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Journal of Stress Physiology & Biochemistry, Vol. 10 No. 3 2014, pp. 153-165 ISSN 1997-0838 Original Text Copyright © 2014 by Khan, ur Rehman, Jamil, Irfan, Waheed, Aslam, Kanwal and Shakir
ORIGINAL ARTICLE
Alleviation of Boron Stress through Plant Derived Smoke Extracts in Sorghum bicolor
112 Pirzada Khan , Shafiq ur Rehman , Muhammad Jamil , Shahid Irfan 1, M.A. Waheed 1, Muhammad Mudassar Aslam 1, Memoona Kanwal 1 and Shakirullah Khan Shakir 1*
1 Department of Botany Kohat University of Science & Technology (KUST), Kohat, 26000 Pakistan
2 Department of Biotechnology & Genetic Engineering Kohat University of Science & Technology (KUST), Kohat, 26000 Pakistan
*E-Mail: shakir [email protected]
Received April 5, 2014
Boron is an essential micronutrient necessary for plant growth at optimum concentration. However, at high concentrations boron affects plant growth and is toxic to cells. Aqueous extract of plant-derived smoke has been used as a growth regulator for the last two decades to improve seed germination and seedling vigor. It has been established that plant-derived smoke possesses some compounds that act like plant growth hormones. The present research was the first comprehensive attempt to investigate the alleviation of boron stress with plant-derived smoke aqueous extract on Sorghum (Sorghum bicolor) seed. Smoke extracts of five plants, i.e. Cymbopogon jwarancusa, Eucalyptus camaldulensis, Peganum harmala, Datura alba and Melia azedarach each with six dilutions (Concentrated, 1:100, 1:200, 1:300, 1:400 and 1:500) were used. While boron solutions at concentrations of 5, 10, 15, 20 and 25 ppm were used for stress. Among the dilutions of smoke, 1:500 of E. camaldulensis significantly increased germination percentage, root and shoot length, number of secondary roots and fresh weight of root and shoot while, boron stress reduced growth of Sorghum. It was observed that combined effect of boron solution and E. camaldulensis smoke extract overcome inhibition and significantly improved plant growth. Present research work investigated that the smoke solution has the potential to alleviate boron toxicity by reducing the uptake of boron by maintaining integrity of plant cell wall. The present investigation suggested that plant derived smoke has the potential to alleviate boron stress and can be used to overcome yield losses caused by boron stress to plants.
Key words: Sorghum, boron toxicity, alleviation, seed germination, plant-derived smoke
ORIGINAL ARTICLE
Alleviation of Boron Stress through Plant Derived Smoke Extracts in Sorghum bicolor
112 Pirzada Khan , Shafiq ur Rehman , Muhammad Jamil , Shahid Irfan 1, M.A. Waheed 1, Muhammad Mudassar Aslam 1, Memoona Kanwal 1 and Shakirullah Khan Shakir 1*
1 Department of Botany Kohat University of Science & Technology (KUST), Kohat, 26000 Pakistan
2 Department of Biotechnology & Genetic Engineering Kohat University of Science & Technology (KUST), Kohat, 26000 Pakistan
*E-Mail: shakir [email protected]
Received April 5, 2014
Boron is an essential micronutrient necessary for plant growth at optimum concentration. However, at high concentrations boron affects plant growth and is toxic to cells. Aqueous extract of plant-derived smoke has been used as a growth regulator for the last two decades to improve seed germination and seedling vigor. It has been established that plant-derived smoke possesses some compounds that act like plant growth hormones. The present research was the first comprehensive attempt to investigate the alleviation of boron stress with plant-derived smoke aqueous extract on Sorghum (Sorghum bicolor) seed. Smoke extracts of five plants, i.e. Cymbopogon jwarancusa, Eucalyptus camaldulensis, Peganum harmala, Datura alba and Melia azedarach each with six dilutions (Concentrated, 1:100, 1:200, 1:300, 1:400 and 1:500) were used. While boron solutions at concentrations of 5, 10, 15, 20 and 25 ppm were used for stress. Among the dilutions of smoke, 1:500 of E. camaldulensis significantly increased germination percentage, root and shoot length, number of secondary roots and fresh weight of root and shoot while, boron stress reduced growth of Sorghum. It was observed that combined effect of boron solution and E. camaldulensis smoke extract overcome inhibition and significantly improved plant growth. Present research work investigated that the smoke solution has the potential to alleviate boron toxicity by reducing the uptake of boron by maintaining integrity of plant cell wall. The present investigation suggested that plant derived smoke has the potential to alleviate boron stress and can be used to overcome yield losses caused by boron stress to plants.
Key words: Sorghum, boron toxicity, alleviation, seed germination, plant-derived smoke
Boron is an essential micronutrient required to maintain structural integrity of plant cell wall and metabolic pathways. The optimum B content for most of the plants is 20-100 ppm. Excess levels of Boron results in B toxicity as the B requirements and toxicity levels varies among plants. Boron availability in soil and in irrigation water is an important factor in agricultural production (Tanaka and Fujiwara, 2007). Boron occurs primarily as boric acid (H3BO3) in soil solution, which can be leached under high rainfall situations (Shorrocks, 1997; Yan et al., 2006). Due to this, B becomes deficient for plants that grow there and plant growth is affected as highly leached soils are not able to retain boron. On the other hand, low rainfall conditions can not sufficiently leach B and therefore may accumulate to levels that are toxic to plant growth (Reid, 2007b).
Plant-derived smoke has the ability to break dormancy and stimulate germination of Audouinia capitata, a fynbos species that grows in a fire-prone habitat (De Lange and Boucher, 1990). Since this discovery originated an idea that aqueous smoke extracts can be used to improve seed germination in a broad range of plants related to many families, irrespective of their fire sensitivity (Dixon et al., 1995; Roche et al., 1997; Brown and Botha, 2004). This phenomenon is potentially applicable in seed technology and has been comprehensively discussed in a number of reviews (Brown and Van Staden, 1997; Van Staden et al., 2000). Recently, a highly active and water soluble germination promoting compound, butenolide, 3-methyl-2H-furo [2, 3-c] pyran-2-one has been identified in smoke of burnt fynbos Passerina
vulgaris and grass Themeda triandra L. (Van Staden et al., 2004) as well as from the combustion of cellulose (Flematti et al., 2004). This compound has the ability to stimulate germination at very low concentrations, i.e. 10 -9 M (Flematti et al., 2004; Van Staden et al., 2004). The present research work is the first systematic attempt to investigate the alleviating effects of plant derived smoke extracts against toxicity caused by boron.
MATERIALS AND METHODS
Seeds Collection
Seeds of Sorghum (Sorghum bicolor), DS-2003 were obtained from NARC (National Agriculture Research Centre) Islamabad, Pakistan.
Preparation of Solutions
Plant materials of M. azedarach, C. jwarancusa, P. harmala, D. alba and E. camaldulensis were collected from Kohat region. Shade dried plant parts of these plants were used for preparation of concentrated aqueous smoke extracts by following methods of De Lange and Boucher (1990) as well as Dixon et al. (1995) with slight modifications. The smoke extracts were diluted up to 1:100, 1:200, 1:300, 1:400 and 1:500 by using distilled water. Treatments
Initially the Sorghum seeds were treated with different boron concentrations, i.e. 5; 10; 15; 20 and 25 ppm respectively. Furthermore, smoke solutions with significant results (1:500) were mixed with boron solutions to prepare “B stress alleviating solutions” for assessment of alleviation potential of plant-derived smoke against B stress.
Seed Germination
Ten seeds of Sorghum per petri plate were moistened and chilled in Petri dish covered with double layer filter papers and then kept at 25 0C for two days to obtain uniform germination. After germination the seeds with equal radical size were placed on a filter paper soaked with respective B stress alleviating solutions. The filter paper was rolled like a cigar and was placed in a growth chamber under controlled conditions at 25 0C for ten days. Seed germination percentage was recorded for ten days of incubation by following Chantachume et al. (1995) method. Root and shoot length as well as fresh and dry weight of seedlings were recorded after ten days of incubation.
Measurement of Boron contents
For determination of B level, oven dried roots and shoots of Sorghum plant were digested in sulfuric acid and hydrogen peroxide in 2:1 ratio and analyzed by spectrophotometer following Azomethine - H method reviewed by Basson et al. (1969).
Statistical analysis
Statistics 9 software was used to analyze the data. One-way ANOVA and LSD- tests were applied to determine the correlation among different variables. P-value less than 0.05 considered as significant value.
RESULTS
Present results revealed that smoke extracts collected from plant materials, improved seed germination (Fig. 1a). Significant increase in germination was observed by treating seeds with different smoke dilutions of C. jwarancusa (1:100 and 1:500), D. alba (1:300, 1:400 and 1:500), M.
azedarach at dilutions (1:100 and 1:500), while all dilutions E. camaldulensis and P. harmala (1:100, 1:200 and 1:500) (Fig. 1b). Significant decrease in final germination of Sorghum with (1:200) dilution of both C. jwarancusa and M. azedarach was resulted. Similarly concentrated smoke extracts of all the plants significantly reduced seed germination percentage.
After 24 hours of germination, smoke treatment (1:500) of E. camaldulensis showed most significant results and increased germination percentage was observed with smoke treatment (Fig 2a), while inhibition resulted with B treatments. With the application of combined solutions of smoke and B, smoke alleviated the inhibitory effects of B on seed germination percentage (Fig. 2b).
Effects on Root and shoot length
Root length of Sorghum was significantly increased with application of E. camaldulensis smoke at dilutions (1:200 and 1:500) and P. harmala at dilution of 1:100 (Fig. 3a) while decreased by the application of D. alba dilutions (1:300, 1:400 and 1:500) and M. azedarach dilutions (1:100, 1:300 and 1:400). Similarly shoot length was significantly increased with the somoke dilutions (1:200 and 1:500) of E. camaldulensis and P. harmala (1:100 and 1:500) while dilutions of Melia and Datura as well as concentrated smoke extracts of plants decreased shoot length (Fig. 3b).
Effects on secondary roots of Sorghum
Number of secondary roots of Sorghum were significantly increased when treated with smoke dilutions (1:200 and 1:500) of E. camaldulensis while 1:400 dilution of P. harmala increased the number of
secondary roots as compared to control. Similarly, 1:100 and 1:500 of C. jwarancusa, all dilutions of D. alba and M. azedarach as well as concentrated smoke extracts of all the plants significantly decreased the number of secondary roots (Fig. 4). Effects on root fresh and dry weight
With application of plant-derived smoke, fresh and dry weight of plant roots were increased. Dilutions of E. camaldulensis (1:100, 1:200 and 1:500) and P. harmala (1:100) significantly increased root fresh weight, while D. alba and M. azedarach decreased root fresh weight at all dilutions. Similarly concentrated smoke extract of all the plants reduced fresh weight of roots. Similarly root dry weight was increased with treatment of E. camaldulensis and P. harmala at the same dilutions as for fresh weight (Table 1). However, smoke solutions of D. alba and M. azedarach as well as concentrated smoke of all the plants decreased root dry weight.
Effects on shoot fresh and dry weight
Fresh and dry weight of Sorghum shoot was increased with the smoke solutions of E. camaldulensis and P. harmala. E. camaldulensis smoke dilutions (1:200 and 1:500) and P. harmala dilution (1:500) showed promotory effects while decrease in fresh and dry weight was observed with
D. alba and M. azedarach (1:300, 1:400 and 1:500). Concentrated smoke extracts of all the plants also resulted inhibition and significantly decreased shoot fresh and dry weights (Table 2).
With increase in B concentration, significant reduction in root and shoot length was found (Fig. 5 -7). However, plant derived smoke significantly alleviated the stress of B on root length (Fig. 5a and 7). Though an improvement was found in shoot length with application of alleviated solutions as compared to B stress but it was not significant (Fig. 5b and 7).
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Figure 1. Effect of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel) and P. harmala (Peg) derived smoke on the germination (%) of Sorghum after 24 (a) and 48 (b) hours. (P < 0.05 S).
Figure 2. Germination (%) of Sorghum treated with boron and alleviating solutions (E. camaldulesis + B) after 24 (a) and 48 (b) hours. Vertical bar shows LSD at P < 0.05 S.
Figure 3. Effects of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel), P. harmala (Peg) and their concentrated (Con) smoke on root (a) and shoot length (b) of Sorghum (P < 0.05 S).
□ DWn1:100 ■ 1:200 □ 1:300 D1:400 □ 1:500 □ Con
Figure 4. Effects of C. jwarancusa (Cym), D. alba (Dat), E. camaldulensis (Euc), M. azedarach (Mel), P. harmala (Peg) and their concentrated (Con) smoke on secondary root of Sorghum (P < 0.05 S).
Figure 5. Effect of B and alleviating solutions on root (a) and shoot length (b) of Sorghum. Vertical bar shows LSD at P < 0.05. Each data point shows mean of three replicates.
Figure 6. Effect of B on root and shoot length of Sorghum
Figure 7. Effect of alleviating solutions on root and shoot length of Sorghum.
Figure 8. B contents of Sorghum root (a) and shoot (b). Each data point represents the mean of three replicates. The vertical bar represents LSD at p < 0.05.
Table 1. Effect of smoke dilution on root fresh and dry weight of sorghum
Root
fresh weight
dry weight
Hoot
Smoke
dilutions
Cym Euc Peg Dat Mel Cym Euc Peg Dat Mel
DW 0.164b 0.164d 0.164b 0.164a 0.164a 0.028bc 0.028c 0.028bc 0.028a 0.028a
1 100 0.160b 0.288Be Q.31&L 0.101b 0.090b 0.053ab 0.082ab 0.103a 0.011b 0.012b
1 200 0.218ab 0.345ab 0.184ab 0.061 be 0.092b 0.054ab 0.085ab 0.059b 0.011b 0.013bc
1 300 0.192ab 0.241cd 0.183ab 0.054bc 0.073b 0.049ab 0.056bc 0.038be 0.010b 0.010b
1 400 0.161b 0.236cd 0.168b 0.081 be 0.066bc 0.041abc 0.054bc 0.030bc 0.010b 0.010b
1 500 0.237ab 0.390a 0.165b 0.078b 0.088b 0.064ab 0.088a 0.034bc 0.011b 0.011b
Cone. 0.011c 0.015e 0.017c 0.018c 0.016c 0.007d 0.006c 0.004d 0.003c 0.002b
LSD 0.0993 0.0969 0.1454 0.0542 0.051 0.0395 0.0250 0.0364 0.0151 0.0154
All the data points shown are mean of three replicates with ten seeds in each Petri dish.
Table 2. Effect of smoke on shoot fresh weight and dry weight of Sorghum
Smoke
diutions
Shoot fresh weight
Cym Euc Peg Dat Mel
DW 0.372a 0.372bc 0.372bc 0.372a 372a
1:100 0.473a 0.559ab 0.750ab 0271 abc 0.247abc
1:200 0.413a 0.837a 0.383bc 0.318ab 0.329ab
Shoot dry weight
Cym Euc Peg Dat Mel
0.090a 0.090bc 0.090bc 0.090a 0.090a
0.106a 0.192b 0244ab 0.025ab 0.025ab
0.109a 0.328a 0.099bc 0.035ab 0.022ab
1:300 0.310a 0.381 be 0.506ab 0.074c 0.086be 0.087a 0.181b 0.147bc 0.0167b 0.015b
1:400 0.350a 0.580ab 0.386bc 0.100bc 0.097bc 0.117a 0.190b 0.127bc 0.021b 0.011b
1:500 0.400a 0.928a 0.876a 0.105b 0.125 be 0.065a 0.415a 0.386a 0.017b 0.019b
Cone. 0.023b 0.012d 0.029d 0.041b 0.0361c 0.009b 0.009d 0.004d 0.004c 0.006b
LSD 0.2540 0.3988 0.4085 02372 0.2447 0.0690 0.1337 0.1637 0.0684 0.0713
All the data points shown in the table 1 are mean of three replicates with ten seeds in each Petri dish.
Table 3L Effecl of boron on root and shoot fresh and dry weight of Sorghum
Treatments Hoot Hoot Slioat Shoot
B (ppm) fresh weight dry weight fresh weight dry weight
DW 0.164a 0.026a 0372a 0.090a
5 0.123ab 0.031a 0.355ab 0.034ab
10 0.062bc 0.013b 0.205abc 0.032a b
15 0.056bc 0 012b 0.262ab 0025ab
20 0.05Sbc 0.0 IDb 0.101c 0 012b
25 0.052c 0011b 0.106bc O.OOSb
LSD 0.0679 0.0127 0.2535 0.0731
All the data paints shows mean af ihrcc replicates a rd each replica contai r ten seeds.
Table 4. Effect of alleviating solution of boron and smoke on root and shoot Fresh and dry weight of Sorghum
Treaments Hoot Hoot Shoot Shoot
B (ppm) Frash weight dry weight fnesh weight dry weight
D.W 0.164a 0.026ab 0.505a 0.053a
5+1:500 0.227a 0.043a 0.451 ab 0.037ab
10+1:50 0.192a 0.040a 0.363ab 0.032ab
15+1:500 0.014b 0011c 0.265bc 0.027b
20+1:500 0.013b 0.011c 0.276bc 0.022b
25+1:500 0.044b 0.010c 0.167c 0.017b
LSD 0.0674 0.0150 0.1776 0.0241
All the data point shows mean of three neplicales and each replica contain ten seeds.
Effect of B stress and alleviating solutions on roots and shoots (Table 4). root and shoot fresh and dry weight
Analysis of B in Sorghum roots and shoots
In case of root fresh and dry weight, there was
Present investigation showed that smoke solution
found no effect at lower concentrations of B but higher
potentially alleviated boron stress probably by
concentrations showed a significant reduction in both
reducing the uptake of B. However B uptake was
the parameters (Table 3). Present results also
reduced significantly in roots as compared to shoot
revealed that combined solutions of B and smoke
(Fig. 6a and b). Though smoke solution reduced the
(alleviating solutions) significantly alleviated the toxic
uptake of B in the shoot, but the reduction was not so
effects of B and improved fresh and dry weight of
significant.
DISCUSSION
This study highlights the effect of C. jwarancusa,
E. camaldulensisa, P. harmala, D. alba and M. azedarach extracted smoke on the germination and seedling vigor of Sorghum in order to alleviate boron stress. Smoke collected from these plants enhanced seed germination significantly (Fig.la). In smoke extracts of the plants, concentrated solutions showed inhibition against all the parameters studied. Present results are in accordance with the investigations of Brown and Van Staden (1997) who reported that smoke extracts at lower concentrations improve seed germination. Improvement in germination at lower concentrations of smoke could be attributed to the removal of germination inhibitors such as ABA and phenolics (Da Cuhna and Casali, 1989; Hilhorst and Karssen, 1992). Similarly in case of root and shoot length, plant derived smoke significantly overcame the inhibitory effects caused by excessive amount of B and co-relates with the results of Sparge et al. (2006). Present investigations also indicated that, smoke solutions resulted an increase in seedling fresh and dry weights (Table 1 and 2). These results are in agreement with that of Kulkarni et al. (2006). Similarly number of secondary roots were significantly increased by eucalyptus at the dilution of 1:200 and 1:500 (Fig. 4).
Excess concentration of B in soil may interrupt growth and development of wheat and other higher plants. The most frequent symptoms of boron toxicity in cereals crops include chlorosis and necrosis (Cartwright et al., 1984), delay in development, reduced shoot growth and plant height (Paull et al.,
1988) and root growth suppression (Huang and Graham, 1990). Significant yield losses due to excess boron have been observed in cereals (Cartwright et al., 1984; Paull et al., 1992). Present work Indicates that treatment of plants with B solutions inhibited both root and shoot growth (Fig. 5 and 6). On the other hand plant derived smoke extracts has the potential to overcome the inhibitory effects of B stress through alleviating solutions. Present investigations revealed that Sorghum has lower B contents in shoots as compared to root (Fig. 8). The selection of smoke dilution i.e. 1:500 of E. camaldulensis for alleviating solution was done on the basis of strong vigour among all the dilutions. The germination (%) and the root and shoot length were significantly increased by smoke as well as alleviating solutions overcome the toxicity of boron stress considerably (Fig. 2 and 5).
A significant relationship was found between diverse factors that could manage B tolerance in Sorghum while treating with alleviating solutions. There was a significant interaction between B stress, alleviating solutions and boron contents of seed and seedlings. In addition, Sorghum seeds treated with boron gave lower seed germination and low root and shoot length. Present results suggested that higher dose of boron retard the growth of Sorghum. Similarly, alleviating effects of smoke extracts against B toxicity is just because smoke has the potential to reduce the uptake of boron which ultimately leads to tolerance of Sorghum to B. However the mechanism by which plant-derived smoke solutions reduce Boron uptake is still a mystery to be unveiled.
The present study investigated that, smoke has
the potential to alleviate phytotoxicity caused by B stress and improved seed germination and seedling vigour in Sorghum under B stress condition. Investigations also revealed that, B toxicity has direct relation to B contents of root and shoot, i.e. more B contents, resulted lower growth of root and shoot while, alleviating solutions of smoke and B, smoke reduced the uptake of B resulted an increase in root and shoot length. Hence, the tolerance to B toxicity could be attributed to lower B uptake. Present work also suggested that, smoke is involved in physiological and metabolic activities that improves plant growth under B toxic condition. It is predicted that this study will provide a basic strategy for reducing the risks of B toxicity and maintaining sustainable plant production.
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