الفهرس 
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Abstract
The present work has been carried out in the labs, and the biocontainment greenhouse of the Institute of Biosciences, Department of Plant Physiology Albert-Einstein-Straße 3, D-18051 Rostock. Germany., during the period 2009-2011.
The main objectives were: (1) To establish an efficient regeneration system for five elite Egyptian rice genotypes using mature embryo culture. As a prerequisite for genetic transformation of these genotypes; (2) To investigate effect of media composition on formation and regeneration of embryogenic calli using mature embryo technique; (3) To transform rice mature embryos using the biolistic particle delivery system and plasmid-DNA containing ggpPS gene and the antibiotic resistant nptII gene as a selectable marker; (4) To investigate regeneration ability of the transformed calli; (5) To confirm the integration of the introduced genes in the genomic DNA of the putatively transformed rice tissues using PCR; (6) To confirm expression of the gene ggpPS by HPLC detection of the end product glucosylglycerol; (7) To transform leaf disks of two potato genotypes by agrobacterium and plasmid-DNA containing ggpPS gene and the antibiotic resistant nptII gene as a selectable marker; (8) To study various characteristics during tissue culture procedures like callus induction, shoot bud formation, shoot formation, and micro tuberization; and (8) To confirm the integration of the introduced genes in the genomic DNA of the putatively transformed potato plants using PCR.
1. Rice Regeneration
Five elite Egyptian rice (Oryza sativa L.) varieties namely Giza177, Sakha101, Sakha102, Sakha103, and Sakha104 were used in this investigation. Sterilized grains were cultured for callus induction on three callus induction medium designate Cia, Cib, and Cic. The first media composed of MS medium basal salts and vitamins, while the other two media were composed of N6 basal medium containing the same composition of N6 vitamins. All media were supplemented with 0.3 g/l Casein hydrolysate, the same concentration of the amino acids L-Proline, L-Glutamine and L-Alanine 0.5 , 0.5 , and 0.45 g/l respectively. The same concentration of sucrose (2.5%) and Sorbitol (0.5%) and 7 g/l Agar. Medium Cia was supplemented with 2.5 mg/l 2,4-D, while Medium Cib supplemented with 2mg/l dicamba, and finally medium Cic was supplemented with 2 mg/l 2,4-D, and 8.5 mg/l Silver nitrate. The two genotypes Sakha 104, and Sakha 101 showed the highest callus induction frequency (CIF) values 84.4 and 80.7, respectively. While, Giza 177 showed a distinctly lower CIF of 52.4. Additionally, the highest CIF was obtained by culturing genotype Sakha 104 on Cic (95.0%), while the lowest CIF (44.3%) resulted from culturing genotype Giza 177 on Cia.
White-, cream-, and yellow-coloured compact organized calli were considered embryogenic calli. The mean embryogenic callus frequencies ECF observed with the five rice varieties was significantly higher on medium Cib (44.7%) as compared to medium Cic (41.3%) and medium Cia (31.7%). In the comparison of the rice cultivars examined here over all three media, Sakha 104 (43.4%) and Sakha 103 (43%) showed the highest mean ECFs, followed by Sakha 101 (40.3), while corresponding mean values for Sakha 102 (35.4%) and Giza 177 (34%) were significantly lower. we found highest ECFs for the three genotypes Giza 177, Sakha 104, and Sakha 103 in combination with Cib (48.3%, 48.0%, and 47.3%, respectively).
Organogenesis was achieved by transferring the embryogenic calli , obtained on callus induction media Cia, Cib, and Cic, to the plant regeneration media Rega, Regb, and Regc. Highest regeneration efficiencies were achieved with Sakha 101 (75.3%) and Sakha 104 (70.7%) on Regb.
2. Rice transformation
A/ Standardization of kill curve:
Control experiments to find out the lethal doses of kanamycine revealed that In the two genotypes Sakha101 and Sakha104 all untransformed calli (control) failed to survive after three cycles of selection on Ci media contain 100mg/l Kanamycin. 75 mg/l kanamycin lead to a large number of escapes, while 125 mg/l stopped the growth of all callus pieces since the first selection cycle.
B/ Transient gus expression:
Various combinations between helium pressure and target distance were tested, highest level of transient expressions of gus in both genotypes Sakha101 and Sakha104 was obtained by 1100 psi helium pressure, and 9 cm target distance.
C/ Genetic transformation of rice calli using helios gene gun.
2400 callus pieces was bombarded (1200 callus pieces from each genotype) by gold particles coated with DNA of the plasmid pGII0229-rd29A-AF_HindIII-EcoRV using previously optimised physical parameters of 1100 psi helium pressure , and 9 cm targit distance.
D/ Selection of putative transgenic rice calli
Bombarded calli was transferred to selection medium containing 100 mg/l kanamycin. After six weeks some calli show stable resistance, excellent shape, and distinctive growth. These pieces were transferred to the preset regeneration medium (Reg b) contain 100 mg/l kanamycine. After about 3 weeks on shooting media some green areas appeared on numerous of callus pieces, but no plant leaves were developed.
E/ Characterization of resistant calli:
36 callus piece resulted from genotype Sakha 101, and 71 callus piece belongs to Sakha 104 gave a positive result with the PCR. These 107 calli were examined by estimating the concentration of glucosylglycerol using HPLC technology. Glucosylglycerol, the end product resulted from the expression of the bacterial gene ggpPS was detected with different concentration in all the 107 calli examined. At the same time no glucosylglycerol were determined inside control calli. Callus transformation frequency for Sakha 101 was 3%, while Callus transformation frequency for Sakha 104 was 5.9%.
3. Agrobacterium mediated transformation of potato:
Two elite potato (Solanum tuberosum L.) genotypes namely Albatros, and Desiree were used in the transformation experiment mediated by Agrobacterium. Leaf discs from virus free in vitro plantlet of potato varieties Albatros and Desiree were excised. The midrib of the leaf blade was wounded several times leaving about one mm gap between the cuts. Wounded leafs was then infected, and co-cultivated with Agrobacterium. Then explants were washed three times with liquid regeneration media having 500 mg/l Cefotaxime.
Callus Induction
leaves were placed on callus induction medium (MGC) and incubated in a growth chamber with a 16 h light, 8 h dark photoperiod under 35 mE m-2 s-1 cool white fluorescent light at 27± 1 ºC for 8 days. Overall, 59.7%, and 68.6% of the explants belonging to the genotypes Albatros, and Desiree respectively developed callus from the cut surface.
Shoot Regeneration
After 8 days, explants were transferred to MGS medium for shoot bud induction, and incubated under the same circumstances. Average of shoot bud formation of the two genotypes Albatros, and Desiree was 46.7% and 39.1%, respectively.
Rooting
Shoots with length of 2–3 cm, were excised and transferred to rooting medium (RM) and incubated under the same temperature and light conditions. transformation efficiency of the two genotypes Albatros, and Desiree was 13.5%, and 9.1%, respectively.
In vitro tuberization
In vitro tuberization was carried out by culturing transformed plants on MS + 6% sucrose + 4 mg/l KIN with 7g/l agar. Tuberization was detected after 10 weeks. from 16 transgenic potato plant belongs to the genotype Albatros 11 plants form micro-tubers, and from 11 transgenic plant resulted from the genotype Desiree, and 2 plants form micro-tubers. DNA extracted from micro-tubers of 11 plants resulted from genotype Albatros, and 2 plants belongs to Desiree, gave a positive result with the PCR.
Complete transgenic plants were produced in a relatively short period. Obtained transformation efficiency of the two genotypes Albatros, and Desiree was 13.5%, and 9.1%, respectively. The resulted plants were morphologically normal, did not show phenotypic changes that may be caused by somaclonal variation.