Breeding of yellow-seeded summer rapeseed (Brassica napus L.) in West Siberia

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1 Tom XXV ROŚLINY OLEISTE 2004 Dmitry A. Potapov, Galina M. Osipova Siberian Research Institute of Fodder Crops, Russian Academy of Agricultural Sciences Krasnoobsk, Novosibirsk Region Key words: Breeding of yellow-seeded summer rapeseed (Brassica napus L.) in West Siberia Hodowla żółtonasiennego rzepaku jarego (Brassica napus L.) w Zachodniej Syberii Brassica napus, canola, yellow seed, interspecific hybridization, inbreeding Yellow seeds of Brassica napus may have more oil, protein and lower fibre content than black seeds. The methods of interspecific hybridization, inbreeding, in vitro propagation, and selections have been used to develop new lines of summer rapeseed suitable for cultivation in West Siberia. The variability of seed colour of inbred lines substantially depended on genotypical features of the initial material. In the sixth generation of inbreeding, several practically stable yellow-seeded lines were identified. These lines demonstrated high seed productivity and early maturing and are currently used in the breeding of the yellow-seeded cultivars of B. napus. Słowa kluczowe: Brasscia napus, canola, żółte nasiona, krzyżowanie międzygatunkowe, chów wsobny Żółte nasiona Brassica napus charakteryzują się większą niż czarne zawartością tłuszczu i białka, a mniejszą zawartością włókna. Ponadto łatwiej jest też określić dojrzałość nasion żółtych, ponieważ chlorofil nie jest maskowany przez czarną okrywę nasienną. Uzyskanie wcześnie dojrzewających, wysokoplonujących, żółtonasiennych odmian Brassica napus jest ważnym celem programów hodowli roślin oleistych prowadzonych na Syberii. Badania nad rzepakiem żółtonasiennym w Syberyjskim Instytucie Hodowli Roślin Pastewnych rozpoczęto w 1987 roku. Wykorzystując metody krzyżowania (hybrydyzacji), chowu wsobnego, rozmnażania in vitro oraz selekcji uzyskano nowe linie rzepaku jarego odpowiedniego do uprawy w Zachodniej Syberii. Do krzyżowania międzygatunkowego wykorzystano żółtonasienne i częściowo żółtonasienne genotypy Brassica campestris, Brassica juncea oraz Sinapis alba. Dla uzyskania zróżnicowania genotypowego materiału hodowlanego stosowano chów wsobny heterozygotycznego materiału rzepaku jarego. Kolor nasion linii wsobnych istotnie zależał od materiału wyjściowego. Stabilne żółtonasienne linie wsobne uzyskano w szóstym pokoleniu. Średnie zawartości tłuszczu, białka i węglowodanów nowej linii SNK-32 przewyższały zdecydowanie wartości dla rzepaku czarnonasiennego SibNIK198 o odpowiednio 7, 8 i 31%, podczas gdy zawartość włókna była o 19% niższa przy plonie nasion większym o 45%. Obecnie obiecujące linie żółtonasienne są wykorzystywane w programach hodowlanych Brassica napus.

2 52 Dmitry A. Potapov... Introduction West Siberia is an important agricultural region of Russia, with nearly 30% of its 15 million population residing in rural areas. The region is characterized by extreme climatic conditions and a short vegetation period. The necessity to maintain acceptable levels of the oilseed production requires the development of early maturing and high yielding crop cultivars which are tolerant to the specific agro-climatic conditions of the region. All cultivars of Brassica napus L. currently grown in Russia are black-seeded. The development of yellow-seeded forms has been proposed as a means to improve the quality of the canola meal (Jönsson 1977). Yellow seeds of B. napus may have more oil and protein than black seeds. Meal from light-pigmented genotypes of rapeseed may have lower fibre content than meal from their dark-pigmented counterparts. In addition, it is much easier to determine the degree of ripening in the yellow seeds, as the occurrence of chlorophyll is not masked by the dark seed coat (Jönsson 1977; Shirzadegan, Röbbelen 1985; Dłużewska et al. 1999; Slominski et al. 1999; Ochodzki, Piotrowska 2002; Bochkaryova et al. 2003; Piotrowska et al. 2003; Relf-Eckstein et al. 2003). Research towards the development of yellowseeded cultivars has been carried on by various research groups around the world. The development of early maturing, high yielding, and yellow-seeded cultivars of Brassica napus is a high priority for the Oilseed Breeding Program of Siberia. Research towards the development of yellow-seeded B. napus has been conducted at the Siberian Research Institute of Fodder Crops since Up to the present, a number of inbred lines with different seed coat colour, have been created (Potapov, Osipova 2003). Here we present selected results of this work. Materials and methods Brassica napus cultivars Andor, Regent (Canada), SibNIIK 198 (Siberian Research Institute of Fodder Crops) and yellow-seeded or light-pigmented genotypes of B. campestris (cultivars Vostochnaya (All-Russia Research Institute of Oil Crops, VNIIMK), Candle, Tobin (Canada)), B. juncea (line 13H-11 from VNIIMK, and line 21 from All-Russia Institute of Plant Industry, VIR), Sinapis alba (line 13H-162 from VNIIMK and line 24 from VIR) have been employed as materials for interspecific crosses. The inbreeding was conducted in combination with selections on our own breeding material, and cultivars of summer rapeseed Agat (VNIIMK), Regent (Canada), Salut (Sweden), and forms from Svalöf Weibull AB (Sweden). The following Brassica species forms were used in different cross combinations: B. juncea: yellow-seeded line 13H-11 (VNIIMK); B. napus: yellow-

3 Breeding of yellow-seeded summer rapeseed seeded line I 3 (No. 615, Sweden), light-brown-seeded lines I 3 (No. 623, 624, Sweden), light-brown-seeded line I 3 (I 0 037, Finland), brown-seeded line I 3 (SNK 196), ochre-seeded line I 7 (Agat, VNIIMK), and black-seeded cultivars: Kubanskiy (VNIIMK), Agat (VNIIMK), and Karat (Sweden). The analysis of segregation was performed with the χ 2 -test; the plants were sorted into two classes, dark- and lightseeded, according to Shirzadegan (1986). Plants were grown, crossed and selfed by conventional techniques. Results and discussion The yellow seed coat character was transferred from other Brassica species through interspecific crosses. During , 72 cross combinations were made. The success of interspecific crosses depends on the relationship between Brassica species according to the triangle of U (1935). The most successful crosses were where B. napus was used as female parent (Osipova, Zimina 1988; Osipova 1995). Forms with different seed coat colour were created and included in the breeding process. The research on creation of inbred lines was concentrated on the character of the yellow seed coat, the seed yield, and the resistance of the plants to the extreme climatic conditions of West Siberia. The maximal diversity of forms was observed in the first generation of inbreeding. In subsequent generations, the variability of the seed coat colour of the lines was substantially reduced (Figure 1). The seed colour variation in the studied lines significantly depended on genotypical features of the initial material (Table 1). The main criteria in the selection among the lines were the low variability of the seed colour and a high fraction of yellow seeds. Out of 1314 studied forms, two practically stable yellow-seeded lines (No. 39 and No. 109) were identified in the sixth generation of inbreeding (Table 2). The analysis of the distributions of the plants across the whole population revealed the following changes from generation to generation. The initial population consisted of 92% plants that produced less than 10% light-pigmented seeds and 8% plants that produced from 10 to 40% light-pigmented seeds. In the first generation, obtained with light-pigmented forms selected from the initial population, the frequency of entirely black-seeded plants was 46% while the frequency of entirely yellow-seeded plants was 18%. In the second generation, the fraction of entirely black-seeded plants decreased while the fraction of entirely yellow-seeded plants increased. The same tendency was observed in all subsequent generations. In the sixth generation, the frequency of entirely yellow-seeded plants reached 39% while 61% of the plants were partially yellow-seeded.

4 54 Dmitry A. Potapov... Table 1 Variability of the seed coat colour (% yellow seeds per plant) in the studied B. napus inbred lines Zmienność koloru okrywy nasiennej (% żółtych nasion na roślinę) w badanych liniach wsobnych Brassica napus Line (pedigree) Linie (pochodzenie) No. 39 (ww-1591) No. 109 (ww-1591) No. 14/162 (Andor Candle) No. 283 (ww-1588) Generation of inbreeding Pokolenie chowu wsobnego Number of plants Liczba roślin Coefficient of variation Współczynnik zmienności [%] Coefficients of variation range Zakresy wartości współczynników zmienności [%] I I I I I I I I I I I I I I I I I I I I I I I

5 Breeding of yellow-seeded summer rapeseed Table 2 Percentage of yellow seeds per plant in the studied B. napus inbred lines Procentowa zawartość nasion żółtych na roślinę w badanych liniach wsobnych B. napus Line Linia Pedigree Pochodzenie Generations of inbreeding Pokolenie chowu wsobnego Number of plants Liczba roślin Yellow seeds per plant Żółte nasiona na roślinę [%] Range Zakres [%] No. 39 ww ± No. 109 ww ± No. 14/162 Andor Candle ± No. 283 ww ± CV, % generations of inbreeding pokolenie chowu wsobnego Fig. 1. Variability of the seed coat colour in the six generations of inbreeding (statistics for 1314 lines). The solid line depicts the average trend in the coefficient of variation value (CV, %); the short and long dashes outline the max and min values observed among the lines, respectively (%) Zmienność barwy okrywy nasiennej pokolenia F 6 chowu wsobnego (dla 1314 linii). Linia ciągła oznacza średnią wartość współczynnika zmienności (CV, %); linie przerywane krótko- i długorodnikowe przedstawiają odpowiednio maksymalne i minimalne wartości pomiędzy liniami (%) The conducted studies and obtained results demonstrate that the method of inbreeding and selection makes it possible to obtain, starting from the sixth generation, practically stable yellow-seeded lines of summer rapeseed. At the same time, the development of genetically stable yellow-seeded forms is limited by the

6 56 Dmitry A. Potapov... complex genetic control of the seed coat colour. The investigations carried out in our group proved that in all different combinations of yellow- and black-seeded forms, F 1 plants produced black seeds. Therefore, black seed colour is dominant over yellow. The F 2 hybrids exhibited 15:1 or 63:1 dark to light seed ratios implying a two- or three-gene inheritance model (Table 3). Segregation of seed colour in F 2 progenies of several cross combinations Segregacja koloru nasion w pokoleniu F 2 kilku kombinacji krzyżówkowych Table 3 Cross combination Kombinacja krzyżówkowa Segregation in seed colour Segregacja koloru nasion observed plants rośliny z nasionami dark-seeded ciemnymi light-seeded jasnymi expected ratio spodziewana proporcja No 623, I 3 Karat : Karat No 623, I : Karat No 624, I : Karat I 0 037, I : Agat No 615, I : Agat, I 7 Kubanskiy : SNK 196, I 5 B. juncea : χ 2 The studies of the influence of environmental factors on the seed coat colour showed that the colour of light-pigmented seeds may vary from yellow to lightbrown, including yellow with darker coloured spots, shades and hilum. To some extent, the observed colour variations may be caused by the meteorological factors. However, the major role, most probably, is played by genes-modifiers, as the influence of these genes varies and changes with environmental conditions (Liu, Gao 1987; Osipova, Potapov 1996). Such modification variability of the seed coat colour complicates the propagation and selection of light-pigmented forms. This problem was mentioned and discussed by other researchers (Shirzadegan 1986; Bechyně 1987; Liu et al. 1991; Van Deynze et al. 1993; Somers et al. 2001). In the initial stages of the breeding work, most yellow-seeded forms of B. napus exhibited low yields and low vitality. In order to accelerate breeding of the most valuable forms, in vitro propagation was used. This method made it possible to increase the number of seeds per plant by 89% and to increase the fraction of yellow seeds per plant by 30% in comparison with the corresponding values for plants propagated in vivo (Table 4).

7 Breeding of yellow-seeded summer rapeseed The efficiency of in vitro method for propagation of yellow-seeded lines Wydajność rozmnażania in vitro linii żółtonasiennych Table 4 Character Cecha in vitro in vivo t* Number of seeds per plant Liczba nasion na roślinę Yellow seeds per plant [%] Żółte nasiona na roślinę 491±43 261± * 88±3 68±5 3.26* * The t-test indicates that the distributions are different at 5% level of confidence test wskazuje, że rozrzut jest istotny (różnice są istotne ) przy 5% poziomie ufności The research on the variability of the new lines, conducted from 1995 to 1998, demonstrated that the selection of yellow-seeded forms, with higher value of seed yield and its components than for the black-seeded forms, is possible (Table 5). The correlation between seed yield and other characters of the yellow-seeded lines was studied in detail (Potapov, Osipova 2003). Six prospective inbred lines were selected for breeding of yellow-seeded cultivars of B. napus. These lines were tested from 1999 to 2002 (Table 6). All these lines demonstrated high productivity and early maturing. The average oil, protein and carbohydrate contents of the new SNK-32 line exceeded the corresponding values of a black-seeded SibNIIK 198 cultivar by 7%, 7% and 31%, respectively; the fibre content was 19% lower and the seed yield was 47% higher. In conclusion, the main result of this long-term work is the development of practically stable yellow-seeded lines of summer rapeseed suitable for cultivation in the extreme conditions of West Siberia. In spite of the limitations associated with the creation of genetically stable yellow-seeded canola, our studies demonstrate that the set of applied methods, including interspecific hybridization, inbreeding, in vitro propagation, and selections, can give satisfactory results. A work on the creation of yellow-seeded canola cultivars is currently underway.

8 Seed yield and its components for yellow- and black-seeded Brassica napus ( ) Plon nasion oraz składniki struktury plonu żółto- i czarnonasiennych Brassica napus ( ) Table 5 Characters Cechy yellow-seeded lines linie żółtonasienne Average Średnia black-seeded lines linie czarnonasienne t yellow-seeded lines linie żółtonasienne Range Zakres black-seeded lines linie czarnonasienne Seed yield per plant [g] Plon nasion na roślinę 26.7 ± ± Number of pods per plant Liczba łuszczyn na roślinę 389 ± ± Number of seeds per pod Liczba nasion w łuszczynie 22 ± 1 26 ± * Weight of 1000 seeds [g] Masa tysiąca nasion 2.7 ± ± * Height of plant Wysokość roślin [cm] 84 ± 2 96 ± * Maturity [days] Dojrzałość [dni] 94 ± ± * The t-test indicates that the distributions are different at 5% level of confidence test wskazuje, że rozrzut jest istotny (różnice są istotne) przy 5% poziomie ufności

9 Average seed yield and seed quality of the prospective yellow-seeded lines of Brassica napus canola Średni plon nasion oraz jakość nasion obiecujących żółtonasiennych linii Brassica napus typu canola Table 6 Line Linia average średnia [tons/ha] Seed yield Plon nasion ( ) relative to standard w stosunku do wzorca average średnia [days] Maturity Dojrzałość ( ) relative to standard w stosunku do wzorca Oil content Zawartość tłuszczu ( ) average średnia [%] relative to standard w stosunku do wzorca average średnia [%] Protein content Zawartość białka ( ) relative to standard w stosunku do wzorca SibNIIK SNK * * * * SNK * SNK * * SNK SNK * * SNK * 1 Standard check cultivar (black-seeded) odmiana wzorcowa (czarnonasienna) * The observed difference is significant at 5% level of confidence istotne różnice przy poziomie ufności 5%.

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