Patent Publication Number: US-2004045047-A1

Title: Method for plant gene transfering by electrical shock and ovary injection

Description:
FIELD OF THE INVENTION  
       [0001] The present invention relates to a technique about gene transferring, and more particularly, to a method for plant gene transferring by electrical shock and ovary injection.  
       BACKGROUND OF THE INVENTION  
       [0002] A conventional way of plant gene transferring includes the following steps: (1) cloning the gene or genes from proper sources and recombining the cloned gene(s) with proper DNA vector (plasmid) used as a gene carrier; (2) transferring DNA carrier harboring the cloned gene(s) into plant cells by proper methods so as to form transgenic cell, including particle gun bombardment method, Agrobacterium-mediated method, microinjection, electroporation, virus-mediated method, and PEG method (polyethylene glycol method) etc.; (3) the above mentioned methods all require the aid of plant tissue culture to transfer the transgenic cells by way of regeneration into transgenic plants having roots, stems, and leaves; (4) examining and screening for the successful and good transgenic plants. The above mentioned methods all have their own characteristics, however, they also have some common shortcomings which are not ready for being overcome. One of the shortcomings is that the efficiency of transferring is not always satisfied. Usually a transgenic process is more likely to be successful, if a tissue culture technique with high regeneration efficiency is available. Unfortunately, in many of the cases the tissue culture technique has not yet been well established so far. This is one of the main reasons making the success of gene transfer so rare in many crops. From this point of view, a new approach of transgenic process being able to enhance transferring efficiency and not relying on the aid of tissue culture will be very valuable and useful.  
       SUMMARY OF THE INVENTION  
       [0003] The object of the present invention is achieved by (1) at a suitable interval of time after pollination, injecting the DNA containing exotic gene(s) into the locule of plant ovary according to a series of operation steps to let DNA surround the ovule(s)inside the locule; (2) by using a controlling device to proceed an electrical shock to the ovary under the controlled voltage, current and timing, in order to form a temporary electric field in the ovary with negative electrode in the center and positive electrode distributing on outside arounding the ovary walll. The injected DNA which is negatively charged in nature will move outward from the central locule toward the directions of ovary wall so as to increase the opportunity to enter into the ovule, and further into the egg cell. This makes DNA recombination between exotic DNA and the fertilized egg&#39;s chromosome (i.e. gene tranfer) possible. The efficiency of gene transfer can be increased.  
       [0004] The present invention will become more obvious from the following description when connected with the accompanying drawings which show, for purposes of illustration only, a preferred embodiment in accordance with the present invention. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0005]FIG. 1 is a flow chart of the steps of the method of the present invention;  
     [0006]FIG. 2 shows parts of a flower of a plant, and  
     [0007]FIG. 3 shows the steps of cutting, penetrating, and injection;  
     [0008]FIG. 4 shows another way of the step of the penetrating; and  
     [0009]FIG. 5 shows the step of insertion, enclosing and electrical conduction of the present invention. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
     [0010] Referring to FIGS.  2  to  5 , the method for transferring gene of plant of the present invention comprises six steps which are cutting step  10 : cutting the stigma off at a suitable interval of time after pollination; penetrating step  20 : making a tiny passage or tunnel by using an injection needle to penetrate from style toward locule; injection step  30 : injecting the DNA from outside into the locule of ovary via the passage; insertion step  40 : inserting electrically conductive piece into the passage to be used as a negative electrode ; enclosing step  50 : enclosing the wall of the ovary with electrically conductive layer so as to form a positive electrode; electrical conduction step  60 : connecting the positive and negative electrodes to an electric device and proceeding an electrical shock to create a special electric field. The six steps are operated continuously.  
     [0011] For the cutting step  10 , referring to FIG. 2 which shows the flower of plant and includes stigma  11 , style  12 , ovary  13 , locule  131 , ovule  132 , placenta  133 , floral stalk  14 , receptacle  15 , calyx  16 , petal  17 , stamens  18 . After being pollinated, when the pollen tubes reach the ovule(s), using a certain tool to cut off the stigma  11  at the position of the style  12  as shown.  
     [0012] For the penetrating step  20 , a tiny passage  21  is defined between the style  12  and the locule  131  by using an injection needle to penetrate into the cutting place of the style  12  and toward the ovary  13  till the locule as shown in FIG. 3, or going further through the locule  131  to penetrate the ovary wall and make an opning  34 on it as shown in FIG. 4, and then pulling the needle out to define the tiny passage  21 .  
     [0013] For the injection step  30 , the DNA  31  is injected into the locule  131  by a needle  33  of the injection device  32  via the passage  21  to allow the DNA  31  to surround the ovule  132 . If the passage  21  is defined by penetration as shown in FIG. 4, some DNA  31  could flow out from the opening  34 , but this does not interrupt the result of gene transfer according to repeated tests.  
     [0014] For the insertion step  40 , inserting an electrically conductive piece  51  such as a Platinum wire, into the passage  13  to form the negative electrode.  
     [0015] For the enclosing step  50 , an electrically conductive layer  51  is enclosed the ovary  13  which is to make a circular positive electrode.  
     [0016] For the electrical conduction step  60 , connecting the positive and negative electrodes as defined in steps  40  and  50  to a special electronic controlling device  61  which will give an electrical shock to form a special electrical field determined by the combination of certain voltage, current and timing.  
     [0017] DNA  31  is negatively charged by its chemical nature and will move outward positive electrode in the electric field. Roughly said, in this situation the DNA molecules inside locule will move from the center to the surroundings. The opportunity for DNA to enter the egg cell inside ovule  132  is increased, especially when the egg-sperm fusion (i.e. fertilization) occurs if the timing is well managed. Around the time of fertilization, some of the egg&#39;s cell wall is thin and the egg cell is somewhat similar to a protoplast. This characteristics makes the DNA  31  easy to penetrate through the cell wall, especially at the moment when ferlization occurs. As a result, the possibility that exotic DNA is combined into the chromosome of the fertilized egg, i.e. gene transfer, can be increased. The transgenic fertilized egg will then become a transgenic seed through nature development, and a transgenic plant can be produced by the transgenic seed through germination and vegetative growth. The whole procedures do not need the aid of tissue culture.  
     [0018] Obviously, in this method the occurrence of gene transfer depends on 5 main factors including: 1. the existence of injected DNA  31  which contains exotic gene(s); 2. sperm cells traveling toward the egg inside the extending pollen tubes after pollination; 3. egg cell(s) inside ovule(s)  132 ; 4. the occurrence of egg-sperm fusion (i.e. fertilization) inside ovule; and 5. electric shock treatment which increases the movement of the exotic DNA  31  inside the locule. To manage all these factors in order to make gene transfer being able to occur with high possibility, an accurate timing is very important for all the steps in the method of the present invention.  
     [0019] The present invention not only achieve the purpose of gene transfer, but also includes the following five advantages:  
     [0020] 1. Increase the rate of success of plant gene transfer: the DNA is injected into the ovary at suitable time after pollination and then treated by electric shock to dramatically increase the rate of success of gene transfer.  
     [0021] 2. Reduce the cost of the research of genetic engineering: the present invention adopts the natural way of plant breeding process to achieve the purpose of gene transfer. It is simple and easy to do. Besides, it is found that using this method the efficiency is higher than the conventional ways used to transfer gene(s) into plants. This method does not need the aid of tissue culture to produce a complete transgenic plant from a transgenic cell. Such a characteristics is quite valuable and helpful for time and expenses saving, especially for those plant species in which the technique of tissue culture has not yet been well established.  
     [0022] 3. Benefit the environmental safety: The method of the present invention neither needs tissue culture technique nor rely on baterium mediation in all the procedures, therefore the problem of environmental contamination and pollution can be greatly reduced. Therte is also no damage to the eco-system, even the working procedures of this invention are all performed in the open field.  
     [0023] 4. Easy to practice and perform: Using the method of the present invention, the practice and skill of gene transfer is easy to learn and teach. Once the technique for a specific plant has been well determined and established, including the timing of DNA injection and the condition of electrical shock, the experiment of gene transfer could become a routine work and be easily performed by an assistant worker who is even without the background of genetics and molecular biology. The main part of experiment work can be done in the field or green house using a syringe of suitable size (or an injection device originally designed for gas chromatography analysis) and an electrical shock inducing device. No expensive instrument and equipment is needed.  
     [0024] 5. Particularly suitable for those plants having multiple seeds produced in one fruit, if the fruit is originated from one single pollinated flower such as tomato and watermelon: Since ovary is the basic unit to deal with in this method, this method is especially useful and valuable for those plants with multiple seeds produced in one single-flower-originated-fruit. Appraently the rate of success can be enhanced, if multiple seeds can be obtained from one single experimental treatment. The more the seeds can be obtained from one treated ovary (flower), the higher the rate of success can be expected. In agriculture, there are many crops bear multiple-seeds-containing fruits which are developed from one single pollinated flower. Their species distribute in different taxonomic families, including Orchidaceae, Cucurbitaceae, Leguminosae, Solanaceae, Rosaceae, Cruciferae, Rutaceae, Myrtaceae, Liliaceae, Passifloracae, Oxalidaceae, Vitaceae, Actinidiaceae, and Caricaceae etc. We estimate that ca. ⅔ of horticultural crops are of this type, and most of these crops still lack good tissue culture research. In fact tissue culture research even has not yet been initiated in many of these crops, and so does the genetic engineering work, therefore, the method of this invention is going to have a huge potential for application in many crops&#39;genetic improvement.  
     [0025] While we have shown and described the embodiment in accordance with the present invention, it should be clear to those skilled in the art that further embodiments may be made without departing from the scope of the present invention.