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

Details Method for introducing two T-DNAS into plants and vectors theref Method for introducing two T-DNAS into plants and vectors theref

1995-08-08

1998-03-24

Robinson, Douglas W.

Chemistry: molecular biology and microbiology

Treatment of micro-organisms or enzymes with electrical or...

Modification of viruses

4352522, 4352523, 43525233, 4353201, 800205, C12N 1505, C12N 1584, C12N 120, A01H 500

Patent

active

057311790

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of PCT/JP94/02049, filed Dec. 6, 1994.


TECHNICAL FIELD

The present invention relates to a method for transforming plants and a vector therefor.


BACKGROUND ART

Methods for introducing foreign genes (methods for transformation) to higher plants are largely classified into direct introduction methods and methods through bacteria belonging to the genus Agrobacterium. The former includes methods in which electric stimulation is utilized (electroporation method and electroinjection method); methods in which chemical treatments such as treatment with PEG are performed; and methods in which a particle gun is employed. The former is widely used for transforming monocotyledons to which the latter method is hardly applied. The latter is the methods in which the abilities of bacteria belonging to the genus Agrobacterium such as Agrobacterium tumefaciens and A. rhizogenes for transforming higher plants are utilized. The latter methods are excellent methods by which DNA fragments having relatively large sizes and having definite ends can be effectively introduced into higher plants, which do not require special culturing techniques such as protoplasts-culturing technique.
Methods for transformation are indispensable to studies of genetic engineering and molecular biology of higher plants, and a method by which a given DNA fragment is efficiently introduced into plant cells and by which a plant containing the DNA fragment is efficiently obtained is demanded. In introduction of a gene, it is necessary to select the plant cells into which the foreign gene is introduced from the plant cells into which the foreign gene is not introduced. Usually, since the number of the latter cells are much larger, it is necessary to utilize a gene which can be easily detected. The most widely used selection markers are drug resistant genes. Examples thereof include antibiotics resistant genes such as kanamycin resistant gene and hygromycin resistant gene; and herbicide resistant genes such as Basta resistant gene and Roundup resistant gene.
In cases where a drug resistance gene is used as a selection marker, a DNA fragment of interest to be introduced into a plant and the drug resistance gene are connected, the connected genes are introduced into plants, drug resistant cells are selected and transformed plants are obtained from the drug resistant cells. In the transformed plants thus obtained, the DNA fragment of interest connected to the drug resistance gene is also simultaneously introduced.
However, this method has the following two problems: introduction of the gene, the selection marker always accompanies the introduced DNA fragment of interest during the subsequent growing step and even in the plants of subsequent generations. Thus, transformed plants containing the unnecessary gene are obtained. In cases where the transformation method is employed in breeding of crops, since varieties which do not contain such an unnecessary gene will have better reputation, the existence of the unnecessary gene is a big problem. Further, when another DNA fragment is introduced into the transformed plants, it is necessary to use another selection marker, which is inconvenient. This is also a big problem. of interest to be introduced into the plant is necessary, one additional step is required in construction of the gene to be introduced.
To avoid these problems, in the direct introduction methods, so called co-transformation method has been developed and widely used (Shimamoto et al., Nature 338:274-276, 1989). In this method, the DNA of the drug resistance gene as a selection marker and a DNA fragment of interest to be introduced into plants are merely mixed without ligation and the mixture is introduced into the plants. In the plants selected according to the drug resistance, some plants contain both the drug resistance gene and the DNA fragment of interest to be introduced into the plants. By controlling the mixing ratio of the two types of DNA, more often than not, the percentage of the drug resistant plants which

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