Abstract
Pinus patula Schiede et Deppe is one of the most important softwood species for pulp production by the South African forestry industry. This study was aimed at developing a protocol for the genetic transformation of Pinus patula embryogenic tissue. This was achieved via the introduction of a bar-GUS cassette under the control of the ubiquitin promoter, through biolistic transfer. A stepwise selection was obtained using MSG3 maintenance medium supplemented with BASTA® herbicide at 1 mg l−1 followed by 3 mg l−1 active ingredient. Transgene delivery was more efficient when the medium was supplemented with 0.25 M sorbitol. Expression of positive histochemical GUS activity in embryonal heads was observed. Positive PCR analysis of both GUS and bar transgenes (40% and 47% transformation efficiency, respectively) indicated successful genetic modification of P. patula embryonal suspensor masses by the pAHC25 plasmid. This indicates that P. patula is amenable to gene transfer.
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Abbreviations
- ABA:
-
abscisic acid
- BA:
-
N 6-benzyladenine
- bar :
-
the bialaphos resistance gene
- DMSO:
-
dimethyl sulphoxide
- EDTA:
-
ethylenediamene tetraacetic acid
- ESM:
-
embryonal suspensor mass
- GUS:
-
β-glucuronidase
- MSG3:
-
maintenance medium (Becwar et al. 1990)
- PCR:
-
polymerase chain reaction
- PEG:
-
polyethelene glycol
- TAE:
-
Tris–acetic acid–EDTA buffer
- Taq :
-
Thermus aquaticus enzyme
- uidA :
-
gene encoding β-glucuronidase from Escherichia coli
- X-glcA:
-
5-bromo-4-chloro-3-indolyl β-D-glucuronide
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Nigro, S.A., Makunga, N.P., Jones, N.B. et al. A biolistic approach towards producing transgenic Pinus patula embryonal suspensor masses. Plant Growth Regul 44, 187–197 (2004). https://doi.org/10.1007/s10725-004-4630-8
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DOI: https://doi.org/10.1007/s10725-004-4630-8