Methods for improving callus formation and regeneration in plants
This disclosure provides methods of improving callus formation in plants. This disclosure further provides genetically engineered plants with improved callus formation.
1. A genetically modified Populus plant, plant cell or plant tissue, wherein the expression of an endogenous suppressor of kinase 1 (SOK1) gene is altered in the Populus plant, plant cell or plant tissue, wherein the alteration comprises inactivation of the endogenous SOK1 gene in the Populus plant, plant cell or plant tissue, resulting in increased callus formation by the Populus plant cell, or increased callus formation in the Populus plant or plant tissue, and wherein the endogenous SOK1 gene encodes an enzyme with at least 98% sequence identity to SEQ ID NO: 23.
2. The genetically modified Populus plant, plant cell or plant tissue of claim 1 , wherein the inactivation of the endogenous SOK1 gene is achieved by introducing a nucleic acid inhibitor of the selected gene to the Populus plant, plant cell or plant tissue.
3. The genetically modified Populus plant, plant cell or plant tissue of claim 2 , wherein the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
4. The genetically modified Populus plant, plant cell or plant tissue of claim 1 , wherein the inactivation of the endogenous SOK1 gene is achieved by genome editing, which is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, ZFNs system and homologous recombination.
5. The genetically modified Populus plant, plant cell or plant tissue of claim 4 , wherein the CRISPR-mediated genome editing comprises introducing into the Populus plant a first nucleic acid encoding a Cas9 nuclease, a second nucleic acid comprising a guide RNA (gRNA), wherein said gRNA is specific to the endogenous SOK1 gene.
6. A method for increasing callus formation, comprising altering in a Populus plant, plant cell or plant tissue the expression of a SOK1 gene or a homolog thereof, wherein the alteration comprises inactivation of the endogenous SOK1 gene in the Populus plant, plant cell or plant tissue, resulting in increased callus formation by the Populus plant cell, or increased callus formation in the Populus plant or plant tissue, and wherein the endogenous SOK1 gene encodes an enzyme with at least 98% sequence identity to SEQ ID NO: 23.
7. The method of claim 6 , wherein the inactivation of the endogenous SOK1 gene is achieved by introducing a nucleic acid inhibitor of the selected gene to the Populus plant, plant cell or plant tissue.
8. The method of claim 7 , wherein the nucleic acid inhibitor is selected from the group consisting of an antisense RNA, a small interfering RNA, an RNAi, a microRNA, an artificial microRNA, and a ribozyme.
9. The method of claim 6 , wherein the inactivation of the endogenous SOK1 gene is achieved by genome editing, which is achieved by a method selected from the group consisting of CRISPR/Cas system, Cre/Lox system, TALEN system, ZFNs system and homologous recombination.
10. The method of claim 9 , wherein the CRISPR-mediated genome editing comprises introducing into the Populus plant a first nucleic acid encoding a Cas9 nuclease, a second nucleic acid comprising a guide RNA (gRNA), wherein said gRNA is specific to the endogenous SOK1.
11. The genetically modified Populus plant, plant cell or plant tissue of claim 1 , wherein said Populus is Populus trichocarpa.
12. The method of claim 6 , wherein said Populus is Populus trichocarpa.