A Rapid and Highly Efficient Method for Transient Gene Expression in Rice Plants

Abstract

Rice is the model plant system for monocots and the sequencing of its genome has led to the identification of a vast array of genes for characterization. The tedious and time-consuming effort of raising rice transgenics has significantly delayed the pace of rice research. The lack of highly efficient transient assay protocol for rice has only added to the woes which could have otherwise helped in rapid deciphering of the functions of genes. Here, we describe a technique for efficient transient gene expression in rice seedlings. It makes use of co-cultivation of 6-day-old rice seedlings with Agrobacterium in the presence of a medium containing Silwet R L-77, acetosyringone and glucose. Seedlings can be visualized 9 days after co-cultivation for transient expression. The use of young seedlings helps in significantly reducing the duration of the experiment and facilitates the visualization of rice cells under the microscope as young leaves are thinner than mature rice leaves. Further, growth of seedlings at low temperature, and the use of surfactant along with wounding and vacuum infiltration steps significantly increases the efficiency of this protocol and helps in bypassing the natural barriers in rice leaves, which hinders Agrobacterium-based transformation in this plant. This technique, therefore, provides a shorter, efficient and cost-effective way to study transient gene function in intact rice seedling without the need for a specialized device like particle gun.

Evaluation of novel surfactants for plant transformation

“…Results presented here may be applied towards these improvements. Surfactants have been shown to improve transformation efficiency for numerous crop species, including but not limited to sugarcane [6], radish [7], soy [8], maize [9], and rice [10,11]. As these investigations all utilized Silwett ® L-77 to enhance transformation, our current results suggest that a 45% increase in the number of transformants may be obtained through the use of BREAK THRU ® surfactants S200, S240, and S279.…”

In planta Female Flower Agroinfiltration Alters the Cannabinoid Composition in Industrial Hemp (Cannabis sativa L.)

“…In previous report, we vigorously studied the effects of surfactant, antioxidant, vacuum time, Agrobacterium, and hemp strain on the efficiency of agroinfiltration (Deguchi et al, 2020a ). To further improve this transient gene expression protocol, keeping plants in the dark after infiltration, a suitable developmental stage and temperature for plant growth would need to be optimized (Burman et al, 2020 ; Zhang et al, 2020 ).…”

MAP kinases may mediate regulation of the cell cycle in rice by E2F2 phosphorylation

“…The cells were harvested and resuspended into the infiltration buffer of 10 m m MES, 10 m m MgCL2, and 200 μ m acetosyringone. The 5‐day‐old germinated seedlings were agro‐inoculated through vacuum infiltration as described previously [39]. The agro‐inoculated seedlings were kept in the dark at 28 °C for 12 h and then transferred into the 16/8 h day/light condition for 3–5 days.…”