The present invention provides a method for the Agrobacterium-mediated transformation of plants, in particular monocotyledonous plants.
The invention is in the field of plant transformation, in particular cereal transformation, specifically in the use of Agrobacterium tumefaciens or any other Agrobacterium species (hereinafter referred to as Agrobacterium). Until recently, only direct transformation methods could be used to produce transgenic cereal plants. Bombardment using a particle gun is the most widely accepted method to this end. More recently, reports have appeared in the literature showing that some of the cereals can be genetically modified using Agrobacterium (Hiei et al, Plant Mol. Biol. (1997) 35:205-218); Ishida et al, Nature Biotechnol. (1996) 14:745-750; Cheng et al, Plant Physiol. (1997) 115:971-980; Tingay et al., The Plant Journal, 11:1369-1376 (1997)).
Transformation efficiencies reported in the literature show wide variability for different cereals. Typically, low figures have been quoted for maize (Ishida 1996), with a system that is highly genotype dependent. With rice, low efficiencies for transformation have also been reported, and particularly low levels have been shown for wheat. In all of these systems, Agrobacterium is applied in vitro, to isolated tissue that is either in the process of de-differentiation or is already de-differentiated.
As described above, systems for Agrobacterium-mediated transformation of cereals have been reported in rice (Hiei, 1997), maize (Ishida, 1996), wheat (Cheng, 1997) and barley (Tingay, 1997). A common feature of these methods is that explants, preferably immature embryos or embryogenic calli derived therefrom, are isolated from a donor plant and inoculated with Agrobacterium in vitro.
Hess and coworkers (Plant Science 72: 233-244, 1990) attempted transformation of wheat by pipetting Agrobacterium into spikelets of wheat. The authors objective in this report was to achieve gene transfer by transformation of pollen and to subsequently recover transformed seed following normal fertilization. Removal of tissue from the inoculated spikelet for subsequent selection and regeneration in culture was not attempted or suggested.
Other workers have reported the Agrobacterium-mediated transformation of maize and rice by inoculation of shoot apices (Gould J (1991) Plant Physiol. 95: 426-434; Park S H (1996) Plant Molecular Biology 32: 1135-1158). Once again, this was with the object of transforming the germ line and thus recovering transformed seed. This pathway of regeneration is distinct from that employed in the method of our invention: in fact, a specified aim of these methods is to avoid any method of plant regeneration going through dedifferentiation of tissue and adventitious regeneration.
U.S. Pat. Nos. 5,177,010 and 5,187,073 (Goldman, et al) disclose a method for transforming corn and Gramineae respectively, comprising wounding newly emerged seedlings and inoculating with Agrobacterium. Once again, the objective of this method is to transform germ line cells in the seedling that will subsequently give rise to reproductive organs in the mature plant and thereby recover transformed pollen from the plant.
Another process that has been studied by those trying to develop cereal transformation is agroinfection. U.S. Pat. No. 5,569,597 (Grimsley, et al) discloses a method of introducing viral DNA into plants using Agrobacterium. Following inoculation of maize seedlings with Agrobacterium having DNA from maize streak virus inserted in its T-DNA, the inventors observed the appearance of disease symptoms, indicating proliferation of virus in plant cells. The Agrobacterium therefore acts as a vehicle to introduce the viral DNA into the plant, after which the virus is able to cause a systemic infection. However, there is no evidence that agroinfection results in plant transformation i.e. transfer of viral DNA to the plant genome. In so far as the patent considers transformation it is, once again, with a view to targeting meristematic tissues in order to achieve transformation of germ cells.