Biolistic transformation of Arabidopsis root hairs: a novel technique to facilitate map-based cloning.
The final stage of map-based gene isolation is complementation of the mutant phenotype with wild-type DNA to determine the exact location of the gene of interest. This usually involves Agrobacterium tumefaciens-mediated transformation, which is reliable and produces stable transformants. However, the process of Agrobacterium transformation may take up to three months to complete. If the mutant phenotype can be seen in a single cell, and the wild-type copy of the gene can act cell autonomously, then complementation of the whole plant is not strictly necessary. We have developed a technique for the biolistic transformation of Arabidopsis thaliana root hairs, and used this to test large insert clones for complementation of two recessive mutant phenotypes, a procedure that takes less than a day. Our results show that biolistic transformation can be used with transient assays to conduct rapid tests for complementation by large insert clones.
Kemp
A
A
School of Biological Sciences, University of Bristol, Woodland Road, Bristol, BS8 1UG, UK. ali.kemp@bristol.ac.uk
Parker
J
J
Grierson
C
C
D016960:Agrobacterium tumefaciens / Q000235:genetics; D017360:Arabidopsis / Q000235:genetics*; D019470:Biolistics; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D010641:Phenotype; D018517:Plant Roots / Q000235:genetics*; D014170:Transformation, Genetic
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins