Regeneration and transformation of the premier UK apple (Malus × pumila Mill.) cultivar Queen Cox
2003
Wilson, F. M. | James, D. J.
A number of factors were assessed for their effects on in vitro shoot proliferation and adventitious shoot regeneration. More in vitro leaves of a quality suitable for use in regeneration and transformation experiments were obtained from shoots on DKW proliferation medium compared with MS medium, and also on MS and DKW media containing phloroglucinol. Compared with MS medium, shoot proliferation was greater on MS with halved levels of NH₄NO₃ and KNO₃. Adventitious shoots were hyperhydric on MS-based but not on DKW-based regeneration medium. More adventitious shoots regenerated on media solidified with Sigma Agargel than on media with Sigma Phytagel or Gelcarin (FMC). Viable transformed shoots were recovered on Sigma Agar or Agargel but not Phytagel. Wounding of leaf explants by stabbing with needles, and stabbing combined with scoring with a scalpel, increased the number of calli regenerating, and these methods, as well as solely scoring with a scalpel, increased the number of calli regenerating shoots compared with the control. Combined stabbing and scoring resulted in more calli producing shoots than solely scoring or stabbing. Vortexing leaf explants with silicon carbide whiskers increased the percentage of subsequently formed calli that regenerated shoots compared with the control. Transformed shoots were regenerated following co-cultivation with Agrobacterium tumefaciens EHA101 harbouring the binary vector pSCV1.6 (with selectable marker gene npt II and GUS reporter gene uid A). The number of transformed shoots as a percentage of explants varied from 0.5% to 2.2%. Molecular analysis of the four extant transformed lines confirmed integration of the transgene and indicated that in three lines there was one integration site, and in one line there were four sites of integration.
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