Mutagenesis method using polyethylene glycol mediated introduction of mutagenic nucleobases into plant protoplasts

Patent metadata

Field	Value
Publication number	US-2016201071-A1
Application number	US-201615084935-A
Country	US
Kind code	A1
Filing date	Mar 30, 2016
Priority date	Dec 21, 2007
Publication date	Jul 14, 2016
Grant date	—

Abstract

Method for targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, comprising combining the duplex acceptor DNA sequence with a donor mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, wherein the method further comprises a step of introducing the donor mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation and the use of PEG protoplast transformation for enhancing the rate of targeted mutagenesis.

First claim

1 - 13 . (canceled)

14 . A method for the production of a plant progeny, wherein the method comprises:
performing targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, wherein the acceptor DNA is from genomic DNA, comprising combining the duplex acceptor DNA sequence with a donor single-stranded (ss) mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor ss mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, the method further comprising a step of introducing the ss mutagenic nucleobase into the plant cell protoplasts using polyethylene glycol (PEG)-mediated transformation; and regenerating a plant progeny of said plant cell protoplast.

15 . The method according to claim 14 , wherein the plant progeny is a plant cell, plant, plant callus, or shoot.

16 . The method according to claim 14 , wherein the donor ss mutagenic nucleobase comprises at least one mismatch with respect to the first DNA sequence of the duplex acceptor DNA sequence to be altered,

17 . The method according to claim 14 , wherein the ss mutagenic nucleobase has a length of between 10-60 nucleotides.

18 . The method according to claim 14 , wherein the ss mutagenic nucleobase comprises Locked Nucleic Acid (LNA) substitutions that are at least one nucleotide removed from the mismatch.

19 . The method according to claim 18 , wherein the ss mutagenic nucleobase comprises two LNAs located at least one nucleotide removed from either side of the mismatch.

20 . The method according to claim 18 , wherein the LNA substitutions are at least 3 nucleotides removed from the 5′ and 3′ ends of the ss mutagenic nucleobase.

21 . The method according to claim 18 , wherein the LNA substitutions are at least 4 nucleotides removed from the 5′ and 3′ ends of the ss mutagenic nucleobase.

22 . The method according to claim 18 , wherein the LNA substitutions are at least 5 nucleotides removed from the 5′ and 3′ ends of the ss mutagenic nucleobase.

23 . The method according to claim 14 , wherein the as mutagenic nucleobase comprises propyne substitutions,

24 . The method according to claim 14 , wherein the as mutagenic nucleobase is conjugated to a nuclear localization signal,

25 . The method according to claim 14 , wherein the donor as mutagenic nucleobase comprises one mismatch with respect to the duplex acceptor DNA sequence to be altered.

Assignees

• Keygene Nv

Inventors

• Bundock Paul
• De Both Michiel Theodoor Jan
• Lhuissier Frank

Classifications

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N15/8206 Primary

  by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title