Method for rapid genome modification in recalcitrant plants

A method of using a regeneration booster or a combination of regeneration boosters, in a method for targeted plant genome modification, preferably in a method according to any of the preceding claims, to

• (a) promote plant cell proliferation and/or assist in the targeted modification and/or provide a positive selection for at least one transiently transformed cell for genome modification and/or for regeneration into at least one modified plant, and/or
• (b) suppress plant cell differentiation if the cell is stably transformed and provide a negative selection for at least one stably transformed cell for regeneration into one transgenic plant, and/or
• (c) provide a dual selection according to (a) and (b) for at least one transformed cell,
• wherein the regeneration booster or combination of regeneration boosters comprise:
• (i) at least one RBP, wherein the at least one RBP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
• 32, 33, 34, 35, 36, 37, 38 and 39, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a catalytically active fragment thereof, or wherein the at least one RBP is encoded by a sequence selected from the group consisting of SEQ ID NOs:
• 20, 21, 22, 23, 24, 25, 26 and 27, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a cognate codon-optimized sequence, and/or
• (ii) at least one PLT or RKD4, wherein the at least one PLT or RKD4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
• 28, 29, 30 and 31, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a catalytically active fragment thereof, or wherein the at least one PLT or RKD4 is encoded by a sequence selected from the group consisting of SEQ ID NOs:
• 16, 17, 18 and 19, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a cognate codon-optimized sequence, and/or
• (iii) at least one further regeneration booster, wherein the further regeneration booster, or the sequence encoding the same is selected from BBM, WUS, WOX, GRF, LEC, or a variant thereof or wherein the further regeneration booster selected from RBP1 to RBP8, wherein the further regeneration booster is different from the first regeneration booster.

A method for regenerating a plant tissue, organ or plant from at least one plant cell- or a single plant cell, wherein the method comprises:

• (i) introducing into the at least one plant cell or the single plant cell at least one regeneration booster, or a sequence encoding the same, wherein the at least one regeneration booster is transiently present, transiently active or transiently expressed in the plant cell, and
• (ii) regenerating a plant tissue, organ or plant from at least one plant cell or the single plant cell, wherein the at least one regeneration booster comprises:
• (a) at least one RBP, wherein the at least one RBP comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
• 32, 33, 34, 35, 36, 37, 38 and 39, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a catalytically active fragment thereof, or wherein the at least one RBP is encoded by a sequence selected from the group consisting of SEQ ID NOs:
• 20, 21, 22, 23, 24, 25, 26 and 27, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a cognate codon-optimized sequence, and/or
• (b) at least one PLT or RKD4, wherein the at least one PLT or RKD4 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
• 28, 29, 30 and 31, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a catalytically active fragment thereof, or wherein the at least one PLT or RKD4 is encoded by a sequence selected from the group consisting of SEQ ID NOs:
• 16, 17, 18 and 19, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a cognate codon-optimized sequence, and/or
• (c) at least one further regeneration booster, wherein the further regeneration booster, or the sequence encoding the same is selected from BBM, WUS, WOX, GRF, LEC, or a variant thereof or wherein the further regeneration booster selected from RBP1 to RBP8, wherein the further regeneration booster is different from the first regeneration booster.