Transformation of guar

Abstract

The invention relates to a method for the transformation and regeneration of legumes of the genus Cyamopsis, in particular Agrobacterium-mediated transformation of guar ( Cyamopsis tetragonoloba, by introducing a recombinant DNA sequence into at least one cell or protoplast and generating genetically modified explants using at least one selection or shoot growth medium comprising at least one compound selected from an auxin inhibitor, e.g. 2-(p-chlorophenoxy)-2-methylpropionic acid (PCIB), a β-lactamase inhibitor, e.g. sulbactam, and an ethylene inhibitor, e.g. silver thiosulfate, so as to obtain a genetically modified plant or part thereof containing in its genome at least one recombinant DNA sequence; to genetically modified plants produced by the method; and to the use of substances such as the β-lactamase inhibitor sulbactam to facilitate transformation of guar and other plants.

1 . A genetically modified plant of the genus Cyamopsis or part thereof, said plant or plant part comprising in its genome at least one recombinant DNA sequence.  
     
     
         2 . A genetically modified plant or plant part according to    claim 1    of the species  C. tetragonoloba.    
     
     
         3 . Seeds, seedlings or plant parts obtained from a plant according to    claim 1   .  
     
     
         4 . A method for producing a genetically modified plant of the genus Cyamopsis or part thereof, comprising the steps of 
 (i) introducing a recombinant DNA sequence carrying a selectable marker into at least one cell or protoplast derived from said genus to obtain a transformed cell or protoplast, and    (ii) generating from said transformed cell or protoplast genetically modified explants using at least one selection medium and/or at least one shoot growth medium comprising at least one β-lactamase inhibitor so as to obtain a genetically modified plant or part thereof containing in its genome at least one recombinant DNA sequence.    
     
     
         5 . A method according to    claim 4    wherein genetically modified shoots are generated using a selection medium and/or a shoot growth medium comprising at least one auxin inhibitor.  
     
     
         6 . A method according to    claim 4    wherein the selection medium and/or the shoot growth medium comprises at least one ethylene inhibitor.  
     
     
         7 . A method according to    claim 5    or    6    wherein the auxin inhibitor is 2-(p-chlorophenoxy)-2-methylpropionic acid (PCIB).  
     
     
         8 . A method according to    claim 4    wherein the β-lactamase inhibitor is sulbactam.  
     
     
         9 . A method according to    claim 6    wherein the selection medium comprises PCIB as the auxin inhibitor in a concentration of 0.01-10 mg/l and sulbactam as the β-lactamase inhibitor in a concentration of 10-1000 mg/l, and the shoot growth medium comprises sulbactam as the β-lactamase inhibitor in a concentration of 10-1000 mg/l.  
     
     
         10 . A method according to    claim 9    wherein the selection medium comprises PCIB in a concentration of 0.05-5 mg/l  
     
     
         11 . A method according to    claim 9    or    10    wherein the selection medium and/or the shoot growth medium comprises sulbactam in a concentration of 20-500 mg/l.  
     
     
         12 . A method according to    claim 4    wherein the selection medium further comprises kanamycin.  
     
     
         13 . A method according to    claim 12    wherein the selection medium comprises kanamycin sulfate in a concentration of 50-300 mg/l.  
     
     
         14 . A method according to    claim 13    wherein explants are transferred from the selection medium to a second selection medium comprising kanamycin sulfate in a concentration lower than that of the first selection medium.  
     
     
         15 . A method according to    claim 6    wherein the selection and/or shoot growth medium comprises silver thiosulfate as an ethylene inhibitor in a concentration of up to 50 μM.  
     
     
         16 . A method according to    claim 15    wherein the selection and/or shoot growth medium comprises silver thiosulfate in a concentration of 0.1-10 μM.  
     
     
         17 . A method according to    claim 4    wherein the selection and/or shoot growth medium further comprises a nickel salt.  
     
     
         18 . A method according to    claim 4    wherein the selection and/or shoot growth medium further comprises a gibberellin.  
     
     
         19 . A method according to    claim 4    wherein the selection and/or shoot growth medium further comprises a penicillin-like antibiotic.  
     
     
         20 . A method according to    claim 19    wherein the penicillin-like antibiotic is selected from carbenicillin and cefotaxime.  
     
     
         21 . A method according to    claim 4    wherein transformation is performed on cotyledons.  
     
     
         22 . A method according to    claim 21    wherein the cotyledons are from seeds that have been germinated for at least 4 days.  
     
     
         23 . A method according to    claim 4    for producing a genetically modified plant of the species  C. tetragonoloba.    
     
     
         24 . A method according to    claim 4    comprising as a further step that genetically modified shoots are grafted onto seedlings or established, rooted plants.  
     
     
         25 . A method for producing a genetically modified plant of the genus Cyamopsis or part thereof, comprising the steps of 
 (i) introducing a recombinant DNA sequence carrying a selectable marker into at least one cell or protoplast derived from said genus to obtain a transformed cell or protoplast, and    (ii) generating from said transformed cell or protoplast genetically modified explants using at least one selection medium and/or at least one shoot growth medium comprising a nickel salt, so as to obtain a genetically modified plant or part thereof containing in its genome at least one recombinant DNA sequence.    
     
     
         26 . A genetically modified plant of the genus Cyamopsis or a part thereof produced by the method of    claim 4    or the method of    claim 25   .  
     
     
         27 . A chimeric plant belonging to the genus Cyamopsis, said plant is capable of producing transgenic seeds and obtained by grafting a genetically modified shoot obtained by the method of    claim 4    and cultivated in vitro onto a non-in vitro cultivated plant.  
     
     
         28 . Chimeric transgenic plants according to    claim 27    belonging to the species  C. tetragonoloba.    
     
     
         29 . A method for producing a genetically modified plant or part thereof of a plant species, comprising the steps of 
 (i) introducing a recombinant DNA sequence carrying a selectable marker into at least one cell or protoplast derived from said genus to obtain a transformed cell or protoplast, and    (ii) generating from said transformed cell or protoplast genetically modified explants using at least one selection medium an/or at least one shoot growth medium comprising at least one penicillin-like antibiotic and at least one β-lactamase inhibitor so as to obtain a genetically modified plant or part thereof containing in its genome at least one recombinant DNA sequence.    
     
     
         30 . A method of producing galactomannan, comprising cultivating the genetically modified plant of    claim 1    or a plant obtained by the method of    claim 4    or    25   , collecting its seeds and isolating galactomannan from said seeds.