Haploid maize transformation
Disclosed are methods for transformation of an androgenic-derived, haploid cell line with a site-specific nuclease. In some embodiments, the androgenic-derived, haploid cell line is a maize microspore-derived plant tissue culture. In addition, the disclosure provides a method for modifying, e.g., by mutating or targeting and integrating donor DNA into, a specific locus of a haploid or dihaploid tissue genome. The disclosure further provides methods for regenerating a whole plant from the haploid or dihaploid tissue that contains either the mutation at a specific genomic locus or a donor DNA integrated within a specific genomic locus may be obtained from the subject disclosure.
1 . A method for modifying a maize genome by site-specific nuclease targeted mutagenesis, by a zinc finger nuclease, the method comprising:
(a) crossing an elite maize line with a different maize line having high microspore culture response to produce a hybrid maize line having elite performance characteristics and high microspore culture response;
(b) producing a microspore derived, transformation competent callus tissue from the progeny of the hybrid maize derived from crossing an elite maize line with a different maize line having high microspore culture response;
(c) isolating a maize microspore-derived, transformation-competent haploid callus comprising a paternal haploid tissue genome from the tissue of the progeny of the hybrid maize line, wherein the callus is assayed and determined to be haploid;
(d) delivering a polynucleotide encoding a site-specific nuclease to the transformation-competent haploid callus resulting in the double strand cleavage of the haploid genome of the callus via particle bombardment; and,
(e) confirming that the haploid callus genome is modified by the encoded site-specific nuclease, wherein the site-specific nuclease's targeting specificity has been engineered to target the modified genomic sequence with at least an 8-fold increased level of targeted mutagenesis as compared to non-transformed callus.
2 . The method of claim 1 , wherein the method further comprises:
(f) delivering a donor polynucleotide and stably integrating the donor polynucleotide in the modified haploid callus genome.
3 . The method of claim 2 , wherein the donor polynucleotide comprises one or two domains and each domain is at least 85% identical to a sequence in the genomic DNA target region of the haploid callus genome.
4 . The method of claim 1 , wherein the method comprises confirming that the haploid callus genome is modified by performing a PCR based assay, Southern blot assay, Northern blot assay, protein expression assay, Western blot assay, ELISA assay, or Next Generation Sequencing assay.
5 . The method of claim 1 , wherein the method further comprises:
(g) treating the haploid callus comprising the modified haploid callus genome with a chromosome doubling agent;
(h) producing dihaploid maize tissue comprising a modified dihaploid maize genome; and,
(i) regenerating the dihaploid maize tissue into a dihaploid maize plant comprising a homozygous modified dihaploid maize genome.
6 . The method of claim 2 , wherein the method further comprises:
(g) treating the haploid callus comprising the modified haploid tissue genome with a chromosome doubling agent;
(h) producing dihaploid maize tissue comprising a modified dihaploid maize genome; and,
(i) regenerating the dihaploid maize tissue into a dihaploid maize plant comprising a homozygous modified dihaploid maize genome.
7 . The method of claim 2 , wherein the method further comprises:
(j) stably integrating the donor polynucleotide into a target region of the haploid tissue genome; and,
(k) confirming that the donor polynucleotide integrated into the target region of the haploid tissue genome.
8 . The method of claim 7 , wherein the integrated donor polynucleotide is expressed within the maize haploid tissue.
9 . The method of claim 7 , wherein the integrated donor polynucleotide imparts an agronomic trait.
10 . The method of claim 1 , wherein the method further comprises:
(l) expressing the site-specific nuclease and introducing a mutation into the haploid genome of maize; and,
(m) confirming that the haploid maize genome comprises a mutation.
11 . The method of claim 2 , wherein the method further comprises:
(n) treating the haploid callus comprising the stably integrated donor polynucleotide in the modified haploid callus genome with a chromosome doubling agent;
(o) producing dihaploid maize tissue comprising stably integrated donor polynucleotide in its dihaploid maize genome; and,
(p) regenerating the dihaploid maize tissue into a dihaploid maize plant that is homozygous for the stably integrated donor polynucleotide.
12 . The method of claim 11 , further comprising:
(q) crossing the dihaploid maize plant with plants of a different parent maize line to produce F1 progeny plants;
(r) selecting one or more F1 progeny plants having the genome modification introduced by the encoded site-specific nuclease to produce one or more maize progeny plants comprising the modification introduced by the encoded site-specific nuclease; and,
(s) optionally, (i) back-crossing F1 progeny plants with the dihaploid maize plant of claim 11 or the different parent maize line to produce backcross progeny plants, (ii) selecting for backcross progeny plants that comprise the genome modification introduced by the encoded site-specific nuclease, and (iii) further optionally repeating steps (i) and (ii) to produce one or more maize progeny plants comprising the modification introduced by the encoded site-specific nuclease.