Enhanced transformation of recalcitrant monocots
View Patent ↗Provided herein are improved methods for transforming monocotyledonous plants, as well as an improved phosphomannose-isomerase (PMI) protein coding region and transformation vectors including the same.
1. A method for increasing the transformation frequency of a monocotyledonous plant tissue, comprising:
introducing a heterologous nucleic acid comprising a coding region having one or more maize-optimized codons into a cell of said plant tissue, wherein said coding region comprises at least 500 consecutive nucleotides of SEQ ID NO:2, or said coding region comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:2, wherein said coding region codes for a phosphomannose isomerase (PMI) protein, thereby producing a transformed monocotyledonous cell comprising the nucleic acid;
whereby said transformation frequency of said monocotyledonous plant tissue is increased as compared to a transformation efficiency without maize-optimized codons in said coding region of said nucleic acid.
2. The method of claim 1 , wherein said introducing step is carried out by Agrobacterium -mediated transformation.
3. The method of claim 2 , wherein said monocotyledonous plant tissue has a 1-, 2-, or 3-fold greater transformation frequency as compared to a transformation frequency without maize-optimized codons in said coding region of said nucleic acid.
4. The method of claim 2 , wherein said monocotyledonous plant tissue has a transformation frequency by Agrobacterium -mediated transformation without maize-optimized codons of less than about 20%, 15%, 10%, or 5% as compared to a transformation frequency with maize-optimized codons in said coding region of said nucleic acid.
5. The method of claim 1 , wherein said monocotyledonous plant tissue is maize, rice, wheat or barley tissue.
6. The method of claim 1 , wherein said monocotyledonous plant tissue is tissue of sugar cane.
7. The method of claim 1 , wherein said monocotyledonous plant tissue is an Indica variety of rice.
8. The method of claim 1 , further comprising: selecting a multiple shoot culture comprising the transformed cell; growing the multiple shoot culture under conditions that promote shoot elongation to produce at least one transformed shoot; and growing the at least one transformed shoot.
9. A method of transforming sugar cane using a phosphomannose-isomerase (PMI) protein as a selectable marker, said method comprising:
(a) introducing a heterologous nucleic acid comprising a phosphomannose-isomerase (PMI) protein coding region having one or more maize-optimized codons, said PMI coding region comprising at least 500 consecutive nucleotides of SEQ ID NO:2, or said coding region comprising a nucleotide sequence having at least 95% identity to SEQ ID NO:2, into a cell of sugar cane plant tissue to thereby produce a transformed sugar cane cell comprising the nucleic acid, wherein said introducing step is carried out by Agrobacterium -mediated transformation; and
(b) regenerating a transformed plant from the transformed cell under conditions selective for PMI protein expression;
to produce said transformed sugar cane.
10. The method of claim 9 , wherein said sugar cane has a 1-, 2-, or 3-fold greater transformation frequency as compared to transformation with an expression cassette comprising a nucleic acid comprising SEQ ID NO:1.
11. The method of claim 9 , wherein the regenerating step comprises:
selecting a multiple shoot culture comprising a transformed cell; growing the multiple shoot culture under conditions that promote shoot elongation to produce at least one transformed shoot; and growing the at least one transformed shoot.