Methods and compositions for enhanced plant cell transformation
View Patent ↗Methods and compositions to increase Agrobacterium transformation efficiency (frequencies) in both dicot and monocot host plants include adding histones to the host plant at most transiently, and using histones and L-cysteine at certain stages in monocot transformation.
1. A method for increasing Agrobacterium transformation efficiency in a host plant, said method comprising:
a. increasing histone levels in the host plant compared to normal levels of histone in the host plant by expressing an exogenous copy of a plant histone gene; and
b. transforming the host plant with Agrobacterium;
wherein the transformation efficiency of the host plant expressing the exogenous plant histone gene is increased compared to that of a plant not comprising the exogenous plant histone gene.
2. The method of claim 1 , wherein the histone is an H2A histone.
3. The method of claim 2 wherein the H2A histone is encoded by Arabidopsis RAT5.
4. The method of claim 1 wherein transformation frequencies are measured by the number of tumors produced in the host plant.
5. The method of claim 2 , wherein the H2A histone is H2A-1.
6. A method of increasing Agrobacterium transformation efficiency in a host plant, the method comprising:
(a) introducing at least one copy of a polynucleotide sequence encoding a plant histone protein to the host plant;
(b) selecting a host plant expressing the polynucleotide sequence encoding a plant histone protein; and
(c) transforming the host plant expressing the polynucleotide sequence encoding a plant histone protein with a DNA molecule of interest using Agrobacterium;
wherein the transformation efficiency of the host plant expressing the polynucleotide sequence encoding the plant histone protein is increased compared to that of a plant not transformed with the polynucleotide encoding the plant histone protein.
7. The method of claim 6 , wherein the host plant is a monocot plant.
8. The method of claim 7 , wherein the monocot plant is maize.
9. The method of claim 6 , wherein the polynucleotide sequence encoding a plant histone protein is a member of an H2A gene family of Arabidopsis.
10. The method of claim 9 , wherein the member of the H2A gene family of Arabidopsis is RAT5.
11. The method of claim 7 further comprising adding L-cysteine at a concentration between about 100 mg/L and about 400 mg/L to media used in cultivating the host plant, wherein the host plant is monocot.
12. A method of increasing Agrobacterium transformation efficiency in a monocot host plant, the method comprising:
(a) introducing a nucleic acid sequence encoding a plant histone H2A into a host plant;
(b) selecting a host plant material expressing the plant histone H2A protein;
(c) infecting the host plant material with a DNA molecule of interest by infection with an Agrobacterium strain;
(d) providing at least one antioxidant in a cocultivation medium, wherein the antioxidant is L-cysteine at a concentration between about 100 mg/L and about 400 mg/L of cocultivation media; and
(e) selecting the infected material for transformants expressing the DNA molecule of interest;
wherein the transformation efficiency of the monocot host plant expressing the nucleic acid sequence encoding the plant histone H2A protein is increased compared to that of a plant not transformed with the nucleic acid encoding the plant histone H2A protein.
13. The method of claim 12 , wherein the monocot plant is maize.
14. The method of claim 12 , wherein the infecting of the host plant in the cocultivation medium is for about 3 days.
15. The method of claim 12 wherein the host plant material is an embryo.