Stable <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.) |
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Authors: | Z N Li F Fang G F Liu M Z Bao |
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Institution: | (1) Key Laboratory of Horticultural Plant Biology, Ministry of Education, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan, 430070, P.R. China |
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Abstract: | London plane tree (Platanus acerifolia Willd.) is an important tree in urban landscaping but it suffers from a number of negative traits which genetic engineering
could be used to address. As with many woody species, P. acerifolia has appeared recalcitrant to genetic transformation. However, the recent development of a method for regenerating shoots
from P. acerifolia leaf explants suggests that such material could be a target for gene-transfer. Using an Agrobacterium tumefaciens strain in which the T-DNA carries the histochemically detected reporter gene β-glucuronidase (GUS), we have followed the
transfer of genes from Agrobacterium to leaf explants of Platanus acerifolia. Using this system, we have identified a set of inoculation and co-cultivation conditions (notably: the pre-treatment of
leaf explants with 0.4 M mannitol, an inoculation period of 10 min, a bacterial OD600 of 0.8–1.0 and a co-cultivation period of 5 days) that permit a good frequency and reliability of transient gene-transfer.
Optimum levels of antibiotics for bacterial elimination and kanamycin-resistant shoot regeneration were also established.
By applying these parameters, we recovered eight independent stably transformed shoots that were kanamycin-resistant and contained
the nptII T-DNA gene, as confirmed by PCR analysis. Furthermore, Southern blot analysis confirmed that, in at least five of these lines,
the transgene was associated with high molecular weight DNA, so indicating integration into the plant genome. |
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Keywords: | Agrobacterium tumefaciens GUS expression High osmotic treatment Platanus acerifolia Transformation |
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