Use of tryptophan indole and anthranilate analogs as plant transformation selection agents
View Patent ↗Methods and compositions are described using tryptophan analogs, indole analogs, and mixtures thereof as agents for selecting plant cells that have been transformed with an anthranilate synthase (ASA2) gene or a fragment thereof, where the ASA2 gene encodes a feedback-insensitive form of anthranilate synthase (AS).
1. A method of selecting a transformed plant cell, the method comprising:
(a) expressing a nucleic acid sequence encoding a feedback insensitive anthranilate synthase (ASA2) in a plastid DNA (ptDNA); and
(b) selecting the plant cell comprising a plastid that expresses the nucleic acid sequence by contacting the plant cell with a medium containing an indole analog, wherein the analog is at a concentration effective to inhibit the growth of a plant cell that does not express the nucleic acid sequence.
2. The method of claim 1 , wherein the indole analog is selected from the group consisting of 4-fluoroindole, 5-fluoroindole, 6-fluoroindole, 6-aminoindole, 4-methylindole, 5 methoxyindole, 7-methoxyindole, and a combination thereof.
3. The method of claim 1 , wherein the concentration of the indole analog ranges in concentration from 1 μM to about 10 mM.
4. A method for selecting a transformed plant cell, the method comprising:
(a) expressing a nucleic acid sequence encoding a feedback insensitive anthranilate synthase (ASA2) or a functional fragment thereof in a plant cell by introducing the nucleic acid sequence in plastid DNA (ptDNA); and
(b) selecting the plant cell that expresses the nucleic acid sequence by contacting the plant cell with a medium comprising indole analogs, at a concentration effective to inhibit the growth of a plant cell that does not express the nucleic acid sequence.
5. A homoplastomic plant regenereated from a plant cell, wherein the transformed plant cell comprises a nucleic acid sequence encoding a feedback insensitive anthranilate synthase (ASA2) in plastids or a functional fragment thereof as the sole selection marker.
6. The homoplastomic plant of claim 5 further comprising a gene of interest that confers one or more of the traits selected from the group consisting of disease resistance, increased biomass, reduced lignin content, increased nutritive value, stress tolerance, and pest resistance.