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Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence
Background: Sweet sorghum is a multi-purpose crop that can be used for human food, animal feed, and bio-fuel production. This crop is drought-tolerant and can therefore be grown in arid and semi-arid regions especially in marginal areas in Egypt. Nevertheless, production of Sweet sorghum cannot coup...
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2021
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| _version_ | 1867613420953534464 |
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| access_status_str | Open Access |
| author | Abushal, Logayn |
| author_browse | Abushal, Logayn |
| author_facet | Abushal, Logayn |
| author_sort | Abushal, Logayn |
| collection | Thesis |
| description | Background: Sweet sorghum is a multi-purpose crop that can be used for human food, animal feed, and bio-fuel production. This crop is drought-tolerant and can therefore be grown in arid and semi-arid regions especially in marginal areas in Egypt. Nevertheless, production of Sweet sorghum cannot coup with the increasing demand in consumption as a food, and fuel due to a lag in scientific research regarding its genetic improvement. Therefore, genetic improvement of Sweet sorghum is innately required so that new traits of economic importance could be introduced; however, this can only be achieved through the establishment of a robust in-vitro tissue culture system. Thus, this study aimed to establish an effective regeneration system for three different Sweet sorghum cultivars namely; Rex, Sugar Drip, and Ramada, using immature inflorescence explants. Subsequently, the cultivar Sugar was selected to examine its transformation efficiency using the microprojectile bombardment technology.
Methods: For callus induction media, explants from immature inflorescences of different lengths (1.5- 16 cm), were grown over a combination of 2,4-D (0, 2, 4 & 6mg/L) with kinetin (0, 0.2 & 0.5mg/L). For regeneration media, BAP was the main cytokinin implemented in the three different treatments along with either: NAA, IAA, or IAA & TDZ, whereas TDZ was employed alone in a fourth treatment. The transformation system of the cultivar Sugar Drip was also developed using immature inflorescence explants. Co-bombardment was performed using the neomycin phosphotransferase II (nptII) gene under the control of maize ubiquitin (Ubi1) promoter. The selection of putative transgenic plants was performed using paromomycin antibiotic.
Results: The best embryogenic callus induction frequency was observed in Rex (77%) on modified MS media supplemented with 4mg/L 2,4-D + 0.2mg/L kinetin after six weeks from culture. However, Ramada and Sugar Drip had their highest callus induction rates of 93 and 94%, respectively, when 6mg/L 2,4-D + 0.2mg/L kinetin was used. The addition of 1mg/L IAA + 0.5mg/L BAP + 0.1mg/L TDZ had its best outcome in terms on shoot induction, shoot/callus number, and root formation. In Sugar Drip transformation, paromomycin eliminated most the non-transgenic plants from the putative transgenic ones. Eventually, 6 out of 348 bombarded samples were found to be transgenic after PCR screening. The percentage of transformation from two independent experiments was around 1.724%.
Conclusion: PCR analysis of putative transformants revealed a transformation efficiency of 1.724%. Therefore, genetic transformation using particle bombardment has shown to be a successful method for the transformation of immature Sweet sorghum inflorescences with relatively high success rates. |
| format | Thesis |
| id | oai:fount.aucegypt.edu:etds-2846 |
| institution | American University in Cairo (Egypt) |
| last_indexed | 2026-06-10T12:35:51.500Z |
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| provenance_str_mv | Harvested via OAI-PMH from AUC Knowledge Fountain — bepress |
| publishDate | 2021 |
| publishDateRange | 2021 |
| publishDateSort | 2021 |
| publisher | AUC Knowledge Fountain |
| publisherStr | AUC Knowledge Fountain |
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| source_str | AUC Knowledge Fountain — bepress |
| spelling | oai:fount.aucegypt.edu:etds-2846 Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence Abushal, Logayn Background: Sweet sorghum is a multi-purpose crop that can be used for human food, animal feed, and bio-fuel production. This crop is drought-tolerant and can therefore be grown in arid and semi-arid regions especially in marginal areas in Egypt. Nevertheless, production of Sweet sorghum cannot coup with the increasing demand in consumption as a food, and fuel due to a lag in scientific research regarding its genetic improvement. Therefore, genetic improvement of Sweet sorghum is innately required so that new traits of economic importance could be introduced; however, this can only be achieved through the establishment of a robust in-vitro tissue culture system. Thus, this study aimed to establish an effective regeneration system for three different Sweet sorghum cultivars namely; Rex, Sugar Drip, and Ramada, using immature inflorescence explants. Subsequently, the cultivar Sugar was selected to examine its transformation efficiency using the microprojectile bombardment technology. Methods: For callus induction media, explants from immature inflorescences of different lengths (1.5- 16 cm), were grown over a combination of 2,4-D (0, 2, 4 & 6mg/L) with kinetin (0, 0.2 & 0.5mg/L). For regeneration media, BAP was the main cytokinin implemented in the three different treatments along with either: NAA, IAA, or IAA & TDZ, whereas TDZ was employed alone in a fourth treatment. The transformation system of the cultivar Sugar Drip was also developed using immature inflorescence explants. Co-bombardment was performed using the neomycin phosphotransferase II (nptII) gene under the control of maize ubiquitin (Ubi1) promoter. The selection of putative transgenic plants was performed using paromomycin antibiotic. Results: The best embryogenic callus induction frequency was observed in Rex (77%) on modified MS media supplemented with 4mg/L 2,4-D + 0.2mg/L kinetin after six weeks from culture. However, Ramada and Sugar Drip had their highest callus induction rates of 93 and 94%, respectively, when 6mg/L 2,4-D + 0.2mg/L kinetin was used. The addition of 1mg/L IAA + 0.5mg/L BAP + 0.1mg/L TDZ had its best outcome in terms on shoot induction, shoot/callus number, and root formation. In Sugar Drip transformation, paromomycin eliminated most the non-transgenic plants from the putative transgenic ones. Eventually, 6 out of 348 bombarded samples were found to be transgenic after PCR screening. The percentage of transformation from two independent experiments was around 1.724%. Conclusion: PCR analysis of putative transformants revealed a transformation efficiency of 1.724%. Therefore, genetic transformation using particle bombardment has shown to be a successful method for the transformation of immature Sweet sorghum inflorescences with relatively high success rates. 2021-12-31T08:00:00Z thesis application/pdf https://fount.aucegypt.edu/etds/1829 https://fount.aucegypt.edu/context/etds/article/2846/viewcontent/Logayn_Thesis.Oct.2021.final.pdf Theses and Dissertations AUC Knowledge Fountain Immature inflorescence length embryogenic callus callus regenerability callus quality plant growth regulators. Biology Biotechnology Life Sciences |
| spellingShingle | Immature inflorescence length embryogenic callus callus regenerability callus quality plant growth regulators. Biology Biotechnology Life Sciences Abushal, Logayn Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title | Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title_full | Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title_fullStr | Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title_full_unstemmed | Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title_short | Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence |
| title_sort | optimization of in vitro tissue culture and transformation system for sweet sorghum cultivars using immature inflorescence |
| topic | Immature inflorescence length embryogenic callus callus regenerability callus quality plant growth regulators. Biology Biotechnology Life Sciences |
| url | https://fount.aucegypt.edu/etds/1829 https://fount.aucegypt.edu/context/etds/article/2846/viewcontent/Logayn_Thesis.Oct.2021.final.pdf |
| work_keys_str_mv | AT abushallogayn optimizationofinvitrotissuecultureandtransformationsystemforsweetsorghumcultivarsusingimmatureinflorescence |