US 12,185,681 B2
Methods for preparing mutant plants
Birgitte Skadhauge, Copenhagen V (DK); Soren Knudsen, Copenhagen V (DK); Gustav Hambraeus, Copenhagen V (DK); Toni Wendt, Copenhagen V (DK); Magnus Rasmussen, Copenhagen V (DK); Jeppe Thulin Østerberg, Copenhagen V (DK); and Ross Fennessy, Copenhagen V (DK)
Assigned to Carlsberg A/S, Copenhagen V (DK)
Filed by Carlsberg A/S, Copenhagen V (DK)
Filed on Apr. 7, 2022, as Appl. No. 17/715,723.
Application 17/715,723 is a continuation in part of application No. PCT/EP2020/078321, filed on Oct. 8, 2020.
Prior Publication US 2022/0312707 A1, Oct. 6, 2022
Int. Cl. A01H 1/04 (2006.01); A01H 1/02 (2006.01); A01H 6/46 (2018.01); C12N 15/10 (2006.01)
CPC A01H 1/04 (2013.01) [A01H 1/026 (2021.01); A01H 6/46 (2018.05); C12N 15/1034 (2013.01)] 18 Claims
 
1. A method for identifying a mutant plant of a predefined species carrying one or more mutation(s) in nucleotide(s) of interest [NOI(s)], in a predetermined target sequence, said method comprising:
a) Providing Np regenerative parts of a parent plant of said species, wherein Np is the Number of regenerative parts to be mutagenized, and
wherein Np is an integer of at least 5,000; and wherein Np further is in the range of 0.7×ONp to 1.3×ONp,
wherein ONp is the Optimal Number of regenerative parts to be mutagenized and ONp=((OLS×100)/Hr)/GECN
where Hr is the expected average harvest rate of said plant species after
mutagenesis in %; and OLS is optimal library size, wherein OLS=log (1−PS/100)/log (1−(1−(1−(Mf×n)
where PS is the probability of success in %, Mf is the mutation frequency, and
n is the number of mutations screened for;
b) subjecting said regenerative parts to a step of random mutagenesis, leading to a mutagenesis frequency of Mf, thereby generating a pool of regenerative parts of generation M0 representing a plurality of genotypes;
c) growing said regenerative parts of generation M0 into mature plants of generation M1 and obtaining regenerative parts of generation M1 from said mature plants;
d) optionally repeating the previous step X times to obtain plants of generation M (1+X) comprising regenerative parts of generation M (1+X);
e) dividing regenerative parts of a plurality of generation M1 or M (1+X) plants into sub-pools, each sub-pool comprising regenerative parts representing a plurality of genotypes, wherein all regenerative parts from one given mature plant are placed in the same sub-pool;
f) preparing DNA samples from each sub-pool;
g) identifying sub-pool(s) comprising DNA comprising the mutation(s) using sensitive detection means; and
h) identifying regenerative parts within said sub-pool comprising said mutation, thereby identifying said mutant plant,
wherein a low rate of mutagenesis in diploid plants is that an average at least 99.8% of all genes are free of non-synonymous mutations and in plants of higher ploidy that an average of at least 98.0% of all genes are free of non-synonymous mutations in a regenerative part of generation M1.