Abstract:
A transformation method, including the following steps: preparing an  Agrobacterium  Ti-plasmid,  Escherichia coli  plasmid, or other DNA vectors carrying exogenous genetic fragments as a genetic donor; collecting a male gamete (pollen) of the plant as a recipient; preparing a 5-50% sucrose solution after aeration and low temperature pretreatment; mixing the pollen with the exogenous genetic fragments in the 5-50% sucrose solution; transferring the exogenous genetic fragments into the pollen in the presence of ultrasonication; pollinating a pistil stigma of the plant with the treated pollen; harvesting seeds at maturity; sowing the seeds in a subsequent growing season; screening a germinating seed and a seedling; and performing PCR amplification and Southern hybridization using DNA samples of plants to further determine transformants.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation-in-part of International Patent Application No. PCT/CN2012/000030 with an international filing date of Jan. 9, 2012, designating the United States, now pending, and further claims priority benefits to Chinese Patent Application No. 201110041484.0 filed Feb. 18, 2011. The contents of all of the aforementioned applications, including any intervening amendments thereto, are incorporated herein by reference. Inquiries from the public to applicants or assignees concerning this document or the related applications should be directed to: Matthias Scholl P. C., Attn.: Dr. Matthias Scholl Esq., 14781 Memorial Drive, Suite 1319, Houston, Tex. 77079. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The invention relates to a plant transformation method. 
         [0004]    2. Description of the Related Art 
         [0005]    At present, two classical methods adopted in plant transformation research are an  Agrobacterium -mediated transformation method, and a particle bombardment method. Both methods require a long and complicated plant tissue culture process and are laborious, costly, and time-consuming As some plant species or varieties are difficult to generate by tissue culture, thus, both methods are highly genotype-dependent, the applications of these two methods have been seriously restricted. Furthermore, somatic variation and death of regenerated plantlets frequently occur during plant tissue culture process, thus the already low transformation ratio would be further reduced. All these defects have greatly limited the wide application of plant transformation technology. Due to the complexity to operate or low efficiency, other plant transformation methods are rarely used in practice despite the reports on their successful transformation. These methods include those involving liposome, PEG, electroporation, microinjection, ultrasonication, ion beam, laser microbeam puncture, and silicon carbide fiber. Therefore, an efficient and simplified plant transformation method has been sought. 
         [0006]    A pollen-tube-pathway method has been applied to a certain extent, and some transgenic lines or varieties have been generated. This method is advantageous in no dependency on tissue culture or plant regeneration, no requirement on well-equipped labs, and its simplicity to operate. However, the transformation efficiency of this method is low and it requires selecting transformants among a large number of progenies. Therefore, the lack of a simple and efficient plant transformation method is still a bottleneck in plant genetic engineering. In a sonication-assisted pollen-mediated plant transformation method, a sonifier cell disrupter is used to treat pollen suspension by 200-300 W ultrasonication power. Fresh pollen is collected and suspended in a 5-15% sucrose solution including foreign DNA having a concentration of at least 40 μg/L. The pollen suspension is treated by ultrasonication, before and after the addition of the foreign DNA, for 5-8 times at an interval of 10 seconds, each for 5 seconds. Pollen is then pollinated to plant stigmas (silks in case of maize), seeds are harvested and sowed in the subsequent season, and transformants are selected from progenies. The method does not require a long and complicated tissue culture process, and is simple, effective, fast, economical, and thus highly practical. This method can be readily integrated into conventional breeding programs and directly used by crop breeders. However, a major shortcoming of this method is its low seed setting rate after pollination, because most pollen grains lose their viability after ultrasonication and fail to complete the fertilization process. Therefore, improving the seed setting rate is the key to a wider application prospect of the method. 
       SUMMARY OF THE INVENTION 
       [0007]    In view of the above-described problems, it is one objective of the invention to provide a sonication-assisted pollen-mediated plant transformation method that remarkably improves a seed setting rate of pollinated plants. 
         [0008]    It is found experimentally that a higher proportion of pollen grains are capable of maintaining their vitality in well aerated sucrose solution at low temperature during pollen treatment, so that a higher seed setting rate is reached after the treated pollen is applied to plant stigmas (corn silks herein). Furthermore, it is favorable to enhance the pollen vitality by preserving newly collected pollen at a low temperature (4° C.) and under dry condition, and using the pollen within 2-48 h. 
         [0009]    The method uses an  Agrobacterium  Ti-plasmid, an  Escherichia coli  plasmid, or other DNA vectors carrying an exogenous genetic fragment as a gene donor; uses male gametes of the plant as intermediate recipients, and the sonication-assisted pollen-mediated gene transfer is achieved in the plant pollination and fertilization process. Under actions of instantaneous high energy release and cavitations of ultrasonication, the foreign DNA fragments are introduced into the plant pollen, then enter plant female gamete embryo sacs along with a growth of pollen tubes to participate the formation of zygotes, and finally incorporated into the target plant genomes. 
         [0010]    Specific process is as follows: a 5-50% sucrose solution is aerated and low temperature treated, and fresh pollen is mixed with the foreign DNA in the 5-50% sucrose solution. The foreign DNA fragments are introduced into the pollen by ultrasonication. The treated pollen is pollinated to plant stigmas. Seeds are harvested at maturity, and then sowed in the subsequent growing season. Transformants are further determined through screening germinating seeds and seedlings with a selector (often but not limited to a herbicide or an antibiotic) and PCR amplification and Southern hybridization on plant DNA samples. 
         [0011]    Before the addition of the pollen and the foreign DNA, the sucrose solution is pretreated with aeration and ice bath. The solution is continuously aerated for more than 20 minutes by a miniature commercially-available aquarium air pump until the air (oxygen) content in the sucrose solution is saturated. Meanwhile, the solution is placed in a 0-4° C. ice bath or a refrigerator. The pollen suspension is always placed in the 0-4° C. ice bath during subsequent operations. The ultrasonic treatment is applied to the pollen suspension before or after the addition of the foreign DNA, with the power of the ultrasonication of 50-500 W, and the time of the ultrasonic treatment of 5 seconds to 2 minutes. 
         [0012]    The fresh pollen can be preserved for 5 days at a temperature of 4° C. with certain pollen viability. The treated pollen is pollinated to the plant stigma, and seeds on the pollinated ears are harvested at the maturity and then sowed in the subsequent growing period. The transformants are determined through screening of the seedlings (the step can be skipped in a marker-free transformation), and PCR amplification, and Southern hybridization of genomic DNA. The transformant is continuously self-pollinated and selected until a stable and homozygous transgenic line is obtained. 
         [0013]    The improved pollen-mediated plant transformation method assisted by ultrasonication is capable to markedly improve seed setting rate following pollination, thereby to increase the overall transformation efficiency. In the method, the exogenous gene can be directly transferred into the recipient genome, thus the complicated plant tissue culture process with demanding technical operation is avoided, and the turnaround time to obtain the transformed seeds is greatly shortened. The method has the merits of high gene transformation efficiency, good reproducibility, less probability of chimera plants, cheap operational requirement, and genotype independency (i.e., wide range of application). Furthermore, this method can be applied to high-throughput transformation systems as it improves the seed setting rate and thus the transformation efficiency. 
     
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0014]    For further illustrating the invention, experiments detailing an improved plant transformation method applied in corns are described below. It should be noted that the following examples are intended to describe and not to limit the invention. 
         [0015]    Except for unavoidable damages under the application of ultrasonication, the cause of low pollen vitality due to the treatment lies partially to the damage of pollen in the suspension, in cases of pollen breakage, and plasmolysis. 
         [0016]    The vitality of pollen can be improved by the modification of the suspension conditions of pollen, of which, sucrose concentration (osmotic pressure), temperature, and air (oxygen) content are three main factors. In view of the above factors, the following experiments are made with the corn variety Zheng 58, and the results are shown in Tables 1-8. 
       EXAMPLE 1 
     Significant Influence of Soaking Time of Corn Pollen in Suspension on In Vitro Germination 
       [0017]    As shown in Table 1, with the extension of the soaking time, the broken rate of corn pollen was increased, and the germination rate was remarkably reduced. After the corn pollen was soaked for 1 hour in suspension without aeration treatment at the temperature of 28° C., the germination rate was reduced to 20%. If the soaking time reached 120 minutes, the pollen germination rate was close to zero. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Reduction of In vitro germination rate of corn pollen with the increase of 
               
               
                 soaking time in the suspension 
               
             
          
           
               
                   
                 Soaking time 
               
             
          
           
               
                   
                 0 min 
                 10 min 
                 20 min 
                 30 min 
                 40 min 
                 50 min 
                 60 min 
                 90 min 
                 120 min 
               
               
                   
               
               
                 Broken rate of 
                 12.8 ± 2.29f 
                 18.6 ± 2.88f 
                 15.7 ± 3.10f 
                 28.2 ± 4.81e 
                 42.3 ± 5.57d 
                 51.1 ± 6.37c 
                 56.3 ± 6.85bc 
                 63.8 ± 6.84b 
                 78.9 ± 6.45a 
               
               
                 pollen (%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Germination rate 
                 81.4 ± 5.52a 
                 78.3 ± 5.76a 
                 75.4 ± 5.23a 
                 65.2 ± 6.18b 
                 48.3 ± 5.97c 
                 32.4 ± 5.54d 
                 20.8 ± 4.17e 
                 15.7 ± 3.26e 
                 0.16 ± 0.22f 
               
               
                 of pollen (%) 
               
               
                   
               
               
                 Note: 
               
               
                 the pollen germination rate was determined after soaking the pollen in a 15% sucrose solution at the room temperature of 28° C. by the incubation of pollen suspension into a culture medium for 30 minutes. The medium was prepared as follow: 15% sucrose + 50 mg/L boric acid + 300 mg/L calcium chloride + 200 mg/L magnesium chloride + 100 mg/L potassium nitrate + 35 mg/L gibberellin. 
               
               
                 Means not sharing the same letters indicate significant difference (P &lt; 0.05). 
               
             
          
         
       
     
       EXAMPLE 2 
     Sucrose Concentration Plays an Important Role in Pollen Germination Rate 
       [0018]    The sucrose was mainly used as an osmotic agent in the solution. As shown in Table 2-1 and Table 2-2, an average breakage rate of corn pollen in the sucrose solution of a low concentration (≦5%) was higher when pollen incubation was carried out at any time. The rate of undamaged pollen was increased with the increase of the sucrose concentration. However, when the concentration of the sucrose was reached up to 50%, the pollen germination rate was remarkably reduced. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2-1 
               
             
             
               
                   
               
               
                 The pollen viability following the In vitro incubation in the culture media 
               
               
                 with different sucrose concentrations (Greenhouse trial) 
               
             
          
           
               
                   
                   
                 Germination 
                   
                   
               
               
                 Sucrose 
                 Broken rate of 
                 rate of pollen 
                 Length of pollen 
               
               
                 concentration 
                 pollen (%) 
                 (%) 
                 tube (μm) 
                 Characteristics 
               
               
                   
               
             
          
           
               
                 1% 
                 72.5 ± 6.85a 
                 7.26 ± 2.37e 
                  200 ± 42.3f 
                 A large amount of pollen 
               
               
                   
                   
                   
                   
                 was broken with internal 
               
               
                   
                   
                   
                   
                 content leaking out into 
               
               
                   
                   
                   
                   
                 the medium; pollen tubes 
               
               
                   
                   
                   
                   
                 were short and slender 
               
               
                 5% 
                 60.3 ± 6.07b 
                 11.1 ± 4.88e 
                  262 ± 48.1f 
                 Same as above 
               
               
                 10% 
                 32.9 ± 4.76c 
                 56.5 ± 5.69c 
                  671 ± 50.2d 
                 Some pollen tubes were 
               
               
                   
                   
                   
                   
                 stretched and broke. 
               
               
                 15% 
                 30.8 ± 4.11cd 
                 60.0 ± 5.82bc 
                 1357 ± 58.4c 
                 Same as above, pollen 
               
               
                   
                   
                   
                   
                 tubes were relatively 
               
               
                   
                   
                   
                   
                 longer. 
               
               
                 20% 
                 26.6 ± 3.73cde 
                 71.2 ± 4.71a 
                 1804 ± 68.7b 
                 Pollen breakage was 
               
               
                   
                   
                   
                   
                 reduced; pollen tubes 
               
               
                   
                   
                   
                   
                 were relatively long, grew 
               
               
                   
                   
                   
                   
                 smooth, straight, and 
               
               
                   
                   
                   
                   
                 evenly. 
               
               
                 30% 
                 25.1 ± 3.51def 
                 65.6 ± 5.63ab 
                 2058 ± 62.4a 
                 Same as above, pollen 
               
               
                   
                   
                   
                   
                 tubes were the longest, 
               
               
                   
                   
                   
                   
                 and grew normally. 
               
               
                 40% 
                 22.4 ± 3.71ef 
                 42.3 ± 5.38d 
                  756 ± 51.7d 
                 Internal content 
               
               
                   
                   
                   
                   
                 aggregated after pollen 
               
               
                   
                   
                   
                   
                 was broken, pollen tubes 
               
               
                   
                   
                   
                   
                 were relatively short. 
               
               
                 50% 
                 17.9 ± 3.24f 
                 12.2 ± 4.61e 
                  380 ± 45.9e 
                 Pollen was seldom 
               
               
                   
                   
                   
                   
                 broken, internal content 
               
               
                   
                   
                   
                   
                 aggregated; pollen tubes 
               
               
                   
                   
                   
                   
                 were chunky with some of 
               
               
                   
                   
                   
                   
                 them deformed. 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2-2 
               
             
             
               
                   
               
               
                 The pollen viability following the in vitro incubation in the culture media 
               
               
                 with different sucrose concentrations (field trial) 
               
             
          
           
               
                   
                   
                 Germination 
                   
                   
               
               
                 Sucrose 
                 Broken rate of 
                 rat of pollen 
                 Length of pollen 
               
               
                 concentration 
                 pollen (%) 
                 (%) 
                 tube (μm) 
                 Characteristics 
               
               
                   
               
             
          
           
               
                 1% 
                 66.4 ± 6.08a 
                 8.69 ± 2.12ef 
                  703 ± 54.2e 
                 A large amount of pollen was 
               
               
                   
                   
                   
                   
                 broken with internal content 
               
               
                   
                   
                   
                   
                 leaking out into the medium; 
               
               
                   
                   
                   
                   
                 pollen tubes were short with a 
               
               
                   
                   
                   
                   
                 tip easily broken 
               
               
                 5% 
                 51.9 ± 5.88b 
                 17.8 ± 5.47d 
                 1428 ± 72.5c 
                 Although the broken rate was 
               
               
                   
                   
                   
                   
                 high, and the germination rate 
               
               
                   
                   
                   
                   
                 was low, pollen tubes were 
               
               
                   
                   
                   
                   
                 long and well grown. 
               
               
                 10% 
                 40.1 ± 5.24c 
                 67.4 ± 5.72b 
                 2206 ± 78.9b 
                 Germination rate was high; 
               
               
                   
                   
                   
                   
                 pollen tubes grew well, and 
               
               
                   
                   
                   
                   
                 were smooth and even. 
               
               
                 15% 
                 23.8 ± 3.83d 
                 80.6 ± 4.94a 
                 2625 ± 65.5a 
                 Broken rate was the lowest, 
               
               
                   
                   
                   
                   
                 germination rate was the 
               
               
                   
                   
                   
                   
                 highest; and the pollen tubes 
               
               
                   
                   
                   
                   
                 were the longest. 
               
               
                 20% 
                 11.4 ± 2.10e 
                 49.6 ± 5.12c 
                 2285 ± 62.0b 
                 Pollen broken rate was sharply 
               
               
                   
                   
                   
                   
                 declined, and the pollen grew 
               
               
                   
                   
                   
                   
                 normally. 
               
               
                 30% 
                 4.07 ± 1.17f 
                 12.8 ± 5.02de 
                 1188 ± 57.6d 
                 Same as above, internal content 
               
               
                   
                   
                   
                   
                 aggregated after the pollen was 
               
               
                   
                   
                   
                   
                 broken. 
               
               
                 40% 
                 2.91 ± 1.00f 
                 2.37 ± 1.21f 
                  200 ± 46.3f 
                 Pollen broken rate was low, 
               
               
                   
                   
                   
                   
                 internal content was 
               
               
                   
                   
                   
                   
                 filamentous, and pollen tubes 
               
               
                   
                   
                   
                   
                 were chunky. 
               
               
                 50% 
                 0.50 ± 0.47f 
                 0 
                 0 
                 Pollen was rarely broken; 
               
               
                   
                   
                   
                   
                 bubble like black circles 
               
               
                   
                   
                   
                   
                 appeared inside the pollen; and 
               
               
                   
                   
                   
                   
                 no germination. 
               
               
                   
               
             
          
         
       
     
         [0019]    From the comparison of in vitro incubation of corn pollen under different growing conditions, the pollen viability from plants grown in different phenological conditions reacted differently to same sucrose concentration. Early-sowed corn was sowed in a greenhouse on Mar. 29, 2010, and pollen was collected from May 28 to June 10. Field corn was sowed in an isolated experimental plot on April 29, and pollen was collected from July 15 to August 5. The study was carried out in Taiyuan, Shanxi, China. As shown in Table 2-1 and Table 2-2, the optimal sucrose concentration for the germination of greenhouse pollen was 20%-30%, and pollen germination was still observed in the 50% sucrose solution although in a rather low rate. the optimal sucrose concentration for field pollen germination was 15%, the sucrose concentration higher than 20% inhibited the germination, and pollen plasmolysis appeared in the 50% sucrose solution which resulted in little pollen germination. Compared with the greenhouse corn, the field corn had lower pollen broken rate in the same sucrose concentration, and has longer length and faster growth of the pollen tube at the same germination rate. 
         [0020]    In conclusion, the lower-concentration sucrose is preferably used as the culture medium for pollen germination of the field-grown corn in Taiyuan, Shanxi China; the higher sucrose concentration is appropriate for pollen germination of corn sowed in greenhouse or other phenological conditions of low temperature and humidity. 
       EXAMPLE 3 
     Effects of Preservation Time and Conditions on Pollen Viability 
       [0021]    Viability of pollen increases in the order in conditions of room temperature humid (25-28° C., RH 50-70%), room temperature dry (25-28° C., RH 30-50%), low temperature humid (10-15° C., RH 70-90%), and low temperature dry (4° C., RH 40-60%). Particularly, pollen germination rate was favorably preserved when the pollen was kept in a Petri dish containing culture medium at 4° C., which is ideal for the sonication-mediated plant transformation. As shown in Table 3-1, the in vitro living time of greenhouse pollen was only 2 h at low temperature and in dry condition; thereafter, the pollen germination rate was reduced substantially, and thus, the greenhouse corn pollen was less ideal. In a Petri dish at 4° C., the field pollen was still viable at a low rate even (Table 3-2) after 5 days&#39; dry preservation. The newly collected pollen was very easy to be broken and had a low germination rate; however, the germination rate was substantially improved after 2 hours&#39; preservation at dry and low temperature conditions, and the pollen had high vitality within 48 hours. The germination rate and the preservation time of corn pollen were related with the quality of the pollen collected on the day. In conclusion, the field corn pollen collected in the normal growing season under good phenological conditions had high tolerance, low pollen broken rate, and fast growth of pollen tube. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 3-1 
               
             
             
               
                   
               
               
                 In vitro germination results of the early-sowed corn under various 
               
               
                 preservation time and preservation conditions (%) 
               
             
          
           
               
                 Preservation 
                 Time for collecting pollen 
               
             
          
           
               
                 condition and time 
                 8:30 
                 10:00 
                 11:30 
               
               
                   
               
             
          
           
               
                 Immediate 
                 18.3 ± 4.33a 
                 56.9 ± 6.72a 
                 69.2 ± 6.79a 
               
               
                 germination 
               
               
                 Low temperature 
                 7.30 ± 2.14b 
                 42.2 ± 5.88b 
                 68.8 ± 6.45a 
               
               
                 dying for 2 h 
               
               
                 Low temperature 
                 0 
                 15.2 ± 4.13c 
                 35.4 ± 5.62b 
               
               
                 humid for 2 h 
               
               
                 Room temperature 
                 0 
                  9.3 ± 3.02cd 
                 12.5 ± 3.67c 
               
               
                 drying for 2 h 
               
               
                 Room temperature 
                 0 
                 0 
                 0 
               
               
                 humid for 2 h 
               
               
                 Low temperature 
                 0 
                  5.2 ± 2.13d 
                 8.26 ± 2.75c 
               
               
                 drying for 4 h 
               
               
                 Low temperature 
                 0 
                 0 
                 0 
               
               
                 drying for 6 h 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3-2 
               
             
             
               
                   
               
               
                 In vitro germination of pollen from field-grown corn at 4° C. dry preservation 
               
             
          
           
               
                   
                 Preservation time 
               
             
          
           
               
                   
                 0.5 h 
                 2 h 
                 4 h 
                 6 h 
                 24 h 
                 48 h 
                 72 h 
                 96 h 
                 120 h 
                 144 h 
               
               
                   
               
               
                 Pollen broken 
                 48.2 ± 
                 26.6 ± 
                 21.5 ± 
                 18.4 ± 
                  16.7 ± 2.55e 
                  24.1 ± 3.34de 
                  46.3 ± 5.15c 
                  52.8 ± 6.28c 
                  79.8 ± 6.91b 
                 90.4 ± 
               
               
                 rate (%) 
                 5.29c 
                 3.86d 
                 3.17de 
                 2.81de 
                   
                   
                   
                   
                   
                 7.87a 
               
               
                 Pollen 
                 35.0 ± 
                 68.3 ± 
                 75.6 ± 
                 80.2 ± 
                  84.3 ± 5.27a 
                  72.4 ± 5.94bc 
                  45.8 ± 4.57d 
                 35.7 ± 4.16e 
                  15.9 ± 3.27f 
                 0 
               
               
                 germination rate 
                 4.52e 
                 5.75c 
                 5.43bc 
                 5.18ab 
                   
                   
                   
                   
                   
                   
               
               
                 (%) 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Length of poller 
                 2000 ± 
                 2250 ± 
                 2500 ± 
                 2750 ± 
                 3000 ± 78.5a 
                 1500 ± 72.3f 
                 1000 ± 68.4g 
                 625 ± 52.8h 
                 250 ± 41.2i 
                 0 
               
               
                 tube (μm) 
                 71.6e 
                 76.5d 
                 73.8c 
                 74.2b 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 4  
     Effects of Suspension Temperature on Pollen Germination 
       [0022]    The temperature of the pollen suspension affected the pollen germination, and low temperature reduced pollen breakage. 
         [0023]    Note: the germination rate was determined after soaking the pollen in the 15% sucrose solution for 5 minutes at a proper temperature, and a few droplets of pollen suspension were transferred and incubated in culture medium to measure the germination rate. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Pollen germination in suspension solution at various temperatures 
               
             
          
           
               
                   
                 temperature 
               
             
          
           
               
                   
                 35° C. 
                 30° C. 
                 25° C. 
                 20° C. 
                 15° C. 
                 10° C. 
                 4° C. 
               
               
                   
               
               
                 Pollen broken 
                 32.2 ± 5.26a 
                 22.4 ± 3.89b 
                 18.5 ± 2.87bc 
                 18.4 ± 2.56bc 
                 16.7 ± 2.23cd 
                 15.1 ± 2.75cd 
                 13.3 ± 2.15d 
               
               
                 rate (%) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Pollen 
                 64.3 ± 4.66b 
                 76.3 ± 5.19a 
                 78.2 ± 5.31a 
                 74.3 ± 5.07a 
                 79.7 ± 5.44a 
                 73.4 ± 5.47a 
                 75.8 ± 5.58a 
               
               
                 germination 
                   
                   
                   
                   
                   
                   
                   
               
               
                 rate* (%) 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 5 
     Effects of Solution Aeration on Pollen Germination 
       [0024]    It should be noted that during the pollen-mediated plant transformation operation, a rapid germination of pollen in the suspension is not favorable for improving the seed setting rate and the transformation ratio, because the germinated pollen tube would probably be damaged during a subsequent pollination, and it is not easy for the germinated pollen tube to grow into the stigma and to complete fertilization. Under the ideal state, the pollen does not germinate or break in the suspension, and its vitality is maintained. Therefore, both pollen germination rate and fertilization rate will be high following pollination. As shown in Table 5, the germination rate was low, and so was the broken rate when pollen suspension was subjected to 20 minutes&#39; aeration treatment. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Effect of suspension aeration on pollen germination* 
               
             
          
           
               
                   
                 Aeration time 
               
             
          
           
               
                   
                 0 min 
                 10 min 
                 20 min 
                 30 min 
                 40 min 
                 50 min 
                 60 min 
               
               
                   
               
               
                 Pollen broken 
                 27.2 ± 4.52a 
                 21.7 ± 3.77b 
                 16.5 ± 2.45c 
                 18.4 ± 2.66bc 
                 14.7 ± 2.13c 
                 16.4 ± 2.73c 
                 13.8 ± 2.24c 
               
               
                 rate (%) 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Pollen 
                 78.4 ± 4.68a 
                 56.3 ± 4.26b 
                 45.1 ± 3.78c 
                 51.4 ± 3.85b 
                 42.7 ± 3.71c 
                 43.5 ± 2.86c 
                 45.3 ± 3.18c 
               
               
                 germination 
                   
                   
                   
                   
                   
                   
                   
               
               
                 rate* (%) 
               
               
                   
               
               
                 Note: 
               
               
                 germination rate was determined by suspending the pollen in the 15% sucrose solution (aerated for at least 20 minutes) for 5 minutes at the temperature of 25° C. and a few droplets of pollen suspension being transferred to culture medium for the measurement. 
               
             
          
         
       
     
       EXAMPLE 6 
     The Effects of Temperature and Aeration on Pollen Germination After Ultrasonication 
       [0025]    Ultrasonication was a key step for the exogenous gene(s) to enter the pollen. The experiment (Table 6) showed that the aeration and low temperature treatment remarkably reduced the pollen broken rate and improved pollen germination rate after ultrasonication. The 11.9% of aeration-treated pollen was able to germinate compared to 3.74% of the untreated control. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Pollen germination in various pretreated sucrose solution before and after 
               
               
                 ultrasonication* 
               
             
          
           
               
                   
                 Pollen before ultrasonication 
                 Pollen after ultrasonication 
               
             
          
           
               
                 Pretreatments 
                   
                 Germination 
                   
                   
                 Germination 
                   
               
               
                 on sucrose 
                 Broken 
                 rate 
                 Length of pollen 
                 Broken 
                 rate 
                 Length of pollen 
               
               
                 solution 
                 rate (%) 
                 (%) 
                 tube (μm) 
                 rate (%) 
                 (%) 
                 tube (μm) 
               
               
                   
               
               
                 Room 
                 43.1 ± 5.11a 
                 62.7 ± 3.43a 
                 2843.8 ± 146.11a 
                 80.1 ± 8.15a 
                 3.74 ± 1.22c 
                 821.88 ± 100.39c 
               
               
                 temperature 
                   
                   
                   
                   
                   
                   
               
               
                 28° C. 
                   
                   
                   
                   
                   
                   
               
               
                 Low 
                 30.7 ± 4.01b 
                 66.4 ± 4.05a 
                 2915.6 ± 151.74a 
                 61.0 ± 6.85b 
                 8.19 ± 1.36b 
                 1378.1 ± 136.56ab 
               
               
                 temperature 
                   
                   
                   
                   
                   
                   
               
               
                 4° C. 
                   
                   
                   
                   
                   
                   
               
               
                 Aerating for 
                 15.4 ± 2.81c 
                 42.1 ± 3.65b 
                 2431.3 ± 143.15b 
                 32.5 ± 4.16c 
                 6.03 ± 1.23b 
                 1221.9 ± 112.95b 
               
               
                 20 min 
                   
                   
                   
                   
                   
                   
               
               
                 Aerating for 
                 13.5 ± 2.49c 
                 40.6 ± 3.67b 
                 2450.0 ± 136.93b 
                 24.5 ± 3.37c 
                 11.9 ± 2.39a 
                 1528.1 ± 150.26a 
               
               
                 20 min at a 
                   
                   
                   
                   
                   
                   
               
               
                 low 
                   
                   
                   
                   
                   
                   
               
               
                 temperature 
                   
                   
                   
                   
                   
                   
               
               
                 of 4° C. 
               
               
                   
               
               
                 Note: 
               
               
                 the pollen was suspended in the 15% sucrose solution of various pretreatments for 5 minutes prior to ultrasonication, and then a few droplets of pollen suspension were transferred to culture medium to germinate. For the germination rate measurement after ultrasonication, a few droplets of pollen were immediately transferred to the culture medium following the ultrasonication treatment. 
               
             
          
         
       
     
       EXAMPLE 7 
     Effects of Various Pollen Treatments on Seed Setting Rate After Pollination 
       [0026]    Pretreated pollen was pollinated to corn silks, and seed setting rates were recorded. As shown in Table 7, aeration treatment was more important than that of low temperature treatment; the combination of aeration and low temperature was the most favorable treatment for seed setting; and the average seed set per ear was improved by 1.27 times (1.65: 0.728). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effects of different treatment on seed setting rate after pollination 
               
             
          
           
               
                   
                   
                 Number of 
                 Seed setting ear 
                   
                 Seed number per 
               
               
                   
                 Number of 
                 seed setting 
                 rate (B/A)% 
                   
                 ear (C/A) 
               
               
                 Treatment 
                 treated ears (A) 
                 ears (B) 
                 Means ± SD 
                 Seed number (C) 
                 means ± SD 
               
               
                   
               
             
          
           
               
                 Ultrasonication 
                 27 
                 27 
                 27 
                 3 
                 4 
                 4 
                 13.58 ± 1.14b 
                 17 
                 19 
                 23 
                 0.728 ± 0.113c 
               
               
                 Ultrasonication + 
                 113 
                 113 
                 113 
                 15 
                 17 
                 19 
                 15.04 ± 1.77ab 
                 96 
                 99 
                 87 
                 0.832 ± 0.055c 
               
               
                 low 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 temperature 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ultrasonication + 
                 315 
                 315 
                 315 
                 51 
                 60 
                 64 
                 18.52 ± 2.15a 
                 392 
                 401 
                 447 
                  1.31 ± 0.096b 
               
               
                 aeration 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Ultrasonication + 
                 280 
                 280 
                 280 
                 40 
                 41 
                 45 
                  15.0 ± 2.45ab 
                 439 
                 451 
                 495 
                  1.65 ± 0.106a 
               
               
                 low 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 temperature + 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 aeration 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 8  
     Transformation Ratio by Various Pollen Pretreatments 
       [0027]    A transformation vector carrying a bar gene was employed in the transformation, which was capable to make transformants to resist the herbicide basta. Therefore, the transformants were preliminarily screened by spraying the herbicide. Corn seeds after transformation were sowed in plots, and sprayed with 2% herbicide basta at a 5-6 leaf stage. Refer to Table 8 for results of herbicide screening for genetically modified seedlings. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
               
                   
               
               
                 Herbicide resistant rate after pollination of different treatments of pollen 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                 Seeding 
                 Seeding 
               
               
                   
                   
                 emergence 
                 emergence 
               
               
                 Treatment 
                 Sowing number 
                 number 
                 rate (%) 
               
               
                   
               
               
                 Ultrasonication 
                  59 (19, 20, 20) 
                  51 (15, 17, 19 
                  86.44b 
               
               
                 Ultrasonication + 
                 215 (70, 72, 73) 
                 186 (58, 63, 65) 
                  86.51b 
               
               
                 low temperature 
               
               
                 Ultrasonication + 
                 222 (76, 80, 66) 
                 197 (68, 69, 60) 
                  88.74b 
               
               
                 aeration 
               
               
                 Ultrasonication + 
                 315 (93, 110, 112) 
                 263 (76, 95, 92) 
                  83.49b 
               
               
                 aeration + low 
               
               
                 temperature 
               
               
                 CK (untransformed) 
                  91 (30, 30, 31) 
                  91 (30, 31, 31) 
                 100a 
               
               
                   
               
             
          
           
               
                   
                   
                 Herbicide 
                   
                   
               
               
                   
                 Herbicide 
                 resistant 
                 PCR 
                 PCR 
               
               
                   
                 resistant 
                 strain rate 
                 positive 
                 positive 
               
               
                 Treatment 
                 strain 
                 (%) 
                 strain 
                 strain rate 
               
               
                   
               
               
                 Ultrasonication 
                  25 (8, 11, 6) 
                 49.02a 
                 10 
                 19.6a 
               
               
                 Ultrasonication + 
                  97 (32, 30, 35) 
                 52.15a 
                 36 
                 19.4a 
               
               
                 low 
               
               
                 temperature 
               
               
                 Ultrasonication + 
                 103 (33, 38, 32) 
                 52.28a 
                 41 
                 20.8a 
               
               
                 aeration 
               
               
                 Ultrasonication + 
                 134 (40, 51, 43) 
                 51.0a 
                 55 
                 20.9a 
               
               
                 aeration + low 
               
               
                 temperature 
               
               
                 CK 
                 0 
                  0b 
               
               
                 (untransformed) 
               
               
                   
               
               
                 Note: 
               
               
                 the resistant plant rate was the number of herbicide resistant plants divided by the number of total seedlings. The PCR positive plant rate was the number of PCR positive plants divided by the number of total seedlings. 
               
             
          
         
       
     
         [0028]    As shown in Table 8, T 0 -generation seeds obtained after various pretreatments did not show significant difference on the ratio of the herbicide resistant plants. Leaves of individual herbicide resistant plants were collected at the 5-leaf stage and total DNA was extracted for PCR analysis. The results showed that about 20% PCR positive plants were obtained no matter which method was used for pollen treatment. Southern hybridization confirmed that all the PCR positive plants were transformants, which indicates that the exogenous gene had been introduced into the recipient plants. 
         [0029]    The above results indicated that the improved method did not produce a significant influence on the transformation ratio while remarkably improved the seed setting rate after pollination, therefore, the number of transformants obtained from each pollination was enhanced remarkably. 
         [0030]    While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects, and therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention.