NL195093C - Method for obtaining CMS-Brassica oleracea plants. - Google Patents

Description

1 195093, Method for obtaining CMS-Brassica oleracea plants

This invention relates to a method for obtaining CMS plants belonging to a Brassica species, which comprises fusing protoplasts of these Brassica species with protoplasts of an Ogura CMS plant that also belongs to this Brassica species, followed by regeneration of the allogeneic cells thus obtained to plants and identification of plants containing mitochondrial DNA that hybridizes to the 1.5 kb Sacl fragment from mitochondrial DNA from Ogura CMS cytoplasm.

Such a method is known from G. Pelletier et al., Proceedings International Symposium Berlin, September 1985, Walter de Gruyter & Co. Berlin, p. 653-661. New cytoplasmic associations in B. napus are described herein. B. napus is an amphidiploid plant (2n = 38) from sexual hybridization between B. oleracea (2n = 18) and B. campestris (2n = 20). This reference describes the restoration of the normal chlorophyll content of rapeseed plants (B. napus) containing a CMS cytoplasm by fusion of a protoplast of a normal B. napus with a protoplast of a CMS B. napus. Reference is made to an article by Bannerot et al., 1974, Proc. Eucarpia Meeting Cruciferae, 52 and in particular the title thereof "Transfer of cytoplasmic male sterility from Raphanus sativus to Brassica oleracea", and it is stated that the plants obtained there exhibit a chlorophyll deficiency, which is assumed to be the result of an interaction between the Brassica cell nucleus and Raphanus chloroplasts.

Male sterility is important in cultivating B. oleracea hybrid seed because normal flowers are self-pollinating. Male sterile lines do not produce viable pollen and cannot pollinate themselves. By eliminating the pollen from one parent variety at a cross, a plant grower is assured of obtaining hybrid seed of uniform quality.

The Ogura CMS cytoplasm from Raphanus sativus, which confers male sterility, can be introduced into B. oleracea by repeated backcrossing. In the obtained Ogura CMS B. oleracea plants, the mitochondria of R, sativus, lead to a CMS phenotype. However, the presence of the R. sativus chloroplasts leads to chlorosis if the plants grow at low temperatures, which consequently leads to yield losses.

The present invention now provides a method by which CMS B. oleracea plants are obtained which contain a core typical of Brassica oleracea, mitochondria of the Ogura CMS cytoplasm and cold-tolerant chloroplasts of normally fertile Brassica oleracea. The plants of the invention have cytoplasmic male sterility, but will not exhibit chlorosis if they grow at a low temperature.

The invention accordingly provides a method as described in the preamble, characterized in that in order to obtain CMS Brassica oleracea plants, protoplasts of B. oleracea 35 are fused with protoplasts of an Ogura CMS B. oleracea plant in which the nuclei are inactivated or have been removed, and, if desired, the CMS B. oleracea cells or plants thus obtained, or their descendants, being used as a starting material for obtaining other CMS B. oleracea plants having a B. oleracea core, B. oleracea chloroplasts and mitochondria of the Ogura CMS cytoplasm using in vitro and / or cross methods.

40 The inactivation or removal of the nuclei from protoplasts is known per se from Genetic Engineering of Plants, National Academy Press 1984, p. 37. This article describes protoplast fusion as an option in agriculture to make plants with specific properties. The assumption is made that certain properties by the cytoplasm could be transferred separately using a donor protoplast from which the nucleus has been removed. This reference does not mention the formation of CMS B. oleracea plants. Nor is it mentioned how the core could be removed.

Plant Cell Culture, R.A. Dixon (1987), p. 61 gives a brief introduction to the inclusion of organelles in protoplasts and notes that "in many instances the protoplast is a most effective delivery system, via fusion, for organelles." This reference does not mention formation of B. oleracea plants.

50 It is also noted that in the late publication Protoplast Symposium, Wageningen, Book

Proceedings, biz. 169-176 (1988), the above-mentioned article by Pelletier et al. Is further discussed. As mentioned above, Pelletier et al. Describes the fusion of a normal B. napus protoplast with a CMS B. napus protoplast. Male-sterile plants would thereby be obtained with a normal chlorophyll content. However, it is stated in the above-mentioned late publication that the offspring of 55 such plants is deformed and therefore in fact not suitable for use in agriculture. This shows that the protoplast fusion method as described in pelletier et al. Was still far from perfect.

It is further noted that the newspaper article Nikkan Kogyo Shinbun, article no. 6676, 4 195093 2 November 21, 1987 (Sakata Seed Corp.) states that Sakata Seed apparently produced new types of cabbage that show CMS but no chlorosis. To this end, this article describes protoplast fusions of B. oleracea with CMS B. campestris, and of B. oleracea with CMS R. sativus. It is not specified which cage plants are thereby obtained.

The present invention is particularly suitable for obtaining CMS in the following B. oleracea varieties: 1 Brassica oleracea L. convar. acephala (DC.) Alef. var. botrytis L. (cauliflower) 2 Brassica oleracea L. convar. capitata (L.) Alef. var. alba DC (white cabbage) 3 Brassica oleracea L. convar. oleracea var. gemmifera DC (Brussels sprouts) 10 4 Brassica oleracea L. convar. acephala (DC.) Alef. var. sabellica L. (kale) 5 Brassica oleracea L. convar. capitata (L.) Alef. var. sabauda L. (Savoy cabbage) 6 Brassica oleracea L. convar. capitata (L.) Alef. var. rubra DC. (red cabbage) 7 Brassica oleracea L. convar. acephala (DC.) Alef. var. gongylodes (kohlrabi) 8 Brassica oleracea L. convar. botrytis (L.) Alef. var. cymosa Duch (broccoli), more particularly in cauliflower, white cabbage, Brussels sprouts and broccoli, as indicated above.

The term Ogura CMS cytoplasm as used herein refers to cytoplasm from Raphanus sativus that contains mitochondria DNA, which confers male sterility to plants. The term Ogura CMS Brassica oleracea plant or plant cell used herein denotes a Brassica oleracea plant or plant cell containing Ogura CMS cytoplasm.

The protoplast fusion according to the invention can be carried out by using polyethylene glycol (PEG) that causes agglutination or caking, in the presence of a fusion buffer, ie a solution with a high pH to "melt" the membranes. Such a somatic hybridization can be performed under the conditions described by Sundberg and med. [Plant Science 43 (1986) 155] for the preparation of interspecific hybrids or modifications thereof.

A suitable method is the following:

The protoplast fusion according to the invention is suitably carried out in a protoplast fusion solution (FS-1), which comprises a buffer such as tris (hydroxymethyl) aminomethane hydrochloride, an osmotic agent, for example a carbohydrate such as mannitol, sorbitol, glucose or sucrose and potassium and calcium salts, contains. The pH can vary from 5.2 to 10 and is preferably about 7.2. The protoplasts of various kinds are mixed and concentrated, suitably to a final density of 105 to 106 protoplasts per ml.

The protoplast mixture is then allowed to stand undisturbed for at least 10 minutes to allow the cells to settle on the bottom of the petri dish. The mixture is then treated with polyethylene glycol (PEG), preferably with a molecular weight of 1500 to 6000. Generally, good results are obtained when, for example, an aqueous solution is used which contains 40% by weight of PEG (FS-2) with a volume ratio from FS-1 to FS-2 from 10: 1 to 1: 1. FS-2 suitably contains an osmotic agent and a calcium salt. The cells are incubated for 1 to 20 minutes in FS-2, depending on the brittleness of the cells.

The fusion is carried out, for example, by washing twice with fusion solutions containing PEG, in a concentration lower than in FS-2, an osmotic agent (for example glucose or sorbitol) in a concentration that gives a lower osmolarity than FS-2 and a magnesium salt, contain.

Temperatures at which the fusion process is suitably carried out are between 20 and 24 ° C and the temperature is preferably 22 ° C.

The concentration of PEG in the "washing solutions" is gradually reduced with each subsequent washing step.

Each washing step will take at least 2 minutes to slowly adjust the protoplasts to the lower osmolarity of the medium, in order to avoid cracking of the cells.

After performing the washing steps, the fused protoplasts are placed in a suitable culture medium. The density of the protoplasts will be in the range of 105 to 106 protoplasts per ml.

It is also possible to perform such a protoplast fusion using electric current.

For the purposes of electrofusion, protoplast chains consisting of lines of up to 8 cells, for example 5 cells, are subjected to a direct current (DC) burst ranging from, for example, 400 to 1000 V / cm with a duration of the current burst of, for example, 10 to 50 ps. The 55 protoplasts thus fused are suitably held in the electric field for some time, for example 1 to 2 seconds, before the electric field is switched off in order to allow the protoplasts to resume their round shape for some time.

The protoplast chains can be obtained in a manner known per se by subjecting the protoplasts to the electric field of an alternating current (AC). Optimal conditions are determined by varying the frequency of the alternating field, for example in the vicinity of 1 MHz and the voltage up to 150 V / cm, so that the cells are arranged within a few minutes.

The fusion products thus obtained can be regenerated in the presence of unfused parent protoplasts, or after an optical selection from the culture. Such an optical selection can be carried out by micro-manipulation of the cells, for example according to the method described by Patnaik et al., Plant Science Letters 24 (1982) 105, for the manual isolation and identification of plant heterokaryons, or using a cell sorter, for example according to the method described by Glimelius and med., Plant Science 45 (1986) 133, for the selection and enrichment of plant protoplast heterokaryons by cell sorting.

When the selection method is used, the parent protoplasts are, for example, stained with fluorescent dyes, for example fluorescein isothiocyanate, wherein, if one of the fusion participants comes from leaves, autofluorescence of the chlorophile can be used for the selection.

The fusion products thus obtained are grown in a suitable culture medium that contains a well-balanced nutritional additive for the growth of the protoplasts, containing micro and macro elements, vitamins, amino acids and small amounts of carbohydrates, for example various sugars, such as glucose. Glucose serves both as a carbon source and as an osmotic agent. The culture medium contains 20 plant hormones (auxins and cytokinins), which are able to control cell division and regenerate shoots. Examples of suitable auxins are α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 3-indole acetic acid (IAA). Examples of suitable cytokinins are 6-benzylaminopurine (BAP) and zeatin (Zea). In general, NAA and 2,4-D are used in combination with BAP to start cell division. The ratio of auxin to cytokinin must then be high, for example greater than 1. After 7 to 10 days, the concentration of auxins is suitably reduced by adding the same culture medium, but without or with considerably fewer auxins. Typical, star-shaped microcalli will usually have developed after 3-4 weeks. Such microcalli will then be transferred to a regeneration medium in order to initiate the shoot formation, preferably after adaptation in an intermediate regeneration medium to the differences in composition and physical properties between the culture medium and the regeneration medium.

For the formation of shoots, the ratio of auxin to cytokinin in the regeneration medium will suitably be low, for example lower than 1:10. In general, it is recommended to use the auxin IAA in combination with the cytokinins Zea and BAP for the shoot regeneration.

The regeneration media, BR-1 and BR-2, are relatively poor media compared to the culture medium. They contain fewer vitamins and the carbon source content is lower, they contain only sucrose and xylose as a carbon source and no amino acids and coconut milk. The regeneration media has a higher viscosity than the culture medium. BR-1 is semi-solid and contains growth regulators NAA, 2,4-D and BAP, the ratio of auxin to cytokinin being lower than 1. BR-2 contains Zea and BAP and optionally IAA.

After regenerating in the BR-1 medium for 4-6 weeks, calli with a diameter of about 3 mm 40 are transferred to the BR-2 regeneration medium which has a low sucrose concentration. At this stage lottery tickets will develop within 2-3 weeks.

The obtained shoots are then rooted in a basic medium, MS, without additional hormones.

The DNA of the nucleus and DNA of the cell organelles of the plants thus obtained can then be identified in a manner known per se, for example using suitable restriction endonucleases and comparing the DNA digestion pattern of the fusion products thus obtained with that of the fusion products. parent lines.

Brassica oleracea protoplasts and inactivated or core-free protoplasts from an Ogura CMS Brassica oleracea plant, used herein as starting material, can be obtained from the corresponding plant cells in a manner known per se.

Cells that do not contain cell walls, i.e. protoplasts, are obtained from green plant material, e.g. leaves, and / or from white plant material, e.g. wilted seedlings, cell suspension cultures, roots or bleached plant material, by conventional methods, e.g. by the method described by Glimelius , Physiologia Plantarum 61 (1984) 38, for the regeneration of hypocotyl protoplasts.

55 If an optical selection of the fused products is envisaged, the starting materials will be suitably selected from a green plant or, if they come from white plant material, they will be suitably colored in order to facilitate the selection.

195093 4

The inactivated or core-free protoplasts of an Ogura CMS Brassica oleracea plant are obtained in a manner known per se from corresponding Ogura CMS Brassica oleracea plant cells or protoplasts, for example by irradiation or according to standard methods known for removing the nuclei from cell material, such as centrifugation.

The inactivation of the nuclei by irradiation can be done with the aid of gamma, ultraviolet or x-rays.

If the radiation is carried out with an X-ray source, the inactivation of the core will generally occur when using a dose of, for example, 10 krad / min for 3 to 20 minutes.

The appropriate dosage of the X-rays can be determined, for example, by determining the minimum X-ray irradiation that kills 100% of the protoplast population; the percentage of dead cells is determined by counting the number of colonies formed after 10-20 days of culture. To obtain optimum conditions for the development of cell colonies at low density, it is desirable to use a nutrient layer, a preconditioned culture medium or an appropriate cell recovery method. To determine the minimum dose required for inactivation of cell divisions, the 15 protoplasts are exposed to five increments of the X-ray dose: the minimum dose, 10 and 20 krad above and below the minimum dose. The protoplasts thus obtained are then used in the method according to the invention.

A satisfactory inactivation of the nuclei can generally also be achieved by irradiation with gamma rays of 60 Co in a dose of 3-30 krad.

Removal of the nuclei can also be performed by incubating protoplasts in a strong osmotic medium to obtain subprotoplasts that do not contain nuclei.

A suitable method for removing nuclei by ultracentrifugation that is suitable for the preparation of protoplast starting material according to the invention that does not contain cores is described by Spangenberg in the EUROPEAN JOURNAL OF CELL BIOLOGY 39 (1985) 41-45. Cytochalasin-B is suitably added to facilitate the release of the nuclei from the cells.

Ogura CMS Brassica oleracea plants can be obtained from B oleracea and CMS Raphanus sativus, as already stated, according to classical breeding methods.

It will be clear that the Brassica plants according to the invention can be used as starting material for obtaining other Brassica oleracea varieties with the desired mitochondria of the Ogura CMS cytoplasm and chloroplasts of normally fertile B oleracea and, if desired, additional desirable characteristics by in vitro and / or cross methods. Such in vitro and cross-breeding techniques are known to those skilled in the art.

Solutions used in the tests: a) TVL solution 0.3M sorbitol 0.05M CaCl2.2H2 O pH = 5.6 - 5.8 b) Enzyme solution 0.6 -1% cellulysine 0.1% macerase

40 dissolved in BR-1 but with 2 x the sucrose concentration, without agarose and 10 x the 2,4-D concentration

pH = 5.4 - 5.8 c) W5 solution (1L) 18.4 g CaCl2.2H2 O

9.0 g of NaCl

45 1.0 g glucose 0.8 g KCl pH = 5.6 - 5.8 d) CPW16S (1L) 16% sucrose 0.0272 g KH2 PO4 50 0.1010 g KN03

1.4800 g CaCl2.2H2 O 0.2460 g MgSO4.2H2 O 0.00016 g KI

0.000025 g CuSO4 .5H2 O pH = 5.5 - 5.8 195093, e) Fusion solution - 1 (FS - 1)

0.15 M sorbitol 0.03 M CaCl 2 .2H 2 O 0.075 M KCl

5 0.05 M tris (hydroxymethyl) aminomethane hydrochloride pH = 7.2 f) Fusion solution-2 (FS-2) 30 - 40% PEG (mw 1500) 0.3 M glucose 10 50 mM CaCl2.2H20 g) Fusion solution-3 (FS-3) 13.3% PEG (mw 1500) 0.1 M glucose 0.067 M sorbitol 15 0.067 M CaCl2.2H20 h) Fusion solution-4 (FS-4) 6.7% PEG (mw 1500) 0.05 M glucose 0.083 M sorbitol 20 0.083 M CaCl 2 .2H 2 O TABLE 1

Composition of the media (mg / l) 25 MS BC-1 BC-2 BR-1 BR-2 KN03 1,900 1,900 1,900 2,500 2,500 NH4NO3 1,650 600 600 250 250

MgSO 4 .2H 2 O. 370 300 300 250 250 KH 2 PO 4 170 170 170

CaCl2.2H2 O 440 600 600 300 300 KCl - 300 300

NaH 2 PO 4 .H 2 O - - 150 150 (NH 4) 2 SO 4 - - - 134 134 35 Kl 0.83 0.75 0.75 0.75 0.75

MnSO 4 .4H 2 O 22.3 10 10 10 10 H 3 BO 3 6.2 3 3 3 3

ZnSO 4 .2H 2 O 8.6 2 2 2 2

NaMo04.2H2 O 0.25 0.25 0.25 0.25 0.25 40 CuSO4.5H2 O 0.025 0.025 0.025 0.025 0.025

CoCl2.6H2 O 0.025 0.025 0.025 0.025 0.025

Fe-EDTA 43 43 43 43 43

Thiamine HCl 0.1 10 10 10 10

Pyridoxine HCl 0.5 1 1 1 1 45 Nicotinic acid 0.5 1 1 1 1

Ascorbic acid -22--

Sodium pyruvate - 20 20

Citric acid - 40 40 - -

Maleic acid - 40 40 50 Fumaric acid - 40 40

Glycine 2 ----

Fructose - 250 250 - -

Ribose - 250 250 - -

Xylose - 250 250 250 250 55 Mannose - 250 250 - -

Rhamnose - 250 250 - 195093 6 TABLE 1 (continued) r

Composition of the media (mg / l) MS BC-1 BC-2 BR-1 BR-2 5 -------

Cellobiose - 250 250 -

Sorbitol - 250 250 -

Mannitol - 250 250 -

Inositol 100 100 100 100 100 10 Saccharose ** 250 250 70,000 50,000

Glucose - 68,400 68,400 - -

Casamino acid - 250 250 - -

Coconut water * - 20 20 - -

Agarose - - - 2,000 4,000 Agar 6,000 ---- NAA - 0.1 0.1 0.1 2.4-D - 1 - 0.1 - IAA - (0.1)

Zeatin - --- 1 20 BAP - 0.5 0.5 0.5 0.5 ml / l ** see the examples

Examples 25

Example 1 a) Seed disinfection and germination i) Seeds of Brassica oleracea, Delira type cauliflower with CMS cytoplasm of Raphanus sativus CMS Ogura (hereinafter referred to as B. oleracea A 61043) were briefly immersed in 20% alcohol and 20% alcohol disinfected in a 1% sodium hypochlorite solution with a rotary shaker at 160 revolutions per minute at 22 ° C. It is then necessary to rinse intensively with sterile, distilled water. The seeds are placed on the MS nutrient medium (see Table 1), with 1% sucrose and without hormones. To obtain green, disinfected plants, the seeds are grown at 22 ° C on replica plates in the light (3000 lux), 16-hour photoperiod. Under the same conditions, the disinfected shoots were grown in subculture in plastic containers.

To obtain white tissue for protoplast isolation, for example hypocotyls, the seeds were grown in Petri dishes in the dark at 22 ° C.

ii) Analogously to the method according to (i), seeds of Brassica oleracea, cultivar SG 121 (*) 40 (cauliflower) were disinfected and allowed to germinate.

(*) deposited on December 8, 1987 at the American Type Culture Collection under the ATCC designation number 40399.

b) Isolation of colored protoplasts from CMS B. oleracea 45 8-day-old disinfected shoots of white tissue according to step a (i) were cut into small pieces and pre-polymerized for about 1 hour in a TVL solution in the dark.

The TVL solution was removed and a bacterial contamination study of the plant material was performed by growing a portion of the incubated TVL solution overnight in a bacterial medium at 22 ° C.

50 To the material was added an enzyme solution containing 0.6-1% cellulysine, 0.1% macerase and 1 pg / ml fluorescein isothiocyanate and the material was incubated at 22 ° C for 16 hours in the dark.

The suspension was then filtered through nylon cloth (70 µm) and washed with half a volume of CPW16s solution by centrifuging for 7 minutes at 750 revolutions per minute. This leads to the 55 intact protoplasts floating. The protoplasts were collected and were first rinsed with W5 solution and then washed with fusion solution-1 by centrifuging for 5 minutes at 500 revolutions per minute. In this way, colored protoplasts that could suitably be selected by hand or by machine were obtained.

7 195093 c) Isolation of (green) protoplasts from B. oleracea

Four-week-old disinfected shoots of plant material according to step a (l1) were treated according to the method of step b, except that no fluorescein isothiocyanate was added during the enzyme treatment. This produced protoplasts from B. oleracea, cultivar SG 121.

d) Irradiation of colored protoplasts from CMS B. oleracea

Freshly isolated CMS protoplasts according to step b) were placed in a 6 cm Petri dish in a 10 W5 solution (2 ml). The protoplasts were irradiated for 5-20 minutes using an X-ray source (Baltobloc CE 100) at a dose of 10 krad per minute. After the irradiation, the inactivated protoplasts were diluted in fusion solution-1 before being used for the fusion tests.

E) Protoplast fusion

Protoplasts according to steps c and d were mixed 1: 1 in a sterile chamber under a sterile air stream to a final concentration of 5 x 10 * protoplasts (pps) / ml of fusion solution-1.

Droplets of 200 µl were placed in an uncoated Petri dish (5-7 drops per Petri dish of 6 cm), after which they were allowed to settle for 15 minutes (the sterile airflow was switched off in order to avoid disturbance of the depositing protoplasts) . Fusion solution-2 (25-100 μΙ per drop) was added to agglutinate for 3-7 minutes.

The sterile air stream was switched on again and the solution was replaced with fusion solution-3 for 5 minutes. The solution was then replaced with fusion solution-4 for 5 minutes. Finally, the fusion solutions were replaced with 1.5 ml of culture medium (BC-1).

F) Selection with micromanipulator and growth of fusion products

Fused cells, which can be visually recognized by the presence of double fluorescence, were picked from the material obtained in step e with a micromanipulator.

Hybrids or cybrids were grown in Biopor filter membranes, diameter 1.1 cm or 3 cm with a pore size of 0.45-3 µm, containing 100 to 105 cells per ml of culture medium-1. The filters were placed in a Petri dish containing 2-2.5 ml feed cells (105 per ml) and the material was incubated in the dark at 22 ° C.

As soon as 20% of the cells divided, culture medium-2 was added.

When typical star-shaped microcalli were developed, they were transferred to a regeneration medium-1 (BR-1) in coated Petri dishes and grown for 4-6 weeks at 22 ° C under 500 lux.

g) Selection using cell sorter and culture of fusion products

The fused protoplasts from the material obtained in step e were selected using a cell sorter equipped with a mercury lamp (HB 100) for the two parameter fluorescence sorting 40.

The fluorescence excitation beam from the cell sorter was adjusted between 488 nm and 500 nm. The emission beam coming from the excited cells was separated into two light beams by color splitting mirrors, one with wavelengths between 560 and 610 nm, the autofluorescence of the chloroplasts, and the other with wavelengths between 500 and 560 nm, the fluorescein isothiocyanate fluorescence. Two photo multipliers were used to measure these signals. The flow-through liquid (carrier liquid used in the cell sorting device for diluting the protoplast sample in a continuous liquid stream) contained W5 medium treated and degassed in an autoclave.

Sorting was based on the principle that fluorescence from unfused parent cells showed only single fluorescence (red fluorescence from leaf mesophyl protoplasts or fluorescein-50 isothiocyanate yellow / green fluorescence from the colored hypocotyl protoplasts), while fused cells double fluorescence (red and yellow / green) will show.

Sorted hybrids or cybrids were grown in a manner identical to that used for cells selected by micromanipulation.

55 h) Cultivation of fusion products without selection

The total fusion mixture according to step e) was kept in the Petri dish, in which the fusion was performed and cultivated in the dark at 22 ° C. After 1-2 days, the cells were pipetted and 195093 8 transferred to a coated Petri dish. After about 7-14 days when 10% of the cells divided, two volumes of culture medium-2 (BC-2) were added. After 10 days, another volume of BC-2 was added. The cells were always grown in the dark and at a temperature of 22 ° C. After 2-4 weeks, when the cells had formed typical, star-shaped microcalli, they were transferred to regeneration medium-1. The microcalli were grown at a low light intensity (500 lux), at a photo period of 16 hours and a temperature of 22 ° C.

i) Plant regeneration

The calli according to steps f and h, which were developed to a dimension with a diameter of 2-5 mm, were transferred to a regeneration medium-2 (BR-2) and at 22 ° C and 3000 lux with a 16-hour photoperiod. cultured. Shoots of approximately 1 cm were transferred to MS medium with 1% sucrose without hormones and were rooted on the same MS medium.

j) Molecular analysis of the fusion products a) Composition of the core DNA

Characterization of the core composition of the fusion products was performed using specific DNA probes. A 0.9 kb Kpn 1 / Bam H1 fragment of the beta-tubulin gene from Arabidopsis thaliana hybridized with different bands in an endonuclease decomposition pattern of DNA from the core of the CMS donor, B. oleracea A 61043. This pattern is specific for B , oleracea A 61043 and differs from the B. oleracea cultivation lines that were used as acceptors for the CMS property (Figure 1A).

b) Composition of mitochondrial DNA

Characterization was performed with pBO 604 clone containing a 1.5 kbp Sac I fragment from the mitochondrial DNA of Ogura CMS cytoplasm of Raphanus sativus. This clone gives hybridization signals with restriction endonuclease residues from mitochondrial DNA from B. oleracea A 61043 Ogura CMS cytoplasms, but not with mitochondrial DNA from fertile cytoplasm from B. oleracea culture lines (Figure 1B).

c) Composition of chloroplast DNA

The DNA present in the chlorosis-sensitive chloroplasts of the Ogura CMS cytoplasm was characterized with the probe lambda Bcp 17. This clone contains a 4.5 kbp Xho I / Sac I fragment from Ogura CMS chloroplast DNA. The clone hybridizes with a 57.5 kbp band in the Sal I digestion of Ogura CMS chloroplast DNA and with a 9.9 kbp band in the Sal I digestion of chloroplast DNA from fertile cytoplasms in B. oleracea culture lines (Figure 1C).

Figures 1A, 1B and 1C are accurate, hand-drawn copies of photographs.

Example 2

Cytoplast isolation by ultracentrifugation

Instead of irradiation (Example 1, step d), core-free protoplasts can also be obtained by ultracentrifugation. This is illustrated in this example for green protoplasts from CMS

B. oleracea.

4-week-old disinfected shoots of plant material according to Example 1, step a (i) were cut into small pieces and preplasolysed in a TVL solution in the dark for about 1 hour.

The TVL solution was removed and a bacterial contamination study of the plant material was performed by growing a portion of the incubated TVL solution overnight in a bacterial medium at 22 ° C.

An enzyme solution was added to the material containing 0.6-1% cellulysine and 0.1% macerase and the material was incubated for 16 hours in the dark at 22 ° C.

The suspension was then filtered through nylon cloth (70 µm) and washed with half a volume of CPW16s solution by centrifuging for 7 minutes at 750 revolutions per minute. This leads to the intact protoplasts floating. The protoplasts were collected and were first rinsed with W5 solution and then washed with fusion solution-1 by centrifuging for 5 minutes at 500 revolutions per minute.

55 A gradient consisting of 10 ml of water saturated with sucrose and a top layer of 4 ml of 1.5 M sorbitol, containing 0.5% dimethyl sulfoxide (DMSO) and 30 pg / ml cytochalasin-B and 2 ml (= 1 x 106) protoplasts in fusion solution-1 was loaded at the top end thereof. The material was added for 15 minutes

Claims (2)

9 195093 »25 ° C centrifuged at 40,000 g, whereby B. oleracea A 61043 protoplasts were obtained without core (cytoplasts). Example 3 Isolation of colored protoplasts of B, oleracea. Instead of green protoplasts from B, oleracea as isolated in Example 1, step c, colored protoplasts can also be isolated. Seeds of B. oleracea, cultivar SG 121 (cauliflower) were disinfected and germinated according to the method of example 1, step a (ii) and eight-day-old hypocotyls thereof were then treated according to the method of example 1, step b, for obtaining colored protoplasts suitable for manual or machine selection. Method of obtaining CMS plants belonging to a Brassica species, which comprises fusing protoplasts of these Brassica species with protoplasts of an Ogura CMS plant that also belongs to this Brassica species, followed by regeneration of the allogeneic cells thus obtained to plants and identification of plants containing mitochondrial DNA that hybridizes to the 1.5 kb Sacl fragment from mitochondrial DNA of Ogura CMS cytopiasma, characterized in that B. oleracea protoplasts are fused to obtain CMS Brassica oleracea with protoplasts from an Ogura CMS B. oleracea plant, in which the nuclei are inactivated or removed, and optionally the CMS B. oleracea cells or plants thus obtained, or their offspring, being used as starting material for obtaining other CMS B. oleracea plants with a B. oleracea core, B. oleracea chloroplasts and mitochondria from the Ogura CMS cyto piasma using in vitro and / or cross methods. Hereby 1 sheet drawing