Cytoplasmic male sterile Brassica oleracea plant and method for obtaining such plant

Abstract: B. oleracea plant, whose cytoplasm is provided, via protoplast fusion, with mitochondria comprising DNA at least partially originating from a B. napus plant, and which is coupled to (the property of) cytoplasmic male sterility (CMS) (i.e. responsible for cytoplasmic male sterility in the B. oleracea plant), and whose cells are provided with species-specific nuclear genome being normal for this plant.

Description: The invention relates to a cytoplasmic male sterile Brassica oleracea plant according to the main claim, i.e. to the development of new parent lines of such a plant. The invention furthermore relates to a method for obtaining said male sterile plant, as well as to hybrid seeds obtained from the new parent lines. Brassica crops have been cultivated for centuries as food crops, while their seed content serves as a basis of various food products (vegetable oil, mustard etc.). Since 1970 various Brassica vegetable crops have been sold as hybrid seeds. Such seeds give hybrid Brassica plants, which, being the crossing products of two strongly inbred parent lines, combine the genetic properties thereof. If either one or both parent lines is (are) (partly) self-pollinating, measures have to be taken to prevent self-pollination in order to obtain pure hybrid Brassica plants. One anti self-pollination measure uses the so-called self incompatibility of one of both parent lines. In this way it is prevented that fertile pollen can grow in the pistil of specific plants, including the pistil of the pollen producing Brassica plant itself. A disadvantage of this method is that in some cases self-pollination still occurs. As a result the seed yield will have a high inbred percentage. Another measure to prevent self-pollination of either one of the parent lines is based on the use of cytoplasmic male sterility (CMS). In radish (Raphanus sativus ) a cytoplasm has been found that induces male sterility, which cytoplasm is also known as Ogura CMS cytoplasm. Through protoplast fusion mitochondria of the Ogura CMS cytoplasm are introduced in Brassica oleracea plants, whereby the nucleus comes from normal fertile B. oleracea plants. In this way CMS B. oleracea plants are obtained. The chloroplasts in the Ogura CMS cytoplasm, which comprise DNA which is responsible for cold sensitivity of plants having this cytoplasm, have been removed through fusions. The purpose of the invention is to obtain a cytoplasmic male sterile B. oleracea plant, in which the disadvantages of the prior art as mentioned before are overcome, and whereby an alternative is given for the Ogura CMS cytoplasm, and in order to achieve that purpose according to the invention a male sterile B. oleracea plant is proposed obtainable by providing its cellular cytoplasm, via protoplast fusion and somatic hybridization, with mitochondrial DNA from a fertile B. napus plant, said DNA in combination with Brassica oleracea-specific nuclear DNA causing cytoplasmic male sterility in the B. oleracea plant. Extensive research has surprisingly shown that, despite the fact that B. napus plants are normal fertile, protoplast fusion can efficiently induce cytoplasmic male sterility in a B. oleracea plant by using the right mitochondrial DNA from a B. napus plant. The aforementioned protoplast fusion is preferably carried out with leaf protoplasts from the B. napus plant (donor) on the one hand and with hypocotyl protoplasts from a B. oleracea plant (acceptor) on the other hand. According to the invention the species-specific mitochondria are introduced through somatic hybridization. In cell fusion the nuclear coded genetic composition of a plant which receives cytoplasm remains unchanged (preferably in its entirety). A backcrossing programme is not necessary, therefore, as cytoplasm having the desired composition is obtained in one go. In practice it has become apparent that B. oleracea plants obtained after protoplast fusion are often tetraploid or aneuploid. Ploidy levels may thereby increase to more than ten times C, with C being haploid genome. Plants with a non-diploid genome are characterized in that their growth is different and in that male as well as female sterility occurs. The use of flow cytometry removes all non-pure diploid plants from a population of B. oleracea plants obtained through fusion. Following this fusion the diploid plants are subject to molecular analysis; through this analysis it is determined which plants contain mitochondrial DNA (or a fragment thereof) which is known to be closely linked genetically to CMS. The cytoplasm of the present B. oleracea plant is further preferably provided with for this plant normal, species specific nuclear genome. Therefore it is a 100% pure product. Further, the cells thereof are preferably provided with species specific mitochondria for B. napus. . In a preferred embodiment according to the invention a B. oleracea plant is chosen from the group consisting of: cauliflower ( B. oleracea L. convar. botrytis (L.) Alef. var. botrytis L.), broccoli ( B. oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch.), Romanesco (B. oleracea L convar. botrytis (L.) Alef. var. botrytis L.), Brussels sprouts ( B. oleracea L. convar. oleracea var. gemnifera DC.), white cabbage ( B. oleracea L. convar. capitata (L.) Alef. var. alba DC.), oxheart cabbage ( B. oleracea L convar. capitata (L) Alef. var. alba DC.). red cabbage (B. oleracea L. convar. capitata (L.) Alef. var. rubra DC.), savoy cabbage (B. oleracea L convar. capitata (L) Alef. var. sabauda L.), turnip cabbage ( B. oleracea L convar. acephala (DC.) Alef. var. gongyloides, curly kale cabbage ( B. oleracea L convar. acephala (DC.) Alef. var. sabellica L.), portugese cabbage ( B. oleracea var. tronchuda syn. costata ). The invention also refers to a cell of a male sterile Brassica oleracea plant obtainable by providing the cytoplasm of the cell, via protoplast fusion and somatic hybridization, with mitochondrial DNA from a fertile Brassica napus plant, said DNA in combination with Brassica oleracea -specific nuclear DNA causing cytoplasmic male sterility in the Brassica oleracea plant. The invention also relates to seeds or plant parts containing a cell as defined above. In accordance with the invention said seeds or plant parts are preferably selected from the group of cauliflower (Brassica oleracea L. convar . botrytis (L.) Alef var. botrytis L.), broccoli (Brassica oleracea L. convar . botrytis (L.) Alef var. cymosa Duch. ) , Romanesco ( Brassica oleracea L. convar botrytis (L.) Alef var. botrytis L.), Brussels sprouts (Brassica oleracea L. convar. oleracea var. gemnifera DC.), white cabbage (Brassica oleracea L. convar. capitata (L.) Alef. Var. alba DC.), oxheart cabbage (Brassica oleracea L. convar. Capitata (L.) Alef. var. alba DC.), red cabbage ( Brassica oleracea L. convar. capitata (L.) Alef. var. rubra DC), savoy cabbage (Brassica oleracea L . convar . capitata (L.) Alef. var. sabauda L .), turnip cabbage (Brassica oleracea L. convar. acephala (DC.) Alef. var. gongyloides ), curly kale cabbage (Brassica oleracea L. convar. Acephala (DC.) Alef. var. abellica L.), and portuguese cabbage (Brassica oleracea var. tronchuda syn. costata ). The invention is also related to a method for producing a male sterile Brassica oleracea plant comprising the step of providing, via protoplast fusion and somatic hubridization, cellular cytoplasm comprising mitochondrial DNA from a fertile Brassica napus plant, said DNA in combination with Brassica oleracea -specific nuclear DNA causing cytoplasmic male sterility in the Brassica oleracea plant. Seeds from both a B. napus plant (donor) and a B. oleracea plant (acceptor) are sterilized and germinated on MS30 medium or the like; for the donor this is carried out in the light, plants are kept through cutting shoots and placing them on fresh growth medium. Seeds from the acceptor are germinated in the dark; from the etiolated plantlets obtained the hypocotyl is used. Protoplasts are isolated by treating leaf and/or hypocotyl material with cell wall-dissolving enzymes such as pectinase and cellulase after pre plasmolysm; this takes place in a plasmolysing solution. After filtration and centrifugation, protoplasts from the donor are irradiated with gamma irradiation. Protoplasts from the acceptor are treated with IOA, after which both protoplast groups are fused. During fusion PEG agent is used to enhance agglutination; high pH during fusion is essential. During fusion protoplasts are left undisturbed in order not to interfere with the fusion process. After fusion the PEG solution is washed out and replaced by a regeneration medium. Regeneration of the protoplasts takes place in this medium, whereby the presence of sugars such as sucrose and hormones like 2,4-D, BA and NAA and the like is of decisive importance. During regeneration the osmotic value of the regeneration medium is lowered in steps by respectively adding medium having a lower sucrose concentration. Micro calli being developed are transferred to solid regeneration medium. Every fortnight said calli are transferred to fresh regeneration medium. Newly developed shoots are placed on rooting medium, in which rooting is enhanced by a phytohormone like IAA. Regenerated plants are examined for their ploidy level by taking leaf samples and determining the relative DNA content ( of the nuclei with a flow cytometer. Plants containing the pure diploid genome are kept, the other plants are destroyed. The remaining plants are further analysed by means of molecular techniques. Using DNA isolated from leaf samples and a probe specifically for mitochondrial DNA, it is investigated whether the right mitochondria are present in the plants. Plants comprising the cytoplasm from the acceptor (to be regarded as escapers) are destroyed. The remaining plants are reproduced by crossing and investigated for usability under glasshouse and field conditions. Matters such as sterility, seed formation and plant quality receive special attention thereby. The invention will be further elucidated with reference to the following examples, which are all preferred embodiments of the invention. Example 1. Surface sterilization of seed Seeds from cabbage ( B. oleracea ) are folded in filtration paper and submerged in a mixture containing 70% ethanol / 30% water for 10 seconds; followed by submersion in sterile water of 55°C for 5 minutes. This treatment is followed by another treatment with 0,3 % (w/v) NaOCl + Tween80 for 20 minutes; this treatment takes place in a laminar flow cabinet. After this treatment the seed package is washed 3 times with sterile water, for 5, 5 and 10 minutes respectively. Example 2. Sowing of parent (starting) material for hypocotyl protoplasts. After a final washing step the seed package is opened and placed on ½MS15 medium in a container. The containers are kept in the dark at a temperature of 25°C. After about 7 days hypocotyls can be used for protoplast isolation. Example 3. Sowing of parent (starting) material for leaf protoplasts. The sterilized seeds are sown according to example 2. The containers are placed in the light at 25°C. After 14-28 days plant leafs can be used for protoplast isolation; shoot tips of the plants are placed on fresh BB; the containers are stored at 25°C in the light. The fully grown leaf is used for protoplast isolation. Example 4. Protoplast isolation. Leaf and hypocotyl material is cut into small pieces and placed in glass petri dishes (Ø 11 cm) or TC-quality petri dishes (Ø 9 cm) containing a thin layer (12 ml) of plasmolysing solution. After said cutting another 12 ml of plasmolysing solution is added. The petri dishes are wrapped in aluminium foil and stored in a laminar flow cabinet for at least one hour. After said one hour the plasmolysing solution is replaced by approximately 24 ml of fresh enzyme solution. The whole is stored overnight in aluminium foil at 25°C; the enzyme mixture for obtaining hypocotyl protoplasts is placed on a shaker, the enzyme for leaf protoplasts, however, is not. The shaker is set to 30 rpm, with an amplitude of 15 mm. After incubation the suspensions are filtered over a Teflon filter holder with two nylon filters of 110µm and 53µm respectively. The filters are re-rinsed with 1/3 volume of CPW16 (± 8 ml). The suspensions are centrifuged at 110 x g for 5 minutes. The little bands that have developed, which contain the protoplasts, are sucked up with a sterile pasteur pipette and transferred to a new centrifuge tube. After carefully adding 9 ml of W5 the protoplast suspensions are centrifuged at 75 x g for 5 minutes. The leaf protoplasts are further treated as follows: the supernatant of the various centrifuge tubes is poured off and the pellets are carefully resuspended in 1-2 ml of W5. The contents of the different centrifuge tubes are combined, and W5 is added to a total of 10 ml. The centrifuge tube containing the protoplasts (donor) is sealed with Parafilm, wrapped in aluminium foil and placed on ice until radiation with 50 kRad takes place. The hypocotyl protoplasts are treated as follows: the supernatant of the various centrifuge tubes is poured off and the pellets are carefully resuspended in 1-2 ml of W5 + 2 mM IOA (4°C). The contents of the different centrifuge tubes are combined and supplemented to a total of 10 ml with W5 + 2mM IOA, and subsequently incubated in a refrigerator at 4°C for 10 minutes. After this the suspension is centrifuged at 75 x g for 5 minutes (the total incubation time in the IOA is 15 minutes, therefore). The pellet is resuspended in 1-2 ml of W5 and the suspension is supplemented with W5 to a total of 10 ml, after which the whole is transferred to a sterile 50 ml flask. This flask, wrapped in aluminium foil, is incubated at 25°C on a shaker (approx. 30 rpm; amplitude 15 mm) during the time that the protoplasts are irradiated. After this the contents of the flask are transferred to a centrifuge tube. The two tubes with both leaf and hypocotyl protoplasts are centrifuged at 75 x g for 5 minutes, after which the pellet is resuspended in 1-3 ml of W5. The density of the protoplast suspensions is determined with a haemocytometer. Example 5. Protoplast fusion. The protoplasts of both suspensions protoplasts are brought together with a density of 9.10 5 protoplasts/ml for fusion. 40µl drops are transferred to petri dishes with a micro litre pipette: 1140µl drops in a Ø 6 cm petri dish or 25 40µl drops in a Ø 9 cm petri dish. The lid is placed on the dishes, after which light is switched off and the laminar flow cabinet is turned off for 15 minutes to enable the protoplasts to adhere to the bottom of the petri dishes. The laminar flow cabinet is switched on again and 60µl of PEG-solution is added to each drop of protoplasts. After 3-5 minutes the following quantity of SV I is added to the petri dishes: 4 ml to a Ø 6 cm dish 9 ml to a Ø 9 cm dish After 3-5 minutes the solution is sucked up and SV II is added: 4 ml to a Ø 6 cm dish 9 ml to a Ø 9 cm dish After 3-5 minutes the solution is sucked and 8p is carefully added: 4 ml in a Ø 6 cm dish 9 ml in a Ø 9 cm dish It is important to add 8p medium carefully, in order to prevent the protoplasts from releasing from the bottom of the petri dish too early. After 3-5 minutes the 8p medium is sucked up and new 8p medium is added. Now protoplasts can release themselves from the bottom of the petri dishes: 4 ml in a 6 cm dish 9 ml in a 9 cm dish The petri dishes are sealed with Parafilm and stored in light of approximately 500 lux. Example 6. Regeneration. On day 8 after fusion 8pA is added to the petri dishes in an amount of up to three times the original volume, that is: 8 ml to a Ø 6 cm dish 18 ml to a Ø 9 cm dish The petri dishes are placed in the light. On day 15 after fusion the micro calli that have developed on the bottom of the dishes are carefully scraped off with a sterile spatula. The contents of the petri dish are distributed over four centrifuge tubes with a 10 ml pipette and centrifuged at 75 x g for 5 minutes. Meanwhile 6 ml of K 3 PPS-V medium is pipetted into four new petri dishes . After centrifuging the supernatant is poured off and the 6 ml of K 3 PPS-V medium is added to the pellet. The pellet consists of dividing cells and micro calli and can easily be resuspended in the K 3 PPS-V medium. The medium and the pellet are sucked up with a 10 ml pipette and pipetted into the petri dishes. From day 21 after fusion the micro calli that have developed are inoculated from the K 3 PPS-V medium to K 3 PPS-R medium. The growing (micro) calli are transferred to fresh K 3 PPS-R medium every 2 weeks. As soon as the calli exhibit shoot development they are transferred to containers with K 3 PPS-R medium. As soon as the shoots are big enough, they are harvested and placed in a rooting medium (BB medium). Example 7 Flow cytrometric determinations Leaf samples of ± 1 cm 2 of a plant that is to be analysed are taken and placed in 2 ml of DAPI solution. This sample is cut into small pieces with a sharp razor blade and filtered through a 15 µm filter. The sample is measured with a Partec CA-II cell analyser according to the De Laat et al . method. The relative DNA content of the plants is determined by comparing the peak positions of the fusion plants with those of diploid plants. Example 8 Molecular determinations B. oleracea plants with cytoplasmic male sterility obtained through somatic hybridization are distinguished from B. oleracea plants with different cytoplasm (different cabbage species including those with ogura, anand or polima cytoplasm) with a DNA probe. This probe consists of a mitochondrial DNA fragment isolated from plants that are phenotypically sterile (CMS) and after digestion with Eco RI cloned in Escherichia coli . The band is characterized in that it can be cut from the E. coli plasmid DNA with Eco RI and hybridizes in a characterizing way with mitochondrial DNA from cabbage (see figure). 200 mg of leaf material from the plants that are to be analysed is collected in a reaction tube and frozen with liquid nitrogen. To these tubes 750µl of extraction buffer is added and homogenized with a Potterstick. The sample is centrifuged for 10 minutes at 13000 x g and the supernatant including the green material is carefully poured off. 125µl of extraction buffer is added to the pellet, after resuspension 135 µl of lysis buffer and 60 µl of lauryl sarcosine solution is added. After briefly mixing the mixture is heated to 65°C for 20 minutes. 375 µl of chloroform/isoamyl alcohol is added and the reaction tube is shaken at least 40 times. Centrifuge for 10 minutes at 13000 x g, transfer the upper fraction to a new reaction vessel and repeat the CHCl 3 /IAA step one more time if necessary. Finally, add 500 µl of isopropanol and shake the reaction tubes a few times. Centrifuge for 5 minutes at 13000 x g. The pellet is resuspended in 500 µl of TE buffer. Digestion of DNA with restriction-endonuclease is carried out as follows: approximately 4 µg DNA is necessary for RFLP analysis. The final solution in which restriction takes place consists of 0.1 volume universal cut-buffer like One-Phor-All Buffer PLUS (Pharmacia), 4 µg DNA, 4 mM spermidine and 2 units restriction enzyme. Incubation takes place at 37°C for at least 3 hours. The reaction is stopped by changing the EDTA concentration to 10mM from a stock solution. The DNA is precipitated by adding 0.1 volume of 3 M NaAc and 2.5 volume ethanol of -20°C. The mixture is kept at -20°C for 2 hours; after centrifugation for 10 minutes at 10000 x g the pellet is washed with ice-cold 70% ethanol/water, dried and solved in TE buffer. Agarose gel electrophoresis of the DNA fragments that are to be separated is carried out as follows: 2 g agarose is weighed and boiled in 250 ml of TAE buffer. After cooling to a lukewarm temperature ehtidiumbromide is added to a final concentration of 0.5 mg/l. The gel is poured and cooled down until it is set. The samples are applied to loading buffer and DNA fragments are separated. Electrophoresis is stopped when the front marker BFB has come to approximately 1 cm from the top of the gel. In preparation to blotting, the gel is treated as follows. 5 minutes in 0.25 M HCl (1x) 15 minutes in 0.5 M NaOH/1,5 M NaCl (2x) 10 minutes in 1 M NH 4 Ac/20 mM NaOH (2x) A Hybond membrane (Amersham) is cut to the same size as the gel and wetted by immersion in 2xSSC for a few seconds and thereafter in 1M NH 4 Ac/20 mM NaOH. Three pieces of Whatman 3MM filtration paper are cut to this size. A setup is made in which the DNA is transferred from the gel to the membrane through liquid transportation for 4 hours. The transfer takes place in 1M NH 4 Ac/20 mM NaOH. After drying of the membrane, the DNA is connected to both sides of the membrane by radiation with UV light for 1 minute. The required DNA probe is labelled as follows: the transferred DNA is denaturated by heating at 95°C for 10 minutes and quick cooling down on ice. The following is mixed in a reaction vessel: 10 ng- 3 µg of DNA 2 µl of hexamucelotide mixture 2 µl of dNTP's (with digoxigenine - dUTP) 1 µl of Klenow polymerase After mixing it is incubated overnight at 37°C. DNA is precipitated by adding 2 µl of 3 M NaAc and 44 µl of ethanol (96%). The mixture is placed at -20°C for 30 minutes. After centrifugation at 10000 x g for 15 minutes, the pellet is washed with 70% ethanol in water (v/v) and the dried pellet is dissolved in 50 µl of TE. Hybridization of the labelled fragments to the membrane: The membrane is placed in 2xSSC for a few seconds. Then the membrane is sealed in a bag with 20 ml of prehybridisation mixture. The membrane is incubated for at least one hour at 60-65°C. The prehybridisation mixture is removed and replaced by 4 ml of hybridisation mixture and incubated overnight. After incubation the membrane is washed: 4 times in washing solution 1 for 2 minutes; 4 times in washing solution 2 for 2 minutes, and 2 times in washing solution 3 (at 60-65°C) for 15 minutes. Detection of the labelled bands takes place at room temperature, the blot is rinsed in rinsing buffer 1 and then sealed with 10 ml of rinsing buffer 2 per 100 cm 2 of membrane. After incubation on a shaker for 30 minutes, rinsing buffer 2 is replaced by 15 ml of anti-dig solution per 100 cm 2 of membrane. After incubation on a shaker for 30 minutes, the membrane is washed: 2 to 3 times in rinsing buffer 2 for 10 minutes; 3 to 4 times in rinsing buffer 1 for 10 minutes, and once in rinsing buffer 3. The membrane is incubated with 10 ml of AMPPD solution per 100 cm 2 of membrane for 20 minutes. The membrane is placed in a cassette with a Polaroid film on top; after sufficient exposure the film is removed and developed. Example 9. Description of floral fenotype of a cytoplasmic male sterile B. oleracea with mitochondria from Brassica napus. Inflorescence: main axis of the bunch towards the top, sometimes shortened or fasciated into bundles, often branched. Bracts seldom present, sometimes turned and rolled. Petioles sometimes shortened or fasciated into bundles. Flower bud: often closed, sometimes open, the sides of the sepals not touching each other and being folded. Smooth to bulged surface, top mostly rolled-in, sometimes pointed. Pistil often outgrows the top. Flower: sepals 1-4mm wide, 4-12 mm long, upright, often slightly turing, sides sometimes bent inwardly, top bent inwardly. Honey glands present. Petals 4, sometimes up to a manifold of this up to 40; 4-7 mm wide, 11-19 mm long. At a multitude of 4 the inner petals are often much shorter. Nail may seemingly be fasciated to a tube. Plate mainly not bent backwards, often strongly rolled sideways, sometimes strongly turned. Colour upper side white or yellow RHS 2A-3A-2C-3C-4A-5A-5B-6A, bottom side white or yellow RHS 2A-2B-2C-3A-3C-4B-4C-4A, side of top often smooth, sometimes toothed. Stamina 6 to 2, and in that case sometimes fasciated 2 by 2, length up to half the sepals, sometimes the same length as the sepals. Anthers, small and triangular, pollen not present. One pistil, sometimes as many as three, one being longer than the others, mainly upright, without pollination often growing to a length of about 30 mm. Bill and pistil sometimes curved, sometimes slightly swollen. Ovary often turning and rolled. Flower in the top of the bunch sometimes almost without sepals, petals and stamina and with strongly turning and rolled ovaries, or seemingly faciated 2 by 2; split open along a fasciation seam, resembling leaf on bract with parallel nervature. Required solutions for cell fusions: Further solutions for cell biology enzyme solution: 1% (w/v) cellulase R-10 in K3PPS-I 0.1% (w/v) pectinase in K 3 PPS-I PEG solution: PEG1500 266.7 mM glucose 300 mM CaCl 2 .2H 2 O 50 mM pH = 7,0 SV I: 50 ml of PEG solution + 100 ml of Pre-plasmolysing solution SV II: 20 ml of PEG solution + 100 ml of Pre-plasmolysing solution IOA (iodacetamide) 2 mM IOA in W 5 ; pH = 5,6 1% agarose solution in MilliQ water. Solutions for molecular biology: extraction buffer: 350 mM of sorbitol 100 mM of Tris-base 5 mM of EDTA add fresh: 20 mM of Na-bisulphite lysisbuffer: 20 mM of Tris-base 50 mM of EDTA 2 M NaCl 2 % (w/v) CTAB N-lauryl sarcosine, Na salt: 10 % (w/v) chloroform/isoamyl alcohol: 24:1 (v:v) isopropanol TE: 10 mM of Tris-HCl; pH 8,0 1 mM of EDTA 4mM of spermidine EDTA stock solution: 1 M 3 M of NaAc ethanol 96 % 70 % ethanol/water v/v ethidiumbromide solution: 10 mg/ml of H 2 O TAE buffer: 4.84 gram Tris base / l 1,14 ml of acetic acid 10 mM of EDTA; pH 8,0 loading buffer: 24 % Ficoll 400 0.1 % SDS 0,1 % xylenecyanole 0,1 % brome phenole blue 0,25 M HCl 0,5 M NaOH/1,5 M NaCl 1M NH 4 Ac/20 mM of NaOH 2xSSC: 30 mM of Na-citrate.2H 2 O; pH 7.2 300 mM of NaCl 0,2 SSC: 10 x diluted 2xSSC 0,1xSSC: 20 x diluted 2xSSC hexanucleotide mix: 10 x concentrated hexanucleotide mix from Boehringer Mannheim dNTP: 0,1 mM of dATP 0,1 mM of dCTP 0,1 mM of dGTP 0,65 mM of dTTP 0,35 mM of digoxygenine-dUTP (Boehringer) blocking reagent: Boehringer Mannheim; art.nr 1175041 prehybridisation mix: 75 mM of Na-citrate 750 mM of NaCl 0,5 % blocking reagent 0,1 % N-lauroyl sarcosine (w/v) 0,2 % SDS hybridisation mix: 75 mM of Na-citraat 750 mM of NaCl 0,5 % blocking reagent 0,1 % N-lauroylsarcosine 0,2 % SDS 40 - 50 ng digoxygenine-dUTP labelled probe/ml washing solution 1: 2xSSC + 0,1 % (w/v) SDS washing solution 2: 0,2xSSC + 0,1 % (w/v) SDS washing solution 3: 0,1xSSC + 0,1 % (w/v) SDS rinsing buffer 1: 100 mM of Tris-HCl pH 7,5 150 mM of NaCl rinsing buffer 2: rinsing buffer 1 0,5 % blocking agens (make fresh) rinsing buffer 3: rinsing buffer 1 50 mM of MgCl 2 anti-dig-AP conjugate: Boehringer Mannheim; art.nr 1175041 AMPPD solution: 10 µl of AMPPD per ml of rinsing buffer 3 (0.26 µM) Used abbreviations: 2,4D: 2,4-dichlorophenoxy acetic acid AMPPD: disodium 3-(4-methoxyspiro(1,2-dioxetane-3,2'-tricyclo-[3.3.1.1 3.7 ]decan}-4-yl)phyenyl phosphate AP: acid fosfatase BA: 6-benzylaminopurine CMS: cytoplasmic male sterility DIG: digoxygenine dATP: desoxyadenosine triphosphate dCTP: desoxycytosine triphosphate dGTP: desoxyguanine triphosphate DNTP: desoxynudeotide triphosphate dTTP: desoxythyminetri phosphate DUTP: desoxyuracyl triphosphate EDTA: ethylenediaminetetra acetic acid g: gravity acceleration (9,8 m.sec -2 ) IOA: iodacetamide kRad: 1000 Rad, unit of ionising radiation µg: microgram µl: microliter mM: millimolar µM: micromolar M: molar MS: Murashige & Skoog NAA: naftalene acetic acid PEG: polyethylene glycol pH: measure of acidity rpm: revolutions per minute SSC: saline sodium citrate SDS: sodiumdodecyl sulphate TC: tissue culture UV: ultraviolet v/v: volume/volume w/v: weight/volume For almost all treatments it can be stated within which limits a pH, a concentration etc. has to be kept and at what level the experiments have to be carried out in practice. The three levels for these solutions/treatments are given below: tissue culture media: ½MS15, MS30 en BB: sugar e.g. sucrosis; concentration 0.5 - 600 mM, normally 200 - 400 mM. pH 5 - 10; normal 5.5 - 6.0. temperature cultivation: 22 - 27°C, normally 25 °C. radiation: 0 - 60 kRad; normally 50 kRad. fusion temperature: 20 - 25 °C; usually 22°C concentrations of plant hormones: 0 - 10 µM; normally 0 - 3µM.

Claims: A male sterile Brassica oleracea plant obtainable by providing its cellular cytoplasm, via protoplast fusion and somatic hybridization, with mitochondrial DNA from a fertile Brassica napus plant, said DNA in combination with Brassica oleracea-specific nuclear DNA causing cytoplasmic male sterility in the Brassica oleracea plant. The Brassica oleracea plant according to claim 1, selected from the group consisting of cauliflower ( Brassica oleracea L. convar. botrytis (L.) Alef var. botrytis L.), broccoli (Brassica oleracea L. convar . botrytis (L.) Alef var . cymosa D uch .), Romanesco (Brassica oleracea L . convar botrytis (L.) Alef var. botrytis L.), Brussels sprouts (Brassica oleracea L. convar. oleracea var. gemnifera DC.), white cabbage ( Brassica oleracea L . convar. capitata (L.) Alef. var. alba DC.), oxheart cabbage (Brassica oleracea L . convar . capitata (L.) Alef. var. alba DC.), red cabbage ( Brassica oleracea L. convar. capitata (L.) Alef. var. rubra DC.), savoy cabbage (Brassica oleracea L. convar . capitata (L.) Alef . var. sabauda L.), turnip cabbage (Brassica oleracea L . convar. acephala (DC.) Alef. var. gongyloides ), curly kale cabbage (Brassica oleracea L. convar. acephala (DC.) Alef. var. abellica L.), and portuguese cabbage (Brassica oleracea var. tronchuda syn. costata ). A cell of a male sterile Brassica oleracea plant obtainable by providing the cellular cytoplasm of the cell, via protoplast fusion and somatic hybridization, with mitochondrial DNA from a fertile Brassica napus plant, said DNA in combination with Brassica oleracea -specific nuclear DNA causing cytoplasmic male sterility in the Brassica oleracea plant. Seeds or plant parts containing a cell according to claim 3. Seeds or plant parts according to claim 4, selected from the group of cauliflower (Brassica oleracea L. convar. botrytis (L.) Alef var. botrytis L.), broccoli (Brassica oleracea L. convar. botrytis (L.) Alef var. cymosa Duch. ) , Romanesco ( Brassica oleracea L. convar botrytis (L.) Alef var. botrytis L.), Brussels sprouts ( Brassica oleracea L. convar. oleracea var. gemnifera DC.), white cabbage ( Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC.), oxheart cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC.), red cabbage (Brassica cleracea L. convar. capitata (L.) Alef . var. rubra DC.), savoy cabbage (Brassica oleracea L. convar. capitata (L.) Alef. var. sabauda L. ), turnip cabbage (Brassica oleracea L. convar. acephala (DC.) Alef. var. gongyloides), curly kale cabbage (Brassica oleracea L. convar. acephala (DC.) Alef. var. abellica L.), and portuguese cabbage (Brassica oleracea var. tronchuda syn. costata). A process for producing a male sterile Brassica oleracea plant comprising the step of providing, via protoplast fusion and somatic hybridization, cellular cytoplasm comprising mitochondrial DNA from a fertile Brassica napus plant, said DNA in combination with Brassica oleracea -specific nuclear DNA causing cytoplasmic male sterility in the Brassica oleracea plant.

Inventors: BARTEN PIETER, BARTEN, PIETER
Applicants: BEJO ZADEN B V, BEJO ZADEN B.V.
Application year: 1997
Main classification: C12N15
IPC codes: A01H1, C12N15, C12N5, A01H4, A01H5