Method for magnetic transfection of maize pollen

1 . A method for magnetic transfection of maize pollen, consisting of the following steps:

(1) preparing plasmid DNA for transfection;

(2) binding magnetic nanoparticles (MNP) nanomagnetic beads to the plasmid DNA at room temperature to form an MNP-DNA complex;

(3) collecting fresh pollen from maize at full flowering and transporting the collected pollen to a laboratory in an insulated icebox isolated from meltwater;

(4) mixing sieved maize pollen with a maize pollen transformation solution pre-cooled at 8° C. to obtain a pollen mixture, wherein the maize pollen transformation solution comprises sucrose 0.5 mol/L, H 3 BO 3 1 mmol/L, KNO 3 1 mmol/L, Ca(NO 3 ) 2 ·4H 2 O 1 mmol/L, MnSO 4 ·H 2 O 1 mmol/L, MgSO 4 ·7H 2 O 1 mmol/L, and GA 3 0.1 mmol/L; and subjecting the pollen mixture to an aperture-opening pre-treatment at 8° C. for 10 min prior to adding the MNP-DNA complex;

(5) after completion of the aperture-opening pre-treatment of step (4), adding the MNP-DNA complex obtained in step (2) to the pollen mixture, mixing and placing the pollen mixture on a magnetic plate pre-cooled at 8° C. for transfection at 8° C. for a total of 20 min; and

(6) after transfection, taking the pollen suspension to a maize field in an ice box and pollinating directly on ears of maize.

2 . The method for magnetic transfection of maize pollen according to claim 1 , wherein step (1) comprises:

extracting plasmid DNA for transfection;

adjusting the extracted plasmid DNA to a concentration of 1000±50 ng/μL with ddH 2 O;

dispensing the adjusted plasmid DNA in microcentrifuge tubes and storing the dispensed plasmid DNA frozen at −20° C. to avoid repeated freeze-thawing; and

prior to magnetic transfection, thawing the plasmid DNA to room temperature.

3 . The method of claim 2 , wherein step (2) comprises:

based on the flowering status of maize inbred lines and plasmid transformation requirement, estimating the transformation trials to be carried out on the day;

allowing the MNP nanomagnetic beads to stand at room temperature for 10 min; and

providing a 200 μL PCR tube, adding and mixing by aspiration 160 μL of ddH 2 O, 7.5 μL of 1 μg/μL of the MNP nanomagnetic beads and 30 μL of 1 μg/μL of the plasmid DNA in the PCR tube, and allowing the PCR tube to stand at room temperature for 20 min or more to obtain the MNP-DNA complex.

4 . The method of claim 3 , wherein step (3) comprises:

collecting fresh pollen in paper bags from maize inbred lines at flowering; and

placing the paper bags with the fresh pollen collected therein in plastic self-sealing bags, sandwiching the plastic self-sealing bags between ice packs, and bringing the plastic self-sealing bags back to a chamber in ice boxes for magnetic transfection.

5 . The method of claim 4 , wherein step (4) comprises:

removing anthers from the fresh pollen obtained in step (3) using a 100 mesh sieve to obtain the sieved maize pollen;

weighing 2 g of the sieved maize pollen and transferring the 2 g of the sieved maize pollen to a 15 mL round-bottom centrifuge tube;

adding 8 mL of the maize pollen transformation solution of claim 1 pre-cooled at 8° C. in the centrifuge tube; and

inverting and mixing the sieved maize pollen and the maize pollen transformation solution in the centrifuge tube, and placing the centrifuge tube horizontally for aperture-opening for 10 min in an 8° C. incubator.

6 . The method of claim 5 , wherein step (5) comprises:

adding the MNP-DNA complex obtained in step (2) entirely to the aperture-opening pre-treatment mixture in step (4) to a total volume of 10 mL;

inverting and mixing the MNP-DNA complex and the aperture-opening pre-treatment mixture;

placing the centrifuge tube on a MagnetoFACTOR-96 plate pre-cooled in an incubator at 8° C., and placing the centrifuge tube on the MagnetoFACTOR-96 plate horizontally for transfection for 10 min;

inverting and mixing the MNP-DNA complex and the aperture-opening pre-treatment mixture once again; and

placing the centrifuge tube on the MagnetoFACTOR-96 plate horizontally for transfection for another 10 min.