الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
 |  | from | 239 |  |  |
| | | from | 239 | | |
Abstract
ABSTRACT
This study was carried out during 2011-2017. The present study aimed to in vitro cultivation of date palm cvs. Medjool and Khalas with espacial emphasis on enhancing embryogenesis (The effect of some macro and micro elements, growth regulators, sucrose glutamine, PEG, silver nitrate and GA3).Also, aimed to isolation and purification, callus induction and formation of somatic embryos from protoplast of two date palm cultivars (Medjool and Khalas). Finally, two simple transformation methods (Agrobacterium tumefaciens and particle bombardment) using Cry 3Aa gene for transgenic date palm development were used.
The morphological response of somatic embryos production is controlled by some internal factors that fall under the influence of the genetic make-up of the plant and specialized for each genotype that is responsible for the production of somatic embryos (Medjool and Khalas cultivars).
Data revealed that, subculture number 2 of Medjool and Khalas cultivars gave highest significant swelling degree of callus (5.00) without significant differences in between. Also, the highest significant value of callus induction degree was observed from Khalas in reculture number 5 (3.53).
In vitro growth and development of produced embryos has been improved by using of full MS salt strength with the modification of some nutrients concentrations, i.e. NH4NO3 (1237.5mg/l) which recorded (13.20 and 9.60 embryos/explant for Medjool and Khalas, respectively) after the third reculture, 1425mg/l KNO3 which gave a higher score of mature somatic embryos (14.40 and 10.00 embryo/culture for Medjool and Khalas respectively) was obtained with 1425mg/l KNO3.
During maturation and germination stages, addition of 0.50mg/l of copper sulphate or o.50mg/l cobalt chloride to the medium was most effective for somatic embryos formation.
The obtained results revealed that thiamin HCl at 10 mg/l was superior in enhancing somatic embryo formation when added to MS medium. Embryogenic callus of both Medjool and Khalas cultivars gave high production of somatic embryos when cultured on the solid medium supplemented with 60 g/l sucrose.
Glutamine had stimulatory effect on date palm somatic embryos formation when added at 200 mg/l. Maximum embryos formation was obtained with the addition of as low as 0.50mg/l ABA to the culture medium. Gradual decreasing of paclobutrazole concentration from 4.00mg/l to 1.00 mg/l in culture medium, stimulated the production of somatic embryos. Somatic embryo number were enhanced using 5gm/l PEG. Supplementation of culture medium with 5mg/l silver nitrate was superior to the other concentrations used.
0.5mg/l GA3 was superior in enhancing elongation of in vitro proliferated shoots. Individual shoots were cultured on modified MS basal medium in addition to IBA (1.0 mg/l), sucrose (30 g/l) and solidified with phyto-agar (8.0 g/l) for more in vitro growth and development .In vitro plantlets were transferred to acclimatization stage in torpedo plastic pots 18x5cm diameter containing peatmoss, perlite and washed sand at equal volume. In vitro date palm plantlets produced from rooting stage grow well in the greenhouse during acclimatization stage without morphological abnormality.
The greatest yield (8.13×105) of Medjool and (7.43×105) of Khalas protoplast was obtained from 2% Cellulase, Hemicellulase, and Pectinase. Additionally, incubating the mixture enzyme with callus for 24 hours was the optimal condition for protoplast isolation. Maximum yield of protoplast was obtained when using 40 and 45 μm stainless steel sieve which was efficient in removing any clumps of undigested tissues and debris of Medjool (7.96x105) and Khalas (7.16 x105). Protoplast was plated on MS medium at a density of 5 × 105 ml containing 10mg/l 2, 4-D and 3.0mg/l 2iP. Obtained micro calli developed after nine months and callus induction after twelve months.
Maximum percentages of explants forming somatic embryos after twenty months were 60 and 30 for Medjool and Khalas date palm cultivars, respectively.
Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The spores contain crystals, predominantly comprising one or more Cry and/or Cyt proteins (also known as δ-endotoxins) that have potent and specific insecticidal activity. One of the advantages of these toxin proteins is their specificity towards certain harmful insects.
Embryogenic callus were recorded 100% mortality when cultured on MS medium containing 100 mg/L kanamycin with different cultivars, thus it was chosen for the selection of transformed explants. Embryogenic callus of Medjool and Khalas were incubated with Agrobacterium tumefaciens strain LBA 4404 for 0.5, 1, 2, 4 and 24 h on LB medium. After the incubation periods, embryogenic callus was transferred to MS medium with 0.1 mg/L NAA, 0.05 mg/L BA, 250 mg/L carbenicillin and 100 mg/L kanamycin for detection of transgenic embryogenic callus. Polymerase chain reaction (PCR) was used for the rapid screening of Cry3Aa gene. For screening, total genomic DNA was isolated from transformants. Using primer specific to Cry3Aa gene (forward and reverse), a PCR product with a size of about 2,000 bp was amplified when all nucleic acid from the transformants were utilized as templates. PCR analysis confirmed the appearance of the transgene of 2,000 bp in one individual plantlet. Presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization. It was found that one transgenic embryogenic callus for both Medjool and Khalas showed a single copy of gene integration. These results signify the successful transfer of Cry3Aa gene into date palm plant.
Our results showed that, when using particles coated with plasmid pCAMBIA 2300(containing Cry 3Aa gene), the highest transformation efficiency was recorded with Medjool (showed two copies of gene integration) than Khalas (showed one copies of gene integration).
Also, the presence and integration of foreign Cry3Aa gene in regenerated kanamycin resistant embryogenic callus was also confirmed by Southern blot hybridization.
Keywords: Agrobacterium tumefaciens, Date palm, PCR, Protoplast, Somatic embryo, Southern blott Analysis, Transformation.