Doubled haploids in oilseed rape (Brassica napus L.) breeding

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1 Tom XXV ROŚLINY OLEISTE 2004 Instytut Hodowli i Aklimatyzacji Roślin, Oddział w Poznaniu Doubled haploids in oilseed rape (Brassica napus L.) breeding Podwojone haploidy w hodowli rzepaku (Brassica napus L.) Key words: androgenesis, Brassica napus, breeding program, doubled haploid, microspore At present, the androgenic induction of haploid is the most economical way to produce haploids in cereals, rapeseed and numerous other crops. There are many factors influencing the development of doubled haploids (DHs) in plant breeding. The paper presents the results of the application of some breeding strategies, including the doubled haploid technique for the winter oil seed rape. It will discuss several examples of taking advantage of doubled haploids in breeding programs. Słowa kluczowe: androgeneza, Brassica napus, program hodowlany, podwojone haploidy, mikropsory Klasyczna hodowla roślin zmienia się obecnie wraz z nowo rozwijającymi się biotechnologiami, które pozwalają na efektywną selekcję genotypów o pożądanych cechach. Nowe biotechnologie wspomagają klasyczną hodowlę roślin uprawych. Jednym z przykładów jest produkcja haploidów i podwojonych haploidów (DH ang. doubled haploid) metodą androgenezy in vitro, w celu szybkiego uzyskania homozygotycznych linii. W badaniach nad rzepakiem problemy związane z indukcją haploidów zostały prawie rozwiązane, pozostaje kwestia ich wykorzystania w schematach hodowlanych. Szczególną korzyścią systemu kultur haploidów jest to, że stanowią one losową próbę genetycznej rekombinacji reprezentowanej w mikrosporze. Duże populacje podwojonych haploidów mogą być źródłem genetycznej zmienności. W pracy przedstawiono metodę uzyskiwania podwojonych haploidów rzepaku ozimego z kultury izolowanych mikrospor. Wykazano, na podstawie wyników prac własnych i danych z literatury, celowość wykorzystania linii DH rzepaku. Podano szereg przykładów celowości zastosowania homozygotycznych linii w hodowli mieszańcowej w oparciu o CMS ogura oraz wykorzystania haploidów i podwojonych haploidów rzepaku w programach hodowlanych rzepaku wysokoerukowego o niskiej zawartości glukozynolanów, czy tworzenia odmian syntetycznych. Konwencjonalna hodowla wymaga prowadzenia materiału hodowlanego przez wiele generacji z każdej kombinacji krzyżówkowej przy dużej populacji roślin dla zagwarantowania prawdopodobieństwa, że pożądana kombinacja wszystkich cech będzie skupiona w jednym osobniku. Przy wykorzystaniu podwojonych haploidów liczba linii ulega znacznemu zmniejszeniu. Linie DH są dla hodowców niezwykle użyteczne także do celów szybkiego nagromadzenia pożądanych cech w niewielkiej liczbie potomstwa (linii).

2 346 At first in vitro anther culture of oilseed rape (Brassica napus L.) had been the technique most often chosen for the production of doubled haploids (DHs), although soon in vitro microspore culture became to be the most economical technique to obtain large populations of doubled haploids. The DH lines represent genetic variability produced by meiosis. The production efficiency of haploids and doubled haploids depends mainly on the efficiency of microspore embryogenesis and plant development from microspore-derived embryos. The development of efficient methods for haploid production is stimulated mainly by potential possibilities of using homozygous lines in breeding programs and basic research. It is important to introduce properly doubled haploid lines into the breeding cycle. The value of doubled haploids for breeding depends also on the value of regenerants representing a random array of gametic recombinations and there should be no preferential regeneration of embryos from microspores (Palmer, Keller 1999). However, segregation distortion patterns have been reported in microspore-derived embryos of Brassica napus L. which may be due to genes regulating embryogenesis (Foisset et al. 1993, Foisset et al. 1997). There is no dominance effect in androgenic plants and also recessive genes are not masked, which makes selection easier and characteristics are more defined. Also population size for selection is smaller than those required for conventional breeding. Many traits important for breeding of oilseed rape are controlled by rare alleles, which can be discovered and fixed using doubled haploids. It is necessary to preserve rare alleles, which may become important in future breeding (Thomas et al. 2003). Production of winter oilseed rape doubled haploids The haploid method is routinely used in the laboratories in many countries for development of new genotypes of winter oilseed rape in various breeding programs. The described below method enables rapid and efficient production of doubled haploids of winter oilseed rape on large scale and it is applied in the Department of Genetics and Breeding of Oilseed Crops in Poznań. This method is used for: development of new genotypes, selection of DH lines with desirable traits for various breeding programs, genetic mapping, development of molecular markers and for quantitative genetic study. The production procedure of doubled haploids of winter oilseed rape used in our Institute consists of the following four major steps: 1 microspore isolation growing conditions of donor plants are: 15 o /10 o C and 16 h light/8 h dark (Cegielska-Taras et al. 2002); 2 in vitro chromosome doubling in isolated microspores incubation for 20 h in NLN medium (Lichter 1982) with 0.5 g/l colchicines. The efficiency of

3 Doubled haploids in oilseed rape a chromosome doubling depends on genotype but is usually over 80% (Cegielska-Taras et al. 1999); 3 in vitro microspore embryogenesis induction of embryogenesis with high temperature at 30 o C for 10 days and then 24 o C (Cegielska-Taras et al. 2002); 4 in vitro embryo conversion into plantlet initiation with low temperature treatment 1 o C, 8 h light/16 h dark for 14 days. The rate of conversion depends also on genotype but in most cases is over 70% (Cegielska-Taras et al. 2002). The time of DH development starting from isolation of winter Brassica napus microspore to androgenic plantlets in soil is 7 8 weeks. Vernalization of androgenic plantlet needs 7 weeks at 4 o C, 8 h light/16 h dark. Winter type of oilseed rape during this procedure requires two times of 7 week vernalization period. The first treatment is done with microspore-donor plants and the second one is with androgenic plantlets. The androgenic plants after vernalization are transferred to the greenhouse conditions. Blooming plants of doubled haploid are bagged for self pollination and seed production (DH 1 a first generation). Doubled haploids are homozygous so all traits are fixed and represent good starting materials for further studies as well as breeding projects (after preliminary field selection) and for basic genetic research. Application of microspore culture system for winter oilseed rape breeding The main advantage of using haploid methods is the rapid and complete homozygousity of the progeny. This makes phenotype selection for quantitative and particularly for qualitative inherited characters much easier, and therefore breeding is more efficient. The question of DH incorporation into breeding schemes is under investigation at present. This method is interesting because it saves time needed for rapid selection of recombinant with new monogenic traits. But much more important are strategies that allow the combination of quantitatively inherited characters controlled polygenically (Wenzel 1998). The probability of selection of progeny which exceed the performance of parents using DH lines is enhanced as compared with conventional breeding methods. This fact becomes particular important in selection for earliness of plants or for small increases in quality traits, for example such as oil content (Szała et al. 2002, Cegielska-Taras 2002). Szała et al. (2003) conducted observations of DH obtained from a cross between two forms differing according to biochemical traits and also had different origin. Obtained DH lines showed that it was possible to get the accumulation effect of genes controlling some polygeny traits in DH 1 generation lines. The initial

4 348 screening of DH lines of winter oilseed rape based on yield components made possible to reduce the investigated DH population to 10% of the studied (selected) population. Selected DH lines were used for further experiments dealing with seed yield. Inbred lines are required to develop parental lines of F 1 hybrids too. Five six generations of inbreeding are required to produce inbred lines. This process can be accomplished in one generation using the DH approach. The techniques of DH lines development from isolated microspore were used in the studies on selection of restorer lines for CMS ogura with low glucosinolate (GLs) content (Fig. 1). DH lines were obtained from hybrids between different winter oilseed rape lines with double low quality and restorer line with high glucosinolate content. Early selection of DH plantlets with restorer gene was carried out with isoenzyme marker PGI-2 (phosphoglucoisomerase) or DNA molecular markers (Bartkowiak-Broda et al. 2003). In this way it was possible to obtain homozygous restorers for CMS ogura with low level of glucosinolates (Fig. 2), (Popławska: personal communication). production of parental lines A A B CMS line (S) rfo rfo maintainer line (F) rfo rfo DH restorer line (S) Rfo rfo DH selfed selection of DH line by use: PGI -2, RAPD OPC 02 (bp 1150), mtdna SCAR approved hybrid seed production A selected DH line selfed A B commercial seeds AB restored F 1 hybrid (S) Rfo rfo according to Popławska 2000 Fig. 1. Hybrid seeds production of restored hybrid cultivars of winter oilseed rape Produkcja nasion zrestorowanych odmian mieszańcowych rzepaku ozimego

5 Doubled haploids in oilseed rape DH BO 20/42 glucosinolates 7.8 µmol g -1 oil 45% DH BO 20/12 glucosinolates 6.1 µmol g -1 oil 43.4% Fig. 2 Characteristic of restorer DH lines of winter oilseed rape developed genotype BO 20 Charakterystyka linii DH restorerów rzepaku ozimego uzyskanych z formy BO 20 Selection system of DH lines of winter oilseed rape with high erucic acid content and low glucosinolates is the other example of combining DH lines and early selection for obtaining desirable genotype. Today the aim of some breeding programs is the development for industrial purposes of high erucic varieties with the increased oil yield and decreased glucosinolate contents. The scheme in Fig. 3 show the process of selection of homozygous line of winter oilseed rape with very high content of erucic acid and low level of glucosinolates in three generations. The early selection on the level of microspore-derived embryos accelerates the selection process for desirable genotype (Cegielska-Taras et al. 1999). The best selected line DH ER contained 57.95% of erucic acid in seed oil and 6.3 µmol g -1 of glucosinolates level (Cegielska-Taras 2002). Fig. 3. Selection system of DH lines of winter oilseed rape with high erucic acid (C 22:1 ) content and low glucosinalates content Schemat selekcji linii DH rzepaku ozimego o wysokiej zawartości kwasu erukowego (C 22:1 ) i niskiej zawartości glukozynolanów Another way of using doubled haploids in breeding is developing the synthetic cultivars. Synthetic cultivars are the result of random matings between the parent lines giving rise to mixture of hybrid. Brassica napus is only partially open

6 350 pollinated plant, so Syn1 seeds produced may become from cross or from self pollination. Therefore only a part of the Syn1 seeds is hybrid origin. Three doubled haploid lines of winter oilseed rape: DH H5-198, DH MR-5, DH W-15 were chosen to make of the synthetic Sn 9. The Syn0 population was made by mixing together equal numbers of seeds of the three parents (DH H5-198, DH MR-5, DH W-15). The generation Syn1 consisted of the seeds harvested from the Syn0s grown in isolation in tent. Syn1 and parental lines were evaluated for seed yield performance in a randomised block with three replicates field experiment, in one locality. The Fig. 4 shows results of the preliminary study carried out with the synthetic Sn 9. The theoretically calculated seed yield for the mixture of three parental DH lines (24,5 dt ha -1 ) was lower than yield of Syn1 (29,5 dt ha -1 ). The synthetic Sn 9 yielded not significantly lower than winter oilseed rape cv. Lisek. The significant increase in seed yield of synthetic Syn 9 of winter oilseed rape was observed as compared to parental lines. Seed yield [dt/ha] Sn 9 29,5 DH MR 5 24,0 DH W 15 24,5 DH H ,0 mean value of DHs 24,5 cv. Lisek 31,5 Fig. 4. Seed yield of synthetic Sn 9 of winter oilseed rape in Syn1 generation and three parental DH line Plon nasion syntetycznej formy Sn 9 w pokoleniu Syn1 oraz trzech rodzicielskich linii DH Final effect of the plant breeding is new improved variety. Usually, 8 10 years are required to develop a new variety using conventional methods such as pedigree or backcross system (5 6 years). The time to produce and release variety is substantially reduced when using the DH system. The DH method reduced that time by about 2 4 years as has been shown by Stringam et al. (1995), (Fig. 5). They presented the work what was attended for the rapid introduction of blackleg resistance from Australian sources (Maluka), into varieties adapted to Western Canada. From the first generation of the cross between Australian and Canadian breeding lines, were obtained DH lines. Selection made on these lines resulted in development new lines with excellent blackleg resistance and superior yield when compared to the best varieties. One of these lines, Quantum (WCC/RRC), was registered as variety after two years.

7 Doubled haploids in oilseed rape Generation Event Year Place P Maluka Linia Greenhouse F1 Hybrid plants 1989 Greenhouse DH1 Production and chromosome Greenhouse doubling of haploid plants DH2 Seed multiplication DH3 Cotyledon bio-assay Visual field evaluation 1991 for screening for agronomic performance blackleg resistance on single row plots Greenhouse and field experiments DH4 Field entry 1992 replicated trials DH5 WCC/RRC entry 1993 Trials according to Stringam et al Fig. 5. Development of blackleg resistant doubled haploid of oilseed rape variety Quantum Schemat hodowli odmiany rzepaku Quantum odpornej na czarną nóżkę As well the link of two biotechnology methods: DHs and marker assisted selection (MAS) can result in substantially reduction of breeding time to obtain desirable genotype as it is presented on the Fig. 6 according Tomas et al For instance it is possible to cross parental lines and conduct field trials of derived DH progeny within 2 years. according to Thomas et al Fig. 6. Comparison of time taken in DH and MAS backross versus conventional backcrossing Porównanie czasu hodowli z wykorzystaniem DH i MAS do czasu hodowli konwencjonalnej The number of varieties produced by doubled haploids listed by Thomas et al. 2003, remains relatively small, for example 47 varieties of Brassica napus L. The list could be incomplete because the breeders are not obliged to inform which methods they use to obtain their variety. There is no doubt that at present doubled

8 352 haploids production is the fastest way to achieve homozygous genotypes in a single generation and can be performed at any stage of breeding program of oilseed rape. Acknowledgement The author thanks Prof. dr. hab. I. Bartkowiak-Broda and Prof. dr hab. Jan Krzymański for critical reading of the manuscript and their valuable comments. Literatura Bartkowiak-Broda I., Popławska W., Fürguth A., Mikołajczyk K Double low restorer lines of winter rapeseed for CMS ogura system. Rośliny Oleiste Oilseed Crops, XXIV: Cegielska-Taras T The use of microspore culture for genetic improvement of winter oilseed rape (Brassica napus L.). Thesis of Plant Breeding and Acclimatization Institute, Radzików, Poland, 2002, 18: (in Polish). Cegielska-Taras T., Szała L., Nałęczyńska A., Kołodziej K., Ogrodowczyk M In vitro selection for high erucic acid content in microspore-derived embryos of winter oilseed rape (Brassica napus L.). J. Appl. Genet., 40, 4: Cegielska-Taras T., Tykarska T., Szała L., Kuraś L., Krzymański J Direct plant development from microspore-derived embryos of winter oilseed rape Brassica napus L. ssp. Oleifera (DC.) Metzger. Euphytica, 124, 3: Foisset N., Delourme R., Lucas M.O., Renard M Segregation analysis of isozyme markers on isolated microspore derived embryos in Brassica napus L. Plant Breeding, 110: Foisset N., Delourme R., Lucas M.O., Renard M In vitro androgenesis and segregation distortion in Brassica napus L.: spontaneous versus colchicine-doubled lines. Plant Cell Reports, 16: Lichter R Induction of haploid plants from isolated pollen of Brassica napus. Z. Pflanzenphysiol. 105: Palmer C.E., Keller W.A., Haploidy. In: Biology of Brassica Coenospecies. Cesar Gomez- Campo (eds). Elsevier, Amsterdam, Popławska W Badania nad formami restorującymi genowo-cytoplazmatyczną męską niepłodność typu polima i ogura u rzepaku ozimego (Brassica napus L. var. oleifera). Thesis of Plant Breeding and Acclimatization Institute, Poznań, Poland (in Polish). Stringam G.R., Bansal V.K., Thiagarajah M.R., Degenhardt D.F., Tewari J.P Development of an agronomically superior blackleg resistant canola cultivar in Brassica napus L. using doubled haploidy. Can. J. Plant Sci., 75: Thomas W.T.B., Forster B.P., Gertsson B Doubled haploids in breeding. In: M. Maluszynski et al. (eds), Doubled Haploids Production in Crop Plants IAEA, Printed in Netherlands, Szała L., Krótka K., Czernik-Kołodziej K., Cegielska-Taras T The characteristic of doubled haploids obtained from cultivar Bor of winter oilseed rape. Rośliny Oleiste Oilseed Crops, XXIII: (in Polish). Szała L., Krzymański J., Wójtowicz M., Cegielska-Taras T The phenotypic variation in the population of doubled haploid lines obtained from F 1 hybrid generation of winter oilseed rape (Brassica napus L.). Rośliny Oleiste Oilseed Crops, XXIV (2): (in Polish). Wenzel G Asexual Cell Genetics. In: Agricultural Biotechnology, edited by Arie Altman, Marcel Dekker Inc.,