Innowacje w metodach hodowli rzepaku ozimego w Czechach dla uzyskania ulepszonego materiału wyjściowego oraz zastosowanie metod biotechnologicznych.

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Tom XXVI

R

OŚLINY

O

LEISTE

2005

Vratislav Kučera1_{, Miroslava Vyvadilová}1_{, Miroslav Klíma}1_{, Radoslav Koprna}2_,

Ivana Macháčková3_{, Vladislav Čurn}4

1_{Research Institute of Crop Production, Prague}

2_{OSEVA PRO Ltd., Department Research Institute of Oilseed Crops at Opava}

3_{SELGEN, Ltd., Breeding Station at Chlumec n/C.}

4_{University of South Bohemia, Faculty of Agriculture, České Budějovice}

Innovations in breeding procedures

of winter oilseed rape for development

of improved initial materials and the use

of biotechnological methods in the Czech Republic

*

Innowacje w metodach hodowli rzepaku ozimego w Czechach

dla uzyskania ulepszonego materiału wyjściowego

oraz zastosowanie metod biotechnologicznych

Key words: oilseed rape, doubled haploids, hybrid breeding, self-incompatibility, CMS,

disease resistance, frost resistance, molecular markers

The research activities in the Czech Republic are directed to increase effectiveness of breeding procedures in winter oilseed rape using innovated methods. Initial plant materials for breeding line and hybrid cultivars, original self-incompatible doubled haploid lines, improved CMS and male fertility restorer lines (Rf) for Ogu-INRA and Shaan 2 systems were obtained by means of doubled haploids. About 1000 microspore culture regenerants have been produced every year. Tests of disease resistance especially against Phoma lingam and Sclerotinia sclerotiorum have been carried out in laboratory and in the field conditions. Frost tolerance laboratory tests of rape plants are performed and a method of in vitro selection has been developed. Molecular methods based on PCR technique are used for identification of self-incompatible genotypes and for detection of materials with modified seed quality and disease resistance. Perspective lines and hybrids are evaluated in breeders’ joint multi-location trials before entering official trials.

Słowa kluczowe: rzepak ozimy, podwojone haploidy, hodowla mieszańcowa, samoniezgodność,

CMS, odporność na choroby, mrozoodporność, markery molekularne

Grupa współpracująca w obrębie związku „Czeski rzepak” składa się z instytucji powiązanych z badaniami genetycznymi i hodowlą rzepaku. Od 2000 roku realizowano wspólne projekty badawcze finansowane przez Ministra Rolnictwa Republiki Czech. Celem tych projektów były badania nad zwiększeniem wydajności metod hodowlanych oraz uzyskaniem perspektywicznych materiałów wyjściowych, dobrze plonujących, charakteryzujących się ulepszonymi cechami jakościowymi oraz odpornych na stresy biotyczne i abiotyczne.

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Vratislav Kučera ... 76

Procedura otrzymywania podwojonych haploidów (DH) została opracowana w Badawczym Instytucie Produkcji Roślinej – Praga Ruzyne (RICP). Każdego roku uzyskuje się tam około 1000 linii DH z materiałów hodowlanych Badawczego Instytutu Roślin Oleistych w Opawie (OP) oraz Stacji Hodowlanej w Slapachu Tabora (SL). Linie DH były testowane w doświadczeniach polowych. Uzyskiwano w ten sposób wstępną ocenę plonu, masy tysiąca nasion (MTN). Przeprowadzano też ocenę odporności na choroby Phoma lingam, Sclerotinia sclerotiorum i Alternaria brassicae. Linie DH były porównywane z grupą linii uzyskaną metodami konwencjonalnej hodowli (L) oraz odmianami z listy rejestrowej (S). Uzyskano linie DH plonujące powyżej linii uzyskanych metodami konwencjonalnymi oraz odmian standardowych, chociaż wyniki te nie były statystycznie istotne. Nie obserwowano wzrostu podatności na choroby podwojonych haploidów rzepaku ozimego. Wyniki badań z kolejnych czterech lat przedstawiają tabele 1–4.

W badaniach nad samoniezgodnością (SI) uzyskano linie DH samoniezgodne o jakości „00”. Otrzymano linie SI DH o zawartości kwasu erukowego poniżej 2% oraz glukozynolanów poniżej 18 µmol/g przy 9% wilgotności nasion (tabela 5).

Przy pomocy markerów molekularnych rozwinięto metodę detekcji genotypów samoniezgod-nych. Badania te prowadzono w Czeskich Budejowicach w South Bohemian University. Podwojone haploidy oceniano pod względem zawiązywania nasion oraz metodą PCR na obecność genu SLG. Badano 36 linii SI DH pod względem obecności genu SLG oraz testu SI na zawiązywanie nasion. W 32 liniach DH wynik analizy PCR był zgodny z testem na zawiązywania nasion. Współczynnik korelacji pomiędzy analizą PCR a testem na zawiązywanie nasion wynosił r = 0,889**. Wskazuje to na istnienie sprzężenia genu I SLG z fenotypem samoniezgodnym.

Dla uzyskania linii CMS Ogu-INRA i Shann2 o ulepszonej jakości wykonywano krzyżowania wielokrotne z donorami typu „00”. Do selekcji linii Rf o niskiej zawartości glukozynolanów (GSL) zastosowano technikę podwojonych haploidów. Wykorzystano także markery molekularne dla weryfikacji roślin z genami Rf. Rezultaty tych badań prezentują tabele 6–7.

W hodowli odpornościowej w celu selekcji genotypów odpornych stosowano metody sztucznej infekcji liścieni isolatami Leptosphera maculans pochodzącymi z różnych miejscowości. Wyselekcjo-nowano genotypy o różnej wrażliwości na Phoma lingam, które następnie były badane metodami molekularnymi

Każdego roku materiały hodowlane oceniano także pod względem odporności na mróz w miej-scowościach, gdzie prowadzono doświadczenia. Posługiwano się również testem laboratoryjnym przeprowadzonym w kontrolowanych warunkach w zakresie temperatur od –10°C do –16°C. Ponadto opracowano metodę wczesnej selekcji in vitro odpornych na mróz mikrospor i mikrosporowych zarodków, opartą na wrażliwości tych form na wysokie stężenie hydroksyproliny (Hyp). Dla selekcji odpornych genotypów optymalne stężenie Hyp wahało się od 14 do 16 mM w pożywce. Wyselekcjonowane genotypy doprowadzano do kwitnienia i zbioru nasion. Wybrane podwojone haploidy będą dalej testowane na odporność na mróz w warunkach kontrolowanych.

Introduction

The team cooperation proceeds within the association “Czech Rape”, including all institutions, which are engaged in oilseed rape genetic research and breeding (Kučera, Vyvadilová 2001). The joint research projects supported by the Ministry of Agriculture of the Czech Republic have been realized since 2000. The aim of the research was to increase the efficiency of breeding procedures and to obtain perspective breeding materials with high parameters of yield, seed quality and with biotic and abiotic stress resistance.

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Innovations in breeding procedures of winter oilseed rape ... 77

Doubled haploid (DH) system was used to produce homozygous lines with stabilized desired traits in the Research Institute of Crop Production Prague-Ruzyně (RICP) (Kučera et al. 1999, 2002). About 1000 microspore lines have been produced and evaluated every year. Some DH lines derived from different hybrid combinations originated from breeding workplaces showed to be prospective for breeding line varieties. Breeding of hybrid cultivars based on CMS Ogu-INRA, Shaan 2 and self-incompatibility systems have been developed and used on a large scale.

The method of frost hardiness improvement by in vitro selection for hydroxy-proline resistant genotypes in microspore culture has been verified. Molecular markers for screening of disease and frost resistant genotypes and genotypes with low linolenic acid content are being developed.

Doubled haploid lines

The DH plants were produced at the RICP by means of microspore culture which was gradually optimized (Klíma et al. 2004). Green cotyledonary embryos about 5 mm large were subcultivated on differentiation medium with benzyl-aminopurine, indolyl acetic acid and 2% sucrose, solidified by 0,8% agar. After 5–7 days two thirds of both cotyledons were cut off prior to transfer on regeneration medium without phytohormones, with 1% sucrose and 1% agar and kept at 22°C under 16/8 h photoperiod for three weeks. Well-developed shoots were rooted on MS plant growth medium with half-strength of mineral salts and NAA phyto-hormone under cultivation temperature 20/15°C.

Evaluation of DH lines

The DH lines were derived from various F1 hybrid combinations originating from the Research Institute of Oilseed Crops Opava (OP) and Breeding Station Slapy u Tábora (SL). DH lines tested in field trials were selected according to the preliminary evaluation of agronomic performance and disease resistance in small plot field trials with two replicates. In the four years from 2001 to 2004, the groups of five to nine different selected DH lines and 21 to 27 conventionally bred lines (F7 – F9 generation of inbreeding) and two to four cultivars registered in the National List of Varieties as standards into four to six location trials were included. In average 35 materials were tested in each year. The experiments were arranged on 10 m2_{plots with four replicates in accordance with the methodology of the State} Registration Trials. The seed yield was corrected to 12 percentage seed moisture.

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Resistance to Phoma lingam, Sclerotinia sclerotiorum and Alternaria brassicae and lodging resistance were scored using a 1–9 scale, with 9 indicating the most favourable score. Thousand seed weight (TSW) was usually determined from two 500-seed samples.

The results of location trials are demonstrated in tables 1–4. Although DH lines differ in yield to some extent from conventionally bred lines (L) and standard cultivars (S), the differences were statistically not significant both within and across localities. In spite of some differences between DH lines and standard cultivars in resistance against individual diseases, there was no indication of any increase in susceptibility of DH lines.

Hybrid breeding

Development of self-incompatible lines with improved 00 quality

The original incompatible lines (SI) with recessive determined self-incompatibility were characterised by high glucosinolate (GSL) and erucic acid content (Havel 1996). DH lines from microspore cultures of the cross population of SI line Tandem 6/85 with high glucosinolate and erucic acid content and „00“ quality donor OP-2051 were derived in 2000. Assessment of antinutritional substances in seeds of DH lines was carried out after verification of SI reaction by seed test. Partial decrease of GSL and erucic acid content was achieved in 10 DH lines derived in 2000 from the first cycle of crossing for seed improvement of original SI lines. Only two lines had distinctly lower erucic acid content than the initial SI material Tandem 6/85. Two breeding lines and two registered varieties Rasmus and Lisek were used as the male lines for the second cycle of quality crossing improvement. Five cross combinations of the best two SI DH lines with „00“ quality donors for deriving of DH lines were created in the year 2002. From these crosses, 87 fertile DH SI lines were obtained in 2004. Nine lines from the analysed set of 45 SI DH lines, derived in 2004, showed erucic acid content lower than 2% and the rest of 36 lines over 2%. In addition, nine plants from ten lines analysed for GSL content, had lower and the only one had higher GSL content than the limit 18 µmol/g by 9% of seed moisture. Table 5 shows seed quality parameters of selected SI DH lines. The method of deriving doubled haploids proved to be applicable for the seed quality improvement of SI lines.

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Tabl e 1 Par am eter s o f an alysed tr aits o f do ub led h ap lo ids (D H) , gr oup s of co nven tio n al lin es (L ) and stand ar d s (S ) in 20 01 — Wa rt oś ci analizow anyc h cech podw oj onyc h haploi dów (DH) oraz ich średn iej i średni ej l in ii w sob nyc h ( L ) w por ów nani u d o o dmi an w zorcow yc h ( S) w 200 1 r ok u Yield — Plon Genoty p e Genotyp [t/ha] [%] * TS W MTN [g] Lodging _Wylegan ie ** Phoma ** Sclerotin ia ** Alternaria ** Origin Pochodzenie OP 1011 (DH) 4.67 96.19 4.82 7. 31 6.56 6.19 6.31 Aztec × Olsen OP 1014 (DH) 4.65 95.78 4. 66 7.44 5.94 6.69 6.75 OP-06 OP 482 (DH) 4.58 94.34 4.21 7.75 6.50 5.94 6.19 Navajo × Olsen OP 1018 (DH) 4.54 93.51 4.74 7.19 6.13 5.75 6.44 Az tec × Olsen OP 1021 (DH) 4.36 89.80 4.48 7.38 6.13 6.56 6.63 OP-06 Rasmus (S) 5.16 106.28 4. 48 7.19 6.25 6.88 6.50 Odila (S) 4.55 93.72 4.43 6.19 6.25 6.75 6.88 DH mean — średnia 4.56 93.92 4.58 7.41 6.25 6.23 6.46 L m ean — śre dnia 4.60 94.75 4.36 6.96 4.59 6.65 6.51 S m ean — śre dnia 4.86 100.00 4.52 6.69 6.25 6.82 6.69 * y ield percentage to mean y ield of standards — plon nasion w % ś redniej wzorców ** nine po int scale (the h igher valu e, th e b etter) — s kala dziewi ęcios topniowa ( najlep szy – 9)

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Tabl e 2 Par am eter s o f an alysed tr aits o f do ub led h ap lo ids (D H) , gr oup s of co nven tio n al lin es (L ) and stand ar d s (S ) in 20 02 — Wa rt oś ci analizow anyc h cech podw oj onyc h haploi dów (DH) oraz ich średn iej i średni ej l in ii w sob nyc h ( L ) w por ów nani u d o o dmi an w zorcow yc h ( S) w 200 2 r ok u Yield — Plon Genoty p e Genotyp [t/ha] [%] * TS W MTN [g] Lodging _Wylegan ie ** Phoma ** Sclerotin ia ** Alternaria ** Origin Pochodzenie SL 617 (DH) 3.63 108.57 4.37 8.31 6. 00 5.85 6.92 S004 × Svalöv 6 290 SL 620 (DH) 3.62 108.28 5.33 7.81 5. 10 5.60 7.33 Jet Neuf × PNG 14 OP 4646 (DH) 3.58 107.08 5.08 7.81 5.38 5.55 7.56 Zorro × Navajo SL 618 (DH) 3.47 103.79 4.60 8.00 5. 50 5.45 6.94 S004 × Svalöv 6 290 SL 614 (DH) 3.46 103.49 4.25 7.19 5. 18 5.82 7.39 S004 × Svalöv 6 290 SL 619 (DH) 3.44 102.89 4.80 7.13 4. 88 5.33 7.33 Jet Neuf × PNG 14 SL 615 (DH) 3.28 98.11 4.48 7.25 5. 22 5.62 7.17 S004 × Svalöv 6 290 SL 616 (DH) 3.05 91.23 4.39 8.00 5. 02 5.67 7.17 S004 × Svalöv 6 290 Rasmus (S) 3.68 110.07 4. 55 7.69 5.61 5.28 7.09 Navajo (S) 3.26 97.51 5. 06 8.06 5.61 4.78 7.36 Orkan (S) 3.09 92.42 5.28 7.00 5.06 4.56 7.09 DH mean — średnia 3.44 102.93 4.66 7.69 5.29 5.61 7.23 L m ean — śre dnia 3.37 100.80 4.65 7.32 5.48 5.74 7.29 S m ean — śre dnia 3.34 100.00 4.96 7.58 5.43 4.87 7.18 * y ield percentage to mean y ield of standards — plon nasion w % ś redniej wzorców ** nine po int scale (the h igher valu e, th e b etter) — s kala dziewi ęcios topniowa ( najlep szy – 9)

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Tabl e 3 Par am eter s o f an alysed tr aits o f do ub led h ap lo ids (D H) , gr oup s of co nven tio n al lin es (L ) and stand ar d s (S ) in 20 03 — Wa rt oś ci analizow anyc h cech podw oj onyc h haploi dów (DH) oraz ich średn iej i średni ej l in ii w sob nyc h ( L ) w por ów nani u d o o dmi an w zorcow yc h ( S) w 200 3 r ok u Yield — Plon Genoty p e Genotyp [t/ha] [%] * TS W MTN [g] Lodging _Wylegan ie ** Phoma ** Sclerotin ia ** Alternaria ** Origin Pochodzenie SL 631 (DH) 4.01 102.89 4.92 7.38 6.27 6.94 6.42 BNW 36 × S 51 25 SL 620 (DH) 3.97 101.86 5.00 7.25 6. 21 7.31 6.25 Jet Neuf × PNG 14 SL 627 (DH) 3.88 99.55 4.31 7.88 5. 43 6.81 6.08 S004 × Svalöv 6 290 SL 626 (DH) 3.76 96.47 4.36 7.63 6. 31 7.13 6.17 S004 × Svalöv 6 290 SL 630 (DH) 3.75 96.22 3.67 8.00 5.46 6.81 5.75 BNW 36 × S 51 25 SL 632 (DH) 3.73 95.70 4.34 7.75 6. 04 6.90 6.42 S004 × Svalöv 6 290 SL 629 (DH) 3.67 94.16 3.90 7.00 5. 94 6.44 5.92 S004 × Svalöv 6 290 SL 633 (DH) 3.67 94.16 4.37 8.13 5. 54 7.00 6.42 S004 × Svalöv 6 290 SL 628 (DH) 3.65 93.65 4.57 7.75 6. 52 7.06 5.92 S004 × Svalöv 6 290 Navajo (S) 4.12 105.71 5. 19 7.20 6.57 7.54 6.18 Aviso (S) 3.97 101.86 4.90 8.00 7.43 7.62 6.36 Rasmus (S) 3.76 96.47 4. 81 7.40 6.57 7.38 6.18 Laser (S) 3.74 95.96 4.91 7.20 6.60 7.67 6.64 DH mean — średnia 3.79 97.18 4.38 7.64 5.97 6.93 6.15 L m ean — śre dnia 3.63 93.14 4.84 7.38 6.40 7.12 6.22 S m ean — śre dnia 3.90 100.00 4.95 7.45 6.79 7.55 6.34 * y ield percentage to mean y ield of standards — plon nasion w % ś redniej wzorców ** nine po int scale (the h igher valu e, th e b etter) — s kala dziewi ęcios topniowa ( najlep szy – 9)

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Tabl e 4 Par am eter s o f an alysed tr aits o f do ub led h ap lo ids (D H) , gr oup s of co nven tio n al lin es (L ) and stand ar d s (S ) in 20 04 — Wa rt oś ci analizow anyc h cech podw oj onyc h haploi dów (DH) oraz ich średn iej i średni ej l in ii w sob nyc h ( L ) w por ów nani u d o o dmi an w zorcow yc h ( S) w 200 4 r ok u Yield — Plon Genoty p e Genotyp [t/ha] [%] * TS W MTN [g] Lodging _Wylegan ie ** Phoma ** Sclerotin ia ** Alternaria ** Origin Pochodzenie SL 638 (DH) 6.47 106.99 5.77 7.75 5. 88 7.13 8.00 S004 × Svalöv 6 290 SL 617 (DH) 6.42 106.16 5.75 7.88 6. 44 7.19 7.00 S004 × Svalöv 6 290 SL 634 (DH) 6.15 101.69 5.61 7.63 5.94 6.81 7.50 Stela OP-4363/03 (DH) 6.06 100.21 5.99 8.00 7.13 7.25 7.00 4124 × Oly m p SL 639 (DH) 6.05 100.04 6.59 8.13 6.44 7.06 7.50 Stela OP-4364/03 (DH) 5.68 93.92 5.89 6.69 5.81 6.94 6.00 4051 × Marita SL 636 (DH) 5.50 90.95 6.61 7.06 6. 56 6.38 7.50 Jet Neuf × PNG 14 SL 635 (DH) 5.33 88.14 6.43 7.47 6.88 6.06 8.00 Stela Navajo (S) 6.25 103.35 6. 38 7.85 6.65 6.30 7.50 Rasmus (S) 6.17 102.03 5. 71 7.70 6.55 6.85 8.00 Laser (S) 6.05 100.04 6.02 7.65 7.35 6.60 8.50 Catonic (S) 5.72 94.58 6. 54 8.30 6.90 6.55 8.00 DH mean — średnia 5.96 98.51 6.08 7.58 6.39 6.85 7.31 L m ean — śre dnia 6.00 99.21 6.01 7.48 6.43 6.37 7.63 S m ean — śre dnia 6.05 100.00 6.16 7.88 6.86 6.58 8.00 * y ield percentage to mean y ield of standards — plon nasion w % ś redniej wzorców ** nine po int scale (the h igher valu e, th e b etter) — s kala dziewi ęcios topniowa ( najlep szy – 9)

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Table 5 Results of seed quality analysis of selected SI DH lines in 2004

Wyniki analizy jakości nasion w wybranych liniach SI DH w 2004 roku

Genotype

Genotyp Numer rośliny Plant number

Glucosinolate content HPLC [µmol/g of seed with 9% moisture]

Zawartość glukozynolanów [µmol/g nasion z 9% wilgotnością]

Erucic acid content

Zawartość kwasu erukowego [%] 2 14,84 0,13 5 3,59 0,37 42 * 1,06 49 * 1,15 53 13,99 1,12 58 13,23 1,38 OP-12 64 9,42 1,39 OP-13 7 * 1,01 OP-14 42 * 1,24

Detection of SI plants by PCR analysis of SLG gene

Molecular markers have been developed for screening of self-incompatible genotypes. In a group of 36 selected doubled haploid plants, seed-set test and PCR for SLG gene detection were performed at South Bohemian University in České Budějovice. Plant genomic DNA was isolated from leaves using Invisorb Spin Plant Mini Kit (Invitek). The DNA fragment was amplified by PCR with the class-I SLG-specific primers, PS5 + PS15 (Nishio 1996). The PCR products were analyzed by agarose-gel electrophoresis with TBE buffer.

Selected 36 SI DH plants with assessed SLG gene and SI reaction according to seed-set test were compared statistically. PCR analysis and seed-set test in the case of 32 plants were identical, in the case of four plants there were different results of SI reaction. The correlation coefficient between PCR and seed-set test for assessment of SI reaction was r = 0.889**. It was shown that class I SLG gene is in a linkage with self-compatible phenotype. A single DNA fragment (approximately 1.3 kb) was amplified from some plants of F2 generation by PCR with the class I SLG-specific primers (Figure 1). PCR amplification of this gene can be used for Marker Assisted Selection. Verification of SI reaction by both methods in the next generation of selected doubled haploid plants is considered.

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Fig. 1. PCR amplification of class I SLG gene in segregating F2 population. Two DNA

fragments (approximately 1.3 kb and 1.0 kb) were amplified. The 1.3 kb fragment (indicated by arrows) was amplified only in some plants and corresponded to class I SLG gene. Self-compatible (SC) plants were marked with 85 % probability. Lane 1-19, plants of segregating F2 population; M , 100 bp DNA ladder — Amplifikacja PCR genu SLG klasy I

w segregującej populacji F2. Dwa fragmenty DNA (w przybliżeniu 1.3 kb i 1.0 kb) zostały

zmodyfikowane. Fragment 1,3 kb (zaznaczony strzałką) został zamplifikowany tylko w niektórych roślinach i odpowiada genowi SLG klasy I. Rośliny samoniezgodne (SC) posiadały ten marker z prawdopodobieństwem 85%. Ścieżki 1–19 rośliny z segregującej

populacji F2, M – wzorzec DNA

Cytoplasmic male sterility

Initial Ogu-INRA and Shaan 2 CMS and particularly their fertility restorers (Rf) also showed a high GSL content. CMS lines with improved seed quality were produced by means of repeated crossings with various 00 quality donors. For the development of Rf lines with decreased GSL content repeated backcrossings with 00 quality donors and DH techniques were used. By the use of these methods some CMS and Rf lines from both male sterility systems were obtained (Table 6, 7). Molecular markers were verified for the detection of plants containing Rf genes.

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Table 6 Glucosinolate content of selected CMS Ogu-INRA and Rf lines in 2004

Zawartość glukozynolanów w wybranych liniach CMS Ogu-INRA oraz liniach Rf w 2004 roku

GSL [µmol/g] GSL [µmol/g] CMS line Linie CMS _{NIRS HPLC} Rf line Linie Rf _{NIRS HPLC} 189-6 10.50 15.30 2097-273 9.32 10.48 558-17 4.52 10.15 2136-281 14.35 15.13 213-29 15.01 15.41 2137-285 8.47 11.79 13-37 10.82 15.03 2141-292 10.57 9.95 303-53 14.43 16.57 323-60 13.02 13.97 82-102 17.85 16.34 114-106 12.10 14.40 523-157 18.40 19.20 551-227 14.18 13.58 Table 7 Glucosinolate content of selected Shaan 2 CMS and Rf lines in 2004 — Zawartość

glukozynolanów oraz skład kwasów tłuszczowych w oleju wyselekcjonowanych linii Shaan 2 CMS oraz linii Rf w 2004 roku

Fatty acids — Kwasy tłuszczowe [%] Genotype Genotyp GSL [_{µmol/g] C} 16:0 C18:0 C18:1 C18:2 C18:3 C20:0 C20:1 C20:2 C22:0 C22:1 S-2 CMS 14.22 3.16 1.08 69.54 18.57 5.59 0.94 0.66 0.10 0.14 0.22 S-3 CMS 26.74 3.06 0.81 71.51 16.62 6.27 0.92 0.41 0.12 0.15 0.14 S-5 Rf 12.96 3.00 0.85 70.66 17.12 6.02 1.09 0.84 0.09 0.16 0.20 S-6 Rf 16.35 3.38 1.04 71.13 16.89 6.25 0.45 0.51 0.06 0.13 0.14 S-7 Rf 13.77 2.49 0.86 74.64 16.07 5.08 – 0.34 0.12 0.16 0.21 S-8 Rf 13.11 2.63 0.92 73.94 16.23 5.34 – 0.53 0.11 0.13 0.17

Breeding for disease and stress resistance

The method of artificial infection of cotyledonary leaves with isolates of the pathogen Leptosphaeria maculans from various localities has been verified and applied. Genotypes with different susceptibility to Phoma lingam were detected and later on characterized by means of DNA fingerprinting.

Frost hardiness of the breeding material included to the location trials is evaluated every year by precise laboratory tests in controlled conditions with four to five freezing temperatures from –10°C to –16°C. The lethal temperature (LT 50) is calculated for each set of plants.

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Microspores and microspore embryos of ten selected breeding materials and cultivars were cultivated on liquid NLN media with different concentrations of hydroxyproline (Hyp) in 2003 and 2004 for in vitro selection of genotypes with increased frost tolerance. The content of free proline in the samples of leaves was measured by using the ninhydrin reaction (Tantau and Dörffling 1991). The hydroxyproline concentrations higher than 16 mM proved to be entirely toxic for all embryos. The toxicity of Hyp negatively affected the development and growth of the survived embryos. The increased proline content was detected only in one genotype derived from microspore culture with Hyp concentration 1 mM.

The optimal Hyp concentrations for selection of resistant individuals ranged from 14 to 16 mM per litre. The lines that have been developed are grown to seeds. The obtained doubled haploid lines will be subjected to laboratory frost tolerance tests under controlled conditions. Their frost injury will be measured by means of electrolyte leakage (Prášil and Zámečník 1998).

Conclusion

The second joint project funded by the Ministry of Agriculture of the Czech Republic aimed at utilisation of innovated methods and unique breeding materials to increase effectiveness of new varieties of oilseed development started in the year 2004. The project includes all of the mentioned activities and its main goal is speeding up the production of initial plant materials for a new line and hybrid cultivar breeding.

References

Havel J. 1996. Získávání autoinkompatibilních linií řepky ozimé. Genet. a Šlecht., 32: 9-18.

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