DEVELOPMENT OF WINTER RAPESEED (B. NAPUS var. OLEIFERA) RESISTANT AGAINST INFECTION OF TURNIP MOSAIC VIRUS (TuMV)
Andrzej Wojciechowski1, Przemysław Lehmann2, Edward Kozubek 2
1Agricultural University of Poznan, Dept. Of Genetics and Plant Breeding, Wojska Polskiego 71c, 60-625 Poznan, Poland, e-mail: ajwoj@au.poznan.pl
2Polish Academy of Sciences, Institute of Plant Genetics, Strzeszynska 34, 60-479 Poznan, Poland, e-mail: pleh@igr.poznan.pl
Transgenic B. napus cv. Westar (spring rapeseed) plants that express the coat protein gene of TuMV (turnip mosaic virus) were generated. Three different responses were observed when the transgenic plant lines were inoculated with TuMV. The most promising transgenic line of cv. Westar was used to breed a few winter rapeseed cultivars to induce the pathogen–mediated resistance in these cultivars. The paper is presenting the results of reciprocal crosses between one transgenic line of spring rapeseed with three cultivars of winter rapeseed. Results of F1, F2, F3 and F4 progenies (in vitro kanamycin tests) show, that transferring of resistance to TuMV via inter-cultivar crosses is possible.
KEYWORDS: transgenic plants, TuMV, coat protein gene
Viruses are the casual agents of important diseases of Brassica crops. For example, in cauliflower (B oleracea), CaMV (cauliflower mosaic virus) causes important yield losses (Passaliegue et al. 1996). In pekinensis cabbage (B. campestris ) an important reduction of yield is observed after epidemic infection of plants by turnip mosaic virus (TuMV). Jun et al (1995) observed that main reason of yield reduction of pekinensis cabbage in Korea were viral diseases first of all caused by TuMV. In the literature TuMV is often mentioned as the casual agent of yield losses in spring rapeseed (B. napus). Walsh and Tomlinson (1985) and Hardwick et al. (1994) found important yield losses of rapeseed due to TuMV.
The preliminary experiments conducted by team of Lehmann (unpublished data) showed that the most winter rapeseed cultivars which are officially approved in Poland, present high susceptibility to TuMV. The objective of our experiment was to determine, if inter-cultivar crosses of transformed spring rapeseed line (A28/1/45) of cv. Westar and three cultivars of winter rapeseed confer resistance to TuMV.
The transgenic lines of cv. Westar resistant to TuMV were generated by using strains of turnip mosaic virus: UK1, CDN1, CZE1 and monoclonal serum EMA 67 kindly supported by John Walsh from Horticulture Research International, Welesbourne, UK (Lehmann et al. 1998). Coat protein gene of TuMV (UK1) was introduced into pJR1 vector. The transformation of cv. Westar was made according to the procedure of Moloney et al. (1988) with applying Agrobacterim tumefaciens LAB4404. The most promising transgenic line was used to breed two winter cultivars: Leo, Mar and one strain, Mah 789 of rapeseed to induce the pathogen-mediated resistance in these cultivars. Different combination of crosses between the spring rapeseed transgenic line A28/1/45 and three genotypes of winter rapeseed were made. In the first step in vitro kanamycin test was utilised to select resistant hybrid plants of F1 – F4 generations. For this purpose one cotyledon from five days old sterile seedlings was placed in Murashige and Skoog medium supported with kanamycin and seedlings with another cotyledon were potted into the soil. In F1 generation almost all hybrid seedlings which were obtained in particular cross combinations were tested. In F2 – F4 generations only thirty seedlings originated from each kanamycin resistant plant were tested. After three weeks of culture thecotyledon explants were examined for the presence of callus and shoots. Well-developed explants were treated as resistance to kanamycin and presumably resistant to TuMV. In the next step plants of F4 progeny which were resistant to kanamycin were infected with TuMV and the level of development of viral disease will be estimated by ELISA based on the amount of virus in the leaves. The data from ELISA test are not presented in this paper.
The data obtained from in vitro kanamycin tests are presented in Tables 1 and 2. No kanamycin resistant plants were found in parent cultivars of winter rapeseed (Table 1). Taking into account numbers of kanamycin resistant plants (KAN+) segregated in particular cross combinations in vitro kanamycin tests showed that KAN+ plants are present in all progenies of all crosses. It is worth to notice that in many cases KAN+ plants, which showed resistance in earlier generation, did not give KAN+ plants in next generations (Table 2). The percentage of KAN+ plants obtained from particular crosses ranged from 6,2 (in A28/1/45 x Mah 789) to 12,4 (in Leo x A28/1/45) (date not included in the tables).
ACKNOWLEDGEMENTS
This work is being funded by the grant no. 5 PO6A/001/11 from Polish Research Council
The authors gratefully acknowledge to excellent assistance of Mrs M. Weigt
REFERENCES
Hardwick N. V., Davies J. M. L., Wright D. M., (1994), Plant Pathol., 43: 1045- 1049.
Jun S. I. Kwon S. Y. M., Paek K. Y., Paek K. H.,(1995), Plant Cell Rep., 14: 620-625
Lehmann P., Kozubek E., Wojciechowski A., (1998), Biotechnologia, 1(40): 129- 139.
Moloney M. M., Walker J. M., Sharma K. K., (1989), Plant Cell Report, 8: 238- 242.
Passaliegue E., Kerlan C., (1996), Plant Sci., 113: 79- 99.
Walsh J. A., Tomlinson J. A., (1985), Ann. Appl. Biol., 107: 485- 495.
Table 1. Segregation of hybrid plants from the reciprocal crosses of spring rapeseed transgenig line A28/1/45 resistant to TuMV with three standard cultivars of winter rapeseed into resistant and sensitive to kanamycin in F1 to F4 generations.
|
Item |
F1 |
F2 |
||||
|
Number of plants |
||||||
|
tested |
KAN- ** |
KAN+ * |
tested |
KAN- ** |
KAN+ * |
|
|
MAH 789 |
30 |
30 |
0 |
|
|
|
|
A28/1/45 x MAH 789 |
160 |
144 |
16 |
180 |
164 |
16 |
|
LEO |
30 |
30 |
0 |
|
|
|
|
A28/1/45 x LEO |
160 |
138 |
22 |
180 |
162 |
18 |
|
MAR |
30 |
30 |
0 |
|
|
|
|
A28/1/45 x MAR |
130 |
113 |
17 |
180 |
163 |
17 |
|
LEO x A28/1/45 |
180 |
165 |
15 |
180 |
149 |
31 |
|
MAH 789 x A28/1/45 |
40 |
34 |
6 |
150 |
140 |
10 |
|
Item |
F3 |
F4 |
||||
|
Number of plants |
||||||
|
tested |
KAN- ** |
KAN+ * |
Tested |
KAN- ** |
KAN+ * |
|
|
A28/1/45 x MAH 789 |
480 |
472 |
8 |
180 |
158 |
22 |
|
A28/1/45 x LEO |
540 |
460 |
80 |
1170 |
1098 |
72 |
|
A28/1/45 x MAR |
450 |
399 |
51 |
900 |
793 |
107 |
|
LEO x A28/1/45 |
390 |
343 |
47 |
|
|
|
|
MAH 789 x A28/1/45 |
300 |
265 |
35 |
750 |
688 |
62 |
*/ plants surviving on the media with kanamycin
**/ plants sensitive to kanamycin
Table 2. Number of plants tested in in vitro kanamycin test in F1 progeny and number of plants KAN+ * & KAN- ** segregated in F1 - F4 progenies from the reciprocal crosses of spring rapeseed transgenic line A28/1/45 resistant to TuMV with three standard cultivars of winter rapeseed.
|
Item |
F1 |
F2 |
F3 |
F4 |
||||
|
Number of plants |
||||||||
|
tested |
KAN+ |
Segregated in next generation into KAN+ & KAN- |
KAN+ |
Segregated in next generation into KAN+ & KAN- |
KAN+ |
Segregated in next generation into KAN+ & KAN- |
KAN+ |
|
|
MAH 789 |
30 |
0 |
|
|
|
|
|
|
|
A28/1/45 x MAH 789 |
160 |
16 |
6 |
16 |
2 |
8 |
5 |
22 |
|
LEO |
30 |
0 |
|
|
|
|
|
|
|
A28/1/45 x LEO |
160 |
22 |
6 |
18 |
9 |
80 |
8 |
72 |
|
MAR |
30 |
0 |
|
|
|
|
|
|
|
A28/1/45 x MAR |
130 |
17 |
6 |
17 |
10 |
51 |
11 |
107 |
|
LEO x A28/1/45 |
180 |
15 |
6 |
31 |
6 |
47 |
|
|
|
MAH 789 x A28/1/45 |
40 |
6 |
4 |
10 |
3 |
35 |
2 |
62 |
*/ plants surviving on the media with kanamycin
**/ plants sensitive to kanamycin