This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
Research article
Zhong-Yi Wu
Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Biotechnological engineering of maize to introduce favorable new traits relies on delivery of foreign DNA into its cells. Current gene delivery methods for maize is limited to specific genotypes, and depend on time-consuming and labor-intensive processes of tissue culture.
Results: Here, we report a new method to transfect maize that is culture-free and genotype independent. Enhanced green fluorescent protein gene (EGFP) or bialaphos resistance gene (Bar) bound with magnetic nanoparticles (MNPs) was delivered into maize pollens, and female florets of five maize varieties were pollinated. Green fluorescence was detected in 92% transfected pollens and 70% immature embryos. EGFP gene detected by PCR ranged from 29 to 68% in T1 generation of these five transfected varieties, and 7-16% of the T1 seedlings showed immunologically active EGFP protein. Moreover, 1.41% of the Bar transfected T1 plants were glufosinate resistant, and heritable Bar gene was integrated into the maize genome effectively (verified by Southern blot), expressed normally and inherited stably in their progenies.
Conclusion: These results demonstrate that exogenous DNA could be delivered into maize efficiently and expressed normally through our genotype-independent pollen transfection system, providing a reliable, fast and large-scale gene delivery choice for most elite maize varieties recalcitrant to tissue culture.
Keywords
magnetic nanoparticles, pollen transfection, maize varieties, genotype independence, green fluorescent protein, bialaphos resistance
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- figS1.png
Profile of plasmid pYBA1132 used for transfection of EGFP gene. Target gene expression cassette is p35S::EGFP::t35S.
- figS2.png
Full scare immunostain images of EGFP and ZmACTIN1 protein. Crude protein was separated and blot through capillary electrophoresis on WES (ProteinSimple), and results were analyzed by Compass software. L: biotinylated ladder. Numbers at top refer to seedling from seeds of EGFP transfected plants. CK: control seedling. EGFP: purified recombinant EGFP protein.
- figS3.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected 178 seeds. (A) EGFP gene detected by PCR. (B) RNA monitored by RT-PCR. (C) Protein analyzed by Western blot. (D) Confocal microscopy shows EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. B: blank, no sample DNA control. P: plasmid of pYBA1132. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS4.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected B73 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS5.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected HZ178 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS6.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected Zheng58 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS7.png
Profile of plasmid pYBA1132-Bar used for transfection of Bar gene. Target gene expression cassette is p35S:: Bar::tNOS.
- TableS1.docx
Primers used in this paper
Loading...
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 02 Nov, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Learn more about our company.
A new preprint design is coming!
We have improved readability, clarity, accessibility, and opportunities for engagement.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research article
Zhong-Yi Wu
Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 02 Nov, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Biotechnological engineering of maize to introduce favorable new traits relies on delivery of foreign DNA into its cells. Current gene delivery methods for maize is limited to specific genotypes, and depend on time-consuming and labor-intensive processes of tissue culture.
Results: Here, we report a new method to transfect maize that is culture-free and genotype independent. Enhanced green fluorescent protein gene (EGFP) or bialaphos resistance gene (Bar) bound with magnetic nanoparticles (MNPs) was delivered into maize pollens, and female florets of five maize varieties were pollinated. Green fluorescence was detected in 92% transfected pollens and 70% immature embryos. EGFP gene detected by PCR ranged from 29 to 68% in T1 generation of these five transfected varieties, and 7-16% of the T1 seedlings showed immunologically active EGFP protein. Moreover, 1.41% of the Bar transfected T1 plants were glufosinate resistant, and heritable Bar gene was integrated into the maize genome effectively (verified by Southern blot), expressed normally and inherited stably in their progenies.
Conclusion: These results demonstrate that exogenous DNA could be delivered into maize efficiently and expressed normally through our genotype-independent pollen transfection system, providing a reliable, fast and large-scale gene delivery choice for most elite maize varieties recalcitrant to tissue culture.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
This is a list of supplementary files associated with this preprint. Click to download.
- figS1.png
Profile of plasmid pYBA1132 used for transfection of EGFP gene. Target gene expression cassette is p35S::EGFP::t35S.
- figS2.png
Full scare immunostain images of EGFP and ZmACTIN1 protein. Crude protein was separated and blot through capillary electrophoresis on WES (ProteinSimple), and results were analyzed by Compass software. L: biotinylated ladder. Numbers at top refer to seedling from seeds of EGFP transfected plants. CK: control seedling. EGFP: purified recombinant EGFP protein.
- figS3.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected 178 seeds. (A) EGFP gene detected by PCR. (B) RNA monitored by RT-PCR. (C) Protein analyzed by Western blot. (D) Confocal microscopy shows EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. B: blank, no sample DNA control. P: plasmid of pYBA1132. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS4.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected B73 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS5.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected HZ178 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS6.png
Analysis of EGFP reporter in T1 seedlings from EGFP transfected Zheng58 seeds. (A) PCR. (B) RT-PCR. (C) Western blot. (D) Confocal merged images of EGFP in leaves. M: DL2000 DNA molecular marker. Numbers at top refer to T1 seedling from seeds of transfected plants. CK: control seedling. P: plasmid of pYBA1132. B: blank, no sample DNA control. L: biotinylated ladder. EGFP: recombinant EGFP protein.
- figS7.png
Profile of plasmid pYBA1132-Bar used for transfection of Bar gene. Target gene expression cassette is p35S:: Bar::tNOS.
- TableS1.docx
Primers used in this paper
Loading...