Plants modified with mini-chromosomes
View Patent ↗The invention is generally related to methods of generating plants transformed with novel autonomous mini-chromosomes. Mini-chromosomes with novel compositions and structures are used to transform plants cells which are in turn used to generate the plant. Methods for generating the plant include methods for delivering the mini-chromosome into plant cell to transform the cell, methods for selecting the transformed cell, and methods for isolating plants transformed with the mini-chromosome. Plants generated in the present invention contain novel genes introduced into their genome by integration into existing chromosomes.
1. A method of making a mini-chromosome, comprising
identifying a centromere nucleotide sequence in a genomic DNA library using a multiplicity of diverse probes, and
constructing a mini-chromosome comprising the centromere nucleotide sequence,
wherein the centromere nucleotide sequence is derived from a crop plant centromere; wherein the centromere comprises at least five copies of a repeated nucleotide sequence that is about 75 base pairs to about 210 base pairs, and
wherein the mini-chromosome has a transmission efficiency during mitotic division of at least 90%.
2. The method of claim 1 wherein the identifying further comprises
determining hybridization scores for hybridization of the multiplicity of diverse probes to genomic clones within the genomic DNA library,
determining a classification for genomic clones within the genomic DNA library according to the hybridization scores for at least two of the diverse probes, and
selecting one or more genomic clones within one or more classifications for constructing the mini-chromosome.
3. The method of claim 1 , wherein at least one probe hybridizes to ribosomal DNA, and a classification comprises a low hybridization score for hybridization to said probe.
4. A method of making an adchromosomal plant, comprising
co-delivering to a plant cell a mini-chromosome and a nucleic acid encoding a growth inducing gene, wherein said nucleic acid is not part of the mini-chromosome and wherein the mini-chromosome comprises a centromeric nucleic acid insert derived from a crop plant centromere and has a transmission efficiency during mitotic division of at least 90%, and wherein the centromere comprises at least five copies of a repeated nucleotide sequence that is about 75 base pairs to about 210 base pairs, and
regenerating a plant.
5. The method of claim 4 , wherein the growth inducing gene is selected from the group consisting of encoding plant growth regulator genes, organogenesis-promoting, embryogenesis-promoting or regeneration-promoting gene.
6. The method of claim 1 wherein at least three different probes are used.
7. The method of claim 6 wherein at least four different probes are used.
8. The method of claim 7 wherein at least five different probes are used.
9. The method of claim 8 wherein at least ten different probes are used.
10. The method of claim 1 wherein at least one probe hybridizes to the centromere region of a chromosome.
11. The method of claim 1 wherein at least one probe hybridizes to satellite repeat DNA.
12. The method of claim 1 wherein at least one probe hybridizes to retroelement DNA.
13. The method of claim 1 wherein at least one probe hybridizes to portions of genomic DNA that are heavily methylated.
14. The method of claim 1 wherein at least one probe hybridizes to arrays of tandem repeats in genomic DNA.
15. The method of claim 1 wherein at least one probe hybridizes to mitochondrial DNA, and a classification comprises a low hybridization score for hybridization to said probe.
16. The method of claim 1 wherein at least one probe hybridizes to chloroplast DNA, and a classification comprises a low hybridization score for hybridization to said probe.
17. The method of claim 1 wherein at least one probe hybridizes to telomere DNA.
18. The method of claim 1 wherein at least one probe hybridizes to a pseudogene.
19. The method of claim 4 wherein the nucleic acid encoding a growth inducing gene is not expressed or not present in the regenerated plant.
20. The method of claim 4 wherein the nucleic acid encoding a growth inducing gene is expressed during regenerating the plant.