Rapid and Efficient Optimization Method for a Genetic Transformation System of Medicinal Plants Erigeron breviscapus

Abstract

Erigeron breviscapus is an important medicinal plant with high medicinal and economic value. It is currently the best natural biological drug for the treatment of obliterative cerebrovascular disease and the sequela of cerebral hemorrhage. Therefore, to solve the contradiction between supply and demand, the study of genetic transformation of E. breviscapus is essential for targeted breeding. However, establishing an efficient genetic transformation system is a lengthy process. In this study, we established a rapid and efficient optimized protocol for genetic transformation of E. breviscapus using the hybrid orthogonal method. The effect of different concentrations of selection pressure (Hygromycin B) on callus induction and the optimal pre-culture time of 7 days were demonstrated. The optimal transformation conditions were as follows: precipitant agents MgCl₂ + PEG, target tissue distance 9 cm, helium pressure 650 psi, bombardment once, plasmid DNA concentration 1.0 μg·μL^-1, and chamber vacuum pressure 27 mmHg. Integration of the desired genes was verified by amplifying 1.02 kb of htp gene from the T0 transgenic line. Genetic transformation of E. breviscapus was carried out by particle bombardment under the optimized conditions, and a stable transformation efficiency of 36.7% was achieved. This method will also contribute to improving the genetic transformation rate of other medicinal plants.

Keywords: Erigeron breviscapus; biolistic; medicinal plant; optimization strategies; regeneration; transformation.

Figure 1

Particle-bombardment-mediated genetic transformation system optimization strategy of E. breviscapus.

Figure 2

Effect of different levels of six parameters on the survival of adventitious buds after three cycles of selection.

Figure 3

Particle-bombardment-mediated transformation, selection, regeneration, and eGFP fluorescent assay: (a–c) transient eGFP expression in leaves, scale bar 100 μm; (d–f) transient eGFP expression in transformed callus, scale bar 50 μm; (g) transformed callus under the first round (2 weeks) of selection; (h) transformed callus under the second round (4 weeks) of selection; (i) transformed callus under the third round (6 weeks) of selection; (j) shoot regeneration; (k) shoot elongation; (l) root formation.

Figure 4

Molecular analysis of transgenic plants. PCR amplification of the htp gene (1026 bp). M: marker 2000; CK+: positive control (plasmid PBI-1300); CK−: negative control (H₂O); WT: wild-type plant, lanes 1–6, putative transformants.