Retrofitting YACs for direct DNA transfer into plant cells.
The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.
Adam
G
G
Plant Science Center, Cornell University, Ithaca, NY 14853-2703, USA. adam@edu2.boku.ac.at
Mullen
J A
JA
Kindle
K L
KL
D018244:Chromosomes, Artificial, Yeast; D015723:Gene Library; D018014:Gene Transfer Techniques; D005800:Genes, Fungal; D017343:Genes, Plant; D005822:Genetic Vectors; D011995:Recombination, Genetic