Abstract:
The present invention relates to a method for breeding a novel plant variety of high fertility  xBrassicoraphanus  with stabilized seed productivity, and discloses a method for breeding a novel plant variety of  xBrassicoraphanus , which involves performing a mutagen treatment on  xBrassicoraphanus  seeds by treating the  xBrassicoraphanus  seeds with 0.01 μg/L of mutagen NMU, sowing the  xBrassicoraphanus  seeds, gathering the  xBrassicoraphanus  seeds, sowing the  xBrassicoraphanus  seeds again, performing artificial crossing in an indoor area to check seed productivity, and checking the uniformity of the variety using an AFLP primer.

Description:
RELATED APPLICATIONS 
       [0001]    This application claims priority from Korean Patent Application No. 10-2009-0044969 filed on Jun. 23, 2009 in the Korean Intellectual Property Office, the entire disclosure of which is incorporated herein by reference. 
       BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    The present invention relates to a novel plant variety of  xBrassicoraphanus  with stabilized seed productivity. 
         [0004]    2. Description of the Related Art 
         [0005]    The present inventors have cultivated ‘ xBrassicoraphanus  (genus, genome: aarr, 2n=38)’, which is an intergeneric hybrid plant between  Brassica campestris  and  Raphanus sativus , and acquired a patent right for the same (Korean Patent No. 0492518, entitled “Novel modified variety of  xBrassicoraphanus  and a method for breeding the same,” patented on May 23, 2005). Thereafter, it was confirmed by the inventors that the foregoing plant may be widely used for sprout vegetables, baby vegetables, wrapping vegetables, young radish vegetables, vegetables for preparation of kimchi, or as a raw material for health supplements. However, common two problems involved in remote-crossed hybrids were not completely overcome. 
         [0006]    Among such problems, one is low seed productivity. In fact, the proposed plant, that is,  xBrassicoraphanus  produces plenty of seeds to make a profit in an aspect of agricultural business (400 to 600 L/ha). However, although about 10 seeds per pod may be generated, only 1 to 3 seeds are ripe while the others atrophy (or are degraded), thus reducing the quantity of seeds to be obtained. The other problem is to be that traits are continuously segregated, thus decreasing uniformity. Although the uniformity is to be a level with no difficulty in agricultural utilization, features of the seed are gradually and continuously segregated through successive generations thereof. From this, there was found a problem in that different traits must thin out during seed gathering and cultivation. 
       SUMMARY OF THE INVENTION 
       [0007]    Therefore, in order to overcome the foregoing problems of the related art, the present invention is directed to breeding and providing a novel plant lineage (‘line’) of which all seeds in a pod are ripe without degradation thereof and which ensures uniformity sufficient to use the seeds as a parent of first filial generation (F1) hybrid variety according to an appropriate breeding method, like general plants, in view of botanical aspect. 
         [0008]    In order to accomplish the above object, the present invention provides a breeding method including: mutagen treatment (that is, mutation); gathering seeds; artificial crossing to check seed productivity; and checking the uniformity of the variety using an AFLP primer. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS  
         [0009]    The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: 
           [0010]      FIG. 1  is a photograph showing a condition of seeds in hydrothecae in each line after gathering seeds of  xBrassicoraphanus  in field; and 
           [0011]      FIG. 2  is photographs showing cultivation of a novel plant variety of  xBrassicoraphanus  to assay horticultural characteristics thereof. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0012]    Experimental Material and Procedure 
         [0013]    Microspore mutation was adopted as a breeding method of a novel plant variety of  xBrassicoraphanus . For microspore culture developed by the present inventors, a rate of blastogenesis was only 0.9 blastocyst per Petri dish (Hong and Lee, 1995). Thus, the foregoing method is unsuitable for inducing mutation. Accordingly, an improved culturing method to produce embryos in larger quantities was established and an experiment to treat the produced embryos with of 0.01 μg/L of a mutagen MNU was executed. Among numerous experiments, a particular one to produce a large quantity of embryos was used and the produced embryos were induced into a plant and a successive one thereof was subjected to seed gathering. As a result, compared to existing  xBrassicoraphanus , individuals with a large seed gathering amount were observed in artificial crossing in an indoor area (see, Tables 15-23, pp. 31-38; Agricultural Research and Development Promotion Center (ARPC) subject, “Improvement of major traits of a novel vegetable,  xBrassicoraphanus  and all year round production thereof as a wrapping vegetable,” final report (2006)). NMU(n-nitroso n-methyl urethane) has 132 of molecular weight and a rapid half life and decomposes by light and so it is produced and treated just before the microspore culture. That is, a solution containing 100,000 microspores per 1 ml culture solution is prepared first. Thereafter, immediately, within the clean bench in which a light is turned off, the concentration of NMU is diluted by the processes as below and then added into the culture solution so that it&#39;s concentration become 0.01 μm. Then, the division of the culture solution is performed into a petridish with a diameter of 60 mm by 2.5 ml and then the divided culture solution is cultured by a general microsphore culture process. That is, during the treatment, NMU does not wash and is cultured at it is. 
         [0014]    The concentration dilution process of NMU: 
       −1M NMU=132 ml NMU/L H 2 O 
       [0015]    −10 mM(×100 dilution) solution production=1.32 ml/L
 
∴0.132 ml=132 μ1=132p/100 ml H 2 O
 
−0.1 mM(×100 dilution) solution production=10 mM solution 1 ml/100 mL H 2 O
 
−1.0 μM(x dilution) solution production=0.1 mM solution 1 ml/100mL H 2 O
 
0.1 μM NMU solution of 1 ml is mixed with the culture solution of 100 mL so that its concentration is adjusted by 0.01 μM and used.
 
         [0016]    Under the inspiration of the foregoing experimental results, a number of intensive and extensive studies have been conducted since 2007. As a result, for four (4) individuals showing a large seed gathering amount in 2007, successive ones were subjected to artificial crossing in an indoor area to identify seed production abilities thereof In an autumn season, they were cultivated in a package to assay uniformity thereof Next, the foregoing lines were cultivated in fields in 2008, to identify seed production abilities in natural environments. Since it was deemed that visual inspection of the uniformity of each of the lines only may be insufficient, comparison assay was performed through amplified fragment length polymorphism (AFLP) using nine (9) primers such as—AAC/M-CAA to CGG. 
         [0017]    Identification and Assay of Uniformity and Seed Productivity 
         [0018]    In an artificial crossing test in an indoor area to identify seed productivity, seeds were harvested in large quantities from each line treated using a mutagen, which were substantially similar to the previous year (TABLE 1). 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Number 
                   
                   
                   
               
               
                   
                   
                 of 
                 Number 
                 Number of 
                   
               
               
                 Systematic 
                 Systematic 
                 hybrid- 
                 of 
                 seeds per 
               
               
                 name 
                 classification 
                 ization 
                 seeds 
                 hybridization 
                 Note 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 BB #1 
                 Preserved line 
                 274 
                 106 
                 0.39 
                 Artificial 
               
               
                   
                   
                   
                   
                   
                 anthesis 
               
               
                   
                   
                   
                   
                   
                 pollination 
               
               
                 BB #11 
                 Mutation line 
                 274 
                 881 
                 3.2 
               
               
                 BB #12 
                 ″ 
                 379 
                 1118 
                 3.0 
               
               
                 BB #13 
                 ″ 
                 227 
                 781 
                 3.4 
               
               
                 BB #14 
                 ″ 
                 231 
                 1009 
                 4.37 
               
               
                 BB #50 
                 ″ 
                 229 
                 786 
                 3.43 
               
               
                   
               
             
          
         
       
     
         [0019]    As a result of cultivating the foregoing lines in autumn, they exhibited remarkably improved uniformity, compared to existing lines. Therefore, in the next year, four new lines as well as two existing lines were cultivated in the same field and compared to each other in respects to seed production ability under the same conditions and in natural environments (see TABLES 2 and  FIG. 1 ). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Comparison of seed gathering ability in field between different 
               
               
                 xBrassicoraphanus lines 
               
             
          
           
               
                   
                 Number of seeds 
               
             
          
           
               
                   
                 Systematic 
                 Number of 
                 Total 
                 Per 
                 Per 
                   
               
               
                   
                 classi- 
                 commercial 
                 amount 
                 hydrothecae 
                 plant 
                 10a 
               
               
                 system 
                 fication 
                 individuals 
                 (mL) 
                 (ea) 
                 (mL) 
                 (L) 
               
               
                   
               
             
          
           
               
                 BB #4 
                 Preserved 
                 21 
                 630 
                 1.6 
                 30.0 
                 60.0 
               
               
                   
                 line 
               
               
                 BB #1 
                 Preserved 
                 39 
                 1.140 
                 2.3 
                 29.2 
                 58.5 
               
               
                   
                 line 
               
               
                 BB #11 
                 Mutation 
                 30 
                 1.790 
                 7.6 
                 59.7 
                 119.3 
               
               
                   
                 line 
               
               
                 BB #12 
                 Mutation 
                 40 
                 3.280 
                 8.8 
                 82.0 
                 164.0 
               
               
                   
                 line 
               
               
                 BB #13 
                 Mutation 
                 ″ 
                 2.810 
                 7.6 
                 70.3 
                 140.4 
               
               
                   
                 line 
               
               
                 BB #14 
                 Mutation 
                 ″ 
                 2.140 
                 8.0 
                 53.5 
                 107.0 
               
               
                   
                 line 
               
               
                   
               
             
          
         
       
     
         [0020]    From the foregoing, a production amount of each of existing lines BB 1 and 4 was about 60L in respects to a total production amount of 10 a , and substantially similar to test results in the art. On the other hand, each of four lines treated using mutagens, that is, BB 11, 12, 13 and 14, respectively, exhibited a production amount of 100L or more. 
         [0021]    Specifically, BB 12 produced about 160L or more, which is substantially similar to an amount of fixed species of  Raphanus sativus  or  Brassica campestris . The reason for such results is found from  FIG. 1  to be because seeds in the pods were almost not degraded but ripe. For BB 1 and 4, most seeds were degraded in brown, while BB 12 showed very little degradation of seeds and most of the seeds were still green. As such, even it was a remote-crossed hybrid, seed productivity was improved to a level completely identical to existing plants. That is, one of the problems of the remote-crossed hybrid was successfully solved. 
         [0022]    Although the second problem (of the remote-crossed hybrid), that is, low uniformity is also concerned, it was found that the uniformity was noticeably increased in package cultivation executed twice, compared to existing lines. More particularly, in outward appearance, the foregoing exhibited substantially similar uniformity to F1 hybrid of  Raphanus sativus  or  Brassica campestris . In order to obviously demonstrate such results, AFLP technique was applied. In the case where all commercial individuals of one line are treated through electrophoresis by AFLP, these will exhibit the same band if they have the same genes. On the other hand, if there is an individual having even one different gene, the individual may show a different band from those of other individuals, that is, a polymorphic band. AFLP was executed on the basis of the foregoing theory and, as expected, BB 4 deemed to have the lowest uniformity was identified to have a polymorphic band rate of 5.4%, which is the highest level among them. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Verification of purity of xBrassicorphanus line by AFLP 
               
             
          
           
               
                   
                 AFLP bands 
                   
               
             
          
           
               
                   
                 Number of 
                   
                 Polymorphism 
                   
               
               
                 Systematic 
                 individuals for 
                   
                 between 
                 Polymorphic 
               
               
                 name 
                 verification 
                 Total 
                 individuals 
                 band rate (%) 
               
               
                   
               
               
                 BB #4 
                 21 
                 919 
                 50 
                 5.4 
               
               
                 BB #1 
                 39 
                 672 
                 30 
                 4.5 
               
               
                 BB #11 
                 30 
                 731 
                 31 
                 4.2 
               
               
                 BB #12 
                 40 
                 673 
                 33 
                 4.9 
               
               
                 BB #13 
                 40 
                 673 
                 25 
                 3.7 
               
               
                 BB #14 
                 40 
                 729 
                 29 
                 4.0 
               
               
                   
               
             
          
         
       
     
         [0023]    However, BB 1 also exhibited the polymorphic band rate of 4.5%, although the above line was considered to have the highest uniformity among existing preserved lines. Further, lines having remarkable improvement in seed productivity among those obtained after mutagen treatment exhibited the seed productivity ranging from 4.0% to 4.9%, which is substantially less than 5.0%. Studies upon use of AFLP techniques as a reference indicating uniformity of (plant) varieties have been executed. Although F1 hybrid with highest uniformity and both parents thereof have different polymorphic band rates along with materials and test equipment, it is known that they mostly show a seed productivity up to 5%. Accordingly, it can be seen that BB 11, 12, 13 and 14 as well as BB 1 may have improved uniformity, which is substantially identical to outward appearance thereof (see  FIG. 2 ). Consequently, the second problem, that is, low uniformity was completely overcome. 
         [0024]    With regard to four (4) lines presumed to be mutated by a mutagen, there may be a question how all traits are fixed in a first modified generation (M1) and become uniform. The foregoing is resulted from experimentations based on a report of Swanson et al. (1989) disclosing that fixed lines having herbicides tolerance have been bred using a mutagen in microspore culture of a rape. That is, the reason for the foregoing question is to be that microspores as haploidic cells and/or microspore-derived haploids show mutation occurred in a division period by mixing a mutagen in a culture medium for incubating microspores and using the mixed medium, which in turn grows into embryos and becomes diploids while being differentiated into a plant. 
         [0025]    As described above, the present invention attains excellent effects in that a remote-crossed hybrid plant of  Brassica campestris  and  Raphanus sativus , that is,  xBrassicoraphanus  exhibits stabilized seed productivity by adopting microspore mutation as a breeding method, thus producing a novel and improved line with high fertility. Moreover, the present invention exhibits additional excellent effects in that alternative novel and modified plant lines of  xBrassicoraphanus  may be bred according to the same breeding method. Therefore, the present invention may be remarkably useful for horticultural plant breeding industrial applications. 
         [0026]    As set forth above, the present invention attains beneficial effects such as improvement in seed productivity since all seeds in each pod of  xBrassicoraphanus  are completely ripe without degradation thereof, as well as maintaining uniformity without segregation of traits.