A Rapid Method for Obtaining the Transgenic Roots of Crassulaceae Plants

Abstract

Crassulaceae plants are valued for their horticultural, ecological, and economic significance, but their genetic improvement is hindered by the absence of efficient and stable genetic transformation methods. Therefore, the development of a tailored genetic transformation method is crucial for enhancing the progress of the genetic improvement of Crassulaceae plants. The results indicate that, in the transformation experiments conducted on Kalanchoe tetraphylla, the K599 strain exhibited the highest transformation efficiency (76.67%), while C58C1 was least efficient (21.43%). An acetosyringone concentration of 100 μM was optimal for the hairy root transformation, and the immersion method yielded the highest efficiency. Additionally, the Silwet L-77 concentration significantly influenced the transformation efficiency, with 0.05% leading to a decrease. Upon four Crassulaceae species, notable differences were observed, with K. tetraphylla exhibiting the highest efficiency of 100%, and Sedum alfredii displaying the lowest efficiency of 5%. The RUBY reporter gene offers a more distinct advantage over GFP in observing the transformation effects. This study developed a simple, feasible, and cost-effective method for obtaining transgenic roots from leaves of Crassulaceae. The methodology provides technical support for the genetic improvement and gene function research of Crassulaceae plants.

Keywords: Agrobacterium-mediated transformation; Crassulaceae; GFP reporter; RUBY reporter; transformation system.

Figure 1

Selection of the optimal conditions for infection. (A) The transformation efficiency of different infection methods (dip, stab, vacuum, and ultrasound) were tested; (B) The transformation efficiency of four A. rhizogenes strains (K599, C58C1, Ar Qual, and Ar 1193) were tested; (C) The transformation efficiency of the different As concentrations (0, 50, 100, 200 μM) were tested. The data presented were the mean ± standard error of three independent replicate experiments. Different lower case letters indicated a significant difference between the explants (p < 0.05).

Figure 2

Effects of different concentrations of Silwet L-77 on rooting rate and transformation efficiency. (A) The rooting rate of Silwet L-77 (0, 0.01%, 0.05%); (B) The transformation efficiency of Silwet L-77 (0, 0.01%, 0.05%). Data presented were the mean ± standard error of three independent replicate experiments. Different lower case letters indicate a significant difference between the explants (p < 0.05).

Figure 3

Workflow for A. rhizogenes-mediated transformation of Crassulaceae plants.

Figure 4

Four Crassulaceae species transformation, GFP reporter observation, and PCR analysis. Four Crassulaceae species used for transformation, from left to right, were K. tetraphylla (A), G. paraguayense (B), S. alfredii (C), and H. spectabile (D), with a scale bar of 5 cm. Transgenic hairy roots from leaf explants of K. tetraphylla (E), G. paraguayense (F), S. alfredii (G), and H. spectabile (H), the top and bottom images in (E–G) were captured using bright filed and fluorescence microscopy, respectively, with a scale bar of 5 mm. (I) Validation of transgenic hairy roots using PCR analysis. M: 2000 marker; P: positive control; N: negative control; L1–L4: hairy roots from K. tetraphylla, G. paraguayense, S. alfredii, and H. spectabile.

Figure 5

Representative photographs of the RUBY reporter applied to Crassulaceae plant transformation. The white arrows represent the hairy roots expressing the RUBY gene. Bar = 2 mm.