Plant transformation and selection
View Patent ↗The present invention relates to a genotype-independent method for transforming and selecting plant explants. The transformation method includes pre-culturing the explants in the presence of an Agrobacterium inducer and exposing the transformed explants to a shoot regeneration media that accelerates shoot development. Plants generated from this transformation method are provided. In particular, methods for obtaining transgenic Eucalyptus and pine cells and regenerating stably transformed Eucalyptus and pine trees are provided. The invention also provides media, methods, and plasmids for selecting and regenerating plants, particularly forest trees.
1. A transformed explant regeneration medium comprising a sulfonylurea or imidazolinone herbicide and a derivative of casein hydrolysate, wherein said medium is free of branched chain amino acids and casein hydrolysate, and wherein said derivative of casein hydrolysate consists of glycine, threonine, serine, tryptophan, tyrosine, methionine, phenylalanine, arginine, and lysine.
2. A method for producing a transgenic plant, comprising (i) pre-culturing an explant on a medium comprising an inducer of Agrobacterium ; (ii) exposing said explant to an Agrobacterium strain harboring a vector capable of transferring a gene to a plant cell; (iii) selecting a transformed explant on the regeneration medium of claim 1 ; and (iv) regenerating a transgenic plant from said explant.
3. A transformed explant regeneration medium comprising a tryptophan analog and a derivative of casein hydrolysate, wherein said regeneration medium is free of tryptophan and casein hydrolysate, and wherein said derivative of casein hydrolysate consists of glycine, threonine, serine, tyrosine, methionine, valine, phenylalanine, arginine, lysine, leucine, and isoleucine.
4. A method for selecting a transformed tree explant cell comprising, (i) transforming a tree explant cell with a vector comprising a gene encoding a herbicide-resistant form of plant acetoacetate synthase (ALS) and a gene involved in lignin biosynthesis or cellulose synthesis, and (ii) growing said transformed tree explant cell on the medium of claim 1 .
5. A method of selecting a transformed tree explant cell comprising, (i) transforming a plant cell with a vector comprising a gene encoding a feedback-insensitive form of anthranilate synthase (AS) and a gene involved in lignin biosynthesis or cellulose synthesis, and (ii) growing said transformed tree explant cell on the regeneration medium of claim 3 .
6. The transformed explant regeneration medium of claim 1 , wherein the medium comprises 0.5 g/L of the derivative of casein hydrolysate and the derivative of casein hydrolysate consists of 1.99 mg/g glycine, 8.52 mg/g threonine, 9.04 mg/g serine, 12.2 mg/g tryptophan, 12.7 mg/g tyrosine, 13.2 mg/g methionine, 25.7 mg/g phenylalanine, 25.8 mg/g arginine, and 50.8 mg/g lysine.
7. The transformed explant regeneration medium of claim 3 , wherein the medium comprises 0.5 g/L of the derivative of casein hydrolysate and the derivative of casein hydrolysate consists of 1.99 mg/g glycine, 8.52 mg/g threonine, 9.04 mg/g serine, 12.7 mg/g tyrosine, 13.2 mg/g methionine, 18.3 mg/g valine, 25.7 mg/g phenylalanine, 25.8 mg/g arginine, 50.8 mg/g lysine, 54 mg/g leucine, and 72.4 mg/g isoleucine.
8. A method for producing a transgenic plant, comprising (i) pre-culturing an explant on a medium comprising an inducer of Agrobacterium ; (ii) exposing said explant to an Agrobacterium strain harboring a vector capable of transferring a gene to a plant cell; (iii) selecting a transformed explant on the regeneration medium of claim 3 ; and (iv) regenerating a transgenic plant from said explant.
9. The method of claim 8 , wherein the transgenic plant is selected from the group consisting of: a transgenic E. occidentalis plant, a transgenic E. dunnii plant, a transgenic E. grandis plant, and a transgenic E. grandis×E. urophylla hybrid plant.
10. The method of claim 2 , wherein the transgenic plant is selected from the group consisting of: a transgenic E. occidentalis plant, a transgenic E. dunnii plant, a transgenic E. grandis plant, and a transgenic E. grandis×E. urophylla hybrid plant.