Abstract
BackgroundThe Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. Despite this transient expression system has been successfully applied in a number of plant species, it is poorly developed in Caragana intermedia.ResultsIn this study, we established an Agrobacterium-mediated transient expression system in C. intermedia leaves and optimized the effect of different Agrobacterial strains, several surfactants and the concentration of Silwet L-77, which would affect transient expression efficiency. Among the 5 Agrobacterial strains examined, GV3101 produced the highest GUS expression level. Besides, higher level of transient expression was observed in plants infiltrated with Silwet L-77 than with Triton X-100 or Tween-20. Silwet L-77 at a concentration of 0.001% greatly improved the level of GUS transient expression. Real-time PCR showed that expression of CiDREB1C was highly up-regulated in transiently expressed plants and reached the highest level at the 2nd day after infiltration. Based on this optimized transient transformation method, we characterized CiDREB1C function in response to drought, salt and ABA treatment. The results showed that transiently expressed CiDREB1C in C. intermedia leaves could enhance the survival rate and chlorophyll content, and reduce the lodging rate compared with the control seedlings under drought, salt and ABA treatments. Furthermore, the rate of leaf shedding of CiDREB1C transient expression seedlings was lower than that of the control under ABA treatment.ConclusionsThe optimized transient expression condition in C. intermedia leaves were infiltrated with Agrobacterial strains GV3101 plus Silwet L-77 at a concentration of 0.001% added into the infiltration medium. Transiently expressed CiDREB1C enhanced drought, salt and ABA stress tolerance, indicated that it was a suitable and effective tool to determine gene function involved in abiotic stress response in C. intermedia.
Highlights
The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants
We developed an efficient transient expression system mediated by Agrobacterium in C. intermedia leaves in order to verify this system in gene function study, we transiently expressed CiDREB1C in C. intermedia leaves based on this optimized method and characterized its function in response to abiotic stress and Abscisic acid (ABA) treatment
GV3101 was the most suitable strain for the transient expression of C. intermedia leaves Studies have shown that the infection capability of A. tumefaciens to plants is varying, and the transient expression efficiency of plants would be significantly affected by the genetic background of A. tumefaciens strains [21]
Summary
The Agrobacterium-mediated transient transformation is a versatile and indispensable way of rapid analyzing gene function in plants. King et al [8] has developed and optimized the Agroinfiltration of soybean, and the results showed that Agrobacterium-mediated GUS reporter gene expression level is increased with vacuum infiltration followed by sonicating, whereas the transient transformation efficiency is not sufficient. Nanjareddy et al [12] used the same method to optimize the transient expression system of Phaseolus vulgaris, and 60%~ 85% of cells on the leaf surface showed GUS staining after infiltrating with suspension containing acetosyringone (a phenolic compound that can attract Agrobacterial cells to the wounded plant tissues via chemotaxis and induces the Vir genes to initiate T-DNA transfer [13]) and Silwet L-77, while the procedure is sophisticated and time-consuming.
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