University of Khartoum

Biology, Tissue Culture, Molecular Systematic, Quantitative Estimation of Zeatin and Potential Uses of Moringa Species in Sudan

Biology, Tissue Culture, Molecular Systematic, Quantitative Estimation of Zeatin and Potential Uses of Moringa Species in Sudan

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Title: Biology, Tissue Culture, Molecular Systematic, Quantitative Estimation of Zeatin and Potential Uses of Moringa Species in Sudan
Author: Hamed, Sarra Ali Saad
Abstract: The research aimed to study the biology, tissue culture, quantitative estimation of zeatin, molecular systematic and potential uses of three Moringa species, namely Moringa oleifera, M. peregrina and M. stenopetala growing in Sudan. Plant samples were collected from their natural localities in the Eastern and Northern states of Sudan. The morphological characteristics of the three plants were studied and the species were identified using standard taxonomical procedures. Tissue cultures were initiated using Murashige and Skoog (MS) medium supplemented with different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) using shoot and root explants taken from sterile seedlings. Micropropagation of M.oleifera and M. peregrina was carried out using MS medium supplemented with kinetin and benzyl adenine (BA) at different concentrations using different explants. Molecular systematic study was conducted using two genetic markers based on the polymerase chain reaction (PCR) technique, Randomly Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) were used for identifying genetic variation within and between the three species individuals, which were assayed for polymorphism. Zeatin content of Moringa spp. leaves, seeds and callus tissues was detected and estimated, using a modified chromatographic method based on Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) techniques. The biological activity of M.oleifera leaf extract was tested using seed germination test with Khaya senegalensis seeds. All obtained data were statistically analyzed, using SAS and STATISTICA computer programs. The morphological characteristics of the vegetative and the reproductive parts confirmed the identity of the exotic M.oleifera and the two indigenous species M. peregrina and M. stenopetala. In tissue culture experiments, shoot explants showed better response than roots for the three species. M.oleifera shoot calluses initiated on MS medium supplemented with 0.5 mg/l 2,4-D gave massive friable tissues with vigorous growth. M. peregrina shoot explants cultured on medium supplemented 0.25 mg/l 2,4-D gave the highest frequency of callus initiation, while M. stenopetala required 0.5 mg/l 2,4-D for the best callus initiation. Shoot and root primordia were observed in all established calluses. Micropropagation results showed that, M.oleifera successful shoot formation and rapid growth was obtained when 6 mg/l BA was added to the medium. The number of shoots, shoot iv length, number of leaves, and number of nodes significantly increased in the regenerated plantlets. When 3 mg/l kinetin was used for M.oleifera, it produced the highest number of roots/explant (3.4) and mean root length about 3.3 cm. Micropropagation of M.peregrina showed that explants grown on media supplemented with 5 mg/l BA gave 2.8 shoots/explant, while 3 mg/l kinetin resulted in the highest number of roots/explant (2.0). The highest value of root length was 3.9 cm. BA failed to initiate roots in the two species. Five RAPD primers (UBC101, UBC103, UBC104, OPA03 and OPA17) were found polymorphic, and gave 32 bands. The average percentage of polymorphism was 79.3% and 25 bands were identified as fingerprints. The distance matrix values ranged between 0.06 and 0.94, which indicates high range of variation between the individuals of the three species. RAPD technique has provided a quick screen for the DNA-sequence polymorphisms. Five ISSR primers (807, 810, 814, 848 and 878) detected polymorphic bands within and between the three Moringa spp. individuals. The average percentage of polymorphism for M.oleifera was 65.2%, and 1band was identified as fingerprint. M.peregrina individuals showed 74.9% polymorphism, and 1band was identified as fingerprint. The average percentage of polymorphism for M. stenopetala was 94.3%. No finger prints were obtained for this species. ISSR primers were tested for their amplification with the 30 Moringa spp. individuals. Five ISSR primers showed clear polymorphism among the three species and detected 34 polymorphic bands with an average of 77.3% polymorphism and 2 bands were identified as fingerprints. The distance matrix values ranged between 0.13 and 1.14, which reflected high range of variation between the accessions of the three species. The UPGMA diagram based on genetic distances using 5 ISSR primers showed that M.oleifera and M. perigrina are more genetically closer than M.stenopetala. The results showed that ISSR genetic markers are more effective and specific than RAPD genetic markers for assessment of genetic diversity of the three species. Zeatin content of the three Moringa species was determined, and M.oleifera, M.peregrina and M.stenopetala fresh leaves tissues contained zeatin in 271.1, 121.8 and 297.34 mg/100gm respectively, while dry leaves extracts contained 145.52, 129.54 and 214.58 mg/100gm respectively. HPLC analysis of seed extracts revealed that the tissues contained lower amounts of zeatin when compared with the leaves tissues, whereas M.oleifera seed extract was devoid of zeatin, while M.peregrina and M.stenopetala seeds contained 78.72 and 59.64 mg/100gm respectively. Fresh callus tissue v extracts analysis, revealed that callus tissues of the three Moringa species have the potentiality to produce zeatin although variations in the content were recorded. M.stenopetala callus showed the highest zeatin concentration (8.73 mg/100gm). The effect of M.oleifera leaf extract on percentage germination of K. senegalensis seeds was stimulatory at the concentration 1:40, v/v (73.3%), followed by 1:30, v/v which resulted in 50.7% and the control seeds gave the least (48%) germination. The study confirmed the identity of Moringa species, showed the regenerative capacity directly through micropropagation and indirectly through callus tissue and the presence of zeatin in appreciable physiological levels, which can be utilized.
Description: 153 Pages
URI: http://khartoumspace.uofk.edu/123456789/27402
Date: 2018


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