.74
Cultivars Narendra Usar Rajendra Pusa BasmatiCSR-30 Dhan-3 Bhagwati 1 94.00±1.15 92.66±1.76 94.66±0.66 92.66±1.33 82.00a±1.55 65.33c±2.40 81.33a±1.33 63.33c±2.40 42.66a±0.66 24.66c±0.66 42.66a±1.33 22.00c±1.15 22.00a±1.15 3.33c±0.66 22.66a±1.33 2.66c±0.66 9.00b±0.57 10.33a±0.33 41.61 30.99 41.94 30.10 2.78 3.97 3.08 3.88 3.71 7.13 4.09 7.17 0.89 1.27 0.99 1.24
the later under salt stress (Table 4). Cultivar CSR30 (57.07%) showed the highest mean STI followed by cvs. Narendra Usar Dhan-3 (51.32%), MTU-7029 (37.09%), BPT-5204 (36.48%), Rajendra Bhagwati (34.07%) and Pusa Basmati-1 (21.64%) respectively (Fig. 3). Finally, the six selected rice cultivars were ranked for their salt tolerance level on their mean salinity tolerance index (STI) based on in vitro seed germination and seedling growth (shoot and root dry weight) under salt stress. Cultivars CSR-30 (49.26%) and Narendra Usar Dhan-3 (46.35%) were found to be the most salt tolerant, cvs. BPT-5204 (34.21%) and MTU-7029 (34.74%) to be moderately tolerant and cvs. R. Bhagwati (29.62%) and Pusa Basmati-1 (22.68%) to be salt sensitive respectively (Fig. 4). DISCUSSION Salt stress shown the inhibitory effect of on seed germination [20], while in paddy the inhibitory effect of salt due to anions [21]. Observation in rice varieties of different groups, a concentration dependent decrease in seed germination under sodium chloride stress as found in present investigation [22]. They also observed differences in salinity tolerance among cultivars based on germination frequency and considered it significant because decline in germination also leads to significant reduction in seedling length, early seedling vigour,
Mean
CD
CV
SE (m)
93.66 73.88 33.88 11.66 3.33
6.99 5.93 3.05 0.84
2.41 5.26 9.73 14.56 14.63
1.30 2.24 1.90 0.98 0.27
speed of germination and dry matter production per plant [23]. Delayed and decreased germination of rice seeds under salt stress observed [24]. High concentration of salt reduces the water potential and hinders water absorption by germinating seeds and thus reduces germination [25]. Salinity may affect seed germination by decreasing the ease with which the seeds take up water because the activity and vents normally associated with germination get either delayed or proceed at a reduced rate [26]. Seedling survival and growth were also important indicators of the salt tolerance of a cultivar [27]. As observed with seed germination, seedling growth was also adversely affected with the increase of salt concentration. The root growth was more affected than the shoot growth. A similar observation on adverse effect on seedling growth due to salt stress and root growth being more affected than shoot growth was observed in rice [28,29]. Adverse effect of salinity on seedling growth of rice has also been reported by Djanaguiraman et al. [30] and Hakim et al. [31]. The adverse effect of salt stress is reflected in the dry matter accumulation of the seedling. Thus the dry matter accumulation of seedling shoot and root indicated by their dry weight can be used as an index to measure the salinity tolerance of the cultivar. Cultivars CSR-30 and Narendra Usar Dhan-3 are known to show high to moderate tolerance to salt stress [32,33]. Cv. BPT-5204 is moderately salt
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Kumari et al. Table 3: Salinity tolerance index (STI) of rice cultivars based on germination frequency under salt stress Cultivar
BPT-5204
MTU-7029
Narendra Usar Dhan-3
Rajendra Bhagwati
CSR-30
Pusa Basmati-1
Salt concentrations (%) 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Germination frequency 93.33 74.66 35.33 9.33 94.66 76.66 36.00 10.00 94.00 82.00 42.66 22.00 9.00 92.66 65.33 24.66 3.33 94.66 81.33 42.66 22.66 10.33 92.66 63.33 22.00 2.66 -
tolerant [34] while Pusa Basmati-1 is salt sensitive cultivar. Thus, the status of selected rice cultivars with respect to salt tolerance was differentiated by in vitro screening through seed germination under salt stress [35]. ACKNOWLEDGEMENT We acknowledge the support of Department of Biotechnology, Government of India, New Delhi for providing Fellowship to author. List of abbreviation used: CRD - complete randomized design, ANOVA - analysis of variance, STI - salt tolerance index, CVS.– Cultivars, CV.Cultivar, DMRT - Duncan’s multiple range test REFERENCES [1] Aggarwal, R.K., Shenoy, V.V., Ramadevi, J., Rajkumar, R. and Singh, L.: Theor. Appl. Genet., 105: 680-690 (2002). [2] Senadhira, D.: Salinity as a concept to increasing rice production in Asia. In: Proc. Regional Workshop in Maintaince of Life Support Species in Asia Pacific Region. NBPGR, New Delhi, (1987).
STI
Mean STI
79.99 37.85 9.99
31.95
80.98 38.03 10.56
32.39
87.23 45.38 23.40 9.57 70.50 26.61 3.59 85.91 45.06 23.93 10.91 68.34 23.74 2.87
41.39
25.17
41.45
23.73
[3] Mass, E.V. and Hoffman, G.J.: J. Irrig. Drain Div., ASCE, 103: 115-134 (1977). [4] Shannon, M.C., Rhoades, J.D., Draper, J.H., Scardaci, S.C. and Spyres, M.D.: Crop Sci., 38: 394-398 (1998). [5] Flowers, T.J. and Yeo, A.R.: New Phytol., 88: 363373 (1981). [6] Lutts, S., Kinet, J.M. and Bouharmont, J.: J. Exp. Bot., 46: 1843-1852 (1995). [7] Yoshida, S.: Fundamentals of Rice Crop Science. IRRI, Los Banos, Philippines (1981). [8] Abbad, A., El-Hadrami, A. and Benchabane, A.: J. Agron, 3: 111-114 (2004). [9] Akbari, G.S., Mohammad, A. and Yousefzadeh, S.: J. Agron., 3: 111-114 (2007). [10] Azza,M.A.M., Fatma EL-Quensi, E.M. and Farahat, M.M.: World J. Agric. Sci., 3: 386-395 (2007). [11] Feizi, M., Aghakhani, A., Mostafazadeh-Frad, B. and Heidarpour, M.: Pak. J. Biol. Sci., 10: 28242830 (2007). [12] Jamil, M., Lee, K.B., Jung, K.Y., Lee, D.B., Han, M.S. and Rha, E.S.: Pak. J. Biol. Sci., 10: 910-914 (2007). [13] Sheng, X., Hu, B., He, Z., Ma, F., Feng, J., Shen, W. and Yang, J.: Int. J. Mol. Sci., 12: 2488-2501 (2011). [14] Ayers, A. D.: Agron. J., 45: 68-71 (1953).
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J. Cell Tissue Research Table 4: Seedling growth of rice cultivars under different salt concentrations Cultivar
BPT-5204
MTU-7029
Narendra Usar Dhan-3
Rajendra Bhagwati
CSR-30
Pusa Basmati-1
Salt concentrations (%) 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0 0 0.5 1.0 1.5 2.0
Fresh wt 15.4 14.07 6.57 4.0 16.33 15.5 14.37 6.66 25 13.66 3.63 3.55 2.5 19.38 10.66 8.16 2.55 20.91 18.00 17.57 10.25 6.5 9.30 5.66 2.33 0.6 -
Shoot Dry wt 2.13 1.61 1.28 0.8 3.75 2.9 2.25 1.16 3 2.1 2.09 1.66 1.5 2.94 2.2 1.91 0.22 3.53 3.4 2.85 1.0 0.5 1.30 0.88 0.33 0.08 -
Seedling growth Root Fresh wt Dry wt STI 14.86 1.60 9.53 0.92 57.50 3.71 0.57 35.62 2.0 0.4 25.00 10.91 2.0 6.5 1.2 60.00 5.12 0.87 43.50 4.16 0.5 25.00 26.91 3.91 18.83 2.25 57.54 11.45 2.0 51.15 9.44 1.2 30.69 3.5 1.0 23.57 11.77 1.5 8.5 0.94 62.66 6.75 0.83 55.33 0.88 0.11 7.33 14.25 2.75 13.63 2.71 98.54 9.0 1.42 51.63 7.50 1.5 54.54 6.0 0.5 18.18 18.46 2.46 9.88 1.22 49.59 3.16 0.5 20.32 0.8 0.1 4.06 -
STI 75.58 60.09 37.55 77.33 60.00 30.93 70.00 69.66 55.33 50.66 74.83 64.96 7.48 96.31 80.73 28.32 28.32 67.69 25.38 6.12
[15] Ali, Y., Aslam, Z., Ashraf, M.Y. and Tahir, G.R.: Int. J. Environ. Sci. Technol., 1: 221-225 (2004). [16] Bewley, J.D. and Black, N.: Physiology and Biochemistry of Seeds in relation to Germination. Springer-Verlag, Berlin (1982). [17] Pushpam, R. and Rangasamy, S.R.S.: J. Ecobiol., 14: 177-182 (2002). [18] Gomez, K.A. and Gomez, A.A.: Statistical Procedure for Agricultural Research. 2nd ed. John Wiley and Sons (1984). [19] Reddy, P.J. and Vaidyanath, K.: Indian J. Agric. Sci., 52: 472-474 (1982). [20] Bhumba, D.R. and Singh, N.T.: Curr. Sci., 31: 9697 (1965). [21] Paliwal, K.V. and Gandhi, A.P.: Indian J. Plant Physiol., 11: 62-67 (1968). [22] Ali, M.S., Karim, M.A. and Hamid, A.: Effect of sodium chloride on seed germination, growth and photosynthesis of rice. Abstracts in 2nd International Crop Science Congress. New Delhi, 55 (1996). [23] Reddy, S.K.N., Reddy, B.M. and Ankaiah, R.: Seed Res, 22: 22-25 (1994). [24] Kazemi, K. and Eskandari, H.: Afr. J. Biotechnol., 10: 17789-17792 (2011). [25] Jamil, M., Lee, D., Jung, K.Y., Ashraf, M., Lee, S.C. and Rha, E.S.: J. Central Europ. Agricul., 7: 273-282 (2006).
Salinity tolerance index (STI) Average STI Mean STI 66.54 47.85 31.55 68.66 51.75 27.96 63.77 60.40 43.01 38.11 68.74 60.14 7.40 97.42 66.18 41.43 23.25 58.64 22.85 5.10 -
36.48
37.09
51.32
34.07
57.07
21.64
[26] Abogadallah, G.M. and Quick, W.P.: Acta Physiol Plant, 31: 815-824 (2009). [27] Garg, A.K., Siddiq, E.A., Sharma, N.P. and Reddy, A.R.: Characterization of rice genotypes on the basis of morphological and biochemical responses to salt stress. Abstracts in 2nd Int Crop Sci Congress, New Delhi, pp 55 (1996). [28] Rahman, M.S., Miyake, H. and Taheoka, Y.: Pak. J. Biol. Sci., 4: 351-355 (2001). [29] Ologundudu, A.F., Adelusi, A.A. and Akinwale, R.O. Note Sci. Biol., 6: 237-143 (2014). [30] Djanaguiraman, M., Ramadass, R. and Durga-Devi, D.: Madras Agric J, 90: 50-53 (2003). [31] Hakim, M.A., Juraimi, A.S., Begum, M., Hanafi, M.M., Ismail, M.R. and Selamat, A.: Afr. J. Biotechnol., 9: 1911-1918 (2010). [32] Sudharani, M., Reddy, P.R. and Reddy, G.H.: Int. J. Sci. Innov. Discover., 3: 22-30 (2013). [33] Surekha Rao, P., Mishra, B., Gupta, S.R. and Rathore, A.: J. Stress Physiol. Biochem., 9: 54-65 (2013). [34] Shanthi, P., Jebaraj, S. and Geetha, S.: Electron. J. Plant Breed., 1: 1208-1212 (2010). [35] Kumari, R., Sharma V.K. and Kumar, H.: Int. J. Pure Appl. Biosci., 3: 191-202 (2015).
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