الفهرس 
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Abstract
The present study was carried out in Department of Genetics, Agriculture Center for Genetic Engineering and Biotechnology (ACGEB), Faculty of Agriculture.
Plant growth promoting rhizobacteria are various soil bacteria that are capable of producing beneficial effects in culture or in a protected environment. They have potential in agriculture use and can lead to increased yields of a wide variety of crops. Azospirillum is one of the most studied PGPR. It reached commercialization in several countries, including Argentina, Mexico, India, Italy, and France. Azospirillum spp. are known for their ability to produce plant hormones, as well as polyamines and amino acids in culture media. Among these hormones, indoles, mainly indole-3-acetic acid, cytokinins and gibberellins . These phytohormones alter metabolism and morphology of plants, leading to better absorption of minerals and water, consequently larger and healthier plants.
In the present study, isolation of Azospirillum spp. (30 isolates) was carried out. Isolates were evaluated for their capability to produce IAA and cytokinin (primary and secondary screening). They were identified depending on biochemical consideration (Bergey’s manual and BIOLOG) and molecular genetics (16SrDNA gene). The most powerful isolate was subjected to genetic improvement based on chemical and physical induction of mutation. The chemical mutagens used were sodium azide, nitrosoguanidine and acridine orange. The physical mutagen used was U.V. light. Mutants were screened for Indoles and cytokinins depending on specific bioassays. The best mutagen in the production of IAA and cytokinin was U.V. light (4.020 µg/ml and 1.81 µg/ml) respectively. Greenhouse experiment were carried out for two seasons on maize plants to investigate the effect of the mutant strains of Azospirillum brasilense on the vegetative parameters of the plant. The fermented broth of the mutants was added to the MS and used in the micropropagation of Paulownia spp. Ex-plants to investigate the effect of the phytohormones especially IAA and cytokinins on the root length of the ex-plant.
The important results of this study could be summarized as follows
1- Primary screening based on concentration of produced IAA gave rise to ten isolates from the thirty isolates which produce high concentration of IAA. The highest concentration recorded in isolate (K16) 2.226 µg/ml.
2- Secondary screening based on concentration of produced cytokinin gave rise to five isolates from the ten isolates which produce high concentration of cytokinin, with the highest concentration in (K6) 1.239 µg/ml, isolate (K16) produce 1.203 µg/ml.
3- The five isolates were identified depending on the biochemical considerations (Bergey’s manual and BIOLOG). Then the results were consigned with molecular identification depending on 16srRNA sequencing.
4- The effect of incubation temperature on the production of IAA was examined and data showed that the optimum temperature was 30° C at which the production of IAA was 1.240 µg/ml.
5- The effect of pH on the production of IAA was examined and results showed that the optimum temperature was 7 and the production was 1.242 µg/ml.
6- The effect of the concentration of L-tryptophan on the production of IAA was examined and results showed that the optimum concentration was 0.2% which gave 1.241 µg/ml, and the concentration of IAA produced decreased by the increase in the concentration of L-tryptophan.
7- Different carbon sources were used in the culture medium for the production of IAA and the best carbon source was L-malate which gave 1.250 µg/ml IAA.
8- Two concentrations of L-malate were used to examine the effect of carbon concentration on the production of IAA, and there were no significant difference between using 2.5 gm/l and 5 gm/l of L-malate.
9- K6 was selected for further investigations, and a preliminary experiment was conducted using U.V. rays as a physical mutagen to detect the optimum dose of U.V. which give 1-5% viability, and results showed that the dose of two minutes gave 4.7 % viable colonies.
10- Another experiment using U.V. rays as a mutagen was conducted for two minutes, and five colonies were picked and IAA was quantified for the five strains, results showed that IAA production was increased by 80 % for the strain U.V.4, 54 % for the strain U.V.2, 13 % for U.V.5 and 7 % for U.V.3 and concentration of IAA was decreased by 10 % in U.V. 1. Cytokinin was quantified for the five strains and concentration of cytokinin was increased by 54 % in U.V.2, 50 % in U.V.4, 4 % in U.V.5 and decreased by 6 % in U.V.1 and there were no significant difference incase of U.V.3.
11- Sodium azide was used with three concentrations (25, 27, 29 mg/ml), and treatment with 27 mg/ml was chosen for further experiments, five colonies were picked and concentration of IAA was quantified for the five strains. Results showed that the concentration of IAA was increased by 61 % in AZ4, 52 % in AZ3, 23 % in AZ1 and 9 % in AZ2, and decreased by 2 % in AZ5. Cytokinin was quantified in the five strains and results showed that the concentration increased by 66 % in AZ3, 62 % in AZ4, 39 % in AZ2 and 5 % in AZ5 and decreased by 16 % in AZ1.
12- Acridine orange was used as a mutagen with concentration 100 µg/ml, and two colonies were picked for further investigations. IAA was quantified and results showed that IAA concentration increased by 24 % in A.O.1 and 23 % in A.O.2, cytokinin concentration was decreased by 6 % in A.O.1, and 8 % in A.O.2.
13- Nitrosoguanidine was used with concentration 100 µg/ml as a chemical mutagen and single colony was selected, IAA concentration and cytokinin were quantified for the mutant strain and result showed an increase with 16 % IAA and decrease with 4 % cytokinin.
14- Greenhouse experiment was conducted for two seasons on maize plants to show the effect of the mutant strains over the vegetative growth and chlorophyll content of the maize plants. Results in the first season showed that inoculation of seeds with AZ3 strain was the most efficient in stem height character at which it gave an increase of 15 % in comparison with the wild type and 56 % in comparison with control. Treatment with AZ4 strain gave the highest record in root length with an increase of 41 % than wild type and 75 % than control. Treatment with AZ3 strain gave the highest record in chlorophyll content using SPAD with an increase 39 % and 17 % than control and wild type treatment respectively. Results in the second season confirmed those obtained in the first season, as the highest record in stem height was in the treatment of seeds with AZ3 with an increase 57 % and 9 % in comparison with control and wild type respectively. The highest record in root length was recorded in treatment U.V.4 with an increase 107 % and 75 % in comparison with control and wild type treatments respectively. The best chlorophyll content was in treatment AZ3 with an increase 64 % and 44 % compared with control and wild type treatments respectively.
15- Two tissue culture experiments for propagation of Paulownia spp. Ex-plants were conducted using fermented broth of mutants (U.V.2, U.V.4, AZ3 and AZ4) and wild type K16 using three concentrations of the broth (10, 20 and 40 ml/l MS), in the first experiment. Growth parameters (root length, number of shoots, number of roots, shoot length and number of leaves) were recorded. Results showed that the concentration of 40 ml/l MS was the best concentration which gave an increase in growth parameters (N.O of roots, root length, N.O of shoots, shoot length and N.O of leaves) (9, 1.56, 1.6, 4.69 and 12 respectively). The second experiment was carried out using the concentration of 40 ml/l MS of the fermented broth of mutants( U.V.2, U.V.4, AZ3 and AZ4) and wild type K16 and positive control using (1ml/l MS IBA and 3ml/l MS Kinetine). Results showed that the best treatment was in using U.V.4 fermented broth which gave a significant increase in growth parameters (N.O of roots, root length and shoot length) (12, 2.03 and 5.5 respectively). The best treatment which gave the best N.O of shoots and N.O of leaves was AZ3 fermented broth. It gaves 4 shoots and 18 leaves.