Abstract
Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.
Highlights
Bread wheat (Triticum aestivum L.) is one of the major economically important crops worldwide that provides around one fifth of the calories to human population (Wang et al, 2014a)
Selection of cell penetrating peptides (CPPs) for In-frame Fusion with mCherry Protein The mCherry protein was chosen as a cargo due to its stability, fast maturation time, high level of fluorescence, and low auto fluorescence level of microspores in the mCherry spectrum range
Four distinct CPPs were picked up for microspore transfection experiments based on their physicalchemical properties (Table 1)
Summary
Bread wheat (Triticum aestivum L.) is one of the major economically important crops worldwide that provides around one fifth of the calories to human population (Wang et al, 2014a). The creation of necessary genetic diversity, introduction of the new traits and the generation of improved cultivars for wheat involves predominantly classical breading. The last, in turn, relies on the techniques which can generate a large number of haploid plants either by pollination of wheat with alien species (Hordeum bulbosum, maize or sorghum) (Barclay, 1975; Laurie and Bennett, 1988a,b) or by microspore and anther culture. Despite some benefits of the wheat pollination technique, the microspore culture produces the large number of haploid plants in the shorter period of time (Touraev et al, 1996; Hul and Kasha, 1997; Hu and Kasha, 1999). Substantial effort has been made to develop methods for transformation of microspores and microspore-derived tissue
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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.
