Method for producing transgenic cereal plants
First Claim
1. A method for producing transgenic cereal plants that will transmit introduced DNA to progeny, comprising the steps of(A) introducing foreign DNA into target cells selected from the group consisting of (i) cells of a meristem that is not enclosed by primordial sheathing leaves and (ii) cells that contribute to said meristem;
- then(B) inducing reorganization of said meristem to increase transgenic sector size, whereby the likelihood that a transgenic sector will contribute to germline transmission is increased, wherein said reorganization is effected through at least one manipulation selected from the group consisting of (i) imposition of a nonlethal selective pressure on said meristem, (ii) mechanically-induced meristem reorganization, and (iii) hormonally-induced shoot multiplication combined with nonlethal selective pressure; and
thereafter(C) exposing said meristem to conditions under which it differentiates to form a plantlet, wherein said plantlet contains said transgenic sector or is homogeneously transformed by said foreign DNA, such that said plantlet can be grown into a transformed cereal plant that will transmit said foreign DNA to progeny.
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Abstract
To obtain a transgenic cereal plant which is stably transformed, an exposed cereal meristem is subjected to biolistic bombardment in order to target non-differentiated meristem cells for transformation. Immature embryos at the early proembryo, mid proembryo, late proembryo, transitional or early coleoptilar stage are harvested for biolistic bombardment. The meristem tissue or cells fated to contribute to the meristem then are manipulated in order to enlarge transgenic sectors, either through selection and/or through effecting a proliferation from the tissue of shoots or multiple meristems per se. The shoot population thus obtained then is screened, by means of a nonlethal enrichment assay, to identify either chimeric sectors that will contribute to germline transmission, or non-sectored, L2 periclinal chimeras that will by definition transmit to progeny. Increased time in culture, under selection, enhances the prospects for sectoral-to-periclinal conversions, and also selects for L1-to-L2 conversions which, through a shift in position, ultimately contribute to the germline. Transgenic sectors also are stabilized during the step of tillering.
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Citations
17 Claims
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1. A method for producing transgenic cereal plants that will transmit introduced DNA to progeny, comprising the steps of
(A) introducing foreign DNA into target cells selected from the group consisting of (i) cells of a meristem that is not enclosed by primordial sheathing leaves and (ii) cells that contribute to said meristem; - then
(B) inducing reorganization of said meristem to increase transgenic sector size, whereby the likelihood that a transgenic sector will contribute to germline transmission is increased, wherein said reorganization is effected through at least one manipulation selected from the group consisting of (i) imposition of a nonlethal selective pressure on said meristem, (ii) mechanically-induced meristem reorganization, and (iii) hormonally-induced shoot multiplication combined with nonlethal selective pressure; and
thereafter(C) exposing said meristem to conditions under which it differentiates to form a plantlet, wherein said plantlet contains said transgenic sector or is homogeneously transformed by said foreign DNA, such that said plantlet can be grown into a transformed cereal plant that will transmit said foreign DNA to progeny. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17)
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Specification