Reinvigoration of diploid strawberry (Fragaria vesca) during adventitious shoot regeneration

Abstract

Diploid strawberry (Fragaria vesca 'Baiguo') is a model plant for studying functional genomics in Rosaceae. Adventitious shoot regeneration is essential for functional genomics by Agrobacterium tumefaciens-mediated transformation. An efficient shoot regeneration method using diploid strawberry leaf explants was conducted on 1/2MS + 1/2B₅ medium that contained 2.0 mg L^-1 TDZ over 14 days of dark culture; this induced the maximum percentage of shoot regeneration (96.44 ± 1.60%) and the highest number of shoots per explant (23.46 ± 2.14) after 11 weeks of culture. The explants considerably enlarged after 12 days; then, turned greenish brown after 30 days, yellowish brown after 36 days, and completely brown and necrotic after 48 days. Large numbers of adventitious shoots were produced from 48 to 66 days, and the shoots elongated from 66 to 78 days; this represents a critical period of reinvigoration, which included 30 days for leaf explant chlorosis, 36 days for adventitious shoot appearance, and 48 days for generation of numerous shoots. During the reinvigoration process, higher expressions of the hormone synthesis-related genes Ciszog1, CKX2, CKX3, CKX7, YUC2, YUC6, YUC10, YUC9, and GA2ox were detected from 30 to 48 days. Our results indicate that these genes may regulate reinvigoration of shoot regeneration.

Figure 1

Adventitious shoot regeneration of strawberry leaf explants. (A) Octoploid strawberry (F. ananassa cv. ‘Honeoye’) leaf explants. (B) Diploid strawberry (F. vesca ‘Baiguo’) leaf explants. Note: Bar, 1 cm.

Figure 2

Dark treatment effects on reinvigoration during adventitious shoot regeneration of diploid strawberry leaf explants. (A) Adventitious shoot regeneration of diploid strawberry F. vesca ‘Baiguo’ leaf explants in the light (DT0) and in the dark for 7 days (DT7), 14 days (DT14), 21 days (DT21), or 28 days (DT28) after 78 days on 1/2MS + 1/2B₅ medium that contained 2.0 mg L⁻¹ TDZ. Arrows indicate the reinvigoration period of adventitious shoot regeneration. (B) Adventitious shoot regeneration on 0, 18, 30, 36, 42, 48, 54, 60, 66, 72, and 78 days of culture under different dark treatments. (C) Culture of the explants including adventitious shoots inoculated on 1/2MS + 1/2B₅ medium contained 0.2 mg L^-1 BA and 0.1 mg L^-1 IBA.

Figure 3

Relationships between hormone transcript levels and morphological characteristics (explant reinvigoration and shoot regeneration). (A) Morphological characteristics of leaf explants in initial 14 days dark treatment during shoot regeneration. (B) Transcript levels of key hormone synthesis-related genes, including Zeatin O-glucosyltransferase (ZOG), cis-zeatin O-glucosyltransferase (Ciszog), cytokinin hydroxylase (CKH), cytokinin oxidase/dehydrogenase (CKX) families, YUCCA (YUC) families, gibberellin 2-beta-dioxygenase (GA2ox), and GA20ox during diploid strawberry ‘Baiguo’ shoot regeneration.