Methods for Introducing Into a Plant a Polynucleotide of Interest

Abstract

The present invention provides methods and compositions which deliver Agrobacterium via microinjection directly into the embryo sac. At the time of injection, the embryo sac can comprise an egg cell, or alternatively, the embryo sac can be fertilized and comprise either a zygote or an embryo. Once inside the embryo sac, the Agrobacterium harboring a T-DNA having a polynucleotide of interest can express of the polynucleotide of interest in the plant. Further, the Agrobacterium can transfer the T-DNA having the polynucleotide of interest to the plant nucleus to produce a transformed plant. The polynucleotide of interest may be stably integrated into the genome of the egg cell, zygote, embryo, or endosperm, and any tissue, plant part, and/or plant generated therefrom.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (Left) shows an immature kernel section (slab) from a 1-day post-pollinated immature kernel containing an embryo sac that was microinjected with Agrobacterium. The T-DNA within the injected Agrobacterium comprises Actin promoter::GUS (PHP22462). Following injection of the Agrobacterium, expression of GUS gene in the embryo sac (stained structure at the bottom of the section, arrow) indicates that the functional Agrobacterium has been delivered and the GUS gene on the T-DNA was expressed. Bar=0.5 mm.

FIG. 1 B (Right) shows an embryo/endosperm structure derived from a 1-day post-pollinated microinjected nucellar slab after 21 days in vitro culture. The nucellar slab, isolated from a transgenic plant stably transformed with PHP10006 (Ubi::FRT1::GFP::35S::MOPAT::FRT1::GUS::FRT5), was injected with Agrobacterium containing PHP17797 (RB::Ubi-FRT1::FLPm::35S::Bar::FRT5::LB). The FLP enzyme expressed from PHP17797 mediated excision of the DNA between the FRT1 sites in PHP10006 in the injected slab, thereby creating an operable linkage of the promoterless GUS gene to the Ubi promoter, resulting in GUS expression. The blue spot (arrow) shows the GUS expression in the embryo area, demonstrating T-DNA transfer and gene expression from the injected Agrobacterium. Bar=1 mm.

Claims

1. A method for expressing a polynucleotide of interest in a plant comprising a) providing an embryo sac from the plant;b) injecting into the embryo sac a composition comprising an effective concentration of an Agrobacterium comprising a T-DNA comprising the polynucleotide of interest operably linked to a promoter active in the plant, wherein said Agrobacterium is capable of T-DNA transfer into a plant cell.

2. The method of claim 1, further comprising recovering from the embryo sac a transgenic plant having the polynucleotide of interest stably integrated into its genome.

3. The method of claim 1, wherein the embryo sac comprises a fertilized embryo sac.

4. The method of claim 3, wherein the fertilized embryo sac comprises an embryo or a zygote.

5. The method of claim 1, wherein the plant is a monocot or a dicot.

6. The method of claim 5, wherein said plant is selected from the group consisting of maize, barley, millet, wheat, rice, soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, and cotton.

7. The method of claim 1, wherein the Agrobacterium comprises Agrobacterium tumefaciens.

8. A method of introducing an Agrobacterium into a plant comprising a) providing an embryo sac from the plant;b) injecting into the embryo sac a composition comprising an effective concentration of an Agrobacterium, wherein the Agrobacterium is capable of T-DNA transfer into a plant cell.

9. The method of claim 8, wherein the Agrobacterium comprises a T-DNA comprising a polynucleotide of interest operably linked to a promoter active in the plant.

10. The method of claim 9, further comprising recovering from the embryo sac a transgenic plant having the polynucleotide of interest stably integrated into its genome.

11. The method of claim 8, wherein the embryo sac comprises a fertilized embryo sac.

12. The method of claim 11, wherein the fertilized embryo sac comprises an embryo or a zygote.

13. The method of claim 8, wherein the plant is a monocot or a dicot.

14. The method of claim 13 wherein the plant is selected from the group consisting of maize, barley, millet, wheat, rice, soybean, canola, alfalfa, sunflower, safflower, tobacco, Arabidopsis, and cotton.

15. The method of claim 8 wherein the Agrobacterium comprises Agrobacterium tumefaciens.

16. A method to identify a fertilized plant embryo sac comprising a) providing pollen from a first plant comprising a polynucleotide encoding a visual marker operably linked to a promoter, wherein promoter is active in the pollen or in an embryo sac;b) providing a population of unfertilized seed, each seed comprising an embryo sac;c) contacting the seed with the pollen; andd) identifying the fertilized embryo sac expressing said visual marker.

17. The method of claim 16, wherein the visual marker is expressed in a central cell of the embryo sac, a zygote of the embryo sac, the pollen, or a pollen tube.

18. The method of claim 16, wherein the visual marker is a fluorescent protein.

19. The method of claim 16, wherein the visual marker is encoded by a polynucleotide having maize preferred codons.

20. The method of claim 16, wherein the fertilized plant embryo is from a monocot.