Preliminary study on resynthesis of winter oilseed rape (Brassica napus L.)

TOM XXXI ROŚLINY OLEISTE OILSEED CROPS 2010 Katarzyna Sosnowska, Laurencja Szała, Anna Olejnik, Teresa Cegielska-Taras Plant Breeding and Acclimatization Institute, National Research Institute Poznań Instytut Hodowli i Aklimatyzacji Roślin PIB, Oddział w Poznaniu Preliminary study on resynthesis of winter oilseed rape (Brassica napus L.) Wstępne badania nad resyntezą rzepaku ozimego (Brassica napus L.) Key words: Brassica napus L., resynthesis, RAPD markers, interspecific hybridization, in vitro culture, genetic distance One of the most widely used methods for resynthesis of rapeseed is crossing the selected subspecies of Brassica rapa L. and Brassica oleracea L. and obtaining plants through in vitro culture of isolated embryos in the early stage of their development. In this study resynthesized rapeseed (RS) was obtained as a result of crosses between Brassica rapa ssp. chinensis var. chinensis (Chinese cabbage pak choy), and Brassica oleracea ssp. acephala var. sabellica (curly kale). Morphological and nuclear DNA cytometric analyses of obtained plants has confirmed their hybrid phenotype and genotype. Molecular analysis carried out using 20 RAPD primers showed that resynthesized plants are distinct from Brassica napus DH lines and from cultivars of winter oilseed rape which are bred and cultivated nowadays. Słowa kluczowe: Brassica napus L., resynteza, markery RAPD, krzyżowanie międzygatunkowe, kultury in vitro, dystans genetyczny Jedną z najczęściej wykorzystywanych metod resyntezy rzepaku jest krzyżowanie wybranych podgatunków Brassica rapa L. i Brassica oleracea L.oraz uzyskanie roślin poprzez kultury in vitro izolowanych zarodków we wczesnym stadium ich rozwoju. Celem pracy było uzyskanie resyntetyzowanego rzepaku ozimego wykazującego odrębność genetyczną od linii i odmian rzepaku obecnie hodowanego i uprawianego. W prezentowanych badaniach resyntetyzowany rzepak (RS) otrzymano w wyniku krzyżowania Brassica rapa ssp. chinensis var. chinensis (kapusta chińska pak choy) i Brassica oleracea ssp. acephala var. sabellica (jarmuż). W wyniku krzyżowania międzygatunkowego uzyskano 294 zarodki we wczesnym stadium rozwoju i wyłożono je na pożywkę MS. Otrzymane rośliny jaryzowano. Wydajność uzyskania roślin resyntetyzowanego rzepaku w stosunku do wyłożonych zarodków wynosiła 10%. Zawartość jądrowego DNA roślin RS analizowano cytometrycznie w celu potwierdzenia ich mieszańcowego charakteru. Wszystkie analizowane mieszańce okazały się allodiploidami (2n). Ocena morfologiczna i cytometryczna jądrowego DNA otrzymanych roślin potwierdziła ich mieszańcowy fenotyp i genotyp. Analiza molekularna przeprowadzona przy wykorzystaniu 20 starterów RAPD wykazała odrębność resyntetyzowanych roślin Brassica napus od linii DH i odmian rzepaku ozimego podwójnie ulepszonego, obecnie hodowanego i uprawianego. Resyntetyzowny gatunek Brassica napus zapewne zwiększy obszar genetycznej zmienności. Biorąc pod uwagę zmienność gatunków podstawowych (Brassica rapa i Brassica oleracea) można się spodziewać, że linie RS Brassica napus przyczynią się do oczekiwanego postępu w hodowli rzepaku.

258 Katarzyna Sosnowska... Introduction Oilseed rape (Brassica napus L.) with the AACC genome (2n = 38) is a natural amfidiploid which is the result of spontaneous hybridization between diploid species Brassica rapa (2n = 20, AA genome) and Brassica oleracea (2n = 18, CC genome). Geographical restriction of oilseed rape cultivation combined with intensive quality breeding (double-low varieties, 00), caused significant limits in the genetic pool of this species. However, both progenitor species: Brassica rapa and Brassica oleracea exhibit high polymorphic features, and therefore can be a source of genetic variation and may be used for further improvements in oilseed rape by distant crosses (resynthesis). In order to obtain the resynthetic oilseed rape (RS) selected subspecies of Brassica rapa and Brassica oleracea are crossed between each other, and hybrid plants are obtained by in vitro culture of isolated embryos. An alternative way to obtain interspecific hybrids is a fusion of protoplasts (Prakash and Raut 1983). Many intergeneric and interspecific hybridizations within seed crops fail due to the inability of pollen germination on another stigma or too short pollen tubes do not reach the germ or cracking of pollen tubes takes place. To overcome these barriers the technique of in vitro culture of ovary and embryos at an early stage of development has been developed (Zenkteler and Guzowska 1967). In addition, in cross-breeding of genetically distant plants there are prezygetic (no pollen germination, short pollen tubes not reaching the germ) and postzygotic (death of embryos) barriers. Olsson, Ellerström (1980) and Wojciechowski (1985) shown that the most common cause of death of embryos within the Brassica is incomplete development of the endosperm. Wojciechowski (1985) also showed that the reason for the decline of embryos developed from the crosses of Brassica rapa and Brassica oleracea was excessive overgrowth of somatic tissue, which resulted in the inhibition of nutrient inputs to the developing embryos. In such cases in vitro culture of ovary or embryo in its early stages appeared to be useful to obtain interspecific hybrids. Progeny generations are obtained more easily when species with a similar gene pool are crossed, but if species are genetically distant the percentage of obtained hybrid plants is generally low. Olsson (1960), Hoffmann and Peters (1958) thought that crosses within the genus Brassica sp. occur more frequently when the maternal parent contains more chromosomes than a paternal. Resynthesis of Brassica napus, using a wide gene pool of the parents, provides a potential opportunity to widen the genetic variability of this species. It also allows the transfer of various desirable traits, such as resistance to diseases, pests, or male sterility (Snowdon et al. 2007). Currently resynthesis of oilseed rape is widely used in yellow seeded oilseed rape breeding, because normally this feature does not occur in Brassica napus (Liu et al. 1991). Therefore it seems possible that

Preliminary study on resynthesis of winter oilseed rape... 259 crossing between progenitor species with yellow or brown seeds will cause introgression of this trait (Zaman 1988). Yellow seeded oilseed rape lines are characterized by a thinner seed coats, which is a source of fiber in the seeds, and tend to have higher fat and protein content than brown or black seeds (Ochodzki and Piotrowska 2002). The crossing of yellow seeded Brassica rapa and bright seeded Brassica oleracea is one of the ways to obtain yellow seeded hybrid of Brassica napus. So far, studies have led to only a few stable forms of yellow seeded oilseed rape (Chen et al. 1988, Liu et al. 1991, Wen et al. 2008). Resynthesis of oilseed rape by crossing the parent forms with the desired characteristics has also enabled the introduction of the trait of earliness of this species (Prakash and Raut 1983, Akbar 1987), so that oilseed rape can develop without delay in flowering in the subtropical zone. There are also more and more studies on the changes in the hybrid (resynthesized) genome at the molecular level (Li et al. 2007). As indicated by Gaeta et al. (2007), the exchange between homologous chromosomes is the most important factor in creating new combinations of alleles and phenotypic variation in resynthesized poliploids of Brassica napus. In addition, the resynthesis of hybrids complements the information about the evolution of species and verifies a taxonomic divisions (Malepszy et al. 1989). The aim of this research was to obtain resynthesized oilseed rape which is genetically distinct from lines and cultivars of Brassica napus bred and cultivated nowadays. Materials and methods For the resynthesis of oilseed rape genetically distant species Brassica rapa ssp chinensis var. chinensis (Chinese cabbage pak choy), and Brassica oleracea ssp. acephala var. sabellica (curly kale) have been selected. Crossing B. rapa B. oleracea was performed by hand. After that the 3-week embryos were transferred on MS medium (Murashige and Skoog 1962) with 3% sucrose and were carried out in in vitro culture. After the explants had turned green, they were transferred onto MS medium with kinetin (10-4 M) and with 2% sucrose. Regenerated shoots were rooted on MS medium supplemented with 2% sucrose and 10 mg/l IBA (Cegielska-Taras and Szała 1997). After the transfer to soil young hybrid plants were put through the nuclear DNA content analysis with the use of flow cytometer. The plants at 4 6 leaf stadium were vernalized in short day conditions in 4 o C for 7 weeks. After that time the number of chromosomes of hybrid plants was doubled by immersing of the roots in colchicines solution (0.05%) for 24 hours. After careful rinsing of roots the plants were transferred back into soil. Further vegetation took place in the greenhouse. Cellophane insolators were imposed on

260 Katarzyna Sosnowska... inflorescences for self pollination of plants (allotetraploids). Fatty acid content and glucosinolates composition were analyzed in the collected seeds. From the leaves of the first five resynthetized, parental plants and genetically diverse DH lines and two cultivars of winter oilseed rape, DNA was isolated applying the modified method of Doyle and Doyle (1990). In the research two cultivars of double low oilseed rape: Monolit and Cabriolet and seven DH lines with different levels of erucic acid in the oil and glucosinolates in meal were used. The analysis of polymorphism of examined genotypes was performed using 20 selected RAPD molecular primers from Operon Technologies. DNA amplification products were separated in 1.8% agarose gel in TBE buffer, at a current of 80 ma and then stained with ethidium bromide. Primers were selected on the basis of previously performed DNA analysis of genetically diverse lines and cultivars of winter oilseed rape. To prove the genetic diversity of resynthetic oilseed rape plants only those RAPD primers were selected which generated a small number (2 6) of polymorphic amplification products in PCR. The value of genetic distance was calculated using the GENDIST program, according to the formula developed by Nei (Nei 1972). Results For the resynthesis of winter oilseed rape genetically distant species Brassica rapa ssp. chinensis var. chinensis (Chinese cabbage pak choy), and Brassica oleracea ssp. acephala var. sabellica (curly kale) have been selected. Chinese cabbage originating from north China is not a headed form and exhibits high tolerance to low temperatures (up to 4 C), and curly kale is characterized by high winter hardiness, it is resistant to several diseases which occur within the genus Brassica and has low soil requirements. As a result of interspecific crosses 294 embryos were obtained, and at an early stage of development were cultured on MS solid medium. After several weeks, the explants were transferred to MS medium with 2% sucrose, initially with the addition of kinetin to ensure the proper development of shoots, and then with the addition of indolilobutyric acid (IBA) for the proper development of the roots. Regenerated plants were vernalized. The efficiency of obtaining of resynthetic oilseed rape plants calculated in relation to cultured embryos was 10% (Table 1). RS plants were analyzed cytometrically for nuclear DNA content to confirm their hybrid nature. All analyzed hybrids turned out to be allodiploids (2n), (Fig. 1), therefore the number of chromosomes of resynthetized oilseed rape plants was doubled using colchicine. Further development of the plants took place in a greenhouse.

Preliminary study on resynthesis of winter oilseed rape... 261 Table 1 Results of hybridization among B. rapa B. oleracea species by means of embryos rescue in vitro culture Wynik krzyżowania międzygatunkowego B. rapa B. oleracea przy wykorzystaniu kultur in vitro izolowanych zarodków we wczesnym stadium rozwoju Series Serie I II III IV V VI Total Razem No of embryos Liczba zarodków 34 25 135 53 17 30 294 No of hybrid plants Liczba roślin mieszańcowych 4 6 8 5 2 4 29 Efficiency Wydajność [%] 10 Brassica rapa Brassica oleracea B. rapa B. oleracea Fig. 1. Cytometric analysis of nuclear DNA in parental forms Brassica rapa i Brassica oleracea and hybrid B. rapa B. oleracea (before doubling of chromosones) Analiza cytometryczna jądrowego DNA form rodzicielskich: Brassica rapa i Brassica oleracea oraz mieszańca B. rapa B. oleracea (przed kolchicynowaniem)

262 Katarzyna Sosnowska... Pollination of hybrid plants by its own pollen was hand-assisted. The collected seeds of RS oilseed rape were analyzed on fatty acids composition and glucosinolate content. The average content of erucic acid in total fatty acid content of oil was 46.8%, and total glucosinolates in the seeds 66.3 µmol/g d.m. The evaluation of morphological features and cytometrical analysis of nuclear DNA of plants obtained from interspecific crosses between Brassica rapa ssp. chinensis var. chinensis Brassica oleracea ssp. acephala var. sabellica confirmed their hybrid phenotype and genotype. Molecular analysis carried out using 20 RAPD primers clearly demonstrated that resynthetized Brassica napus plants derived from crosses between Chinese cabbage and curly kale are distinct from the DH lines and varieties of oilseed rape cultivated nowadays. Sample image separation of DNA amplification products using primer OPG-11 in agarose gel electrophoresis is shown in Figure 2. Genetic distance value was calculated using the GENDIST program with a formula of Nei (1972). The greatest genetic distance was found between B.chiniensis and B.oleracea 1.2669 and the lowest among interspecies hybrids RS-1 and RS-4 0.0358. Graphic relationship tree was drawn using the Ward method in STATISTICA program. Created dendrogram based on 284 RAPD markers divided tested genotypes into two main groups (Fig. 3). The first group is the Chinese cabbage and curly kale and hybrids obtained from them, and the other the variety of "00" type and DH lines of winter oilseed rape. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 1 marker 2 B. rapa 3 B. oleracea 4 11 linie RS 12 Monolit 13 Cabriolet 14 20 linie DH EG 21 marker Fig. 2. DNA amplification products using primer OPG-11 for B.rapa parent, B.oleracea parent, hybrid plants Brassica napus cultivars and breeding lines Produkty amplifikacji DNA przy użyciu startera OPG-11 form rodzicielskich (B. rapa, B. oleracea), roślin mieszańcowych RS, odmian i linii hodowlanych B. napus

Preliminary study on resynthesis of winter oilseed rape... 263 Discussion The most commonly used subspecies for interspecific croning of the species Brassica rapa L. ssp. oleifera are pekinensis, Chinensis, rapifera; and of the species Brassica oleracea L: ssp capitata, acephala, italica, alboglabra (Seyis et al. 2005, Zhang and Zhou 2006). In the present study an objective was to produce the RS winter genotypes, therefore species characterized by tolerance to low temperatures have been selected. In many publications, the methodology of obtaining interspecific hybrids is similar. The main difference concerns the stage of development at which the structure formed after pollination ovaries or embryos at early stage of development are transferred to regeneration medium (Wojciechowski 1985, Seyis et al. 2005, Wen et al. 2008). In the presented study the embryos created 21 days after pollination were carried out in in vitro cultures. The medium in the first phase of embryo growth was not supplemented with casein hydrolysates, or coconut milk as proposed by Inomata (1978). Fig. 3. Dendrogram genetic distance among B. rapa parent, B. oleracea parent, hybrid plants (RS) Brassica napus and cultivars Monolit, Cabriolet and DH lines using 284 RAPD markers Produkty amplifikacji DNA przy użyciu startera OPG-11 form rodzicielskich (B. rapa, B. oleracea), roślin mieszańcowych RS, odmian i linii hodowlanych B. napus.

264 Katarzyna Sosnowska... For regeneration of shoots and roots previously developed method for obtaining androgenic plants of Brassica napus was used. Oilseed rape is characterized by good reaction to a high concentration of kinetin in the course of shoots regeneration (Cegielska-Taras and Szała 1997). Efficiency of plants obtained in relation to the cultured embryos was comparable with other reports. It is believed that the fewest hybrids are created when the mother is Brassica oleracea (Stewart 2004). However, Takeshita et al. (1980) showed that obtaining interspecific hybrids is more likely to succeed in the culture of embryos when Brassica oleracea is mother. As expected, the RS lines obtained in the presented studies were characterized by a high content of erucic acid in oil and high glucosinolate content in seed because the phenotype of a hybrid depends on the quality of parental forms. Seyis et al. s research (2005) confirms that the use of species characterized by a low content of erucic acid to the resynthesis results in almost zero erucic acid RS genotypes of Brassic napus. Resynthesized Brassica napus extends the range of genetic variability of this species. Considering the variation in parental species (Brassica rapa and Brassica oleracea) we can expect that resynthesized Brassic napus will meet some demands of the advanced oilseed rape breeding. Literature Akbar M.D. 1987. Artifical Brassica napus flowering in Bangladesh. Theor. Appl. Genet., 73: 465-468. Cegielska-Taras T., Szała L. 1997. Regeneracja roślin z mikrosporowych zarodków rzepaku ozimego (Brassica napus L.). Rośliny Oleiste Oilseed Crops, XVIII: 21-30. Chen B-Y., Heneen W.K. 1989. Resynthesized Brassica napus L.: A review of its potential in breeding and genetic analysis. Hereditas, 111: 255-263. Doyle J.J., Doyle J.L. 1990. Isolation of plant DNA from fresh tissue. Focus, 12: 13-15. Gaeta R.T., Pires J.Ch., Iniguez-Luy F., Leon E., Osborn T.C. 2007. Genomic changes in resynthesized Brassica napus and their effect on gene expression and phenotype. The Plant Cell, 19: 3403-3417. Hoffmann W., Peters R. 1958. Versuche zur Herstellung synthetischer und senisynthetischer Rapsformen Züchter, 28: 40-51. Inomata N. 1978. Production of interspecific hybrids between Brassica campestris and Brassica oleracea by culture in vitro of excised ovaries II. Effect of coconut milk and casein hydrolisate on the development of excised ovaries. Japan J. Genet., 53: 1-11. Li J., Fang X., Wang Z., Luo L., Li J., Hu Q. 2007. Observation on chromosome behavior during meiosis of resynthesiezed Brassica napus L. Proceedings of the 12th International Rapseed Congress, Wuhan, China. I, 320-322. Liu H., Han J., Hu X. 1991. Studies on the inheritance of seedcoat color and other related characters of yellow-seeded Brassica napus. Proceedings of 8th International Rapeseed Congress, Saskatoon, Canada, 1438-1444.

Preliminary study on resynthesis of winter oilseed rape... 265 Malepszy S., Niemirowicz-Szczytt K., Przybecki Z. 1989. Biotechnologia w genetyce i hodowli roślin. Wydawnictwo Naukowe PWN, Warszawa. Rozdział 3: 81-150. Murashige T., Skoog F. 1962. A revised medium for rapid growth and bioassays with Tobacco tissue cultures. Plant Physiol., 15: 473-497. Nei M. 1972. Genetic distance between populations. American Naturalist., 106: 283-291. Ochodzki P., Piotrowska A. 2002. Właściwości fizyczne i skład chemiczny nasion rzepaku ozimego o różnym kolorze okrywy nasiennej. Rośliny Oleiste Oilseed Crops, XXIII (2): 235-241. Olsson G. 1960. Species Crosses within the genus Brassica. II. Artificial Brassica napus L. Hereditas, 46: 351-386. Olsson G., Ellerström S. 1980. Polyploid breeding in Europe. In: Brassica Crops and Wild Allies, Biology and Breeding. Eds. S. Tsunoda et al., Jap. Sci. Soc. Press, Tokyo, 167-190. Prakash S., Raut R.N. 1983. Artificial synthesis of Brassica napus and its prospects as an oilseed crop in India. Indian. J. Genet., 43: 282-290. Seyis F., Friedt W., Lühs W. 2005. Development of resynthesized Rapeseed (Brassica napus L.) Forms with Low Erucic Acid Content Through in ovulum Culture. Asian Journal of Plant Sciences, 4, 1: 6-10. Snowdon R., Lühs W., Fried W. 2007. Brassica Oilseed. In: Genetic Resources, Chromosome Engineering, and Crop Improvement. Edited by Ram J. Singh, CRC Press Taylor & Francis Group. Chapter 7: 195-230. Stewart A. 2004. A review of crossing relationship between cultivated Brassica species. Cruciferae Newsl., 25: 25-26. Takeshita M., Kato M., Tokumasu S. 1980. Application of ovule culture to the production of intergeneric and interspecific hybrids in Brassica and Raphanus. Japan J. Genet., 55: 373-387. Wen J., Tu J.-X, Li T-D, Fu T.-D, Ma C.-Z, Shen J.-X. Shen 2008. Improving ovary and embryo culture techniques for efficient resynthesis of Brassica napus from reciprocal crosses between yellow-seeded diploids B. rapa and B. oleracea. Euphytica, 162: 81-89. Wojciechowski A. 1985. Interspecific hybrids between Brassica campestris L. and B. oleracea L. I. Effectiveness of crossing, Pollen Tube Growth, Embriogenesis. Genetica Polonica, 26, 4: 423-436. Zaman M.W. 1988. Limitations for introgression of yellow seed coat colour in Brassica napus. Sveriges Utsadesförenings Tidskrift, 99: 205-207. Zenkteler M., Guzowska I. 1967. O niektórych zagadnieniach eksperymentalnej embriologii roślin. Wiadomości Botaniczne, XI, 4: 281-289. Zhang G.Q., Zhou W.J. 2006. Genetic analyses of agronomic and seed quality traits of synthetic oilseed Brassica napus produced from interspecific hybridization of B. campestris and B. oleracea. Journal of Genetics, 85: 45-51.