Date of Award

12-11-2025

Degree Type

Thesis

Degree Name

Master of Science (M.S.)

Department

Agricultural and Environmental Sciences

First Advisor

Christine Ondzighi-Assoume

Abstract

Sorghum (Sorghum bicolor L.) is a C4 crop of significant agricultural and industrial importance, known for its high biomass yield, drought tolerance, and adaptability to marginal lands. However, the high lignocellulosic biomass content of sorghum, particularly its lignin component, poses a significant challenge for efficient biomass conversion. Genetic engineering offers a powerful approach to achieve such improvements by targeting the modification of lignin biosynthetic pathways. However, progress in sorghum genetic improvement has been hindered by the crop’s recalcitrance to genetic manipulation, transformation, and regeneration. To overcome these limitations, we developed a four-in-one genetic engineering technology for sorghum by establishing an efficient in vitro cell suspension culture system capable of maintenance, cryopreservation, transformation, and regeneration to produce genetically modified plants. Our system provides a foundation for the application of advanced molecular tools such as CRISPR/Cas9-mediated gene editing to improve sorghum for bioenergy applications. The impressive achievements of our study include successfully establishing embryogenic cell cultures of three varieties: Tx430, Topper 76-6, and Dale, for both grain and sweet sorghum. Our cell cultures exhibited cell density, viability, and regeneration potential ranging from 10% to 88%, demonstrating their potential for sustained growth and the capability for genetic manipulation. Regeneration was achieved efficiently from stem-derived calli, with a maximum regeneration efficiency of up to 76%. Furthermore, through Agrobacterium tumefaciens–mediated transformation, the three varieties of cell lines exhibited transformation efficiency ranging from 56 to 78%. Our study provides a robust foundation for future gene-editing efforts aimed at enhancing sorghum’s biomass quality and bioenergy production.

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