Methods for isolating cells without the use of transgenic marker sequences

A method for isolating at least one modified plant cell or at least one modified plant tissue, organ, or whole plant comprising the at least one modified plant cell, without stably integrating a transgenic selectable marker sequence, the method comprising:

• a. introducing at least one first targeted codon deletion modification into a first plant genomic target site of at least one plant cell to be modified using at least one first site-specific effector, comprising a nuclease, a recombinase, or a DNA modification reagent, wherein the at least one targeted codon deletion modification causes expression of at least one phenotypically selectable trait;
• b. introducing at least one second targeted modification into a second plant genomic target site of the at least one plant cell to be modified, wherein the at least one second targeted modification is introduced using at least one second site-specific effector to create the at least one second targeted modification at the second plant genomic target site, wherein the at least one second targeted modification is introduced simultaneously or subsequently to the introduction of the at least one first targeted base modification into the same at least one plant cell to be modified, or into at least one progeny cell, tissue, organ, or plant thereof comprising the at least one first targeted modification to obtain at least one modified plant cell;
• and
• c. isolating at least one modified plant cell, tissue, organ, or whole plant, or isolating at least one progeny cell, tissue, organ, or plant thereof by selecting
• i. for the at least one phenotypically selectable trait caused by the at least one first targeted codon deletion modification at the first plant genomic target site, and optionally by further selecting
• ii. for the at least one second targeted modification in the second plant genomic target site,
• d. optionally:
• crossing at least one modified plant or plant material comprising the at least one first and the at least one second targeted modification with a further plant or plant material of interest to segregate the resulting progeny plants or plant material to achieve a genotype of interest, optionally wherein the genotype of interest does not comprise the at least one first targeted modification.

A method of generating a genetically modified plant by genome editing, the method comprising the steps of:

• a) providing cells or tissues of the plant to be genetically modified;
• b) providing a first genome editing system and a second genome editing system, wherein the first genome editing system can target and modify a plant selectable marker gene, and the second genome modification system can target and modify a gene of interest in the plant;
• c) co-transforming the cells or tissues with the first and second genome editing systems;
• d) regeneration plants from said transformed cells or tissues, preferably without selection pressure;
• e) selecting plants in which the selectable marker gene has been modified from the plants regenerated in step d);
• and
• f) identifying a plant whose target gene is modified from the plants selected in step e).