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cloning_trafo_protocol.md 1.48 KiB

Transformation of barley plants

Cloning of transformation vectors:

  • cloning of transformation vectors was performed according to the protocol by Kumar et al. (2018)
  • sgRNAs were cloned into recipient vector pMGE599
  • two sgRNA combinations:
approach covered CDS sequence (from start codon) sgRNA sequence (incl. PAM, 5'-3') construct name
1 7-26, 97-116 GGCGGTGGCGCAGCCGCGGATGG, GAGATCTATACCTACGAGGCCGG pMGE599/7-97
2 1182-1201, 1222-1241 GGTCAGCTACCCAGCCTGACTGG, TAATAAACTGCAGATTCTCA GGG pMGE599/182-1222

Transformation:

  • GP-fast plants were cultivated in long-day conditions (16h day / 8 night) in control temperature (20°C / 16°C)
  • immature embryos were used for transformation according to Hensel et al. (2009)
  • DNA was extracted from regenerated plants using QIAGEN_DNeasy-Plant-Mini protocol
  • successful insertion of the transformation vector into the genome was tested by PCR using the primers Hyg-156 (5'-ACGCACAATCCCACTATCC-3') and Hyg-047 (5'-GTGTCGTCCATCACAGTTTG-3')

References:

Hensel G, Kastner C, Oleszczuk S, Riechen J, Kumlehn J (2009) Agrobacterium-mediated gene transfer to cereal crop plants: Current protocols for barley, wheat, triticale, and maize. International Journal of Plant Genomics, 2009: 835608.

Kumar N, Galli M, Ordon J, Stuttmann J, Kogel K-H, Imani J (2018) Further analysis of barley MORC1 using a highly efficient RNA-guided Cas9 gene-editing system. Plant Biotechnology Journal, 16(11): 1892–1903.