Comparison of Salt Tolerance in Brassicas and Some Related Species

In this paper the salt tolerance in Brassicas and some related species was compared. When seedlings germinated on sand cultures with liquid MS medium were considered, the relative germination rate, root length, shoot length and fresh seedling weight were significantly correlated with each other (P < 0.01), and only the relative shoot lengths were significantly different among the tested genotypes (P < 0.05); When both seedlings germinated on MS and MS plus 0.4% NaCl were considered, only the relative shoot length of seedlings germinated on MS was significantly different from that germinated on MS + 0.4% NaCl (P < 0.05), and also only the relative shoot lengths were significantly different among the tested genotypes (P < 0.01). Raphanus sativa cv. Changfeng, B. juncea cv. JC and Brassica napus cv. ZS 10 showed low salt tolerance in terms of relative germination rate, root length, shoot length and fresh seedling weight; B. oleracea cv. JF-1, Sinapis alba cv. HN-2 showed high salt tolerance in terms of relative germination rate, root length, shoot length and fresh seedling weight. Based on our result we suggest that relative shoot length might be convenient to rank the salt tolerance but cluster analysis based on multiple parameters of relative germination rate, root length, shoot length and fresh seedling weight might be more accurate in screening for salt tolerance in Brassicas and related species.


Introduction
Soil salinity is an issue that affects an estimated 6% of the world's land surface area or 12,780 million hectares (Mha) and secondary salinization from irrigation impacts an estimated 20% of irrigated land or 1474 Mha [1].According to the United Nations reports, 20% of agricultural land and 50% of world cropland are salt affected [2].Furthermore, there is also a dangerous trend of a 10% per year increase in the saline area throughout the world [3].China has a large area of inland saline-alkali land, approximately 8.1 MHa, equivalent to 40% of the total cultivated land in the country [4,5].
The Brassicaceae family consists of many important field crops such as oilseed rape, the Brassica oleracea vegetable group (cauliflower, broccoli, brussels sprouts, kale) as well as other species such as Brassica rapa and Raphanus sativus [6].However, their growth, yield, and oil production are markedly reduced due to salinity [7].
The most common adverse effect of salinity on the crop of Brassica is the reduction in plant height, size and yield as well as deterioration of the product quality [8,9].
In previous studies, the Brassicas were usually grown in salt conditions to compare parameters such as relative germination rate, root length, fresh seedling weight or seed yield [10][11][12][13][14][15][16][17].In the present study the seeds of Brassicas and related species were grown on sand cultures with and without salt to explore an efficient index to compare the salt tolerance in Brassicas and related species and to determine variations in degree of salt tolerance at inter-and intra-specific levels particularly at the germination and seedling stages.

Materials
Seeds of Sinapis alba (cv.HN1, HN2), Brassica carinata cv.EJ and Isatis indigotica cv.HN were provided by Pro.Dr. Zaiyun Li of Huazhong Agricultural University, China.The Brassica napus seeds were from the Oil Crops Institute, CAAS, China.The other seeds were provided by Lab of Plant Genetics and Breeding of Hubei University, China.

Methods
Sixty healthy seeds for each genotype were inoculated on sand cultures with liquid MS and MS + 0.4% NaCl.Germination rates were scored 4 -5 days after inoculation.The germinated seeds on MS and MS + 0.4% NaCl were transferred onto sand cultures with MS and MS + 0.4% NaCl for 3 -4 days and then root length, shoot length and fresh seedling weight were measured.The temperature was set at 25˚C with a 16-hr photo period under 2000 lx.The experiment was arranged in a completely randomized design with three replicates.Salt tolerance indices were calculated according to Zeng et al. (2002) [18] and further used to classify genotypes using cluster analysis as described by El.Hendawy et al. (2005) [19].Cluster analysis was carried out based on Euclidean distance of the salt tolerance indices.The cluster group rankings were obtained from the average of means of the multiple parameters in each cluster group.A sum was obtained by adding the number of cluster group rankings in each genotype.The genotypes were finally ranked based on the sums, such that those with the smallest and largest sums were ranked respectively as the most and least tolerant genotypes in terms of relative salt tolerance.Variance analyses, multiple comparisons and cluster analyses were carried out on SPSS.

Correlations among Relative Germination
Rate, Root Length, Shoot Length and Fresh Seedling Weight From Table 1 it was found that when seeds were germinated on sand cultures with liquid MS medium, the relative germination rate, root length, shoot length and fresh seedling weight were significantly correlated with each other (P < 0.01); When seeds were germinated on sand cultures with liquid MS medium plus 0.4% NaCl, the relative germination rate was only significantly correlated with shoot length (P < 0.05), the relative root length was significantly correlated with shoot length and fresh seedling weight (P < 0.01), and relative shoot length was significantly correlated with fresh seedling weight (P < 0.01).

Effect of Germination Condition on Relative Root Length, Shoot Length and Fresh Seedling Weight
When only seeds germinated on MS medium were con- sidered, variance analysis indicated that the difference of relative shoot lengths among the tested genotypes was significant (P < 0.05), while that of relative root length and fresh seedling weight were not significant (data not shown).
When both seeds germinated on MS and MS + 0.4% NaCl were considered, variance analysis indicated that the relative shoot length of seedlings germinated on MS was significantly different from that germinated on MS + 0.4% NaCl (P < 0.05), while that of relative root length and fresh seedling weight were not significant; the relative shoot lengths were significantly different among the tested genotypes (P < 0.01), while that of relative germination rate, root length and fresh seedling weight were not significant (data not shown).
From Table 2 it was found that based on relative shoot length B. carinata cv.EJ, B. oleracea cv.JF-1 and Eruca sativa cv.hub12 showed high salt tolerance while Raphanus sativus cv.changfeng and B. napus cv.ZS 10 were identified as most salt-sensitive.

Cluster Analysis of Salt Tolerance
From Table 3

Discussion
Although salt stress affects all growth stages of a plant, seed germination and seedling growth stages are known to be more sensitive in most plant species [8,20,21].[13].Our preliminary studies also showed that seed germination, root length, shoot length and fresh seedling weight of Brassicas were inhibited by 20% -60% when the NaCl concentration was 0.4%.Therefore we used 0.4% NaCl for seed inoculation and seedling growth to compare the salt tolerance.
In earlier screening of Brassica species for salt tolerance, the superiority of amphidiploid species B. carinata, B. juncea, and B. napus over the diploid species, B.rapa, B. nigra, and B. oleracea was proposed from different tudies [8,[22][23][24].It has been further suggested that the s salt tolerance of amphidiploids has been acquired from the A (B. rapa) and C (B. oleracea) genomes [10].However, there were also different conclusions as significant inter-and intraspecific variation for salt tolerance exists within Brassicas, which can be exploited through selection and breeding for enhancing salt tolerance of the crops [9,12,13,[25][26][27].For example, of turnip cultivars, Shaljum desi surakh was highest in seed germination, while it was lowest in seedling shoot dry biomass production.However, Neela Shaljum having lower seed germination percentage produced maximum seedling shoot dry biomass.In the same way, cv.Desi of radish with minimum seed germination had highest shoot dry weight under saline conditions [12].Therefore efficient and accurate indices and criteria for identifying salt-tolerance in Brassicas and related species are needed.The multivariate cluster analysis method was recommended as the best criteria for the identification of salt tolerance in crop species such as rice [18,28,29], green gram [30], wheat [19], tomato [31], sugarcane [32], peanut [33], cauliflower [34].As pointed out by Khrais et al. [35] and Zeng et al. [18], the advantages of using a multivariate analysis in the evaluation of salt tolerance are hat it allows: 1) a simultaneous analysis of multiple t  3.
parameters to increase the accuracy of the genotype ranking; 2) the ranking of genotypes even when plants are evaluated at different salt levels and salt tolerance varies with salinity levels, especially when the salt tolerance indices are averaged across salt levels; and 3) a more convenient and accurate estimation of salt tolerance among genotypes by simply adding the numbers in cluster group ranking at different salt levels.
In the present study, when seedlings germinated on sand cultures with liquid MS medium were considered, the relative germination rate, root length, shoot length and fresh seedling weight were significantly correlated with each other (P < 0.01), and the relative shoot lengths were significantly different among the tested genotypes (P < 0.05), while the relative root length and relative fresh seedling weight were not significantly different; when seedlings germinated on liquid MS medium plus 0.4% NaCl were considered, the relative germination rate was only significantly correlated with shoot length (P < 0.05), the relative root length was significantly correlated with shoot length and fresh seedling weight (P < 0.01), and relative shoot length was significantly correlated with fresh seedling weight (P < 0.01).When both seedlings germinated on MS and MS plus 0.4% NaCl were considered, the relative shoot length of seedlings germinated on MS was significantly different from that germinated on MS + 0.4% NaCl (P < 0.05), suggesting that germination in salt condition had some selection effect for shoot length, while relative root length and fresh seedling weight were not significantly different; the relative shoot lengths were significantly different among the tested genotypes (P < 0.01), while the relative germination rate, root length and fresh seedling weight were not significantly different.
Based on relative shoot length B. carinata cv.EJ, B. oleracea cv.JF-1 and E. sativa cv.hub12 were classified as highly salt-tolerant while R. sativus cv.Changfeng, and B. napus cv.ZS 10 were identified as most salt-sensitive.R. sativa cv.changfeng, B. juncea cv.JC and B. napus cv.ZS 10 showed low salt tolerance in terms of relative germination rate, root length, shoot length and fresh seedling weight; B. oleracea cv.JF-1, S. alba cv.HN-2 showed high salt tolerance in terms of relative germination rate, root length, shoot length and fresh seedling weight; B. carinata cv.EJ showed high salt tolerance in terms of root length, shoot length and fresh seedling weight but very low salt tolerance in terms relative germination rate.
Based on our result we suggest that relative shoot length might be convenient to rank the salt tolerance but cluster analysis based on multiple parameters of relative germination rate, root length, shoot length and fresh seedling weight might be more accurate in screening for salt tolerance in Brassicas and related species.

Figure 1 .
Figure 1.Hierarchical cluster analysis of the genotypes based on multiple salt tolerance indices of seedlings germinated on MS medium (Euclidean distance).The genotypes that the numbers represent were indicated in Table3.

Table 1 . Correlations among relative germination rate, root length, shoot length and fresh seedling weight.
Note: Left Bottom: Correlations of relative germination rate, root length, shoot length and fresh weight of seedlings germinated on MS + 0.4% NaCl; Top Right: Correlations of relative germination rate, root length, shoot length and fresh weight of seedlings germinated on MS; ** P < 0.01; * P < 0.05.

Table 2 . Multiple comparison of relative shoot length among the tested genotypes when seeds germinated on MS were con- sidered.
it was found that R. sativa cv.changfeng, B. juncea cv.JC and B. napus cv.ZS 10 showed low salt tolerance in terms of relative germination rate, root length, shoot length and fresh weight; B. oleracea cv.JF-1, S. alba cv.HN-2 showed high salt tolerance in terms of relative germination rate, root length, shoot length and fresh seedling weight; B. carinata cv.EJ showed high salt tolerance in terms of root length, shoot length and fresh seedling weight but with very low salt tolerance in terms relative germination rate.B. carinata cv.EJ and Sinapis alba cv.HN-2 were identified as highly salt-tolerant.S. alba cv.HN-1, I. indigoica cv.HN and B. rapa cv.xiaobaicai were identified t The genotypes were classified into 5 groups.Genotypes in the same group are not significantly different from each other.
From Figure1it was found that B. oleracea cv.JF-1, as salt-tolerant, R. sativa cv.changfeng and B. napus cv.ZS 10 were identified as most salt-sensitive.