Abstract
Few studies have been conducted on the links between histological and hormonal variation during shoot regeneration. Therefore, we investigated this link in strawberry (Fragaria × ananassa cv. ‘Honeoye’). Main plant growth regulators were measured using reverse-phase liquid chromatography–tandem mass spectrometry. Histological observations were conducted to understand the pattern of adventitious shoot regeneration from the leaf segments. After 14 days of dark culture, a mean shoot regeneration frequency of 94.7 % was obtained on MS medium supplemented with 2.0 mg L−1 thidiazuron, 30 g L−1 sucrose, and 6 g L−1 agar (pH 5.9). During shoot regeneration, indole acetic acid (IAA) concentrations increased, abscisic acid (ABA) decreased, and gibberellic acid (GA3) and zeatin showed peaks. The results could be correlated with the cell division and differentiation that occurred during shoot regeneration. Histological observation showed that the adventitious shoots were derived from subepidermal cells and the epidermal cells of the midrib near the cut. The meristemoids, primordia and shoots were formed sequentially on day 6, day 12, and day 18 after culture. During the meristemoids formation on day 6 after culture, IAA rapidly increased from 1.49 to 1.72 ng g−1 fresh weight (Fw), ABA rapidly decreased from 52.61 to 13.47 ng g−1 Fw, and zeatin increased from 1.68 to 5.98 ng g−1 Fw. During primordia formation on day 12 after culture, IAA rapidly increased to 1.88 ng g−1 Fw, ABA rapidly decreased to 2.69 ng g−1 Fw, GA3 peaked at 73.91 ng g−1 Fw, and zeatin peaked at 7.69 ng g−1 Fw. Our results suggested that the histological variations were consistent with the plant hormonal changes during shoot regeneration, and that changes in hormone concentration could be used as a reference to characterize the mode of shoot regeneration.
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Abbreviations
- ABA:
-
Abscisic acid
- GA3 :
-
Gibberellic acid
- HgCl2 :
-
Mercuric chloride
- BA:
-
6-benzyladenine
- IBA:
-
Indole-3-butyric acid
- TDZ:
-
Thidiazuron
- IAA:
-
Iindole-3-acetic acid
- BSAA:
-
3-Benzo[b]selenienyl acetic acid
- IPA:
-
N6(2-isopentenyl)adenosine
- 2IP:
-
N6(2-isopentenyl)adenine
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Acknowledgments
This study was funded by Beijing Innovation of Science and Technology (KJCX20140202), Young Foundation of Beijing Academy of Agriculture and Forestry Sciences (QNJJ201507), National High Technology Research and Development Program of China (2011AA100204), Beijing Nova program (Z121105002512036), and Nonprofit research project (20120307501-4). We are grateful to B.A. Hong Zhang and Mingming Zhao for their useful techniques throughout the experiments.
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11240_2015_851_MOESM1_ESM.tif
Fig. S1 Adventitious shoot regeneration derived from subepidermal cells of the midrib near the cut in transverse sections of leaf explants of ‘Honeoye’ by histological observation. a A leaf explant at the time of culture initiation (day 0). b A leaf explant on day 3 of culture. Some subepidermal cells on the adaxial side had dedifferentiated; these cells contained dense cytoplasm and a conspicuous nucleus. c The meristemoids (MD) and vascular structures (VS) formed on day 6 of culture. d A leaf explant on day 9 of culture. e The shoot primordium (SP) appeared on day 12 of culture. f A leaf explant on day 15 of culture. g A leaf explant on day 17 of culture. h, i A leaf explant on day 18 of culture, adventitious shoots (ADS) included in the square are enlarged in i. j–o Leaf explants on day 21 of culture, ADS and VS included in the squares are enlarged in k, l, n, o. EC epidermal cells. Bars = 100 μm. (TIFF 40805 kb)
11240_2015_851_MOESM2_ESM.tif
Fig. S2 Adventitious shoot regeneration derived from epiderma cells of the midrib near the cut in transverse sections of leaf explants of ‘Honeoye’ by histological observation. a A leaf explant at the time of culture initiation (day 0). b A leaf explant on day 3 of culture. c The meristemoids (MD) formed on day 6 of culture. d A leaf explant on day 9 of culture. e A leaf explant on day 12 of culture. f The shoot primordium (SP) appeared on day 15 of culture. g A leaf explant on day 17 of culture. h, i A leaf explant on day 18 of culture, adventitious shoots (ADS) included in the square are enlarged in i. j–o Leaf explants observed on day 21 of culture, ADS included in the squares are enlarged in k, l, n, o. EC epidermal cells. Bars = 100 μm. (TIFF 40551 kb)
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Wang, H., Li, M., Yang, Y. et al. Histological and endogenous plant growth regulators changes associated with adventitious shoot regeneration from in vitro leaf explants of strawberry (Fragaria × ananassa cv. ‘Honeoye’). Plant Cell Tiss Organ Cult 123, 479–488 (2015). https://doi.org/10.1007/s11240-015-0851-y
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DOI: https://doi.org/10.1007/s11240-015-0851-y