Generation of transgenic plants by Agrobacterium Tumefaciens-mediated transformation
This protocol is extracted from research article:
MaMAPK3-MaICE1-MaPOD P7 pathway, a positive regulator of cold tolerance in banana
BMC Plant Biol, Feb 17, 2021; DOI: 10.1186/s12870-021-02868-z

The full-length cDNA of MaMAPK3 was subcloned into the pMD18-T vector (TaKaRa, Dalian, China) (Fig. S10A). Plasmid pMD18-T containing MaMAPK3 was amplified using primers (GSP1) harboring SpeI and BamH I restriction sites. The PCR product was digested with these enzymes and introduced into the pCAMBIA 1301-GUS vector (Fig. S10B) to generate the fusion construct 1301-MaMAPK3-GUS under the control of the Ubipromoter. The double-stranded RNA interference (dsRNA) construct (Fig. S10C) was generated via a PCR-mediated method using the amplification products from a unique N-terminal region (300 bp) spanning a portion of the 5′-untranslated region and adjacent coding region of the MaMAPK3 gene. The sense strand was then amplified using a primer combination harboring BamH I and Hind III restriction sites on the opposed ends of the product, whereas the antisense strand was amplified using a primer combination harboring Pst I and Mul I restriction sites on the opposite ends of the product. These two products were introduced into pYL-RNAi under the control of the Ubi promoter. The newly constructed 1301-MaMAPK3-GUS plant expression vector and RNAi vector were introduced into A. tumefaciens strain EHA105 by heat shock [41]. The over-expression vector was adopted for the transformation of ‘Dajiao’ suspension cultured cells (ECSs), and the RNAi vector was used for the transformation of ‘Dajiao’ ECSs as previously described [41, 67]. Hygromycin-resistant plants were selected and identified by PCR (HPT and MaICE1) using two pairs of primers (GSP5 and GSP6, Table S5). Only those yielding the expected PCR fragments by both primers were regarded as positive. Moreover, the expression of MaMAPK3 at the mRNA level was examined by qRT-PCR (primer set GSP6, Table S5). The MaACT1 gene (primer set GSP7, Table S5) was selected as a housekeeping gene. Positive banana plants were multiplied vegetatively using meristems of in vitro plantlets. The rooted plantlets were hardened in the greenhouse and used for further analyses.

Specific primers (GSP2) containing Spe I or BamH I restriction sites were used to amplify MaICE1 cDNA. The PCR product was digested with SpeI and BamHI, and then ligated into binary vector pOx driven by the Zea mays L. polyubiquitin promoter. The constructed binary vector was denoted as pOx-MaICE1. The newly constructed pOx-MaICE1 plant expression vector was introduced into A. tumefaciens strain EHA105 by heat shock [37]. The over-expression vector was adopted for the transformation of Cavendish banana ECSs as previously described (Dou et al., 2016; Hu et al., 2013). Hygromycin-resistant plants were selected and identified by PCR using two pairs of primers (GSP5and GSP8). Only those yielding the expected PCR fragments by both primers were regarded as positive. Moreover, the expression of MaICE1 at the mRNA level was examined by qRT-PCR (primer set GSP9, Table S5). The MaACT1 gene (primer set GSP7) was selected as a housekeeping gene. Positive banana plants were multiplied vegetatively using meristems of in vitro plantlets. The rooted plantlets were hardened in the greenhouse and used for further analyses.

The RNAi construct for MaMPAK3 gene suppression was amplified using the primers GSP10 and GSP11, then the PCR products were cloned into pYLRNAi, which was kindly provided by Dr. Yao-Guang Liu from College of Life Sciences, South China Agricultural University, China. The following procedure was carried out as previously described.

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