Methods and tissue culture media for inducing somatic embryogenesis, agrobacterium-mediated transformation and efficient regeneration of cacao plants

Abstract

The present invention relates to improved methods of (i) inducing somatic embryogenesis from cacao tissue explants and (ii) regenerating cacao plants from somatic embryos. The invention further relates to cacao somatic embryos and plants obtained according to the methods of the invention. Novel tissue culture media adapted for use in the above-identified methods are also within the scope of the invention. The novel media of the invention include primary callus growth medium, secondary callus growth medium, embryo development medium, primary embryo conversion medium, secondary embryo conversion medium and plant regeneration medium.

What is claimed is:  
     
         1 . A method of inducing somatic embryogenesis in a cacao tissue comprising the steps of: 
 ( a ) providing a cacao tissue explant;    ( b ) culturing said explant on a primary callus growth medium, said medium having the property of inducing callus growth on said explants;    ( c ) culturing the callus produced in step ( b ) on a secondary callus growth medium, said medium having the property of inducing homeostatic growth and bipolar callus cell development; and    ( d ) culturing the callus produced in step ( c ) on an embryo development medium, said medium having the property of inducing embryo differentiation.    
     
     
         2 . The method of    claim 1    wherein the primary callus growth medium comprises a basal medium, a carbon source, and a growth regulator.  
     
     
         3 . The method according to    claim 2    wherein said basal medium is a DKW basal medium.  
     
     
         4 . The method of    claim 2    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         5 . The method of    claim 2    wherein such growth regulator is thidiazuron, 2,4-dichlorophenoxyacetic acid or a combination thereof.  
     
     
         6 . The method of    claim 1    wherein said secondary callus growth medium comprises a basal medium having a low salt concentration, a carbon source, and a growth regulator.  
     
     
         7 . The method of    claim 6    wherein said basal medium is a WPM low salt basal medium.  
     
     
         8 . The method of    claim 6    wherein said carbon source is glucose, sucrose, or a combination thereof.  
     
     
         9 . The method of    claim 6    wherein said growth regulator is kinetin, 6-benzyladenine, 2,4-D, or any combination thereof.  
     
     
         10 . The method of    claim 1    wherein said embryo development medium comprises a basal medium and a carbon source.  
     
     
         11 . The method of    claim 10    where said basal medium is a DKW basal medium.  
     
     
         12 . The method of    claim 10    wherein said carbon source is sucrose.  
     
     
         13 . The method of    claim 1    wherein said explant is a staminode or a petal base explant.  
     
     
         14 . A method for regenerating cacao plantlets comprising the steps of: 
 ( a ) providing a cacao somatic embryo;    ( b ) germinating said somatic embryo on a primary embryo conversion medium; and    ( c ) regenerating cacao plantlets on a secondary embryo conversion medium from germinated embryos produced in step ( b ).    
     
     
         15 . The method of    claim 14    wherein said primary embryo conversion medium comprises a basal medium, a source of potassium and nitrate ions, and a carbon source.  
     
     
         16 . The method of    claim 15    wherein said basal medium is a DKW basal medium.  
     
     
         17 . The method    claim 15    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         18 . The method of    claim 14    wherein said secondary embryo conversion medium comprises a diluted basal medium and a carbon source.  
     
     
         19 . The method of    claim 18    wherein said carbon source is sucrose, glucose or a combination thereof.  
     
     
         20 . The method of    claim 14    wherein said cacao somatic embryo is produced according to    claim 1   .  
     
     
         21 . A method of regenerating cacao plantlets comprising the steps of: 
 ( a ) providing a cacao somatic embryo;    ( b ) culturing said somatic embryo on a plant regeneration medium.    
     
     
         22 . The method of    claim 21    wherein said plant regeneration medium comprises a diluted basal medium, a carbon source and a growth regulator.  
     
     
         23 . The method of    claim 22    wherein said diluted basal medium is a WPM low salt basal medium.  
     
     
         24 . The method of    claim 22    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         25 . The method of    claim 22    wherein said growth hormone is a gibberellic acid.  
     
     
         26 . The method of    claim 21    wherein said cacao somatic embryo is produced according to    claim 1   .  
     
     
         27 . A primary callus growth medium comprising a basal medium, a carbon source, and a growth regulator, said medium having the property of inducing callus growth on cacao tissue explants.  
     
     
         28 . The medium of    claim 27    wherein said basal medium is a DKW basal medium.  
     
     
         29 . The medium of    claim 28    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         30 . The medium of    claim 28    wherein said growth regulator is thidiazuron, 2,4-dichlorophenoxyacetic acid or a combination thereof.  
     
     
         31 . A secondary callus growth medium comprising a basal medium having a low salt concentration, a carbon source, and a growth regulator, said medium having the property of inducing homeostatic growth and bipolar cacao callus cell development.  
     
     
         32 . The medium of    claim 31    wherein said basal medium is a WPM low salt basal medium.  
     
     
         33 . The medium of    claim 31    wherein said carbon source is glucose, sucrose, or a combination thereof.  
     
     
         34 . The medium of    claim 31    wherein said growth regulator is kinetin, 6-benzyladenine, 2,4-D, or any combination thereof.  
     
     
         35 . An embryo development medium comprising a basal medium and a carbon source, said medium having the property of inducing embryo differentiation.  
     
     
         36 . The medium of    claim 35    wherein said basal medium is a DKW basal medium.  
     
     
         37 . The medium of    claim 35    wherein said carbon source is sucrose.  
     
     
         38 . A primary embryo conversion medium comprising a basal medium, a source of potassium and nitrate ions, and a carbon source.  
     
     
         39 . The medium of    claim 38    wherein said basal medium is a DKW basal medium.  
     
     
         40 . The medium of    claim 38    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         41 . A secondary embryo conversion medium comprising a diluted basal medium and a carbon source.  
     
     
         42 . The medium of    claim 41    wherein said carbon source is sucrose, glucose or a combination thereof.  
     
     
         43 . A plant regeneration medium comprising a diluted basal medium, a carbon source and a growth regulator, said medium having the property of inducing regeneration of cacao plantlets from cacao somatic embryos.  
     
     
         44 . The medium of    claim 43    wherein said diluted basal medium is a WPM low salt basal medium.  
     
     
         45 . The medium of    claim 43    wherein said carbon source is glucose, sucrose or a combination thereof.  
     
     
         46 . The medium of    claim 43    wherein said growth hormone is a gibberellic acid.  
     
     
         47 . A cacao somatic embryo produced according to    claim 1   .  
     
     
         48 . A cacao plant produced according to    claim 14   .  
     
     
         49 . A cacao plant produced according to    claim 20   .  
     
     
         50 . A cacao plant produced according to    claim 21   .