Whisker-mediated transformation of plant cell aggregates and plant tissues and regeneration of plants thereof

Abstract

Plant cell aggregates and plant tissues can be transformed by elongated, needle-like structures called “whiskers”. The process comprises the agitation of plant cell aggregates and plant tissues of the plant to be transformed in the presence of DNA and whiskers, whereby DNA uptake and integration thereof in facilitated. The process may be applicable to other plant cell aggregates and plant tissues which have not proven easily transformable by other techniques.

1 . A method for producing fertile transgenic plants comprising the steps of: (i) establishing a regenerable callus culture from a plant to be transformed wherein said callus culture is selected from the group consisting of Type I, Type II, hypocotyl-derived, and cotyledon-derived callus culture; (ii) selecting plant cell aggregates therefrom for transformation; (iii) transforming said plant cell aggregates or plant tissues with DNA by whisker-mediated transformation; (iv) identifying transformed cell lines therefrom; and (iv) regenerating fertile transgenic plants therefrom. 
     
     
         2 . The method of  claim 1  wherein said Type I callus cultures are established from  Zea mays  or  Oryza sativa.    
     
     
         3 . The method of  claim 1  wherein said Type II callus cultures are established from  Zea mays.    
     
     
         4 . The method of  claim 1  wherein said hypocotyl-derived cultures are established from  Gossypium hirsutum.    
     
     
         5 . The method of  claim 1  wherein said cotyledon-derived cultures are established from  Gossypium hirsutum.    
     
     
         6 . The method of  claim 1  wherein said plant cell aggregates are initiated on solid medium. 
     
     
         7 . The method of  claim 1  wherein said plant tissues are selected from the group consisting of meristems, pollen, cotyledons, and germ cells. 
     
     
         8 . The method of  claim 7  wherein said meristems, pollen cotyledons, and germ cells are selected from the group consisting of  Zea mays, Oryza sativa , and  Gossypium hirsutum.    
     
     
         9 . The method of  claim 1  wherein said DNA comprises a selectable marker gene or a reporter gene. 
     
     
         10 . The method of  claim 9  wherein said selectable marker gene imparts herbicide resistance to said fertile transgenic plant. 
     
     
         11 . The method of  claim 1  or  10  wherein said fertile transgenic plant is selected from the group consisting of  Zea mays, Oryza sativa , and  Gossypium hirsutum.    
     
     
         12 . A fertile, transgenic plants produced by the steps comprising: (i) establishing a regenerable callus culture from a plant to be transformed wherein said callus culture is selected from the group consisting of Type I, Type II, hypocotyl-derived, and cotyledon-derived callus culture; (ii) selecting plant cell aggregates therefrom for transformation; (iii) transforming said plant cell aggregates or plant tissues with DNA by whisker-mediated transformation; (iv) identifying transformed cell lines therefrom; and (iv) regenerating fertile transgenic plants therefrom. 
     
     
         13 . The fertile transgenic plant of  claim 12  wherein said Type I callus cultures are established from  Zea mays  or  Oryza sativa.    
     
     
         14 . The fertile transgenic plant of  claim 12  wherein said Type II callus cultures are established from  Zea mays.    
     
     
         15 . The fertile transgenic plant of  claim 12  wherein said hypocotyl-derived cultures are established from  Gossypium hirsutum.    
     
     
         16 . The fertile transgenic plant of  claim 12  wherein said cotyledon-derived cultures are established from  Gossypium hirsutum.    
     
     
         17 . The fertile transgenic plant of  claim 12  wherein said plant cell aggregates are initiated on solid medium. 
     
     
         18 . The fertile transgenic plant of  claim 12  wherein said plant tissues are selected from the group consisting of meristems, pollen, cotyledons, and germ cells. 
     
     
         19 . The fertile transgenic plant of  claim 18  wherein said meristems, pollen, cotyledons, and germ cells are selected from the group consisting of  Zea mays, Oryza sativa , and  Gossypium hirsutum.    
     
     
         20 . The fertile transgenic plant of  claim 12  wherein said DNA comprises a selectable marker gene or a reporter gene. 
     
     
         21 . The fertile transgenic plant of  claim 20  wherein said selectable marker gene imparts herbicide resistance to said fertile transgenic plant. 
     
     
         22 . The fertile transgenic plant of  claim 12  or  claim 21  wherein said fertile transgenic plant is selected from the group consisting of  Zea mays, Oryza sativa , and  Gossypium hirsutum.    
     
     
         23 . The  Zea mays  fertile transgenic plant of  claim 22  further comprising the seed and progeny thereof. 
     
     
         24 . The  Oryza sativa  fertile transgenic plant of  claim 22  further comprising the seed and progeny thereof. 
     
     
         25 . The  Gossypium hirsutum  fertile transgenic plant of  claim 22  further comprising the seed and progeny thereof. 
     
     
         26 . A DNA construct functional in plant comprising in the 5′ to 3′ direction of transcription, a transcriptional regulatory region functional in said plant cell and having a DNA sequence according to SEQ ID NO:1, and a gene of interest, said gene being either in the sense or antisense orientation. 
     
     
         27 . The transcriptional regulatory region according to  claim 26  comprising the DNA sequence SEQ ID NO:1.