Use of Thidiazuron for High-Frequency Callus Induction and Organogenesis of Wild Strawberry (Fragaria vesca)

Abstract

Strawberry, belonging to the Fragaria genus, is an important crop that produces popular fruits globally. F. vesca, known as wild strawberry, has great characteristics, such as a robust and powerful aroma, making it an important germplasm resource. The present study aims to establish an efficient regeneration method for the in vitro propagation of F. vesca. Firstly, leaf explants were used to induce callus formation on a Murashige and Skoog medium with combinations of cytokinins (thidiazuron (TDZ) and 6-benzylaminopurine (BA)) and auxin (2,4-dichlorophenoxyacetic acid (2,4-D)). Among them, 0.45-4.54 µM TDZ combined with 0.45-4.53 µM 2.4-D exhibited a high induction rate after 4 weeks of culturing. Different explants were examined for their ability to form a callus, and whole leaves on the medium containing 2.27 µM TDZ and 2.27 µM 2,4-D showed the highest callus induction rate at 100% after 2 weeks of culturing in darkness. The highest shoot regeneration ability through organogenesis from the callus was obtained at 0.44 µM BA. After 2 weeks of culturing, the shoot regeneration rate and shoot number per explant were 96% and 19.4 shoots on an average, respectively. Rooting of shoots was achieved using indole-3-butyric acid (IBA) or an α-naphthaleneacetic acid (NAA)-containing medium, and the resulting plantlets were acclimatized successfully and formed flowers eventually. In this report, we demonstrated that shoot organogenesis was derived from the meristematic cells of calli and by transferring the induced calli to a 0.44 µM BA medium, the regeneration period can be shortened greatly. A protocol for the efficient regeneration of wild strawberry was established, which might be useful for their large-scale propagation or obtaining transgenic plants in the future.

Keywords: Fragaria vesca; plant growth regulator; plant regeneration; wild strawberry.

Figure 1

Callus induction from the leaf culture of strawberry after a 4-week culture (bar). (A) Thidiazuron (TDZ) 2.27 µM and 2,4-dichlorophenoxyacetic acid (2,4-D) 0.45 µM combination (1 mm); (B) TDZ 4.54 µM and 2,4-D 2.27 µM combination (1 mm).

Figure 2

Callus induction and shoot regeneration using different explants (bar). After a 2-week culture, calli were found on (A) the whole leaf and (B) the petiole explants under the thidiazuron (TDZ) 4.53 µM and 2,4-dichlorophenoxyacetic acid (2,4-D) 2.27 µM combination (1 mm) in the dark condition. After a 4-week culture, both calli and shoots were observed. More calli were seen in (C) the whole leaf and (D) the petiole explants culture under the TDZ 2.27 µM and 2,4-D 2.27 µM combination (1 mm). The shoots started to develop in (E) the whole leaf and (F) the petiole explants culture under the TDZ 4.54 µM and 2,4-D 0.45 µM combination (1 mm).

Figure 3

Histological observation of the shoot regeneration from wild strawberry (Fragaria vesca) calli after 2 weeks of culturing, with their corresponding development stages (bar). (A) Meristematic cells originated from the cells of the callus explants (500 μm); (B) shoot organogenesis with leaf primordium (LP) and shoot apical meristem (SAM) (500 μm); (C) the corresponding developmental stage of the callus explants corresponding to (A) (1 mm); (D) the stage of adventitious shoot (AS) development corresponding to (B) (1 mm).

Figure 4

Root induction from wild strawberry (Fragaria vesca) shoots under the dark condition after 8 weeks of culturing (bar). (A) the indole-3-butyric acid (IBA) 0.41 µM treatment (10 mm); (B) the IBA 4.14 µM treatment (5 mm).

Figure 5

Acclimatization of wild strawberry (Fragaria vesca) plants. (A) The strawberry after three months of cultivation; (B) flowers blooming after strawberry cultivation.