الفهرس 
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Abstract
The insecticides cause mammalian toxicity, the appearance and spread of resistant insect pest species, the ground water contamination and environmental pollution, that restrict their use. Therefore, the moment towards the use of entomotoxin producing by bacteria such as Bacillus thuringiensis as a microbial control agent must be encouraged.
Bacillus thuringiensis (Bt) is a spore-forming bacterium well-known for its insecticidal properties associated with its ability to produce crystal inclusions during sporulation. These inclusions are proteins encoded by cry genes and have shown to be toxic to a variety of insects and other organisms like nematodes and protozoa.
Lepidoptera and Coleoptera are of the most devastating insect pests for important crops in Egypt; their control is a priority as well as a necessity.
In this context, use of environmentally safe technology to reduce crop damage would be extremely valuable. Consequently, this study was planned to establish the genetic improvement of Bacillus thuringiensis strains to control potato tubers moth; (Phthorimea operculella) and cotton leaf worm (Spodoptera litoralis) using protoplast fusion technique
The obtained results can be summarized as the following:
1- Identification and characterization experiments were done on the two parental Bacillus strains obtained from M.Sc. that were registered at the NCBI under accessions number: LC070660 for Elsharkia (Sh) and LC070661 for Damietta (D) strains.
2- Plasmids pattern were used to characterize the two strains.
3- The antibiotic resistance patterns of the Sh and D strains showed that the two strains were resistant to Ampicillin (AMP), while they were sensitive to the other 25 different antibiotics. The Sh strain was sensitive to Cefpodoxime (CPD), and resistant to Nitrofurantoin (NIT) and D strain was resistant to Cefpodoxime (CPD), and sensitive to Nitrofurantoin (NIT).
4- One attempt of protoplast fusion technique was used between Bt Sh and Bt D strains. During the two hours of fusion time, one fusion product was obtained on the selective medium from the 2 strains.
5- SDA-PAGE was applied on the two parental strains Sh and D and their fusant
6- Detection of cry genes (cryI, cryII, cryIII, cryIV and cryV) confirmed that Bt Sh was tolerant to heat and salt and contained cry gene type II (589 bp), Bt D that was sensitive to heat and salt contained cry gene type I and II (490 bp and 589 bp) and fusion product was tolerant to heat and salt and contained cry gene type I and II (490 bp and 589 bp).
7- Bt Sh contained cry gene type IIAb. Bt D contained cry gene type IAb and IIAb and the fusion product contained cry gene type IAb and IIAb.
8- The two strains Sh and D and their fusant were bio-assayed against the first instar larvae of cotton leaf worm; Spodoptera littorals and potato tubers moth; Phthorimea operculella. The results showed that the new fusion strain caused gradual mortality to potato tubers moth with feeding on treatment potato after 21 days with high mortality% which recorded 80% for potato tubers moth; Phthorimea operculella. The new fusion strain caused gradual mortality to cotton leaf worm; Spodoptera littorals with feeding on treatment artificial diet after 7 days with mortality percentage recorded to 80% for cotton leaf worm; Spodoptera littorals.
The results recommend using Bt D strain to control the cotton leaf worm Spodoptera littorals and the strain resulting from the fusion of Bt D and Bt Sh to control potato tuber moth Phthorimaea operculella.
4- One attempt of protoplast fusion technique was used between Sh (Bt Sh) and D strains. During the two hours of fusion time one fusion product
was obtained on the selective medium from Bt Sh==Bt D.
5- SDA-PAEG was applied on the two parental strains; Sh and D and their one fusant
6- Detection of cry genes (cryI, cryII, cryIII, cryIV, cryV gene) confirmed that Bt Sh which was tolerant to heat and salt and contains cry gene type II (500 bp), Bt D that was sensitive to heat and salt contains cry gene type I & II ( 450 bp & 500 bp) and fusion products was tolerant to heat and salt and contains cry gene type I & II (450 bp & 500 bp).
7- Bt Sh contains cry gene type IIAb, Bt D contains cry gene type IAb & IIAb and fusion product was t contains cry gene type IAb & IIAb.
8- The two strains; Sh and D and their fusant were bioassayed against the first instar larvae of cotton leaf worm; Spodoptera littorals and potato tubers moth; Phthorimea operculella. The results showed that the new fusion strain caused gradual mortality to potato tubers moth with feeding on treatment potatos after 21 days high precentage of mortality was recorded to 80% potato tubers moth; Phthorimea operculella, the result. The new fusant fusion caused gradual mortality to cotton leaf worm; Spodoptera littorals with feeding on treatment artificial medium after days about 80% of cotton leaf worm; Spodoptera littorals.