Abstract:
This invention relates to a hybrid breeding technology for crop plants in the family Brassicaceae characterized in that F 1  seed is produced by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible line. By the breeding technology of this invention, selfed seeds contamination can be prevented in F 1  breeding and F 1  seed production of crop plants in the family Brassicaceae and, moreover, the cost of seed production can be reduced through an improved seed production efficiency.

Description:
BACKGROUND OF THE INVENTION 
     This invention relates to a hybrid breeding method for crop plants in the family Brassicaceae. 
     The invention relates to a breeding method for an F 1  variety which, particularly in rape, is double-low (low erucic acid-low glucosinolate content) and improved in yield, oil content and quality, and disease and pest resistance. 
     Referring to rapeseed (Brassica napus, n=19), which is self-compatible, the utilization of F 1  has not been made to this day partly because the production of F 1  seed through utilization of self-incompatibility is not feasible and partly because a stable male sterile line which is not affected by temperature or day length and the fertility restoring gene for the male sterility have not been discovered as yet. As to other crop plants in the family Brassicaceae, too, several plants and varieties are unstable in the expression of self-incompatibility in the production of F 1  by the utilization of self-incompatibility and there is the problem that F 1  is occasionally contaminated with selfed seeds (hereinafter referred to as &#34;intra&#34;) as well as the problem that the cost of F 1  seed production inclusive of the cost of bud pollination for parent seed production is high. 
     Meanwhile, labor conservation is a major objective in the fields of stock seed production, F 1  seed production, seed cleaning and cultivation and, as one aspect of this recent trend, the requirement in regard to the purity of seed is getting more and more stringent. 
     SUMMARY OF THE INVENTION 
     The object of this invention is to provide a hybrid breeding method of improved efficiency which helps to prevent intra contamination and contributes to cost reduction in the production of F 1  seed from crop plants in the family Brassicaceae. 
     For the F 1  breeding of rape, the inventors of this invention envisaged the development of lines possessing stable male sterility and fertility restoring genes for male sterility and the introduction of self-incompatible genes from related species and conducted a large amount of research. As a result, the inventors discovered a combination of cytoplasm with male sterility showing a very stable expression of male sterility and fertility restoration with a fertility restoring gene and utilizing the combination developed an F 1  rape variety  No. 9122! of spring type which is double-low and promises an increased yield. Furthermore, for cost reduction through increased seed yield, the inventors developed an F 2  variety of said rape, namely  T-410!. The inventors further developed an F 1  variety  No. 9123!, using a new female parent derived from a B line which was excellent in disease resistance and seed production efficiency. In addition, by introducing several kinds of self-incompatible genes, the inventors succeeded in the development of rape lines possessing various excellent characters. Then, the inventors did further research for the prevention of intra contamination in the production of F 1  seed and the reduction of seed production cost and have developed a highly efficient hybrid breeding method for crop plants in the family Brassicaceae, which is based on a combination of male sterility and self-incompatibility (the selection and development of lines compatible with carbon dioxide treatment). 
     A first hybrid breeding process for crop plants in the family Brassicaceae in accordance with this invention is characterized in that F 1  seed is produced by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible line (FIG. 13). 
     This process is most effective for the prevention of intra contamination associated with unstable self-incompatibility and a large difference in flowering time between the parents, among other causes. When the self-incompatibility of the male parent is unstable, the male parent is cut off and the seed is not harvested from the male parent for the prevention of intra contamination. When the self-incompatibility of the male parent is stable, there is no seed formation on the male parent so that the male parent need not be cut off but both the male and female parents can be reaped indiscriminately, with the result that a remarkable cost reduction is realized. Thus, mix-sowing of male and female parents, mechanical sowing and mechanical harvesting are made possible. This process is useful for radish, cabbages and Chinese cabbages. 
     A second process according to this invention is characterized in that F 1  seed is obtained by crossing the female parent of a male sterile line introduced self-incompatibility with the male parent of a self-incompatible or self-compatible line possessing fertility restoring gene (FIG. 17). 
     The first process is not suitable for the breeding of crop plants which their seeds were utilized such as rape, for F 1  shows sterility and self-incompatibility. The second process, which overcomes this drawback, is characterized in that a fertility restoring gene for restoration of pollen fertility in F 1  and a self-incompatible or self-compatible gene are introduced into the male parent. While the advantages of this second process are similar to those of the first process, it has the additional advantage that because of the consequent restoration of pollen fertility, the utilization of F 2  becomes feasible, in particular, and a still greater seed yield and a more remarkable cost reduction can be realized. Particularly the reduction of seed production cost is a matter of top priority in rape F 1  breeding and this process as well as a fourth process to be described below is a very effective technique and these processes can be selectively used according to the characteristics and F 1  combining abilities of the lines. This process is particularly effective for the utilization of F 2  of crop plants in the family Brassicaceae. 
     A third process of this invention is characterized in that F 1  seed is obtained by crossing the female parent of a male sterile line introduced self-compatibility with the male parent of a self-compatible line or the male parent of a self-incompatible line (FIG. 32). 
     This process is effective for crop plants having no self-incompatibility or crop plants which are dominantly self-incompatible but have strong self-compatibility or for the development of F 1  varieties of these lines. For example, this process is effective for karashina (mustard plant), takana (leaf mustard), radish, cabbages, Chinese cabbages and so on. 
     A fourth process according to this invention is characterized in that F 1  seed is obtained by crossing the female parent of a male sterile line introduced self-compatibility with the male parent of a self-incompatible line possessing fertility restoring gene (FIG. 36). 
     In the second process mentioned above, the female parent is self-incompatible and male sterile but when a self-compatible and male sterile line is used as the female parent as in this process, the F 1  breeding of highly self-compatible crops and varieties, reduction of F 1  seed production cost, and improved F 2  seed production efficiency can be realized. Particularly, when the number of self-incompatible genes is increased for four-way crossing, the effect of open flower crossing in the utilization of F 2  is remarkable and the seed production capacity is increased. This process is effective for rape in the main. Moreover, even in single crossing or three-way crossing, too, the incorporation of a self-incompatibility in a male parent possessing fertility restoring gene dispenses with the need for cutting off the male parent, thus enabling omnibus reaping. The method of crossing can be selected according to the characteristics and F 1  combining ability of the lines. 
     In accordance with the breeding technology of this invention, intra contamination in the F 1  breeding and F 1  seed production of crop plants in the family Brassicaceae can be prevented and, moreover, the cost of seed production can be reduced through an improved seed production efficiency. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a diagrammatic representation of the breeding process according to an embodiment of this invention; 
     FIG. 2 is a diagrammatic representation of the breeding process according to another embodiment of this invention; 
     FIG. 3 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 4 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 5 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 6 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 7 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 8 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 9 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 10 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 11 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 12 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 13 is a diagrammatic representation of the breeding method according to an embodiment of this invention; 
     FIG. 14 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 15 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 16 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 17 is a diagrammatic representation of the breeding method according to another embodiment of this invention; 
     FIG. 18 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 19 is a diagrammatic representation of a part of the breeding process according to the same embodiment corresponding to FIG. 18, which is sequential to the bottom of FIG. 18; 
     FIG. 20 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 18 and 19, which is sequential to the right of FIG. 18; 
     FIG. 21 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 18-20, which is sequential to the bottom of FIG. 20 and the right of FIG. 19; 
     FIG. 22 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 23 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 22, which is sequential to the bottom of FIG. 22; 
     FIG. 24 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 22 and 23, which is sequential to the right of FIG. 22; 
     FIG. 25 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 22-24, which is sequential to the bottom of FIG. 24 and the right of FIG. 23; 
     FIG. 26 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 27 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 26, which is sequential to the bottom of FIG. 26; 
     FIG. 28 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26 and 27, which is sequential to the right of FIG. 26; 
     FIG. 29 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-28, which is sequential to the bottom of FIG. 28 and the right of FIG. 27; 
     FIG. 30 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-29, which is sequential to the right of FIG. 28; 
     FIG. 31 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 26-30, which is sequential to the bottom of FIG. 30 and the right of FIG. 29; 
     FIG. 32 is a diagrammatic representation of the breeding method according to still another embodiment of this invention; 
     FIG. 33 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 34 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 35 is a diagrammatic representation of the breeding process according to still another embodiment of this invention; 
     FIG. 36 is a diagrammatic representation of the breeding method according to still another embodiment of this invention; 
     FIG. 37 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 38 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 37, which is sequential to the bottom of FIG. 37; 
     FIG. 39 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 37 and 38, which is sequential to the right of FIG. 37; 
     FIG. 40 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 37-39, which is sequential to the bottom of FIG. 39 and the right of FIG. 38; 
     FIG. 41 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 42 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 41, which is sequential to the bottom of FIG. 41; 
     FIG. 43 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 41 and 42, which is sequential to the right of FIG. 41; 
     FIG. 44 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 41-43, which is sequential to the bottom of FIG. 43 and the right of FIG. 42; 
     FIG. 45 is a diagrammatic representation of a part of the breeding process according to still another embodiment of this invention; 
     FIG. 46 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIG. 45, which is sequential to the bottom of FIG. 45; 
     FIG. 47 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45 and 46, which is sequential to the right of FIG. 45; 
     FIG. 48 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-47, which is sequential to the bottom of FIG. 47 and the right of FIG. 46; 
     FIG. 49 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-48, which is sequential to the right of FIG. 47; 
     FIG. 50 is a diagrammatic representation of a part of the breeding process according to the embodiment corresponding to FIGS. 45-49, which is sequential to the bottom of FIG. 49 and the right of FIG. 48. 
    
    
     In FIGS. 1-4, FIGS. 10-12, FIGS. 14-16, FIGS. 18-31, FIGS. 33-35 and FIGS. 37-50, ∘ stands for F (male fertility). In FIGS. 5-9, ∘ stands for an individual. In FIGS. 1-12, FIGS. 14-16, FIGS. 18-31, FIGS. 33-35 and FIGS. 37-50, ∘ stands for MS (male sterility), x for Cross (crossing), Δ for r (fertility restoring gene), ,  and ▴ for test individuals, □ for individual seed production, ▪ for mass seed production, S a , S b , S d  and S e  for self-incompatible genes. In FIGS. 13, 17, 32 and 36, S 1 , S 2 , S 3  and S 4  stand for self-incompatible genes, S f1  and S f2  for self-compatible genes, ms for cytoplasm with male sterility, r for fertility restoring gene, and * for seed production by CO 2  treatment or bud pollination. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The breeding processes according to this invention and the advantages of the breeding technology of the invention are now described in detail with reference to examples. 
     1. Breeding of rape F 1  variety  No. 9122! by the utilization of male sterility (FIGS. 1-3) 
     Breeding process: The F 1  obtained by the utilization of male sterility was slightly unstable in the expression of male sterility in the winter variety and was difficult to breed. Therefore, the breeding of a spring variety with stable male sterility was attempted. 
     1) Development of AB line  60To-AB! (FIG. 1) 
     A selected line  60To! could be developed as a maintainer for the male sterile line  MS-N1! discovered from among  N-1!s which were spring varieties in 1987. This was later made  60To-B!, subjected to continuous backcrossing, individual selection during 5 generations and, then, mass selection. Selections were carried out, with emphasis on spring growing habit and double-low feature, in regard to the size and shape of the pod, plant posture and disease resistance, among others. 
     2) Development of C line  62WeB-C! (FIG. 2) 
     The fertility restoring gene for  MS-N1!-derived cytoplasm with male sterility was discovered in the winter variety  IM line! and named  IM-B!. In 1988, this was crossed with a spring double-low line  62We!. Then, with the homozygotic presence of a fertility restoring gene being confirmed, selection breeding was carried out, with emphasis on spring growing habit and double-low feature, in regard to the size and shape of the pod, plant posture, and alignment in flowering time with the male sterile AB line on the female parent side. 
     3) Development of F 1   No. 9122! (FIG. 3) 
     By testing a number of F 1  combinations, the parent lines with the highest combining ability were selected from said AB and C lines and F 1   No. 9122! was developed. 
     The result of investigation of the seed yield of this F 1   No. 9122! is shown in Table 1. It is apparent from the table that the seed yield of F 1   No. 9122! in 1992 was fairly high as compared with the control variety  OAC Triton!. The increased seed yield of F 1   No. 9122! contributes to a reduced cost of seed production of F 2   T-410!. 
     
                                           TABLE 1__________________________________________________________________________Comparison of seed yields of F.sub.1  No. 9122! and control rapeTakii Plant Breeding and Experiment Station, Kosei-cho, Koka-gun, ShigaPrefecture(Sowing: November 25, 1991; investigation: July 3, 1992)                               Yield,                                     SeedPlant-      Amount of                 Flowering                      Flowering                               l/10 a                                     produc-Spring,ing   Number       Seed seed per                 began                      ended                           Degree                               (on a 40                                     tion1992 area   of  produced            plant                 (month/                      (month/                           of bee                               thousand                                     indexVariety(m.sup.2)   plants       (l)  (ml) date)                      date)                           visit                               plant basis)                                     (%)__________________________________________________________________________No. 91223.75   143 1.8  12.6 4/16 5/20 Excel-                               504   171(F.sub.1)                       lentOAC  7.05   272 2.0  7.4  4/17 5/20 Excel-                               294   100Triton                          lent(commonvariety)__________________________________________________________________________ 
    
     Table 2 shows the glucosinolate contents and fatty acid compositions of F 1   No. 9122!, main Canadian varieties (3 varieties), registered varieties Asaka-no-natane (registration no. Natane Norin 46) and Kizaki-no-natane (registration no. Natane Norin 47) developed at Tohoku Agricultural Experiment Station. Asaka-no-natane and Kizaki-no-natane, both of which are domestic varieties, are close to the international level in erucic acid content but are by far higher in glucosinolate content, namely, single-low. In contrast, F 1   No. 9122! can be regarded as a double-low line with its glucosinolate and erucic acid contents being both comparable to the international levels. 
     
                                           TABLE 2__________________________________________________________________________The glucosinolate contents and fatty acid compositions of F.sub.1  No.9122! and control cultivars 1992  Gluco-  sino-      Fatty acid (%)  late      Myrist-          Palmit-              Stear-                  Ole-                      Linol-                          Linolen-                               Arachidon-                                     Eicosan-                                          Behen-                                              Eruc-Cultivar  (μM/g)      ic acid          ic acid              ic acid                  ic acid                      ic acid                          ic acid                               ic acid                                     ic acid                                          ic acid                                              ic acid__________________________________________________________________________No. 9122.sup.1)  16.8      0.0 5.0 1.5 63.7                      20.4                          7.7  0.5   1.2  0.0 0.0Tobin.sup.2)  20.1      0.0 3.4 2.4 56.1                      24.4                          11.6 0.5   1.5  0.0 0.0OAC Triton.sup.2)  15.8      0.0 3.9 2.6 54.4                      22.0                          9.6  0.6   2.9  0.3 3.6Westar.sup.2)  17.0      0.0 3.9 2.8 61.6                      20.5                          7.8  0.6   2.0  0.3 0.5Asaka-no  &gt;50     4.6     61.2                      21.9                          8.7        1.3      0.2natane.sup.3)Kizaki-no  &gt;50     4.7     63.7                      18.8                          8.9        1.3      0.1natane.sup.3)__________________________________________________________________________ .sup.1) Assayed by Nippon Oil and Fat Testing Association (glucosinolate contents were determined at Takii Plant Breeding and Experiment Station) .sup.2) Determined at Takii Plant Breeding and Experiment Station .sup.3) Assayed by Tohoku Agricultural Experimental Station 
    
     2. Breeding of rape F 1   No. 9123! by the utilization of male sterility (FIG. 4) 
     For further enhancement of disease resistance, lodging resistance and seed yield of the F 1  variety  No. 9122!, a new maintainer (B line) was developed for the breeding of F 1  variety  No. 9123!. The maintainer was  2DR-B! obtained by the serial selection and breeding carried out since 1990. 
     Table 3 shows the seed yield data for F 1   No. 9122!. Table 4 shows the seed yield data for F 1   No. 9123!. In the spring of 1992, a field trial of F 1   No. 9122! was carried out in Canada. Then, in the spring of 1993, field trials of F 1   No. 9122! and  No. 9123! were carried out in the Netherlands. 
     The comparison of seed yields of F 1   No. 9122! and F 1   No. 9123! is presented in Table 5. 
     
                                           TABLE 3__________________________________________________________________________Seeds yields of rape F.sub.1  No. 9122!Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido,1991-1992        1991           1992           Seed           Seed   Line      Area           produced    Area                          producedLine   combination        (m.sup.2)           (l)  Remarks                       (m.sup.2)                          (l)  Remarks__________________________________________________________________________AB 60To-AB × 60To-B        20 10.0        230                          30.0B  60To-B    10 5.7         87 15.0C  62WeB-C   18 6.5         60 20.0F.sub.1   60To-AB × 62WeB-C        50 17.0 Field trial                       460                          50.0 Field trial in                in Canada      the Netherlands                (spring, 1992) (spring, 1993)__________________________________________________________________________ 
    
     
                                           TABLE 4__________________________________________________________________________Seeds yields of rape F.sub.1  No. 9123!Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido,1991-1992        1991           1992           Seed           Seed   Line      Area           produced    Area                          producedLine   combination        (m.sup.2)           (l)  Remarks                       (m.sup.2)                          (l)  Remarks__________________________________________________________________________AB 60To-AB × 2DR-B        -- --          9.0                          2.0B  2DR-B     -- --          4.5                          0.7F.sub.1   (60To-AB × 2DR-B) ×        -- --          9.0                          2.0  Field trial in   62WeB-C                          the Netherlands                               (spring, 1993)__________________________________________________________________________ 
    
     
                                           TABLE 5__________________________________________________________________________Comparison of seed yields of F.sub.1  No. 9122! and F.sub.1  No. 9123!Takii Naganuma Breeding Station, Naganuma-cho, Yubari-gun, Hokkaido(Sowing: May 1, 1994)  Planting Susceptibility                  Seed Seed Seed produc-Spring, 1994  area      Number of           to blackleg                  produced                       produced                            tion indexCultivar  (m.sup.2)      plants           (++ - -)                  (l)  (l/20 m.sup.2)                            (%)__________________________________________________________________________No. 9122 (F.sub.1)  20.0      1800 ∓   9.4  9.4  98No. 9123 (F.sub.1)  20.0      1800 ∓   9.6  9.6  100Westar 10.0       320*           ++     1.2  2.4  25OAC Triton  10.0       460*           +      2.2  4.4  47__________________________________________________________________________ *: The high incidence of blackleg resulted in a decreased plant population. 
    
     3. Breeding of a rape variety with introduced self-incompatibility 
     Starting with a line which was mainly spring type and double-low, breeding was performed for the purpose of introducing the self-incompatible genes of cabbages (B. oleracea, n=9) and Chinese cabbages (B. campestris, n=10). 
     The breeding processes of main 4 lines are described below. 
     1) Development of  59ReS a  ! (FIG. 5) 
     In order to introduce one of the self-incompatible genes of cabbage (factor a) into the spring type, double-low line  59Re! (B. napus, n=19), a synthetic napus (B. napus, n=19), viz. an amphidiploid, was developed from komatsuna (B. campestris, n=10) and cabbage (B. oleracea, n=9) and, further, after hybridization with nabana (B. campestris, n=10), crossing with a selected line of  59Re! was performed. The objective of crossing with nabana was as follows. Because of the use of a synthetic napus between green vernalization type cabbage and seed vernalization type komatsuna, it was considered necessary to bring them closer to spring types with weak low temperature response and strong day length response. Then, using a line selected with regard to spring habit and double-low characteristic, self-pollination was repeated 4 times to develop a rape line  59ReS a  ! having the self-incompatibility factor a from the synthetic napus. Moreover, reciprocal crossing of the line for reconversion to rape cytoplasm was also carried out. 
     2) Development of  59ReS b  ! (FIG. 6) 
     The first half of the breeding process was substantially the same as for the development of  59ReS a  ! and one of the self-incompatible genes of cabbages (factor b, different from factor a) was introduced. Here, using a selected line from rape line  59Re! as the pollen parent, crossing was carried out once and, then, using a selected line of  59Re! as the female parent, crossing was carried out twice. Thus, the conversion from komatsuna cytoplasm to rape cytoplasm was made to introduce the stability of rape phenotype and a line  59ReS b  ! of low glucosinolate content having factor b was obtained. 
     3) Development of  62WeS b  ! (FIG. 7) 
     By the same procedure as the development of  59ReS b  !, factor b was introduced into the spring type, double-low line  62We!. 
     4) Development of  H-Bi-S d  ! (FIG. 8) 
     A synthetic napus was developed from komatsuna and cabbage as an amphidiploid and crossed with nabana, and its progeny was backcrossed with the spring type, double-low rape line  62We! twice, and then a line was developed by self-pollinating and selection. On the other hand, one line of synthetic napus obtained by cell fusion between a cabbage line  ER159! and Chinese cabbage  Green Rocket 70! was crossed with rape line  LE112-82!, followed by crossing with a selected one from rape line  60To! to develop a crossing line. 
     These two lines were crossed to develop a double-low line having the factor d derived from cabbage  ER159!, self-incompatibility and improved cold resistance. 
     4. Breeding of a rape line which has both a fertility restoring gene for male sterility and a self-incompatible gene 
     1) Development of  H-Bi-S d  B! (FIG. 9) 
     By crossing three lines, viz. a line obtained by introducing a rape line  IM!-derived fertility restoring gene for rape line  MS-N1!-derived cytoplasm with male sterility into a selected one from rape line  59Re!, a selected crossing line between said synthetic napus and rape line  60To!, and a selected one from rape line  EG1-83!, a double-low rape line  H-Bi-S d  B! having a fertility restoring gene for male sterility and self-incompatibility factor d was developed. 
     5. Breeding of a line by using a combination of the male sterility with self-incompatibility in Brassicaceae plants other than rape 
     This breeding process is now described with reference to radish (Raphanus sativus, n=9) and karashina (mustard plant) (B. juncea, n=18). 
     1) Development of radish AB lines  62Z 55  -AB! and  62Z 56  -AB! (FIG. 10) 
     Referring to  62Z 55  -AB!, in case that a radish male sterile line  R-5! was first crossed with the parent line  Z 55  ! of an established radish F 1  variety which had homozygotically one incompatible gene (factor 5), in the next generation  1-2165! all the progeny was male-sterile. Therefore, using  Z 55  ! as a maintainer, continuous backcrossing was carried out. In 1991,  3-70021! was subjected to CO 2  treatment for temporary overthrow of self-incompatibility and  62Z 55  -AB! was developed by mass seed production with bees for crossing. 
     As to  62Z 56  -AB!, the breeding process up to  3-70021! in 1991 was the same as for  62Z 55  -AB! but this  3-70021! was crossed with  Z 66  ! which was substantially equivalent to  Z 55  ! genetically but had a different self-incompatible gene. Because of the different self-incompatible gene, CO 2  treatment was unnecessary in this case. Another difference from  62Z 55  -AB! was that an increased seed yield was obtained because of the hybrid vigour due to crossing with  Z 66  !. 
     2) Development of karashina (mustard plant) AB line  1PP-AB! (FIG. 11) 
     For the F 1  breeding of karashina (B. juncea, n=18) which is self-compatible, a karashina line  1PP-B! confirmed to act as a maintainer for the rape (B. napus, n=19) male sterile line  60To-AB! was selected and continuous backcrossing was initiated. For  60To-AB!, nucleus substitution using  1PP-B! was carried out. 
     3) Development of a radish self-compatible AB line  OK-AB! (FIG. 12) 
     The radish self-compatible line  OK! was found to be a maintainer providing all the progeny with male sterility for a male sterile line  R-5! (genetically a cytoplasmic male sterile line) and development of a radish self-compatible male sterile line was started in 1987. As a result,  OK-AB! was obtained in 1992. 
     The method of F 1  breeding by the combination of male sterility and self-incompatibility using the above lines is now described. 
     6. Production of F 1  seed using the female parent of a male sterile line introduced self-incompatibility and the male parent of a self-incompatible line (FIG. 13) 
     This experiment was performed on radish in which the production of F 1  seed is conventionally carried out mostly by four-way crossing utilizing self-incompatibility. Regarding radish, intra contamination is a frequent problem and, moreover, the number of seed grains per pod is small. Therefore, the seed production cost is high and a demand exists for cost reduction. The development of several lines in which the nucleus substitution of cytoplasmic male sterile line with the parent lines of F 1  was carried out, was already completed. As regards seed production of self-incompatible line, lines permitting seed production by carbon dioxide treatment were utilized. 
     1) Production of F 1  seed by single crossing (FIG. 14) 
     It was confirmed in 1987-1988 that the parent line  Z 55!  of F 1  which had already been developed acts as a maintainer (B line) for the male sterile line  R-5! and continuous backcrossing with  Z 55  ! was started. The 1991  3-70021! line with about 95% nucleus substitution (corresponding to  msS 1  ! at top left in 1 of FIG. 13) was crossed with  Z 55  ! (corresponding to  S 1  !) by carbon dioxide treatment and as a result,  62Z 55  -AB! (4-2143) was obtained in 1992. This  62Z 55  -AB! corresponds to  msS 1  ! in the center in 1 of FIG. 13. Using this line as the female parent, F 1  (corresponding to  msS 13  ! in FIG. 13) was obtained by crossing it with  TM 22  ! (corresponding to  S 3  ! in 1 of FIG. 13) which was a separately developed parent line confirmed to have an excellent F 1  -combining ability with respect to  Z 55  !. 
     2) Production of F 1  seed by three-way crossing (FIG. 15) 
     The process was substantially the same as the above production of F 1  seed by single crossing but was different in that, in 1991, the  3-70021! female parent was crossed with  Z 66  ! which was substantially equivalent to  Z 55  ! genetically but differed from the latter in the self-incompatible gene. In this case, because of the difference in incompatibility factor, CO 2  treatment was unnecessary. In 1992,  62Z 56  -AB! (4-2147) was obtained and F 1  was developed by crossing with  TM 22  ! as in the single crossing described in 1).  62Z 56  -AB! corresponds to  msS 12  ! in 2 of FIG. 13. 
     This procedure is different from the single crossing described above in 1) in that CO 2  treatment is not required and that the seed yield of  62Z 56  -AB! exceeds that of the single-crossed hybrid  62Z 55  -AB!. 
     3) Production of F 1  seed by four-way crossing (FIG. 16) 
     The female parent side was the same as that used in the three-way crossing in 2) but  TM 11  !, a line which was genetically equivalent to  TM 22  ! but had a different incompatibility factor, was added to the male parent side.  TM 22  ! corresponds to  S 3  ! in 3 of FIG. 13. Similarly,  TM 11  ! corresponds to  S 4  ! and  TM 21  ! corresponds to  S 34  !. 
     The foregoing is a description of the processes 1-3 of FIG. 13, taking radish as an example. In actual practice, for cabbages, Chinese cabbages, turnips, etc. which yield large amounts of seed per pod and are comparatively easy to increase seed yields, it is unnecessary to develop a line which is genetically equivalent but has a different incompatibility factor and the procedure 1) (single crossing) and procedure 2) (three-way crossing), both shown in FIG. 13, are suitable. For radish and other crops which are rather poor in seed yield, the procedure 3) (four-way crossing) is most suitable. 
     7. Production of F 1  seed by using the female parent of a male sterile line introduced self-incompatibility and the male parent of a self-incompatible or self-compatible line possessing fertility restoring gene (FIG. 17) 
     1) Production of F 1  seed by three-way crossing utilizing a male parent of a self-compatible line (FIGS. 18-21; FIG. 19 is sequential to the bottom of FIG. 18, FIG. 20 is sequential to the right of FIG. 18, and FIG. 21 is sequential to the bottom of FIG. 20 and the right of FIG. 19) 
     Referring to 1 of FIG. 17,  msS 1  ! corresponds to 1993  5-32008!;  S 2  ! corresponds to 1992  4-80151!;  msS 12  ! corresponds to 1993  5-82407!; and  rS f1  ! corresponds to 1992  4-80005!. 
     The male sterile line  60To-AB! (1991-3ND-42001) was crossed with  59ReS b  ! having a self-incompatible gene (factor b) 4 times since 1991 to obtain  5-32008! in 1993. This line was crossed with  59ReS a  ! (1992-4-80151) having a different self-incompatible gene (factor a) to develop the female parent of a male sterile line introduced self-incompatibility. Then, using the self-compatible male parent possessing fertility restoring gene  WeB-C! (1992-4-80005), F 1   msS 12  ·rS f1  ! was obtained. 
     2) Production of F 1  seed by three-way crossing utilizing a male parent of a self-incompatible line (FIGS. 22-25. FIG. 23 is sequential to the bottom of FIG. 22; FIG. 24 is sequential to the right of FIG. 22; and FIG. 25 is sequential to the bottom of FIG. 24 and the right of FIG. 23). 
     Referring to 2 of FIG. 17,  msS 1  !,  S 2  ! and  msS 12  ! correspond to the respective lines mentioned for the three-way crossing described in 1), and  rS 3  ! in 2 of FIG. 17 corresponds to  H-Bi-S d  B! (1992-4-84004). Because the male parent introduced self-incompatibility was used, the male parent produced no seed and omnibus cutting was possible at the production of F 1  seed, thus contributing to cost reduction. 
     3) Production of F 1  seed by four-way crossing (FIGS. 26-31. FIG. 27 is sequential to the bottom of FIG. 26; FIG. 28 is sequential to the right of FIG. 26; FIG. 29 is sequential to the bottom of FIG. 28 and the right of FIG. 27; FIG. 30 is sequential to the right of FIG. 28; and FIG. 31 is sequential to the bottom of FIG. 30 and the right of FIG. 29) 
     The female parent line was the same as used in the three-way crossings in 1) and 2) above, but a different male parent line was used. Referring to 3 of FIG. 17,  rS 3  ! corresponds to  H-Bi-S d  B! (1992-4-84004),  rS 4  ! corresponds to  H-En-S e  B! (1992-4-84010), and  rS 34  ! corresponds to 1992  4-31201!. Thus, 1993  5-82407! was crossed with 1992  4-31201! to develop F 1 . By the combination of some self-incompatibility factors, not only the seed production capacity of F 1  was increased but also the utilization of F 2  was facilitated. By this procedure, mass seed production and cost reduction can be realized. 
     8. Production of F 1  seed using the female parent of a male sterile line introduced self-compatibility and the male parent of a self-compatible line or the male parent of a self-incompatible line (FIG. 32) 
     Karashina (B. juncea, n=18) and radish (R. sativus, n=9) were used. While karashina is self-compatible, radish may be self-compatible or self-incompatible. 
     1) Production of F 1  seed by utilizing the male parent of a self-compatible line (FIG. 33) 
     As it was found that the  1PP-B! line of karashina (n=18) acts as a maintainer for  60To-AB!, i.e. a male sterile line of rape (n=19), nucleus substitution is performed by continuous backcrossing. Backcrossing through 2-3 generations from the 1993  5-2256! is necessary. In this way the karashina AB line  1PP-AB! is obtained. Then, using  62CbAe-C! under developing, for instance, as C line, F 1  breeding is performed. 
     Referring to 1 of FIG. 32,  msS f1  ! corresponds to  1PP-AB! (progeny of 1993-5-2256) and  S f2  ! corresponds to  62CbAe-C! (progeny of 1993-5-102). 
     2) Production of F 1  seed by utilizing the male parent of a self-incompatible line: karashina (FIG. 34) 
     Referring to 2 of FIG. 32,  msS f1  ! corresponds to  1PP-AB! (progeny of 1993-5-2256) and  S 1  ! corresponds to  62CBS a  ! (progeny of 1993-5-704). This  62CBS a  ! is a line obtained by crossing  62Cb! with a Chinese cabbage line (B. campestris, n=10) to introduce a self-incompatible gene and given n=18 chromosome number and improved characters through selection and breeding. F 1  is developed using this line as the male parent of a self-incompatible line. 
     3) Production of F 1  seed by utilizing the male parent of a self-incompatible line: radish (FIG. 35) 
     When a self-compatible radish line  OK! (1987-62-4582) was crossed with  R-5! (1987-62-2072) having cytoplasm with male sterility, it was found that  OK! acts as a maintainer. Therefore,  OK-AB! (1992-4-2155) was developed by continuous backcrossing. This line was crossed with the established parent line  TM 12  ! to develop F 1  (corresponding to  msS f1  S 1  ! in 2 of FIG. 32). 
     9. Production of F 1  seed by utilizing the female parent of a male sterile line introduced self-compatibility and the male parent of a self-incompatible line possessing fertility restoring gene (FIG. 36) 
     1) Production of F 1  seed by single crossing (FIGS. 37-40. FIG. 38 is sequential to the bottom of FIG. 37; FIG. 39 is sequential to the right of FIG. 37; and FIG. 40 is sequential to the bottom of FIG. 39 and the right of FIG. 38). 
     The line corresponding to  msS f1  ! in 1 of FIG. 36 is  60To-AB! (1992-4-92005) and the line corresponding to  rS 1  ! is  H-Bi-S d  B! (1992-4-84004). Because of the introduction of self-incompatibility into the male parent line, omnibus reaping of female and male parents in the production of F 1  seed was feasible. 
     2) Production of F 1  seed by three-way crossing (FIGS. 41-44. FIG. 42 is sequential to the bottom of FIG. 41; FIG. 43 is sequential to the right of FIG. 41; and FIG. 44 is sequential to the bottom of FIG. 43 and the right of FIG. 42) 
     Referring to 2 of FIG. 36,  msS f1  ! corresponds to  60To-AB! (1991-3ND-42001);  S f2  ! corresponds to  2DR-B! (1991-3N-40514);  msS f12  ! corresponds to 1992  4-82072!; and  rS 1  ! corresponds to  H-Bi-S d  B! (1992-4-84004). For enhancement of disease resistance, lodging resistance and seed yield,  2DR-B! possessing such characteristics was introduced. 
     3) Production of F 1  seed by four-way crossing (FIGS. 45-50. FIG. 46 is sequential to the bottom of FIG. 45; FIG. 47 is sequential to the right of FIG. 45; FIG. 48 is sequential to the bottom of FIG. 47 and the right of FIG. 46; FIG. 49 is sequential to the right of FIG. 47; and FIG. 50 is sequential to the bottom of FIG. 49 and the right of FIG. 48) 
     The above process is different from the three-way crossing described in 2) in that  H-En-S e  B! (1992-40) corresponding to  rS 2  ! in 3 of FIG. 36 was introduced into the male parent side. By this procedure, the mass production of  rS 12  ! was facilitated and the utility of F 2  was enhanced.