Berseem Cultivars Salinity Tolerance & Early Seedling

By: Dr. P. Kumar

Salinity tolerance of some cultivars of berseem (Trifolium alexandrinum L.) during germination and early seedling growth

Shipra Agarwal(1), Neha Agarwal(2), Alka(3) and P. Kumar(4)*

Botany Department, Hindu College, Moradabad- 244001, India

(1), (2)

(3), (4)

* Corresponding author


The salinity tolerance of 20 cultivars of berseem (Trifolium alexandrinum L.) was tested under varying salt stresses (3, 6, 7.2, 10, 12 and 14 dSm-1) during germination and early seedling stage. Germination was differentially affected at different salinity levels. Genotypes JB 1, JHB 04 2, SAIDI and WARDAN had shown minimum inhibition in % germination at 10, 12 and 14 dSm-1 while cultivars BL 10, UPB 110, FAHLI and JHB 05 2 had expressed maximum inhibition. Dry weight of shoot decreased with increase in salinity levels but significant reductions were obtained only at higher salinity levels (7.2 - 14 dSm-1). All values for SS, PR and SSI indicated positive correlations with that of NS of shoot dry weight. Perfect positive correlations (r = 1**, P = 0.01) were obtained between SSI and PR of shoot dry weight. Dry weight of root and seedling also declined with increase in salinity but significant reductions were noted at 6 - 14 dSm-1 in all cultivars tested. Seedling dry weight indicated that all genotypes registered significant reductions at 6 to 14 dSm-1. Correlation coefficient (r values) indicated strong positive correlation between SS & NS and perfect positive correlation was observed between SSI and PR for seedling dry weight. On the bases of SSI, SHSI and seedling dry weight cv. BL 22 and WARDAN have been proved to be tolerant while BL 10 and JHB 05 1 have been proved sensitive on the bases of SSI and SHSI. Cultivars HFB, MESCAVI, BB 2 and JHB 05 2 also proved as

sensitive on the bases of SSI and seedling dry weight.


Trifolium alexandrinum, salinity, germination, seedling growth


The salt tolerance of crop plants is a very acute problem in agriculture. It has attracted the attention of many investigators and practical agricultural workers because of the need to increase yield on saline soils and to develop and utilize new saline areas. Rapidly increasing soil salinity has multifarious effects on plant growth and productivity. Salt affected land comprises of 19% of the 2.8 billion hectares of arable land on earth, and an increase in this menace is posing a serious threat to agriculture globally. Excessive salinity adversely affects plants through ion toxicity and by decreasing the uptake of water, which is usually limited under rangeland conditions. Plants resist salinity through mechanisms conditioning either avoidance or tolerance (Levitt, 1980; Johnson, 1991). Increased productivity of forage crops under saline condition is a desired characteristic in irrigated areas. Clover species are often used in single or as mixed stands. Salinity impairs seed germination, reduces nodule formation, retards plant development and ultimately reduces crop yield (Greenway and Munns, 1980).

The commercial use of berseem clover (Trifolium alexandrinum L.) is relatively new in United States. It has been an important crop in the Mediterranean, Near East, and India for many years. Because of its importance as a field crop in Egypt, it is commonly called "Egyptian clover". Berseem clover is a winter annual legume with oblong leaflets and hollow stems. It grows upright and produces yellowish-white flowers. The plants may grow as tall as 18 to 30 inches. Legumes are considered a relatively salt sensitive family (Mass and Hoffman, 1977) within which limited variability for salinity tolerance has been detected (Johansen et al., 1990). The purpose of present study is to determine some physiological basis of salt tolerance of available genotypes of berseem (Trifolium alexandrinum L.) at early seedling growth.

Materials and Methods:

Seeds of 20 varieties of berseem (Trifolium alexandrinum L.) were obtained from Indian Grassland and Fodder Research Institute (IGFRI), Jhansi, India to test their salt tolerance on the bases of germination and early seedling growth.

The following varieties namely cv. UPB110, cv. JB1, cv. BB3, cv. BL1, cv. HFB, cv. BL 22, cv. MESCAVI, cv. BL 42, cv. BL10, cv. WARDAN, cv. JHB146, cv. JHB04-1, cv. JHB04-2, cv. JHB04-3, cv. JHB05-1, cv. JHB05-2, cv. JHB05-3, cv. BB2, cv. FAHLI and cv. SAIDI were procured for the present investigation. A Petriplate culture experiment was conducted to examine the tolerance of the genotypes to salt stress considering germination and vegetative growth characteristics at seedling stage.

First of all, seeds were sorted out manually selecting healthy, uniform sized and uninfected seeds. These seeds were surface sterilized with 0.1% mercuric chloride (HgCl2) solution for one minute and washed thoroughly with distilled water to remove the traces of HgCl2. Petridishes (3" diameter) which were used in the present experiment were cleaned with teepol, washed repeatedly with distilled water and were sterilized in hot oven at 800C for 24 hours. The surface sterilized seeds were placed on Whatman no.1 filter paper lined in the Petridishes. Twenty-five seeds were kept at equidistance in each Petridish. The filter papers were moistened with saline solutions of 3, 6, 7.2, 10, 12 and 14 dSm-1. Distilled water moistened Petridishes served as control. Saline solutions of different EC levels were prepared by mixing the salts of NaCl, CaCl2, NaHCO3 and Na2SO4 as described by U. S. Salinity Laboratory Staff handbook (1954).

The Petridishes having seeds were kept in BOD incubator at a constant temperature of 25+ 20C. The saline water evaporated from the Petridishes in the last 24 hours was set of compensated by adding an equal quantity of distilled water with the help of dropper. Each salinity treatments in each cultivar was maintained in triplicate to eliminate the experimental error. The observations were recorded on % germination as well as length and dry weight of shoot and root at 240 hours after imbibition. For the purpose, the samples were collected following completely randomized design considering three replicates (Brunning and Kintz, 1977). The dry weights were measured after keeping the fresh plant samples in hot air oven at 700C for 48 hours. Seedling dry weight was also computed by adding the dry weights of root and shoot. The seedling height considered the sum of length of shoot and root and Seedling height stress index (SHSI) was calculated by the following formula: SHSI = (Seedling height of stressed seedlings/Seedling height of control seedling) X 100. The data were analyzed statistically.

Statistical Analysis

The data obtained from the laboratory experiment were combined into a single analysis, which was performed by minitab statistical program (Minitab Inc., state college, P.A.). The data were subjected to analysis of variance and means were separated by least significant difference (LSD), if the F-test was significant between genotypes (Brunning and Kintz, 1977) at P = 0.05. Percentage reduction (PR) due to salinity stress in relation to the non-stressed environment was also determined for all traits. The salinity intensity index (SII) was calculated for dry weight of root, shoot and seedling for each genotype with the help of SII = (1-Xss/Xns), where Xss and Xns are the means of all genotypes under SS and NS environment respectively (Fisher and Maurer, 1978).

Salinity susceptibility index (SSI) for seedling dry weight, root dry weight and shoot dry weight for each genotype was also calculated as follows: SSI = (1-Yss/Yns)/SII, where Yss and Yns are the means of a given genotype in SS and NS environment respectively. The index of salinity tolerance provides data on the relative effects of increasing salt concentration on each genotype.

The Pearson product- moment correlation (r) is used to determine if there is a relationship between two sets of paired numbers.

t = r Ö(n-2)/ Ö(1-r2), which follows student's t- distribution with (n-2) degree of freedom. If the value of t comes out to be significant, we reject h0 at the level of significance adopted and conclude that r ? 0, i.e. 'r' is significant of correlation in the population. If t comes out to be non- significant then H0 may be accepted and we conclude that variables may be regarded as uncorrelated in the population.

Results and Discussion:

Effect of salinity on seed germination

The evaluation of various cultivars of berseem to salt tolerance was conducted under salt stressed and non-stressed conditions. In general, the seed germination was not affected significantly in WARDAN, JHB 04 2, JHB 04 3 and SAIDI upto 10 dSm-1 while JB 1 and JHB 146 were not affected significantly at any salinity level (Table 1), therefore these varieties expressed better salinity tolerance than BB 3, BL 1, BL 10, JHB05 2, FAHLI and BL 42 which exhibited significant reductions. West and Francoise (1982), Murillo-Amador and Troyo-Dieguez (2000), Alka et al. (1981), Zurayk et al. (1998), and Jamil et al. (2005) have also reported salinity-induced inhibition in seed germination in cowpea, barley, chickpea and Brassica species respectively.

Table 1

The remaining varieties had been differentially affected at different salinity levels, however, in these varieties the germination remained unaffected at 3 and 6 dSm-1 but significant reductions were observed at 10, 12 and 14 dSm-1. Cultivars JB 1, JHB04-2, SAIDI and WARDAN registered minimum inhibition in seed germination at 10, 12 and 14 dSm-1 while cultivars BL 10, UPB 110, FAHLI and JHB05-2 expressed maximum inhibition at these salinity levels (Table 1). Dantas et al. (2005) have also reported that percent germination decreased at 100 and 200 mol m-3 NaCl concentration in some cultivars of cowpea. The deleterious effects of high salt concentrations are due to osmotic effect, specific ion effect and altered ion activities (Kurian et al., 1967). Dell and Quilla (1992) also reported that high salt concentrations reduced water potential of the soil, which subsequently and adversely affected water absorption and final germination percentage.

Effect of salinity on seedling growth characteristics:

Salinity invariably inhibited seedling growth in different genotypes of berseem which is expressed as seedling height stress index (SHSI), dry weight of root, shoot and seedling, and salt susceptibility index (SSI) (Table 2).

Table 2, 2a, 2b

Perusal of the data presented in table (2), indicates differential responses in different varieties. On the basis of salinity height stress index (SHSI), JB 1, MESCAVI, WARDAN, BL 22, JHB 04 3, FAHLI and SAIDI had shown better performance at 6 to 10 dSm-1 while BL 10 and JHB 05-1 had expressed poorest performance. The remaining varieties expressed mixed responses at 6 - 10 dSm-1. Dantas et al. (2005) have reported decreased hypocotyl length at 100 mol m-3 and decreased root and total seedling length at 50 mol m-3 NaCl salinity in some cowpea genotypes. They also reported that 10 mol m-1 NaCl enhanced hypocotyl length which might have occurred due to possible priming effect caused by low NaCl concentration.

Analysis of variance revealed that the shoot dry weight was not affected significantly at 3 dSm-1 in twelve genotypes viz UPB 110, JB 1, BB 3, BL 22, JHB 146, JHB04-1, JHB04-3, JHB05-1, JHB05-2, JHB05-3, FAHLI and SAIDI. Correlation coefficient (r values) also indicates strong positive correlation between PR of SDW and seedling dry weight and SSI of SDW and PR of seedling dry weight but negative weak correlation was obtained between SS of SDW and PR of seedling dry weight. Strong positive correlations were also observed for PR and SSI of SDW with that of SSI of seedling dry weight but negative weak correlations were obtained between SS of SDW and SSI of seedling dry weight (Table 2b). Significant reductions in dry weight of shoot were observed at 6 to 14 dSm-1 in all genotypes except for BB 3, BL 22 and FAHLI at 6 dSm-1. It is further confirmed from the data that genotypes JHB05-1 (23 - 54%), JHB05-2 (19 - 50%) and BB 2 (21 - 55%) recorded maximum inhibition in dry weight of shoot whereas BL 22 (4 - 22%), WARDAN (11 - 31%) and JHB04-2 (8.6 - 47.8%) recorded minimum at 6 - 12 dSm-1 (Table 2). According to several authors salt stress decreased root and shoot growth of the seedling (Viegas et al., 1999; Camara et al., 2000; Murillo-Amador & Troyo-Dieguez, 2000). The reduction in seedling dry weight under salt stress may be attributed to the inhibited hydrolysis of reserved food and its translocation to the growing shoots (Singh et al., 2001).

Dry weight of root was significantly and invariably inhibited in all the genotypes from 6 to 14 dSm-1 (Table 2) but varieties behaved differently at 3 dSm-1. Somewhat similar trend was exhibited by seedling dry weight at various salinity levels. The overall picture indicates that MESCAVI, BL 10, JHB 04-1, JHB 05-2 and BB 2 registered more than 30% reductions at 6 dSm-1, while lesser reductions were recorded in BL 1, BL 22, JHB 05-1 and SAIDI ( 60%) at 10 dSm-1, while cultivars UPB 110, BL 22, BL 1 and JHB 05-1 registered less than 35% reduction in root dry weight. The varieties behaved slightly different at 12 dSm-1, where HFB, MESCAVI, BL 42, WARDAN, JHB 04-3, JHB 05-2 and FAHLI administrated more than 70% reductions, indicating greater sensitivity while other varieties suffered lesser. Neumann (1995) reported that salinity can rapidly inhibit root growth and hence reduced its capacity of water uptake and essential mineral nutrition from soil. Salinity induced inhibition in shoot and root growth of cowpea has also been reported by Murillo-Amador & Troyo-Dieguez (2000).

Statistical analysis of data on seedling dry weight indicates that all genotypes registered significant reductions at 6 to 14 dSm-1 as compared to their respective controls. Correlation coefficient (r values) indicates strong positive correlation between SS & NS and perfect positive correlation was observed between SSI and PR for seedling dry weight (Table 2a). Positive correlations between stressed and non stressed seedling dry weight supported similar findings by Foolad (1996). Slight negative correlation was observed between PR & SS and SSI & SS for seedling dry weight (Table 2a). Correlation coefficients were also worked out for NS, SS, PR and SSI between shoot dry weight and seedling dry weight (Table 2b). These values indicate strong positive correlation between NS of SDW and NS & SS of seedling dry weight. Values also exhibited strong positive correlation between SS of SDW and SS of seedling dry weight but negative weak correlations were obtained for PR & SSI of SDW and SS of seedling dry weight (Table 2b). Slight variations were recorded at 3 dSm-1 where BL 22, WARDAN, JHB 146, JHB 04-2, JHB 05-2 and SAIDI were not affected significantly while other varieties expressed significant reductions in seedling dry weight. The magnitude of reduction in seedling dry weight differed in different varieties expressing their relative salt tolerance under different salinity regimes. Table (2) indicates that HFB, MESCAVI, JHB 05-2 and BB 2 experienced maximum inhibitions (25%) at 6 dSm-1 while genotypes BL 1, MESCAVI, BL 10, JHB 146, JHB 04-3, JHB 05-1, JHB 05-2, BB 2 and FAHLI recorded more than 30% inhibitions at 7.2 dSm-1. The inhibitory effects were further aggravated at 10 and 12 dSm-1. More than 40%

inhibition was noticed in HFB, JHB 146, JHB 05-1, JHB 05-2, BB 2 and FAHLI at 10 dSm-1, while genotypes HFB, MESCAVI, JHB 04-2, JHB 05-1, JHB 05-2, BB 2 and FAHLI recorded more than 50% inhibitions at 12 dSm-1, therefore data indicate that maximum inhibition in seedling dry weight was recorded in HFB, MESCAVI, BB 2 and JHB05-2 at 6 to 12 dSm-1.

Table (2) also indicates that genotypes BL 22, BL 42, WARDAN, JHB 04-1 and JHB 04-2 recorded minimum inhibition of around 10% at 6 dSm-1 and these cultivars also exhibited similar behavior at 7.2 dSm-1 while remaining varieties registered higher reductions in seedling weight. The behavior of the varieties slightly differed at 10 dSm-1. Here UPB 110, BL 22, WARDAN and JHB 04-2 recorded least reductions (less than 30%). In addition to these varieties, BB 3, JB 1 and BL 1 also proved better at 10 dSm-1 where the minimum reduction was around 30%. Present findings confirmed the earlier reports of Dantas et al. (2005) who reported decreased seedling growth in cowpea under NaCl salinity. It was formally thought that excess of salt retards the absorption of water and suppresses the growth through osmotic effect. Kirkham et al. (1969) concluded that growth of salt stressed bean and barley plant was reduced not by lack of water but by the lower osmotic potential. It may be that, either the adjustment process or the low osmotic potential is the causes of the growth suppression. Munns (2003) reported that suppression of plant growth under saline condition might either be due to the decreased availability of water or to the increasing toxicity of NaCl with increasing salinity. Correlation coefficients for shoot dry weight of stressed and non-stressed plants were also worked out (table 2b). All values for SS, PR and SSI indicated positive correlations with that of NS of SDW, however, strong positive correlations were obtained between SS and NS of SDW. Negative weak correlations were obtained between PR & SS and SSI & SS of SDW Interestingly, perfect positive correlations were obtained between SSI and PR of shoot dry weight (Table 2b).

Salinity tolerance in terms of salt susceptibility index (SSI)

The tolerance of varieties to salinity stress was also accessed on the basis of SSI for their dry weight of root, shoot and seedling at 6, 7.2 and 10 dSm-1 (Table 2) as earlier proposed by Bayuelo-Jimenez et al. (2002) who tested the salt tolerance of different traits of Phaseolus species at seedling stage and accordingly the genotypes have been grouped into two categories. In first category, cultivars having SSI values less than 0.83 are marked as salt tolerant/moderately tolerant and in second category, cultivars having SSI values more than 1.0 are termed as sensitive. On the basis of SSI values for seedling dry weight, the first category of tolerant cultivars is comprised of BL 22, JHB 04-2, WARDAN, BL 42, BB 3, JHB 04-1, JHB 05-3 and SAIDI and second category of sensitive cultivars is comprised of JHB 05-1, JHB 05-2, BB 2, HFB, MESCAVI, BL 10, JHB 146 and JHB 04-3 (Table 2).

On the basis of SSI for shoot dry weight, varieties BL 22, BL 42, WARDAN, JHB 04-2, UPB 110 and BB 3 are grouped under tolerant category while cultivars BL 1, JHB 146, JHB 05-1, JHB 05-2 and BB 2 are grouped under sensitive category (Table 2). Therefore, the data on seedling dry weight and shoot dry weight do not agree in toto and had exhibited differential salt tolerance of the genotypes. It is interesting to state that the values of SSI for shoot dry weight at 6, 7.2 and 10 dSm-1 are in accordance with the values of SSI for seedling dry weight in genotypes JB 1, BB 3, BL 22, BL 42, WARDAN, and JHB 04-2 which have been proved as tolerant/moderately tolerant whereas cultivars JHB 146, JHB 05-1, JHB 05-2 and BB 2 have been proved as sensitive.

The values of SSI for root dry weight of different genotypes tested for their salt tolerance were compared with that of the values of SSI of seedling dry weight. It is evident that the data are not in total accordance with the SSI of shoot and seedling dry weight. However, at many places these are in agreement with each other. SSI values for root dry weight have also corresponded with the SSI values of seedling dry weight in genotypes UPB 110, HFB, BL 22, MESCAVI, BL 10, JHB 04-3, JHB 05-2, JHB 05-3, BB 2, SAIDI and FAHLI at 6, 7.2 and 10 dSm-1 whereas other varieties differed at some salinity levels (Table 2).

On the bases of SSI, SHSI and seedling dry weight cv. BL 22 and WARDAN have been proved as tolerant while BL 10 and JHB 05 1 have been proved as sensitive on the bases of SSI and SHSI. Cultivars HFB, MESCAVI, BB 2 and JHB 05 2 also proved as sensitive on the bases of SSI and seedling dry weight. The varietal differences exhibited by berseem cultivars in relation to seedling growth can be attributed to the fact that some varieties are more capable of adjusting to the osmotic stress by regulating uptake and accumulation of ions and synthesis of organic molecules.


The authors are thankful to Dr. S. N. Singh, Principal and Dr. A. K. Chaudhry, Head, Department of Botany, Hindu College, Moradabad for providing the laboratory facilities and to Prof. V. K. Sharma, Head, Department of Statistics for statistical analysis. Thanks are also due to Dr. A. K. Roy, Plant Improvement Division, Jhansi for providing the pure line seeds of berseem.


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Table 1 Effect of salinity on germination in some cultivars of berseem

GenotypeGermination (%)

Salinity level (dSm-1)

0367.2101214CD at 5%

cv. UPB 11083.480*80*76.677066.6760.64.10

cv. JB 19090*90*90*90*86.67*86.67*4.10

cv. BB 393.339090909090803.26

cv. BL 196.67909083.3383.3383.3383.333.74

cv. HFB9696*95.33*93.33*9080804.91

cv. BL 2296.6793.33*9393.33*86.6786863.35

cv. MESCAVI96.6796.67*96.33*909083.3383.334.43

cv. BL 4286.6783.33*808076.3376.3373.335.13

cv. BL 1096.6786.6783.3383.3376.6770703.35

cv. WARDAN9696*92*92*92*90904.62

cv. JHB 14696.196*96*96*95*92*92*4.17

cv. JHB 04-110096*96*96*9288845.24

cv. JHB 04-29896*96*96*96*88843.51

cv. JHB 04-39896*96*96*95.4*88845.35

cv. JHB 05-19292*88*868484804.37

cv. JHB 05-29692*88888476765.07

cv. JHB 05-39288*88*88*8080805.99

cv. BB 29692*92*908580805.13

cv. FAHLI9696*84848282804.43

cv. SAIDI9696*96*96*92*92*885.79

* = Non significant

Table 2 Effect of salinity on dry weight (mg/seedling) of shoot (SDW), root (RDW) and seedling (TDW) and seedling height stress index (SHSI) in different genotypes of berseem

(PR- Percent reduction; SSI- Salt susceptibility index)

GenotypeSalinity(dSm-1)SHSIShoot growth SDW PR SSIRoot growth RDW PR SSITotal dry weight TDW PR SSI

cv. UPB 11001.900.662.56


693.81 1.60-15.790.990.52-





CD at 5%0.260.0410.20

cv. JB 102.040.4552.50







CD at 5%0.1660.0060.029

cv. BB 302.801.34.10







CD at 5%0.5270.0940.079

cv. BL 102.000.6672.67







CD at 5%0.3110.00340.027

cv. HFB02.000.6672.67







CD at 5%0.3180.05010.02

cv. BL 2201.500.6672.17







CD at 5%0.0740.0260.021

cv. MESCAVI02.640.463.10







CD at 5%0.0950.0440.071

cv. BL 4201.930.6672.60






CD at 5%

cv. BL 1001.930.462.39






CD at 5%0.0250.020.038

cv. WARDAN01.930.462.39






CD at 5%0.0340.0240.26

cv. JHB 14602.570.633.20







CD at 5%0.0580.0330.077

cv. JHB 04-102.990.5403.53







CD at 5%0.0380.0150.024

cv. JHB 04-202.550.763.31







CD at 5%0.0490.0580.17

cv. JHB 04-302.250.4552.71






CD at 5%0.0770.0150.04

cv. JHB 05-102.600.623.22






CD at 5%

cv. JHB 05-202.600.603.20







CD at 5%0.3180.00840.48

cv. JHB 05-302.300.4552.76







CD at 5%0.3040.00390.041

cv. BB 202.460.723.18






CD at 5%0.070.0420.029

cv. FAHLI02.301.23.50






CD at 5%0.3310.0780.045

cv. SAIDI03.101.224.32







CD at 5%0.070.0320.063

* = Non significant, SHSI = Seedling height stress index, SDW = Shoot dry weight, PR = Percent reduction, SSI = Salt susceptibility index, RDW = Root dry weight, TDW = Total dry weight

Table 2a Correlation coefficient among non-stressed (NS), salt-stressed (SS), percent reduction (PR) and salt susceptibility index (SSI) for seedling dry weight (TDW) of different berseem genotypes.

Total dry weight

TDW NS SS PR SSI Salinity level (dSm-1)


NS .959**.906**.803**0.0260.1700.3420.0270.1660.342










** Indicates values of r tested highly significant at P= 0.01 using t- test.

Shoot dry weight

TDW NS SS PR SSI Salinity level (dSm-1)























Table 2b Correlation coefficient among non-stressed (NS), salt-stressed (SS), percent reduction (PR) and salt susceptibility index (SSI) for shoot dry weight (SDW) and seedling dry weight (TDW) of different Berseem genotypes

**Indicates values of r tested highly significant at P= 0.01 using t- test.


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