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.
School of Sciences and Engineering
MS in Biotechnology
Walid M. Fouad
Shireen K. Assem
Committee Member 1
Committee Member 2
Institutional Review Board (IRB) Approval
Approval has been obtained for this item
(2021).Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence [Master's Thesis, the American University in Cairo]. AUC Knowledge Fountain.
Abushal, Logayn. Optimization of In-Vitro Tissue Culture and Transformation System for Sweet Sorghum Cultivars Using Immature Inflorescence. 2021. American University in Cairo, Master's Thesis. AUC Knowledge Fountain.
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