An Easy and Rapid Transformation Protocol for Transient Expression in Cotton Fiber

Abstract

Cotton fiber is the most important natural textile material in the world. Identification and functional characterization of genes regulating fiber development are fundamental for improving fiber quality and yield. However, stable cotton transformation is time-consuming, low in efficiency, and technically complex. Moreover, heterologous systems, such as Arabidopsis and tobacco, did not always work to elucidate the function of cotton fiber specifically expressed genes or their promoters. For these reasons, constructing a rapid transformation system using cotton fibers is necessary to study fiber’s specifically expressed genes. In this study, we developed an easy and rapid Agrobacterium-mediated method for the transient transformation of genes and promoters in cotton fibers. First, we found that exogenous genes could be expressed in cotton fibers via using β-glucuronidase (GUS) and green fluorescence protein (GFP) as reporters. Second, parameters affecting transformation efficiency, including LBA4404 Agrobacterium strain, 3 h infection time, and 2-day incubation time, were determined. Third, four different cotton genes that are specifically expressed in fibers were transiently transformed in cotton fibers, and the transcripts of these genes were detected ten to thousand times increase over the control. Fourth, GUS staining and activity analysis demonstrated that the activity profiles of GhMYB212 and GhFSN1 promoters in transformed fibers are similar to their native activity in developmental fibers. Furthermore, the transient transformation method was confirmed to be suitable for subcellular localization studies. In summary, the presented Agrobacterium-mediated transient transformation method is a fast, simple, and effective system for promoter characterization and protein expression in cotton fibers.

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L

“…The efficiency of Agrobacterium -mediated transient expression is affected by concentration, infiltration time, and acetosyringone (AS) concentration, whereas the efficiency of biolistic-mediated transient expression is affected by gold particle concentration, bombardment distance, and helium pressure [ 6 ]. In recent years, transient expression systems have ceased to be exclusive to model plants, such as Arabidopsis [ 8 ] and Nicotiana benthamiana [ 9 ], and individual transient expression systems for many economically important plants, such as cotton [ 10 ], Ricinus [ 11 ], citrus [ 12 ], Chenopodium quinoa [ 13 ], and Vigna unguiculata [ 14 ], have been established. However, compared to these economic crops, the establishment of transient expression systems in medicinal plants are very much behind, which may leave the functions of genes incomplete and hinders the development and utilization of these natural medicinal values by humans.…”

Revealing Genetic Differences in Fiber Elongation between the Offspring of Sea Island Cotton and Upland Cotton Backcross Populations Based on Transcriptome and Weighted Gene Coexpression Networks

“…Cotton fiber quality is an important agronomic trait; therefore, significant efforts have been made to study its biological mechanisms [ 31 , 32 , 33 , 34 , 35 ]. In recent years, several key genes involved in fiber development have been reported [ 36 , 37 , 38 , 39 , 40 ], which have greatly contributed to the study of fiber development mechanisms, and studies have shown that hormones, functional genes, and transcription factors form a complex regulatory network that coordinates the dynamic fiber development process [ 3 ].…”

The establishment of transient expression systems and their application for gene function analysis of flavonoid biosynthesis in Carthamus tinctorius L. (safflower)

“…[6]. In recent years, the transient expression system is no longer exclusive to model plants such as Arabidopsis [8] and Nicotiana benthamiana [9], many economic plants such as cotton [10], ricinus [11], citrus [12], Chenopodium quinoa [13], and Vigna unguiculata L. [14] have also established their own transient expression systems. However, there are still few medicinal plants possessing their own transient expression system.…”