2.8 Agrobacterium-Mediated Transformation of A. thaliana
The ORFs of the CsHSP24.6 were ligated into the entry vector
pDONR207 using the Gateway BP Enzyme mix (Invitrogen, Carlsbad, CA,
USA). The primers used for vector construction are listed in Table S1.
After sequence confirmation, the entry vectors were introduced into the
destination vector pCB2004 using the Gateway LR Clonase enzyme
(Invitrogen, Carlsbad, CA, United States). The pCB2004 vectors carryingCsHSP24.6 were transformed into A. tumefaciensstrain GV3101 through electroporation. The A. tumefacienswere inoculated on solidified LB medium with 50 mg L−1kanamycin and 50 mg L−1 rifampicin at 28 °C for
approximately 48 h. A positive GV3101 colony harboring the vector
pCB2004-CsHSP24.6 was confirmed through PCR. Then, the A.
tumefaciens were cultured in liquid LB medium until the
OD600 of the cell suspension reached 0.6-0.8. TheA. tumefaciens cells were centrifuged at 5000 ×g at 4 °C
for 10 min and used for genetic transformation of A. thaliana by
using the floral-dip method as previously described45.
Seeds harvested from A. tumefaciens –infected plants were
sterilized and plated on an MS basic medium containing 15 μg
mL−1 glufosinate ammonium. Herbicide-resistant
seedlings were transplanted to soil and grown in a greenhouse. TheA. tumefaciens -mediated transformation of CspTAC5 was
performed by using an identical method.