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Improving Salinity Tolerance in Brassica (Brassica napus var. Bsa and Brassica campestris var. Toria) by Exogenous Application of Proline and Glycine Betaine.


Soil salinity along with a variety of environmental stresses is now a very serious problem all over the world due to its adverse effects on plant growth and physiology (Taie et al., 2013). Salt stress is a great challenge for agriculture. Yield of the crop is reduced because crops fail to cope with salinity stress (Aymen and Cherif, 2013). There is 22 million hectares arable land in Pakistan. About 24% of crops of Pakistan are grown on rainfed land, whose area is about 4.6 million hectares (Muhammad and Muhammad, 2007). All over the world 35% agricultural production has been decreased due to salinity. Salinity affects 7% of the world's entire land area (Chaum et al., 2012). Salinization of arable land is increasing day by day and it is expected that after 25 years 30% of the total land area will face the problem ofsalt stress (Latefand Chaoxing, 2014; Kapoor et al., 2013). Crop growth and productivity are decreased by soil salinity (Cominelli et al.,2013).

Plants protect themselves from injurious and destructive effects of salt stress by producing different compatible osmoprotectant metabolites such as proline and glycine betaine (Chelli-Chaabouni et al., 2010). These osmolytes gather in the plant and protect tissue and cellular membranes of the plant (Anjum et al., 2012). It has been reported that foliar spray of proline and glycine betaine is valuable for plants in mitigating salt induced injuries (Ahmad et al., 2012; Hoque et al., 2007).

Proline and glycine betaine are also source of carbon and nitrogen. They stabilize the structures of membranes. Proline metabolism has a main role in storage and transfer of energy (Gilberti et al., 2014). The effects of salinity stress can be decreased by foliar application of the osmolytes i.e. proline and glycine betaine. In the plants stress tolerance quality is enhanced by the application of foliar spray and it is also a beneficial plan (Ali and Ashraf, 2011).

Brassica species are present in family Cruciferae. Members of the family Cruciferae are known as mustard plants. The petals of the plants belonging to this family are in a cross manner i.e. four petals are cross shaped. Canola (Brassica napus and Brassica campestris L.) is an important oil seed crop, its world average is 1,820 kg/ha (Chambo et al., 2014). 13% of the world's demand of oil is obtained from canola. Oil content of canola seeds is 40% (Snowdon et al., 2007). The experiment was conducted to determine the effect of proline and glycine betain on brassica under saline conditions.

Materials and Methods

The pot culture experiment was conducted in Department of Botany, University of Sargodha, Sargodha Pakistan. Proline and glycine betaine were applied exogenously to improve salinity tolerance in plants. The experiment was laid out in completely randomized design (CRD) with three replications.

Physiological parameters. Chlorophyll content. Method proposed by Davies (1976) was used to compute the chlorophyll contents. The extraction from the 0.5 cm chopped leave pieces was done with 5 mL acetone (80%) and kept at 10[degrees]C. The absorbance of the supernatant was measured at 645 and 663 nm on spectrophotometer. Chl a, Chl b and total chlorophyll were calculated by using the following formula:

Chl. a = [12.7 (OD 663) -2.69 (OD 645)] x V/1000 x W Chl. b = [22.9 (OD 645) -4.68 (OD 663)] x V/1000 x W Total Chl. = [20.2 (OD 645) + 8.02 (OD 663)] x V/100 x W V = Volume of the extract W = Weight of the sample

Total free amino acids. Method proposed by Hamilton and Van Slyke (1943) was used to compute the total free amino acids. Chopped segments of leaves were extracted with 0.2M phosphate buffer of 7.0 pH. 1 mL from extract, 10% pyridine and 2% ninhydrin solution were put in the test tube. Distilled water was used to make the volume upto 50 mL. The optical density of this coloured solution was seen at 570 nm on spectrophotometer (Hitatchi, 220, Japan). A standard curve was made with Leucine and then calculation for free amino acids was done by this formula:

Total amino acids (mg/g fresh wt) = Graph reading of sample x volume of sample x dilution factor/Weight of fresh tissue x 100

Nitrate reductase activity (NRA). Method proposed by Sym (1984) was used to compute the nitrate reductase activity.

Procedure. Phosphate buffer with the molarity of 0.02 M was added in leaf sample. From this mixture 1 mL was taken out and 0.02M KN[O.sub.3] solution was entered in it. The amount of KN[O.sub.3] used for this purpose was 1 mL and 1-naphthyl ethylene diamine dihydrochloride (0.02%) was added in the solution after vigorous shaking of 1-naphthyl ethylene diamine dihydro-chloride. With N[O.sub.2] diazocomplex, a pink colour was produced. Spectrophotometer was used to determine the absorbance at 542 nm.

Total soluble sugars. Method proposed by Yemm and Willis (1954) was used to compute the total soluble sugars.

Procedure. In the test tubes of 25 mL, plant extract (0.3 mL) was added. 6 mL of anthrone reagent was added in the test tubes. Then the test tubes were warmed in the boiling water bath for 10 min. These test tubes were cooled down by placing them in chilled water for 10 min and then incubated for 20 min by maintaining the temperature at 25[degrees]C. Spectrophotometer was used to measure the optical density at 625 nm (Hitatchi, 220, Japan). Standard curve was developed for the calculation of soluble sugars.

Na and K analysis. Digestion. In the digestion tubes concentrated H2SO4was added with 0.5 g of ground material (Wolf and Stahl, 1982). 35% hydrogen peroxide was added in the digestion tubes. After this they were heated at 350[degrees]C in the digestion block. This process of heating was continued for 30 min. 0.5 mL of hydrogen peroxide was added in it. For making the digested material colourless 0.5 mL hydrogen peroxide was added and the tubes were placed again in the digestion block. This step was done again and again until the solution became colourless. The volume was kept 50 mL in volumetric flasks by adding distilled water.

Estimation of cations ([Na.sup.+] and K+). Method proposed by Jenway (PFP 7) was used to determine sodium ([Na.sup.+]) and potassium (K+) with the help of flame photometer PFP7 (Yilmaz and Yavuz, 1999).

Calcium determination. Method proposed by salinity laboratory (Kunze and Dixon, 1986) was used to determine calcium.

Yield and yield components. Plant height was recorded using a meter rod. Data for yield and yield components were recorded at maturity.

Statistical analysis. Analysis of variance of the data from each attribute was computed using three factor factorial design (Steel et al., 1997).

Results and Discussion

As shown by Table 1 that salinity stress has significantly reduced the growth and production of brassica but the exogenous application of proline and glycine betaine minimizes the effect of salinity. Salinity has decreased plant height (Caption: Fig. 1), shoot fresh weight (Caption: Fig. 2), shoot dry weight (Caption: Fig. 3), root fresh weight (Caption: Fig. 4), root dry weight (Caption: Fig. 5), Chl a (Caption: Fig. 6), Chl b (Caption: Fig. 7), total Chl (Caption: Fig. 8), total soluble sugars (Caption: Fig. 9), protein (Caption: Fig. 10), total free amino acids (Caption: Fig. 11), NRA (Caption: Fig. 12), potassium (Caption: Fig. 13), calcium (Caption: Fig. 14) and sodium (Caption: Fig. 15) increased under salt stress. As shown that 8 mM and 10 mM concentration of proline and glycine betaine were more effective to reduce the effect of salinity as compared to 4 mM and 5 mM concentrations of proline and glycine betaine. Sodium ions present in the growth mediums showed antagonistic effect on calcium and potassium ions.

Figure 1-15 shows the effect of exogenous application of proline and glycine betaine on plant height, shoot and root fresh and dry weight, shoot, Chl a, Chl b, total Chl, protein, total free amino acids, NRA, sodium, calcium, potassium of brassica under saline and nonsaline conditions.

Salinity tolerance in brassica increased by exogenous application of proline and Glycine betaine. Glycine betaine content was observed to be increased under salt stress (Amandeep et al., 2014).

Glycine betaine was found effective to decrease salt stress damage when it was exogenously applied on canola (Athar et al., 2015). With glycine betaine, proline also play role in improving plant growth. The application of 1 and 5 mM proline improved the growth of brassica varieties (Posmyk and Janas, 2007). Salinity levels increases uptake of [Na.sup.+] in all plant parts including root, stem and leaf. Simultaneously it decreases the calcium and potassium uptake due to antagonistic effects. The addition of [Na.sup.+] in plant parts increased as the level of salinity was increased. The amount of proline, soluble carbohydrates and reduced sugar increased as salinity increased (Mostajeran and Gholaminejad, 2014).

Proline is a widespread compatible solute. There are many roles for proline in saving plants from harmful and damaging effects of salinity. It can stabilize the membranes and guard them from harmful ions which can destroy their structure (Khan et al., 2009). Proline and glycine betaine can be used to check the salt tolerance ability of different plant species (Ahmad et al., 2009).

Foliar spray of proline and glycine betaine enhances the development of both salt affected and non-stressed plants of canola varieties. Same results have been observed in maize (Nawaz and Ashraf, 2007) and wheat (Raza et al., 2007).

It is clear from the outcomes of current study that salinity stress reduced plant growth, plant height, dry biomass and fresh weight. Growth of all the plants increased by applications of proline and glycine betaine, either the plants were facing salinity or were grown under non saline conditions. Chl a, Chl b and total chlorophyll were also reduced under salt stress but their value increased when proline and glycine betaine were applied on the plants. The 4 and 5 mM levels of proline and glycine betaine are less effective to reduce salinity than 8 and 10 mM levels of proline and glycine betaine. Proline and glycine betaine decreased the harmful effects of salinity stress and enhanced the growth of plants. The encouraging effects are clear from the above results.

(received August 29, 2016; revised March 10, 2017; accepted March 14, 2017)


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Ameer Khan (a), Hira Fatima (b), Abdul Ghani (a), Muhammad Nadeem (c), Abida Aziz (b), Mujahid Hussain (a) * and Muhammad Ikram (a)

(a) Department of Botany, University of Sargodha, Sargodha, Pakistan

(b) Department of Botany, The University of Lahore, Sargodha, Pakistan

(c) Institute of Food Science and Nutrition, University of Sargodha, Sargodha, Pakistan

* Author for correspondence;


Caption: Fig. 1. Concentration of proline/glycine betaine.

Caption: Fig. 2. Concentration of proline/glycine betaine.

Caption: Fig. 3. Concentration of proline/glycine betaine.

Caption: Fig. 4. Concentration of proline/glycine betaine.

Caption: Fig. 5. Concentration of proline/glycine betaine.

Caption: Fig. 6. Concentration of proline/glycine betaine.

Caption: Fig. 7. Concentration of proline/glycine betaine.

Caption: Fig. 8. Concentration of proline/glycine betaine.

Caption: Fig. 9. Concentration of proline/glycine betaine.

Caption: Fig. 10. Concentration of proline/glycine betaine.

Caption: Fig. 11. Concentration of proline/glycine betaine.

Caption: Fig. 12. Concentration of proline/glycine betaine.

Caption: Fig. 13. Concentration of proline/glycine betaine.

Caption: Fig. 14. Concentration of proline/glycine betaine.

Caption: Fig. 15. Concentration of proline/glycine betaine.
Table 1. Salinity effects on Brassica growth and production


sov             DF   Plant       Shoot      Root        Shoot
                     height      fresh      fresh       dry
                                 weight     weight      weight

Variety (V)     1    2361.92**   14.10**    20544.7**   297.47**
Treatment (T)   2    3667.83**   263.18**   131470**    1786.21**
Chemicals (C)   2    188.26**    12.16**    1477.30**   36.41**
VxT             2    115.95**    0.14NS     372.45**    6.71**
V X C           2    1.93NS      0.26NS     6.01**      0.14NS
TxC             4    2.63*       0.32NS     9.36**      0.46NS
V X T X C       4    0.54NS      0.09NS     3.33*       0.34NS
Error           36
Total           53


sov             DF   Root        Chi a       Chi b       Total
                     dry                                 Chi

Variety (V)     1    482.23**    20.49**     20.72**     142841**
Treatment (T)   2    5189.66**   2516.62**   6440.18**   668953**
Chemicals (C)   2    464.87**    32.59**     195.34**    157850**
VxT             2    38.32**     0.00NS      0.00NS      109487**
V X C           2    10.59**     0.00NS      0.00NS      110148**
TxC             4    19.05**     1.91*       13.17**     137291**
V X T X C       4    5.42**      0.00NS      0.00NS      121428**
Error           36
Total           53


sov             DF   Sugars      Proteins    A.A         NRA

Variety (V)     1    1202.56**   8517.43**   538452**    7618.79**
Treatment (T)   2    10245.7**   99259.5**   6194365**   226283**
Chemicals (C)   2    137.93**    5292.14**   1349845**   13771.5**
VxT             2    274.53**    892.85**    279862**    2045.37**
V X C           2    5.51**      67.22**     3070.57**   123.20**
TxC             4    1966.87**   112.45**    73593.8**   928.13**
V X T X C       4    16.84**     93.59**     39303.2**   816.04**
Error           36
Total           53


sov             DF   Na          Ca          K

Variety (V)     1    4593750**   80.60**     4511001**
Treatment (T)   2    1.1E+08**   4918.25**   4.0E+07**
Chemicals (C)   2    1.4E+07**   379.26**    2998814**
VxT             2    656250**    0.00NS      377554**
V X C           2    0.00NS      0.00NS      12138.5**
TxC             4    143569**    13.89**     92754.2**
V X T X C       4    0.00NS      0.00NS      2969.79**
Error           36
Total           53
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Author:Khan, Ameer; Fatima, Hira; Ghani, Abdul; Nadeem, Muhammad; Aziz, Abida; Hussain, Mujahid; Ikram, Muh
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Mar 1, 2018
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