Abstract
A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2–5 mg dm−3) and naphthaleneacetic acid (NAA; 0.05–0.15 mg dm−3). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm−3 BA and 0.1 mg dm−3 NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm−3 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.
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Abbreviations
- BA:
-
6-benzylaminopurine
- CIM:
-
callus induction medium
- CTAB:
-
cetyltrimethyl ammonium bromide
- 2,4-D:
-
2,4-dichlorophenoxyacetic acid
- GUS:
-
β-glucuronidase
- MS:
-
Murashige and Skoog
- NAA:
-
naphthaleneacetic acid
- SIM:
-
shoot induction medium
- STS:
-
silver thiosulphate
- X-Gluc:
-
5-bromo-4-chloro-3-indolyl β-D-glucuronide
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Acknowledgements
The research was financially supported by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Zhejiang province, P.R. China and the German research foundation DFG (grant No. SFB617-A19). Authors thank the German Academic Exchange Service (DAAD) for providing a short visiting scholarship.
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Tang, G.X., Knecht, K., Yang, X.F. et al. A two-step protocol for shoot regeneration from hypocotyl explants of oilseed rape and its application for Agrobacterium-mediated transformation. Biol Plant 55, 21–26 (2011). https://doi.org/10.1007/s10535-011-0003-0
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DOI: https://doi.org/10.1007/s10535-011-0003-0