Abstract
Genotype Nisqually-1 is the first model woody plant with an available well-annotated genome. Nevertheless, a simple and rapid transformation of Nisqually-1 remains to be established. Here, we developed a novel shoot regeneration method for Nisqually-1 using leaf petiole and stem segment explants. Numerous shoots formed in the incision of explants within two weeks. The optimized shoot regeneration medium (SRM) contained 0.03 mg l−1 6-benzylaminopurine, 0.02 mg l−1 indole-3-butyric acid and 0.0008 mg l−1 thidiazuron. Based on this, Agrobacterium-mediated genetic transformation of stem explants was examined using the vector pBI121 that contains the β-glucuronidase (GUS) as a reporter gene. Consequently, factors affecting transformation frequency of GUS-positive shoots were optimized as follows: Agrobacteria cell suspension with an OD600 of 0.4, 20 min infection time, 2 days of co-cultivation duration and the addition of 80 µM acetosyringone into Agrobacteria infective suspension and co-cultivation SRM. Using this optimized method, transgenic plantlets of Nisqually-1 – with an average transformation frequency of 26.7% – were obtained with 2 months. Southern blot and GUS activity staining confirmed the integration of the foreign GUS gene into Nisqually-1. This novel transformation system for Nisqually-1 was rapid, efficient, and simple to operate and will improve more genetic applications in this model tree.
Highlights
Forests provide humanity with many benefits including clean air, lumber, and fuels, etc
Transgenic calli were induced from stem segment explants by naphthaleneacetic acid (NAA) and 2iP plant growth regulators (PGRs) and shoots were regenerated from transgenic calli undergoing two types of MS media supplemented with different concentration of thidiazuron (TDZ)
Five groups of PGR combinations among TDZ, 6-BA, NAA and IBA were tested for their effects on shoot regeneration (Supplementary Table S1)
Summary
Forests provide humanity with many benefits including clean air, lumber, and fuels, etc. Forest trees evolve many specific traits such as large wood formation, perennial growth and adaptability of seasonality[1] Many of these features distinguish the trees from herbaceous plants and present challenges to the study of the genetic, cellular and molecular mechanisms that underlie the unique tree biology[2, 3]. The lack of studies in Nisqually-1 could be attributed to difficult or time-consuming genetic transformation for Nisqually-1 unlike many Populus hybrids Such recalcitrance of this model tree has been an impediment to the utilization of its well-annotated genomic resource. A short-period transformation method simple to operate would be valuable for facilitating molecular and genetic studies on the unique traits of this first model tree
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
