Notebook

Summary

Establishment of engineered sweet potato is a time-consuming task as the seedling regeneration and the accumulation of starch in the storage roots require a relatively long period of time. The following is a record of the process of our project experiment. For detailed operation procedures and experimental results, please refer to the Protocols and Engineering Success pages.

1 March

  1. Embryogenic calli induction of sweet potato
  2. sgRNA target selection
  3. sgRNA design
  4. sgRNA oligos synthesis

2 April

  • Embryogenic calli proliferation of sweet potato

    Fig. 1 Embryogenic callus culture of sweet potato

  • Construction of the backbone vector

    Fig. 2 Digestion result of psgR-Cas9-At

    Fig. 3 E. Coli transformed with backbone vector of sgRNA (IbGBSSI)

    Fig. 4 PCR product of sgRNA (IbGBSSI)

  • Construction of the plant expression vector

    Fig.5 Digestion result of psgR-Cas9-sgRNA and pCAMBIA1301s

  • Agrobacterium tumefaciens transformation

    Fig. 6 A. tumefaciens transformed with the vector psgR-Cas9-sgRNA(IbGBSSI)-p1301s

    Fig. 7 PCR result of A. tumefaciens transformed with psgR-Cas9-sgRNA(IbGBSSI)-p1301s

  • Agrobacterium-mediated transformation for sweet potato

    Fig. 8 Embryogenic callus co-culture with A. tumefaciens transformants (A) and selection of positively transformed callus with hygromycin (B)

  • Screening and selection of calli mutant

3 May

  • Regeneration and sub-culture of transgenic sweet potato seedlings

  • GUS detection for regenerated seedlings

    Fig. 9 Results of GUS staining

  • PCR detection for regenerated seedlings

    Fig. 10 Detection results of PCRs for gene Hyg and gene Cas9.

  • Transplantation of transgenic seedlings

4 June

  • Literature review

5 July

  • Determination of chlorophyll content in leaves

    Fig. 11 chlorophyll contents of the transgenic lines

6 August

  • Storage roots harvest

    Fig. 12 Phenotypes of the IbGBSSI-knockout lines planted in greenhouse

  • Determination of the expression level of IbGBSSI in storage roots

    Fig. 13 Q-PCR result of the relative expression level of IbGBSSI in root tubers

  • Extraction of total starch in storage roots

  • Starch analysis of transgenic sweet potato storage roots

    • Qualitative detection of the starch components by iodine staining

      Fig. 14 Component detection of total starch by iodine staining

    • Quantitative detection of the starch composition

      Fig. 15 Amylose content (%) of total starch of root tubers