Selective ablation of diploid embryos
View Patent ↗Methods of selecting haploid embryos are disclosed. Methods of producing haploid embryos and non-viable diploid embryos on a plant are provided. Methods for selecting haploid embryos produced from haploid inducer maize lines are provided. Methods for producing improved maize haploid inducer lines are disclosed. Maize haploid inducer lines comprising transgenes causing ablated or abnormal diploid embryos are disclosed.
1. A method of selecting for haploid embryos comprising:
a) pollinating a first maize plant with the pollen from a maize haploid inducer line to produce embryos wherein said maize haploid inducer line comprises:
i) an embryo expressed lethal polynucleotide operably linked to an embryo-preferred promoter, wherein said embryo-preferred promoter is modified with tet operator sequences,
ii) an alpha-amylase polynucleotide that inhibits pollen transmission operably linked to a pollen-specific promoter, and
iii) an embryo expressed inhibitor polynucleotide encoding a tet repressor that prevents the lethality of said embryo expressed lethal polynucleotide;
wherein said embryo expressed inhibitor polynucleotide inhibits transcription of said embryo expressed lethal polynucleotide and further wherein said alpha-amylase polynucleotide and said embryo expressed inhibitor polynucleotide are linked so that they will segregate together;
b) producing haploid embryos and non-viable diploid embryos; and
c) selecting haploid maize embryos.
2. A method of developing a transformed maize haploid inducer line comprising the following steps:
a) obtaining a transformed cell by transforming a cell from a maize inducer line with a first and second expression cassette wherein said first expression cassette comprises an embryo expressed lethal polynucleotide operably linked to an embryo-preferred promoter, wherein said embryo-preferred promoter is modified with tet operator sequences, and wherein said second expression cassette comprises an alpha-amylase polynucleotide that inhibits pollen transmission operably linked to a pollen-specific promoter and an embryo expressed inhibitor polynucleotide encoding a tet repressor that prevents the lethality of said embryo expressed lethal polynucleotide by binding to the embryo-preferred promoter operably linked to the embryo expressed lethal polynucleotide and inhibiting transcription of said embryo expressed lethal polynucleotide;
b) regenerating a transformed plant from said transformed cell;
c) selfing said transformed plant to obtain a transformed maize haploid inducer line.
3. A transgenic maize haploid inducer line comprising a first and second expression cassette wherein said first expression cassette comprises an embryo expressed lethal a-polynucleotide operably linked to an embryo-preferred promoter, wherein said embryo-preferred promoter is modified with tet operator sequences, and wherein said second cassette comprises an alpha-amylase polynucleotide that inhibits pollen transmission operably linked to a pollen-specific promoter and an embryo expressed inhibitor polynucleotide encoding a tet repressor that inhibits the lethality of said polynucleotide that is lethal to embryos by binding to the embryo-preferred promoter operably linked to the embryo expressed lethal polynucleotide and inhibiting transcription of said embryo expressed lethal polynucleotide.
4. The transgenic maize haploid inducer line of claim 3 wherein said embryo preferred promoter is a lec1 promoter.
5. The transgenic maize haploid inducer line of claim 3 wherein said pollen-specific promoter is a PG47 promoter.