Method for introducing two T-DNAS into plants and vectors therefor

A hybrid vector comprising:

• a first T-DNA containing (1) a selection marker gene which functions in a plant, and (2) a second T-DNA having a restriction site;
• wherein there is sufficient distance on said hybrid vector between said first T-DNA and said second T-DNA to allow said first T-DNA and said second T-DNA to be independently inherited,
• said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacterium belonging to the genus Agrobacterium;
• said acceptor vector containing at least
• (a) a DNA region having a replication origin allowing replication of a plasmid in both a bacterium belonging to the genus Agrobacterium and in Escherichia coli,
• (b) a DNA region containing virB gene and virG gene in virulence region of Ti plasmid pTiBo542 of Agrobacterium tumefaciens, and
• (c) a DNA region which is homologous with a part of said intermediate vector, which is subjected to homologous recombination in said bacterium belonging to the genus Agrobacterium;
• said intermediate vector containing at least
• (i) a DNA region having a replication origin allowing replication of a plasmid in Escherichia coli, which does not function in said bacterium belonging to the genus Agrobacterium,
• (ii) a DNA region which is homologous with a part of said acceptor vector, which is subjected to homologous recombination in said bacterium belonging to the genus Agrobacterium, and
• (iii) a DNA region which constitutes at least a part of said second T-DNA.

A method for transforming and cultivating a plant using a bacterium belonging to the genus Agrobacterium, comprising co-transforming plant cells with a first T-DNA (1) and a second T-DNA (2);

• and selecting the cells which acquired drug resistance;
• said first T-DNA (1) containing a gene giving said drug resistance, which functions in said plant;
• said second T-DNA (2) containing a desired DNA fragment to be introduced into said plant, the second T-DNA (2) being contained in a hybrid vector;
• said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in said bacterium belonging to the genus Agrobacterium;
• said acceptor vector being pSB3 or pSB4;
• and
• said intermediate vector being pSB21, pSB22, pSB24, pTOK170, pYS151, pTOK235, pTOK245 or pTOK246;
• obtaining a plant transformed with said drug resistance gene and said desired DNA fragment;
• and
• cultivating said plant and selecting a plant in the next generation, which contains said desired DNA fragment but does not contain said drug resistance gene.

A hybrid vector comprising:

• a first T-DNA containing (1) a gene giving a drug resistance, which functions in a plant;
• and
• (2) a second T-DNA having a restriction site;
• wherein there is sufficient distance on said hybrid vector between said first T-DNA and said second T-DNA to allow said first T-DNA and said second T-DNA to be independently inherited,
• said hybrid vector being prepared by homologous recombination between an acceptor vector and an intermediate vector in a bacterium belonging to the genus Agrobacterium;
• said acceptor vector being pSB3 or pSB4;
• and
• said intermediate vector being pSB21, pSB22, pSB24, pTOK170, pYS151, pTOK235, pTOK245 or pTOK246.