Date of Award
Master of Science
C. Neal Stewart Jr.
Tarek Hewezi, Scott C. Lenaghan
Transient gene expression in plant protoplasts has been widely utilized for rapid functional screening of promoters. This project describes a novel soybean cell suspension culture and protoplast system for rapid and predictive analysis of promoter activity. Since recovery of transgenic lines is low in soybean stable transformation, high-throughput studies of promoter activity have intrinsic limitations. First, I describe a novel cell suspension culture system from leaf-derived callus. Once the cell culture is established, it is easy to maintain, sterile, and produces fast-growing cells amenable for protoplast production. Second, I describe an efficient and simple protoplast preparation procedure. The results revealed that, the highest yield was 2.82 ± 0.94×108 protoplasts/g fresh weight with high viability (77.73% ± 7.83%) using 4-d-old cells. Third, protoplast transformation efficiency was optimized with 20 min PEG incubation, which resulted in a transformation frequency of 30.65% ± 2.70%. These transfected protoplasts could be assessed for reporter gene activity 48 hr post-transfection. Finally, to determine whether the system had predictive value with regards to promoter activity in soybean tissues, six well-characterized soybean promoters were used to direct the expression of a green fluorescent (GFP) gene among protoplasts derived from cell cultures, leaves, stems, and immature cotyledons. From the results, all promoters displayed similar expression profiles in four different tissues derived protoplasts with correlation coefficient for leaf and cell culture is 0.99, for stem and cell culture is 0.96 and for immature cotyledon and cell culture is 0.96. Cell culture derived protoplasts expression were also compared with transcript abundance data of endogenous tissues using qRT-PCR assays. Overall, a reliable and renewable cell culture was developed with consistent results, which can provide convenient alternative to leaf tissue as well as other soybean tissues, which would be necessary in high throughput automated screens.
Sultana, Mst Shamira, "Predictive promoter activity in soybean using a novel cell-suspension culture and transient expression analysis in protoplasts. " Master's Thesis, University of Tennessee, 2018.