Physiological and Molecular Studies on the 24-Epibrassinolide Mechanism(s) in Ameliorating Effects of Salted Phaseolus vulgaris L. cv. Brunco Tissues
Rabha Mohammed Abd-Alsid Mansur;
Abstract
In vitro regeneration of common bean (Phaseolus vulgaris L.) is
a requirement for genetic transformation, which involves induction and development to the whole plant.
Several studies have shown that brassinosteroids attenuate the effects of salt stress in plants. In order to assess the influence of exogenous application of varying concentrations of NaCl and 24-epibrassinolide (24-Epi) each alone or in combination into MS culture media in inducing salt tolerance in vitro P. vulgaris L. plantlets different physiological, biochemical, hormonal and molecular parameters were investigated. The decline in several components of plant metabolism, induced by salt, was attenuated by 24-epibrassinolide application indicating the importance of brassinosteroid synthesis for plants growing under salinity and that 24-Epi improvement might have contributed to the salt tolerance.
1) This part of the study established a promising protocol for callus induction and regeneration of Phaseolus vulgaris L. cv. Brunco. Whereas callus was induced on leaf, stem and root explants cultured on MS medium fortified with 1.5 mg L-1 2, 4-D in combinated with 0.1 mg L-1 of 24-Epi , leaf explants showed the best result. Direct and indirect somatic embryogenesis was achieved from stem and leaf explants while root failed in recovery with all tested media. The best performance of direct and indirect somatic embryogenesis were observed on MS medium supplemented with 0.2 mg L-1 TDZ + 0.05 or 0.1 mg L-1 of 24-Epi, while highest percentage of embryogenic frequency 88.9 and 72 were recorded for stem explants. However, the greatest embryogenic calli fresh weights 14.7 and 9.7 (mg/jar) were derived from leaf explants. The highest regeneration percentage was achieved for stem explants compared to leaf and recorded 88.9 and 66.7 for direct and indirect regeneration, respectively, cultured on MS medium augmented with 2 mg L-1 TDZ + 0.1 mg L-1 of 24-Epi. Hence 24-Epi (0.1 mg L-1) additively improved 2, 4-D and TDZ role in Brunco plantlets regeneration.
2) This part also aimed to optimize an in vitro shoot lets regeneration system via direct and indirect organogenesis using 24-Epi. Stem and stem-derived calli recorded the best results of shoot lets proliferation percentage (90% and 70%) compared with leaf and leaf derived calli (83% and 50%) respectively. However, roots and roots derived calli completely failed in shoot lets regeneration. In general, the data point out that addition of 2 mg L-1 TDZ + 0.1 mg L-1 24-Epi to MS medium greatly improved most vegetative growth criteria concerned to shoot lets regeneration of P. vulgaris via direct and indirect organogenesis during the whole growth period regardless of the explants and the pathway. The healthy regenerated shoot lets (direct and indirect pathway) previously cultured on S4 (2 mg L-1 TDZ) were in vitro transferred and sub cultured on different rooting culture media for rooting stage. Concerning medium composition generally, 0.5 mg L-1 IAA + 0.1 mg L-124-Epi (R7) medium appears the best for rooting efficiency compared with the other used media.
From the obtained results it can be recognized that, the behavior and trends of 24-Epi on in vitro P. vulgaris cultures media may be oriented and arranged to the behavior of auxins in the nutrient medium for roots formation and as a cytokinin in shoot lets proliferation.
The development of effective plant regeneration pro¬tocols of P.vulgaris through this study would provide exciting opportunities for further studies regarding the effects of the physiological and molecular events during in vitro culture, and will promote the application of plant tissue culture technology in the area of selection resistance, and genetic transformation.
3) The present study also aimed to investigate physiological, biochemical, hormonal and molecular responses of P. vulgaris plantlets to salinity stress concomitant with investigating the role of exogenous hormonal application strategy (24-Epi) in ameliorating the deleterious effect of salt stress. In vitro-produced plantlets of P. vulgaris were treated with varying levels of NaCl (50, 100 and 150 mM) added to MS solid media and/or a long with 24-Epi (0.1 mg L-1).
Plantlets were analyzed for photosynthetic pigments (chl a, chl b and carotenoids), carbohydrate fractions (soluble sugar, sucrose and starch), nucleic acids, nitrate reductase (NR) activity and total soluble protein assessments. In addition proline , antioxidant enzymes activities e.g., superoxide dismutase (SOD), catalase (CAT), ascorbic peroxidase (APX) , glutathione reductase (GR) ,ascorbic acid oxidase (AAO), peroxidase (POD), polyphenol oxidase (PPO), hydrogen peroxide levels, malondialdehyde content (MDA)were estimated. Also, nutrients like N, K, Ca, p, Na, Cl ions and endogenous hormones like, idole-3-acetic acid (IAA), naphthalene acetic acid (NAA), gibberellic acid(GA3), cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA) were investigated. On the other hand, at the molecular level, sodium dodecyl sulfate polyacrylamide gel electrophoresis technique (SDS-PAGE) for protein profile, Native-PAGE technique for peroxidase and polyphenol oxidase isozymes banding patterns was done. Moreover, Random Amplified Polymorphic DNA by polymerase chain reaction (RAPD-PCR) was applied to investigate any molecular genetics differences between regenerated P. vulgaris plantlets under the present study conditions.
Phaseolus vulgaris shoots and roots of plantlets exposed to NaCl especially (100 and 150 mM levels) exhibited a significant decline in photosynthetic pigments ,carbohydrate fractions ,nucleic acids ,NR activity , soluble proteins, antioxidant enzymes activity like SOD, CAT, APX, AAO (in roots harvested after three days only), GR, POD and PPO activities. Similar retard response was recorded in some ions contents likes' nitrogen, potassium, calcium, phosphorus, and in certain endogenous hormones as naphthalene acetic acid, gibberellic acid, and in cytokinins accumulation. Moreover, analyzing changes in SDS-PAGE protein banding patterns revealed total number of 30 bands with molecular weights ranging from 14.5 KDa to 90.9 KDa. Fourteen bands were observed as monomorphic, while sixteen bands were polymorphic, giving 53.33% polymorphism. Proteins banding were increased under salt stress compared to control induced as salt marker proteins. Peroxidase and polyphenol oxidase isozymes banding patterns increased under salt stress and polymorphism percentage was 75 and 85.7 respectively indicating priority role for PPO than for POD.
On contrast, P. vulgaris shoots and roots sub cultured in salinized MS in vitro and harvested three and ten days later showed increase in proline content, ascorbic acid oxidase (especially after ten days), hydrogen peroxide and malondialdehyde and in plantlets endogenous hormones like indole –3-acetic acid, abscisic and jasmonic acid.
Whereas, addition of 24-Epi (0.1 mg L-1) to the MS media alone or along with different salt levels especially the lowest salt dose significantly mitigated salt stress deleterious effect by enhancing chlorophylls a and b ,soluble sugars, sucrose , starch and proline accumulation manifesting osmotic adjustment. Growth was encouraged via enhancing nucleic acids synthesis, nitrate reductase activity and rising proteins content concomitant with increasing cytokinins, jasmonic, indole acetic acids, gibberellic acid and maintain membrane integrity and affects their selectivity resulting in rebalance ions toxicity via discarding sodium and chlorine ions simultaneously with enhancing nitrogen, potassium, calcium and phosphorus ions inside cells . 24-Epi alone or along with salts can potentially ameliorate oxidative stress and so protect membranes and vital bio compounds from free radicals attack by regulating antioxidant enzyme gene expression expressed as increment in their activity. Also increased carotenoids to assist in scavenge free radicals. In contrast, AAO activity was decreased, and H2O2 concomitant with MDA levels declined. Also at the hormonal levels ABA recorded retard accumulation. At the molecular level, SDS-proteins revealed unique 24-Epi protein with molecular weight 77.6 KDa. On the other hand salts 50 and 100 or 150 mM NaCl in combination with 0.1 mg L-124-Epi, lead to disappearance of proteins with molecular weights 84.3 and 55.2, 18.1 KDa referred to control.
a requirement for genetic transformation, which involves induction and development to the whole plant.
Several studies have shown that brassinosteroids attenuate the effects of salt stress in plants. In order to assess the influence of exogenous application of varying concentrations of NaCl and 24-epibrassinolide (24-Epi) each alone or in combination into MS culture media in inducing salt tolerance in vitro P. vulgaris L. plantlets different physiological, biochemical, hormonal and molecular parameters were investigated. The decline in several components of plant metabolism, induced by salt, was attenuated by 24-epibrassinolide application indicating the importance of brassinosteroid synthesis for plants growing under salinity and that 24-Epi improvement might have contributed to the salt tolerance.
1) This part of the study established a promising protocol for callus induction and regeneration of Phaseolus vulgaris L. cv. Brunco. Whereas callus was induced on leaf, stem and root explants cultured on MS medium fortified with 1.5 mg L-1 2, 4-D in combinated with 0.1 mg L-1 of 24-Epi , leaf explants showed the best result. Direct and indirect somatic embryogenesis was achieved from stem and leaf explants while root failed in recovery with all tested media. The best performance of direct and indirect somatic embryogenesis were observed on MS medium supplemented with 0.2 mg L-1 TDZ + 0.05 or 0.1 mg L-1 of 24-Epi, while highest percentage of embryogenic frequency 88.9 and 72 were recorded for stem explants. However, the greatest embryogenic calli fresh weights 14.7 and 9.7 (mg/jar) were derived from leaf explants. The highest regeneration percentage was achieved for stem explants compared to leaf and recorded 88.9 and 66.7 for direct and indirect regeneration, respectively, cultured on MS medium augmented with 2 mg L-1 TDZ + 0.1 mg L-1 of 24-Epi. Hence 24-Epi (0.1 mg L-1) additively improved 2, 4-D and TDZ role in Brunco plantlets regeneration.
2) This part also aimed to optimize an in vitro shoot lets regeneration system via direct and indirect organogenesis using 24-Epi. Stem and stem-derived calli recorded the best results of shoot lets proliferation percentage (90% and 70%) compared with leaf and leaf derived calli (83% and 50%) respectively. However, roots and roots derived calli completely failed in shoot lets regeneration. In general, the data point out that addition of 2 mg L-1 TDZ + 0.1 mg L-1 24-Epi to MS medium greatly improved most vegetative growth criteria concerned to shoot lets regeneration of P. vulgaris via direct and indirect organogenesis during the whole growth period regardless of the explants and the pathway. The healthy regenerated shoot lets (direct and indirect pathway) previously cultured on S4 (2 mg L-1 TDZ) were in vitro transferred and sub cultured on different rooting culture media for rooting stage. Concerning medium composition generally, 0.5 mg L-1 IAA + 0.1 mg L-124-Epi (R7) medium appears the best for rooting efficiency compared with the other used media.
From the obtained results it can be recognized that, the behavior and trends of 24-Epi on in vitro P. vulgaris cultures media may be oriented and arranged to the behavior of auxins in the nutrient medium for roots formation and as a cytokinin in shoot lets proliferation.
The development of effective plant regeneration pro¬tocols of P.vulgaris through this study would provide exciting opportunities for further studies regarding the effects of the physiological and molecular events during in vitro culture, and will promote the application of plant tissue culture technology in the area of selection resistance, and genetic transformation.
3) The present study also aimed to investigate physiological, biochemical, hormonal and molecular responses of P. vulgaris plantlets to salinity stress concomitant with investigating the role of exogenous hormonal application strategy (24-Epi) in ameliorating the deleterious effect of salt stress. In vitro-produced plantlets of P. vulgaris were treated with varying levels of NaCl (50, 100 and 150 mM) added to MS solid media and/or a long with 24-Epi (0.1 mg L-1).
Plantlets were analyzed for photosynthetic pigments (chl a, chl b and carotenoids), carbohydrate fractions (soluble sugar, sucrose and starch), nucleic acids, nitrate reductase (NR) activity and total soluble protein assessments. In addition proline , antioxidant enzymes activities e.g., superoxide dismutase (SOD), catalase (CAT), ascorbic peroxidase (APX) , glutathione reductase (GR) ,ascorbic acid oxidase (AAO), peroxidase (POD), polyphenol oxidase (PPO), hydrogen peroxide levels, malondialdehyde content (MDA)were estimated. Also, nutrients like N, K, Ca, p, Na, Cl ions and endogenous hormones like, idole-3-acetic acid (IAA), naphthalene acetic acid (NAA), gibberellic acid(GA3), cytokinins (CKs), abscisic acid (ABA), jasmonic acid (JA) were investigated. On the other hand, at the molecular level, sodium dodecyl sulfate polyacrylamide gel electrophoresis technique (SDS-PAGE) for protein profile, Native-PAGE technique for peroxidase and polyphenol oxidase isozymes banding patterns was done. Moreover, Random Amplified Polymorphic DNA by polymerase chain reaction (RAPD-PCR) was applied to investigate any molecular genetics differences between regenerated P. vulgaris plantlets under the present study conditions.
Phaseolus vulgaris shoots and roots of plantlets exposed to NaCl especially (100 and 150 mM levels) exhibited a significant decline in photosynthetic pigments ,carbohydrate fractions ,nucleic acids ,NR activity , soluble proteins, antioxidant enzymes activity like SOD, CAT, APX, AAO (in roots harvested after three days only), GR, POD and PPO activities. Similar retard response was recorded in some ions contents likes' nitrogen, potassium, calcium, phosphorus, and in certain endogenous hormones as naphthalene acetic acid, gibberellic acid, and in cytokinins accumulation. Moreover, analyzing changes in SDS-PAGE protein banding patterns revealed total number of 30 bands with molecular weights ranging from 14.5 KDa to 90.9 KDa. Fourteen bands were observed as monomorphic, while sixteen bands were polymorphic, giving 53.33% polymorphism. Proteins banding were increased under salt stress compared to control induced as salt marker proteins. Peroxidase and polyphenol oxidase isozymes banding patterns increased under salt stress and polymorphism percentage was 75 and 85.7 respectively indicating priority role for PPO than for POD.
On contrast, P. vulgaris shoots and roots sub cultured in salinized MS in vitro and harvested three and ten days later showed increase in proline content, ascorbic acid oxidase (especially after ten days), hydrogen peroxide and malondialdehyde and in plantlets endogenous hormones like indole –3-acetic acid, abscisic and jasmonic acid.
Whereas, addition of 24-Epi (0.1 mg L-1) to the MS media alone or along with different salt levels especially the lowest salt dose significantly mitigated salt stress deleterious effect by enhancing chlorophylls a and b ,soluble sugars, sucrose , starch and proline accumulation manifesting osmotic adjustment. Growth was encouraged via enhancing nucleic acids synthesis, nitrate reductase activity and rising proteins content concomitant with increasing cytokinins, jasmonic, indole acetic acids, gibberellic acid and maintain membrane integrity and affects their selectivity resulting in rebalance ions toxicity via discarding sodium and chlorine ions simultaneously with enhancing nitrogen, potassium, calcium and phosphorus ions inside cells . 24-Epi alone or along with salts can potentially ameliorate oxidative stress and so protect membranes and vital bio compounds from free radicals attack by regulating antioxidant enzyme gene expression expressed as increment in their activity. Also increased carotenoids to assist in scavenge free radicals. In contrast, AAO activity was decreased, and H2O2 concomitant with MDA levels declined. Also at the hormonal levels ABA recorded retard accumulation. At the molecular level, SDS-proteins revealed unique 24-Epi protein with molecular weight 77.6 KDa. On the other hand salts 50 and 100 or 150 mM NaCl in combination with 0.1 mg L-124-Epi, lead to disappearance of proteins with molecular weights 84.3 and 55.2, 18.1 KDa referred to control.
Other data
| Title | Physiological and Molecular Studies on the 24-Epibrassinolide Mechanism(s) in Ameliorating Effects of Salted Phaseolus vulgaris L. cv. Brunco Tissues | Other Titles | دراسات فسيولوجية وجزيئية علي آليه 24- برازينوليد في تلطيف الإجهاد الملحي بأنسجة الفاصوليا صنف برانكو | Authors | Rabha Mohammed Abd-Alsid Mansur | Issue Date | 2015 |
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