METHOD FOR MAGNETIC TRANSFECTION OF MAIZE POLLEN

Abstract

The present invention discloses an improved method for magnetic transfection of maize pollen, including: (1) preparing plasmid DNA for transfection; (2) binding MNP nanomagnetic beads to the plasmid DNA at room temperature to form MNP-DNA complex; (3) collecting fresh pollen from maize at full flowering and bringing it rapidly back indoors under ice box storage conditions insulated from water; (4) mixing a maize pollen transformation solution with sieved maize pollen and being subjected to aperture-opening pre-treatment at low temperature; (5) adding the MNP-DNA complex to aperture-opening pre-treatment solution, mixing gently and placing on a low temperature pre-cooled magnetic plate for transfection; (6) after transfection, taking the pollen suspension to field in ice box and pollinating directly on ears of maize. The present invention directly uses maize grown in the field for transformation, requires only conventional refrigeration equipment, is simple and inexpensive to operate and is applicable to all maize varieties.

Claims

1. An improved method for magnetic transfection of maize pollen, characterized in comprising the following steps of: (1) preparing plasmid DNA for transfection; (2) binding MNP nanomagnetic beads to the plasmid DNA at room temperature to form MNP-DNA complex; (3) collecting fresh pollen from maize at full flowering and bringing it rapidly back indoors under ice box storage conditions insulated from water; (4) mixing a maize pollen transformation solution with sieved maize pollen and being subjected to aperture-opening pre-treatment at low temperature; (5) adding the MNP-DNA complex obtained in step (2) to aperture-opening pre-treatment solution of step (4), mixing gently and placing on a low temperature pre-cooled magnetic plate for transfection; (6) after transfection, taking the pollen suspension to field in ice box and pollinating directly on ears of maize; wherein, in step (4) and step (5), the low temperature is 6 to 10° C.

2. The improved method for magnetic transfection of maize pollen according to claim 1, characterized in that the low temperature is 8° C.

3. The improved method for magnetic transfection of maize pollen according to claim 2, wherein: step (1) specifically comprises the following steps: high purity plasmid DNA for transfection was extracted in bulk, adjusted to a concentration of 1000±50 ng/μL with ddH.sub.2O, dispensed in small tubes and stored frozen at −20° C. to avoid repeated freeze-thawing, and the plasmid DNA was taken out and thawed to room temperature prior to magnetic transfection.

4. The improved method for magnetic transfection of maize pollen according to claim 3, wherein: step (2) specifically comprises the following steps: based on the flowering status of maize inbred lines and plasmid transformation requirement, the transformation trials to be carried out on the day were estimated; the nanomagnetic beads MNP were allowed to stand at room temperature for 10 min, a 200 μL PCR tube with the transformation number written on the cap was added with 160 μL of ddH.sub.2O, 7.5 μL of 1 μg/μL of magnetic nanobeads and 30 μL of 1 μg/μL of DNA, mixed by gentle aspiration and allowed to stand at room temperature for 20 min or more to obtain the MNP-DNA complex.

5. The improved method for magnetic transfection of maize pollen according to claim 4, wherein: step (3) specifically comprises the following steps: fresh pollen was collected in paper bags from maize inbred lines at flowering, and the powdered paper bags were taken in plastic self-sealing bags, sandwiched between ice packs and quickly brought back to the chamber in ice boxes for magnetic transfection.

6. The improved method for magnetic transfection of maize pollen according to claim 5, wherein: step (4) specifically comprises the following steps: the anthers were removed from the pollen obtained in step (3) using a 100 mesh sieve, approximately 2 g of the sieved pollen were weighed and transferred to a 15 mL round-bottom centrifuge tube with the transformation number marked on the wall, 8 mL of maize pollen transformation solution pre-cooled at 8° C. were added, covered tightly and mixed thoroughly upside down, and the tube was left horizontally for aperture-opening for 10 min in an 8° C. incubator, The content of each component in the maize pollen transformation solution was: sucrose 0.5 mol/L, H.sub.3BO.sub.3 1 mmol/L, KNO.sub.3 1 mmol/L, Ca(NO.sub.3).sub.2 1 mmol/L, MnSO.sub.4.Math.H.sub.2O 1 mmol/L, MgSO.sub.4.Math.7H.sub.2O 1 mmol/L, GA.sub.3 0.1 mmol/L.

7. The improved method for magnetic transfection of maize pollen according to claim 6, wherein: step (5) specifically comprises the following steps: the MNP-DNA obtained in step (2) was entirely added to the aperture-opening pre-treatment solution in step (4), total volume approximately 10 mL, gently inverted and mixed, placed on a MagnetoFACTOR-96 plate pre-cooled in an incubator at 8° C., and placed horizontally for transfection for 10 min, then gently inverted and mixed once, and again placed horizontally for transfection for 10 min, for a total of 20 min.

8. The improved method for magnetic transfection of maize pollen according to claim 7, wherein: step (6) specifically comprises the following steps: after magnetic transfection, the pollen centrifuge tube was clamped in a pre-cooled ice bag at 8° C. and quickly taken to the field in an ice box; the pollen was gently shaken upside down and pollinated on 10 ears of pre-bagged maize inbred lines that were spitting; a drop of pollen suspension was added to the maize filaments truncated to approximately 2 cm long with a de-tipped 1 mL tip, 1 mL/ear, and the pollen suspension was spread evenly with gloves, After pollinating the pollen of one plasmid, the pollen of the next plasmid was pollinated with a new glove, 20 days after the pollination, fruiting seeds were visible.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Additional objectives, functions, and advantages of the present invention will be set forth in the description of embodiments which follow, with reference to the accompanying drawings in which:

[0025] FIG. 1 shows a schematic diagram of collecting fresh maize pollen at full flowering in the method of the present invention;

[0026] FIG. 2 shows a schematic diagram of screening out anthers in the method of the present invention;

[0027] FIG. 3 shows a schematic diagram of low temperature magnetic transfection in the method of the present invention;

[0028] FIG. 4 shows a schematic diagram of applying a pollen suspension to maize filaments in the method of the present invention;

[0029] FIG. 5 shows a schematic diagram of setting of maize 20 days after pollination with magnetically transfected pollen in the method of the present invention;

[0030] FIG. 6 shows direct germination of fresh maize pollen in the method of the present invention;

[0031] FIG. 7 shows germination of maize pollen after treatment at 4° C. for 30 minutes in the method of the present invention;

[0032] FIG. 8 shows germination of maize pollen after treatment at 8° C. for 30 minutes in the method of the present invention;

[0033] FIG. 9 shows germination of maize pollen after treatment at 12° C. for 30 minutes in the method of the present invention;

[0034] FIG. 10 shows germination of maize pollen after treatment at 16° C. for 30 minutes in the method of the present invention;

[0035] FIG. 11 shows germination of maize pollen after treatment at room temperature for 30 minutes in the method of the present invention;

[0036] FIG. 12 shows statistics of the germination rate of maize pollen after 30 minutes of treatment in transfection solutions at different temperatures in the method of the present invention;

[0037] FIG. 13 shows maize setting of maize inbred line HZ178 magnetically transfected at low temperature (8° C.) in the method of the present invention;

[0038] FIG. 14 shows maize setting of maize inbred line HZ178 magnetically transfected at room temperature in the method of the present invention;

[0039] FIG. 15 shows maize setting of maize inbred line Jing 92 magnetically transfected at low temperature (8° C.) in the method of the present invention;

[0040] FIG. 16 shows maize setting of maize inbred line Jing 92 magnetically transfected at room temperature in the method of the present invention;

[0041] FIG. 17 shows maize pollen apertures at room temperature in the method of the present invention;

[0042] FIG. 18 shows that some maize pollen apertures after the operculum have been induced to open after pretreatment with transformation solution for 10 minutes at 8° C. in the method of the present invention;

[0043] FIG. 19 shows fluorescence expression of pollen transfected with nanomagnetic beads (MNP) only by pretreatment with transformation solution for 10 minutes at 8° C., followed by magnetic transfection in the method of the present invention; RFP: red fluorescent field; Merge: red fluorescent field and bright field overlay image; Bright: bright field;

[0044] FIG. 20 shows fluorescence expression of pollen transfected with RFP plasmid DNA only by pretreatment with transformation solution for 10 minutes at 8° C., followed by magnetic transfection in the method of the present invention; RFP: red fluorescent field; Merge: red fluorescent field and bright field overlay image; Bright: bright field;

[0045] FIG. 21 shows fluorescence expression of pollen transfected with both MNP and RFP plasmid DNA by pretreatment with transformation solution for 10 minutes at 8° C., followed by magnetic transfection in the method of the present invention; RFP: red fluorescent field; Merge: red fluorescent field and bright field overlay image; Bright: bright field;

[0046] FIG. 22 shows fluorescence expression of maize pollen magnetically transfected with MNP and RFP plasmid DNA directly for 30 minutes at 8° C. without pretreatment with transformation solution for 10 minutes in the method of the present invention; RFP: red fluorescent field; Merge: red fluorescent field and bright field overlay image; Bright: bright field;

[0047] FIG. 23 shows a schematic diagram of the structure of the vector pUbi-RFP in the present invention;

[0048] FIG. 24 shows a schematic diagram of the structure of the vector pYBA1132-bar in the present invention;

[0049] FIG. 25 shows performance of T1 generation seedlings obtained by introduction into the maize inbred line Zheng 58 by means of magnetic pollen transfection in the present invention;

[0050] FIG. 26 shows the results of five glufosinate-resistant transgenic maize plants tested by bar/PAT rapid transgenic test strips in the present invention;

[0051] FIG. 27 shows the results of Southern hybridization of T1 generation maize plants in the present invention;

[0052] FIG. 28 shows the results of Southern hybridization of T2 generation maize plants in the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0053] An improved method for magnetic transfection of maize pollen:

[0054] Nanomagnetic beads (MNP, item no. 9006) and magnetic plates (item no. 9008-96) were purchased from Chemicell, Germany.

[0055] The maize inbred lines were 178, HZ178, Jing 92 and Zheng 58.

[0056] The specific maize pollen magnetic transfection process is shown in FIG. 1 to FIG. 5.

[0057] (1) High purity plasmid DNA for transfection was extracted in bulk, adjusted to a concentration of 1000±50 ng/μL with ddH.sub.2O, dispensed in small tubes and stored frozen at −20° C. to avoid repeated freeze-thawing, and the plasmid DNA was taken out and thawed to room temperature prior to magnetic transfection.

[0058] (2) Based on the flowering status of maize inbred lines and plasmid transformation requirement, the transformation trials to be carried out on the day were estimated; the nanomagnetic beads MNP were allowed to stand at room temperature for 10 min, a 200 μL PCR tube with the transformation number written on the cap was added with 160 μL of ddH.sub.2O, 7.5 μL of 1 μg/μL of magnetic nanobeads and 30 μL of 1 μg/μL of DNA, mixed by gentle aspiration and allowed to stand at room temperature for 20 min or more to obtain the MNP-DNA complex.

[0059] (3) Fresh pollen was collected in paper bags from maize inbred lines at flowering (FIG. 1), and the powdered paper bags were taken in plastic self-sealing bags, sandwiched between pre-cooled ice packs at 8° C. and quickly brought back to the chamber in ice boxes for magnetic transfection.

[0060] (4) The anthers were removed from the pollen obtained in step (3) using a 100 mesh sieve (FIG. 2), approximately 2 g of the sieved pollen were weighed and transferred to a 15 mL round-bottom centrifuge tube (marked with the transformation number on the wall), 8 mL of maize pollen transformation solution pre-cooled at 8° C. were added, covered tightly and mixed thoroughly upside down, and the tube was left horizontally for aperture-opening for 10 min in an 8° C. incubator. The content of each component in the maize pollen transformation solution was: sucrose 0.5 mol/L, H.sub.3BO.sub.3 1 mmol/L, KNO.sub.3 1 mmol/L, Ca (NO.sub.3).sub.2 1 mmol/L, MnSO.sub.4.Math.H.sub.2O 1 mmol/L, MgSO.sub.4.Math.7H.sub.2O 1 mmol/L, GA.sub.3 0.1 mmol/L.

[0061] (5) The MNP-DNA obtained in step (2) was entirely added to the aperture-opening pre-treatment solution in step (4) (total volume approximately 10 mL), gently inverted and mixed, placed on a MagnetoFACTOR-96 plate (up to four 15 mL round-bottom centrifuge tubes per plate) pre-cooled in an incubator at 8° C., and placed horizontally for transfection for 10 min, then gently inverted and mixed once, and again placed horizontally for transfection for 10 min, for a total of 20 min (FIG. 3).

[0062] (6) After magnetic transfection, the pollen centrifuge tube was clamped in a pre-cooled ice bag at 8° C. and quickly taken to the field in an ice box; the pollen was gently shaken upside down and pollinated on 10 ears of maize inbred lines (pre-bagged) that were spitting; a drop of pollen suspension was added to the maize filaments truncated to approximately 2 cm long with a de-tipped 1 mL tip, 1 mL/ear, and the pollen suspension was spread evenly with gloves (FIG. 4). After pollinating the pollen of one plasmid, the pollen of the next plasmid was pollinated with a new glove. 20 days after the pollination, fruiting seeds were visible (FIG. 5).

Low Temperatures can Better Maintain Maize Pollen Viability.

[0063] 1) Effect of transformation solution treatment and temperature on the viability of maize pollen. Pollen viability was observed by germination of maize pollen in 15 mm diameter glass-bottomed dishes, with fresh maize pollen as control (-). Approximately 5 mg of fresh maize pollen (-) was added to 200 μL of pollen germination culture solution (15% PEG4000, 150 g/L sucrose, 300 mg/L Ca(NO.sub.3).sub.2.Math.4H.sub.2O, 100 mg/L H.sub.3BO.sub.3, 200 mg/L MgSO.sub.4-7H.sub.2O, 100 mg/L KNO.sub.3 and 0.1 mM GA.sub.3); 20 μL of pollen suspension (pre-treated for 30 minutes at 4° C., 8° C., 12° C., 16° C. and room temperature in transformation solution, equivalent to 2×10.sup.4 pollen grains) were also added in 180 μL of pollen germination culture solution; stirred gently and incubated for 3 hours at 25° C. in the dark before observing pollen germination under a microscope and taking photographs for counting. The results of pollen germination of the maize inbred line Jing 92 are shown in FIGS. 6 to 12. At 4° C., 8° C., 12° C., 16° C. and room temperature, the pollen germination rates for 30 minutes of pretreatment with the transformation solution were 48% (153/318), 54% (579/1077), 21% (130/623), 25% (136/552) and 12% (113/935) respectively. The pollen pretreated in the transformation solution for 30 minutes at 8° C. had the highest germination rate and maintained 75% of the viability of pollen pretreated without transformation solution (64%, 719/1121). A similar effect was observed in four maize varieties, Jing 92, HZ 178, Zheng 58 and 178, where pollen pretreated in transformation solution for 30 minutes at 8° C. had a higher germination rate than room temperature pretreatment (Table 1).

TABLE-US-00001 TABLE 1 Pollen germination rate of four maize inbred lines with/without transformation solution pretreatment at 8° C. and at room temperature Maize Transformation liquid Total pollen Germinated Germination variety treatment for 30 minutes Temperature count pollen count rate (%) Jing 92 − − 1121 719 64.14 ± 9.97 + 8° C. 1077 579 53.76 ± 4.96 + room temperature 935 113 12.09 ± 2.62 − − 657 482 73.36 ± 8.62 HZ178 + 8° C. 701 280 39.94 ± 2.81 + room temperature 760 95 12.50 ± 2.38 − − 533 350  65.67 ± 11.78 Zheng 58 + 8° C. 693 381 54.98 ± 5.37 + room temperature 601 46  7.65 ± 2.50 178 − − 785 473 60.25 ± 3.89 + 8° C. 548 174 31.75 ± 1.17

[0064] 2) Higher fruit-setting in low temperature magnetically transfected maize. At the end of the magnetic transfection, the transfected pollen was pollinated to the ears of maize, and after the seeds had matured, they were harvested, photographed and the fruit-setting rate counted. The results are shown in FIGS. 13 to 16 and in the table below. Maize pollinated by magnetically transfected pollen at 8° C. had a fertility rate of 40 to 68 seeds/ear, which was significantly higher than that of maize pollinated by room temperature magnetically transfected pollen (9-10 seeds/ear), indicating that low temperature magnetic transfection was more conducive to maintaining pollen viability, improving fertility and obtaining more maize seeds for screening and identification.

TABLE-US-00002 Magnetic Number Total Ear average seed Maize transfection of ears seeds setting rate variety temperature harvested count (seed/ear) HZ178 8° C. 5 199 39.8 room temperature 5 51 10.2 Jing 92 8° C. 6 407 67.83 room temperature 3 28 9.33
The aperture opening of maize pollen is essential for the introduction and expression of exogenous genes.

[0065] To investigate the effect of pollen apertures on DNA introduction into maize pollen, we first looked at the surface structure of maize pollen apertures under scanning electron microscopy. Maize pollen usually has only one pollen aperture with operculum (FIG. 17), which prevents the entry of external material. This is common in cereal pollen. Secondly, we studied the opening or otherwise of pollen apertures at 8° C.. Pretreatment with transformation solution at 8° C. for 10 minutes induced the opening of the operculum of some maize pollen (FIG. 18), removing the barrier to entry of exogenous DNA into the pollen. Subsequently, we studied the pollen aperture opening rates of four maize inbred lines, Jing 92, HZ 178, Zheng 58 and 178, after treatment with transformation solution at room temperature or 8° C. for 10 minutes. As shown in the table below, pollen treated at 8° C. maintained an opening rate of 40-50%, which, although slightly lower than the room temperature treatment, was sufficient for magnetic transfection of maize pollen.

TABLE-US-00003 Total number of Number of open aperture pollen Opening Maize Opening visible pollen (transformation solution treated for rate variety temperature apertures 10 minutes) (%) Jing 92 room 1276 695 54.47 ± 9.13  temperature 9512 3838 40.35 ± 10.88 8° C. HZ178 room 7706 4748 61.61 ± 13.40 temperature 11853 6044 50.99 ± 18.20 8° C. Zheng room 4726 2496 52.81 ± 12.77  58 temperature 6542 3224 49.28 ± 14.52 8° C. 178 8° C. 11068 4479 40.47 ± 4.39 

[0066] We studied the transient expression of exogenous genes in maize pollen using red fluorescent protein (RFP) as a reporter gene (driven by the maize ubiquitin (Ubi) promoter, FIG. 23). After pretreatment with transformation solution for 10 min and magnetic field transfection for 20 min, maize pollen was transferred into the expression culture (20% PEG4000, 150 g/L sucrose, 300 mg/L Ca(NO.sub.3).sub.2.Math.4H.sub.2O, 100 mg/L H.sub.3BO.sub.3, 200 mg/L MgSO.sub.4.Math.7H.sub.2O, 100 mg/L KNO.sub.3) and incubated at 25° C. for 20 h. Red fluorescence was not detected in pollen transfected with nanomagnetic beads (MNP) only (FIG. 19) or RFP plasmid DNA only (FIG. 20), but was detectable in 22% (129/590) of pollen transfected with both MNP and RFP plasmid DNA (FIG. 21). On the other hand, red fluorescence was detected in approximately 3% (6/208) of maize pollen that had been magnetically transfected with MNP and RFP plasmid DNA directly for 30 min at 8° C. without pretreatment with transformation solution for 10 min (FIG. 22). These results suggest that pretreatment with the transformation solution for 10 min was more effective in inducing pollen opening for exogenous gene entry and transient expression compared to direct transfection.

[0067] The herbicide screening marker gene bar can be efficiently introduced into the maize genome and stably inherited by magnetic pollen transfection.

[0068] To screen stable transformed progeny, we introduced the herbicide selection marker gene bar (vector shown in FIG. 24) into the maize inbred line Zheng 58 by magnetic transfection of pollen. After screening at the three-leaf stage with 200 mg/L glufosinate, 1.41% (5/355) of the T1 generation seedlings showed herbicide resistance (FIG. 25). These five glufosinate resistant transgenic maize plants tested positive (with both detection and control lines) by the bar/PAT rapid transgenic test strip (FIG. 26), indicating that the bar gene was successfully introduced into maize by magnetic transfection of pollen and expressed normally. In addition, Southern hybridization results showed that these T1 generation maize plants had about 2-5 copies integrated in their genomes (FIG. 27) and all five transgenic lines inherited the bar gene to the T2 generation, where Southern hybridization testing revealed genetic segregation in the T2 generation (FIG. 28). The above findings show that by our magnetic transfection of maize pollen, exogenous genes were efficiently integrated into the maize genome and are normally expressed and stably inherited in the progeny.

[0069] Existing methods of transfecting maize pollen are carried out at room temperature, but maize pollen is highly susceptible to germination, inactivation and rupture at room temperature. We found that the room temperature magnetically transfected maize pollen maintained only about 10% viability and the pollinated maize ears had a fertility rate of only 10 seeds/ear. In contrast, maize pollen can be about 75% viable after magnetic transfection at a low temperature of 8° C., and its pollinated female maize ears could have an increased fertility of up to 60 seeds/ear. In this way, low-temperature magnetic transfection of maize pollen can dramatically increase fertility and yield more transformed seeds for subsequent screening and identification. In addition, existing maize pollen transfection methods are not pre-treated for aperture opening. Because the pollen apertures of maize are usually capped, when direct magnetic transfection is performed, the efficiency of nanomagnetic beads to introduce exogenous DNA into pollen is only 2%. However, at a low temperature of 8° C., which can maintain the viability of maize pollen, 40 to 50% of the maize pollen aperture operculum will be opened after pretreatment with transformation solution for 10 minutes, at which point the efficiency of the nanomagnetic beads to introduce exogenous DNA into pollen could be increased to 22%. Based on the above two aspects, the improved maize pollen magnetic transfection method in this application can not only maintain the vitality of most of the maize pollen through low temperature environment, but also improve the efficiency of exogenous DNA introduction through aperture opening pretreatment, and has a wide application prospect.

[0070] The foregoing embodiments are merely illustrative of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various variations and modifications made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention are embraced in the protection scope of the present invention as defined by the appended claims.