Production of Transgenic Poinsettia

Abstract

Since its introduction into North America over 170 years ago, the poinsettia has become a major ornamental potted plant, and is an important component of the U.S. floral industry. Susceptibility to insect pests and diseases caused by pathogens remains a problem for poinsettia production, even under greenhouse conditions. While chemical treatment can control certain insect pests and disease pathogens, such treatment can also have an adverse effect upon poinsettias. The methods described herein provide a means to genetically engineer transgenic poinsettia that express macromolecules capable of protecting the plant against the insects and pathogens. The production of transgenic plants can also be used to enhance the commercial value of poinsettia by controlling characteristics such as flower color.

1 . A method for in vitro regeneration of poinsettia plants comprising the steps of: 
 (a) incubating poinsettia plant tissue explants capable of producing reddish epidermal callus on callus induction medium;    (b) subculturing reddish epidermal callus to embryo induction medium comprising casein hydrolysate to form embryogenic callus;    (c) culturing said embryogenic callus on developmental medium;    (d) culturing said embryogenic callus on maturation medium; and    (e) recovering poinsettia plants from said embryos.    
   
   
       2 . The method of  claim 1 , wherein said callus induction medium comprises about 0.5-0.8 mg/liter 1-naphthalene acetic acid, about 0.2-0.4 mg/liter 6-benzylaminopurine and about 1 gm/liter casein hydrosylate.  
   
   
       3 . The method of  claim 1 , wherein said embryo induction medium comprises about 0.5-0.8 mg/liter 1-naphthalene acetic acid, about 0.2-0.4 mg/liter 6-benzylaminopurine 400 to 1700 mb/liter NH 4 NO 3 , 1900 to 3500 mb/liter KNO3 and about 1 gm/liter casein hydrosylate.  
   
   
       4 . The method of  claim 1 , wherein said developmental medium comprises about 0.05 mg/liter 6-benzylaminopurine, and about 10 gm/liter mannitol.  
   
   
       5 . The method of  claim 1 , wherein said maturation medium comprises about-5-20 μM abscisic acid, about 30-100 gm/liter sucrose, about 1 gm/liter casein hydrosylate, and about 10 gm/liter mannitol.  
   
   
       6 . A method for producing transgenic poinsettia plants, comprising the steps of: 
 (a) incubating poinsettia plant tissue explants capable of producing reddish epidermal callus on callus induction medium;    (b) culturing reddish epidermal callus on embryo induction medium comprising casein hydrolysate to form embryogenic callus;    (c) introducing an expression vector into said incubating embryogenic callus to produce transformed embryogenic callus, wherein said expression vector comprises a selectable marker gene and a second foreign gene, or    (c′) introducing two expression vectors into said incubating embryogenic callus to produce transformed embryogenic callus, wherein one of said expression vectors comprises a selectable marker gene, and wherein the second of said expression vectors comprises a second foreign gene,    (d) culturing said transformed embryogenic callus on selection medium;    (e) culturing said transformed embryogenic callus containing embryos on developmental medium;    (f) culturing said transgenic embryos on maturation medium; and    (g) recovering transgenic plants from said transgenic embryos.    
   
   
       7 . The method of  claim 6 , wherein said callus induction medium comprises about 0.5-0.8 mg/liter 1-naphthalene acetic acid, about 0.2-0.4 mg/liter 6-benzylaminopurine and about 1 gm/liter casein hydrosylate.  
   
   
       8 . The method of  claim 6 , wherein said embryo induction medium comprises about 0.5-0.8 mg/liter 1-naphthalene acetic acid, about 0.2-0.4 mg/liter 6-benzylaminopurine and about 1 gm/liter casein hydrosylate.  
   
   
       9 . The method of  claim 6 , wherein said developmental medium comprises about 0.05 mg/liter 6-benzylaminopurine, and about 10 gm/liter mannitol.  
   
   
       10 . The method of  claim 6 , wherein said maturation medium comprises about 5-20 μM abscisic acid, about 30-100 gm/liter sucrose, about 1 gm/liter casein hydrosylate, and about 10 gm/liter mannitol.  
   
   
       11 . The method of  claim 8 , wherein said embryo induction medium further comprises about 400 to 1700 mg/liter NH 4 NO 3  and about 1900 to 3500 mg/liter KNO 3 .  
   
   
       12 . The method of  claim 6 , wherein said poinsettia plant tissue explants are selected from the group consisting of immature embryos, mature embryos, shoot tips and stem segments.  
   
   
       13 . The method of  claim 6 , wherein said selectable marker gene is selected from the group consisting of a neomycin phosphotransferase gene, a hygromycin phosphotransferase gene, a phosphinothricin gene, a dihydrofolate reductase gene, a 5-enolpyruvylshikimate-3-phosphate synthase gene, an acetohydroxyacid synthase gene, a chloramphenicol acetyltransferase gene, a 3″-adenylyltransferase gene, a gentamicin acetyltransferase gene, a streptomycin phosphotransferase gene, and an aminoglycoside-3′-adenyl transferase gene.  
   
   
       14 . The method of  claim 13 , wherein said selectable marker gene is hygromycin phosphotransferase and said selection agent is hygromycin.  
   
   
       15 . The method of  claim 6 , wherein said expression vector that comprises said second foreign gene further comprises a promoter, wherein said promoter is selected from the group consisting of Cauliflower Mosaic Virus (CaMV) 35S promoter, the enhanced 35S promoter, the UBQ3 promoter, the UBQ10 promoter, the UBQll promoter, the UBQ14 promoter, the TEFA 1 promoter, the rolc promoter, and the Commelina Yellow Mottle Virus promoter, wherein the expression of said second foreign gene is under the control of said promoter.  
   
   
       16 . The method of  claim 15 , wherein said promoter is selected from the group consisting of the CaMV 35S promoter, the enhanced 35S promoter, the UBQ3 promoter, and the UBQ10 promoter.  
   
   
       17 . The method of  claim 6 , wherein the expression of said second foreign gene confers resistance to disease caused by an organism selected from the group consisting of virus, bacterium, fungus, and insect.  
   
   
       18 . The method of  claim 17 , wherein said second foreign gene disrupts the function of said virus, and wherein said virus-disrupting gene is selected from the group consisting of viral coat protein, 2′-5′ oligonucleotide synthetase, viral genome antisense RNA, and pokeweed antiviral protein.  
   
   
       19 . The method of  claim 6 , wherein said second foreign gene confers resistance to an insect, and wherein said insect resistance gene is selected from the group consisting of tryptophan decarboxylase, lectin, and  Bacillus thuringiensis  toxin.  
   
   
       20 . The method of  claim 19 , wherein said lectin is  Galanthus nivalis  lectin.