HU204561B - Process for producing hybrid brassicaceae with citoplasmic male sterility - Google Patents

Abstract

Brassica oleracea plants containing mitochondria of the Ogura CMS (male sterility) cytoplasm and chloroplasts of normal fertile Brassica oleracea. Method of production by protoplast fusion.

Description

Scope of the description: 8 pages (including 1 sheet)

EN 204561

The invention relates to cytoplasmic male sterility

Brassica is working on developing a new birth line for oleracea. Patch lines can be used to produce hybrid seeds. This invention enables a plant cultivator to incorporate cytoplasmic male sterility (CMS) into a commercially desirable variant of B.oleracea.

Male sterility is a valuable property in the breeding of B.oleracea hybrid seed, since normal flowers are self-pollinating. Male sterile lines do not produce viable pollen and are unable to self-pollinate. By eliminating the pollen of one of the parent variants, the crop generator can ensure that he receives a hybrid seed of uniform quality. Currently, cytoplasmic male sterility is not present in variants of B. oleracea. Hybrid seeds use self-incompatibility systems within industrial-commercial-producer nuclei to avoid self-pollination during seed production. This system results in inaccurate and non-pure hybrid seed items. In addition, it is time-consuming and labor intensive, and it is therefore costly to introduce this genetic system into all breeding lines. Raphanus sativus has discovered a cytoplasm that transmits male sterility. This cytoplasm is known as Ogura CMS cytoplasm, and DNA from mitochondrials and chloroplasts in the cytoplasm is genotypically different from the DNA present in the cytoplasm of prolific B.oleracea plants. The Ogura type CMS cytoplasm can be introduced into B.oleracea by repeated crosscutting. In the resulting Ogura CMS 3 B. oleracea plants, R. sativus mitochondria produce a CMS phenotype. However, the presence of chloroplasts of R.somatic results in chlorine when the plants grow at low temperatures, which in turn causes a reduction in production. Therefore, such 3 Ogura CMS B.oleracea plants are little used in industrial-commercial hybrid seed production.

The present invention relates to B. oleracea plants which have Ogura CMS cytoplasmic mitochondria and normal fertilized B.oleracea cold curd chloroplasts (Roplasts of the present invention. The plants according to the invention have cytoplasmic male sterility but do not show chlorosis when growing at low temperatures.

The present invention also relates to a method for producing B.oleracea plant material having cytoplasmic male sterility and industrially commercially desirable nuclear characteristics and which does not exhibit chlorosis when grown at low temperature. Such CMS B.oleracea plants are fused by fusion of industrially-commercially desirable nucleated B.oleracea protoplasts and Ogura CMS B.oleracea plants with inactivated or nuclear-free protoplasts, and regeneration of the resulting allogeneic cells into plants.

The method according to the invention for CMS is mainly available in the following B.oleracea variants:

1. Brassica oleracea L., acephala variant (DC), botrytis L. subvariant (cauliflower),

2. Brassica oleracea L., capitata variant (L), alba (DC) sub variant (white cabbage),

3. Brassica oleracea L., oleracea variant, gemmifera (DC) sub variant (Brussels sprouts),

4. Brassica oleracea L., acephala variant (DC), sabellicaL. subvariant (fodorkel),

5. Brassica oleraceaL., Capitata variant (L), sabauda

L. subvarian (curled cabbage),

6. Brassica oleracea L., capitata variant (L), rubra (DC) sub variant (red cabbage),

7. Brassica oleracea L., acephala variant (DC), 10 gongylodes sub-variant (turnip),

8. Brassica oleracea L., botrytis variant (L), cymosa

Duch subvariant (broccoli), preferably cauliflower, white cabbage, brussels sprouts and broccoli.

The Ogura CMS term, as used above, refers to cytoplasm of Raphanus sativus origin; it contains mitochondrial DNA that transmits male sterility to plants. The term Ogura CMS Brassica oleracea plant or plant cell, aho, is used herein to refer to a Brassica oleracea plant or plant cell containing Ogura CMS cytoplasm.

The protoplast fusion in the method according to the invention is carried out by the application of agglutinating polyethylene glycol (PEG) in the presence of a fusion buffer, i.e. a high pH solution, to allow fusion of the membranes. Such somatic hybridization may be carried out under the conditions described by Sundberg et al. (Plant Science 43, 155 (1986)) for the production of interspecific hybrids or their modifications.

In the method according to the invention, protoplast fusion is preferably performed in a protoplast fusion solution (FS-1) which is for a buffer, e.g. tritium (hydroxymethyl) aminomethane hydrochloride, an osmotic agent, e.g. carbohydrates, e.g. mannitol, sorbitol, glucose or sucrose, and potassium and calcium salts. The pH may range from 5.2 to 10, preferably pH 7.2. Protoplasts of different origins are mixed and concentrated, suitably at the final density of KP-10 ⁶ protoplast / ml.

The protoplast mixture is then allowed to stand 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 polyethylene glycol of from 1500 to 6000. Generally, good results are obtained when e.g. An aqueous solution containing 40% PEG was used (FS-2) in a volume ratio of 10: 1 to 1: 1 FS-1 / FS-2. Preferably, FS-2 also contains an osmotic and a calcium salt. Cells were incubated for 1 minute to 20 minutes in FS-2, depending on cell fragility.

The fusion is completed by e.g. twice washing with a fusion solution containing PEG lower than FS-2, an osmotic agent (e.g. glucose or sorbitol) at a concentration lower than osmotic pressure of FS-2 and a magnesium salt.

The fusion process is preferably carried out at a temperature between 20 ° C and 24 ° C, preferably at 22 ° C.

EN 204561

The concentration of PEG in the "washing solutions" is gradually reduced in each successive wash step (see, e.g., Example 9 and fusion solutions 3 and 4).

The individual washing steps must take at least 2 minutes to allow the protoplasts to slowly slow down the osmolarity of the medium to prevent the cells from cracking.

Upon completion of the washing steps, the fused protoplasts are transferred to a suitable culture medium. The density of protoplasts is in the range of 10 ⁵ to 10 ⁶ protoplasts / ml.

According to another method, the protoplast fusion can be performed by electrical voltage.

For electrofusion of up to 8 cells, e.g. 5-cell protoplast chains are subjected to direct current (DC) pulses in the range of 400-1000 V / cm, e.g. 10-50 psec pulse duration. The protoplasts thus fused are suitably for a period of time, e.g. For 1-2 seconds, we keep it in an electric field before turning off the electric field to give the protoplasts some time to regain their circular shape.

The protoplast chains can be prepared in a manner known per se, and the protoplasts are exposed to an AC field. Optimal conditions are determined by varying the frequency of the alternating field, e.g. At a voltage of about 1 MHz and a voltage of up to 15 V / cm, so that the cells are queued within a few minutes.

The resulting fusion products can be regenerated in the presence of non-fused parent proteases or after optical selection from the culture. Such optical selection may be accomplished by micromanipulation of the cells, e.g. by the method described by Patnaik et al. (Plant Science Letters, 24, 105 (1982)) for manual isolation and identification of plant heterocarions, or using a cell sorting apparatus, e.g. by the method described by Glimelius et al., Plant Science 45,133 (1986) for the selection and enrichment of plant protoplast heterocarions by cell sorting.

When using the selection strategy, the parent protoplasts are fluorescent dyes, e.g. staining with fluorescein isothiocyanate; if one of the fusion partners is of leaf origin, the autofluorescence of chlorophyll can be used for selection.

The resulting fusion products are cultured in a culture medium that provides a well-balanced medium for protoplast growth; it includes micro- and macro-elements, vitamins, amino acids and small amounts of carbohydrates, e.g. various sugars such as glucose. Glucose serves as a carbon source and osmotic. The culture medium contains plant hormones (auxins and cytokinins) that can regulate cell division and regeneration. Examples of suitable auxin include naphthyl acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4D) and indole acetic acid (IAA). Examples of suitable cytokinins include benzylamino-purine (BAP) and zeatin (Zea).

Generally, NAA and 2,4-D are used in combination with BAP to initiate cell division. The auxin / cytokinin ratio should then be high, e.g. Greater than 1.

After 7-10 days, the concentration of auxins is suitably diluted by the addition of the same culture medium containing no or significantly less auxins. Usually, after 3-4 weeks, typical star-shaped microcells are developed. Such microcells are then transferred to a regenerating medium to initiate the shoot formation, preferably in an intermediate regeneration medium after adaptation to differences in composition and physical properties between the culture medium and the regeneration medium.

Preferably, the auxin / cytokinin ratio for shoot formation in the regeneration medium is low, e.g. 1:10. For regeneration, it is generally preferred to use the IAA auxin in combination with Zea and BAP cytokinins.

BR-1 and BR-2 regeneration media are relatively poor media relative to the culture medium. They contain fewer vitamins, the carbon content is lower, they contain only sucrose and xylose as carbon sources and do not contain amino acids and coconut milk. The viscosity of the regenerating media is also higher than that of the culture medium. BR-1 is semi-solid and NAA; It contains 2,4-D and BAP growth regulators with auxin / cytokinin ratio of less than 1. BR-2 contains Zea and BAP, and optionally IAA.

After 4-6 weeks of regeneration in BR-1 medium, callipers with a diameter of about 3 mm are transferred to BR-2 regenerating medium containing a low sucrose concentration. At this stage, shoots develop within 2-3 weeks.

The resulting shoots are then rooted in non-hormone-free MS medium.

The DNA of the nuclear nucleus thus obtained and the DNA of the cellular organ can then be identified by known methods, e.g. using the appropriate restriction endonucleases and the DNA digestion sample of the resulting fusion products is compared with the DNA digestion pattern of the parent lines.

Brassica oleracea protoplasts and inactivated or nuclear-free protoplasts of the Ogura CMS Brassica oleracea plant can be obtained from known plant cells in a known manner.

Cell-free cells, i.e. protoplasts, are extracted from plant material, e.g. from leaf materials and / or white plant materials, e.g. yellowed germs, cell suspension cultures, roots or bleached plant materials can be prepared by conventional methods, e.g. by the method described by Glimelius [Physiologia Plantarum 61, 38 (1984)] for the regeneration of pre-cotyledon (hypocotyl) protoplasts.

If the fusion products are to be selected by optical selection, the starting materials are preferably removed.

EN 204561 is removed from the green plant, or if it is derived from white plant material, it is preferably stained to facilitate selection.

Inactivated or nuclear-free protoplasts of the Ogura CMS Brassica oleracea plant alone are produced from the corresponding Ogura CMS Brassica oleracea plant cells or protoplasts, e.g. irradiation or other standard methods known to remove nucleus from cellular material; such as centrifugation.

Inactivation of the nucleus can be accomplished by gamma, UV or X-rays.

Where irradiation is effected by means of an X-ray source, inactivation of the core is generally effected e.g. The dose is 15 to 10 minutes per minute for 3 to 20 minutes.

A suitable dose of X-ray, e.g. determining the level of minimal X-ray radiation that kills 100% of the protoplast population; the number of dead cells, estimated from the number of colonies formed in the culture, is estimated to be 10 to 20 days later. In order to obtain optimal conditions for the development of cell sites at low density, it is desirable to use a nourishing layer, a preconditioned culture medium, or a suitable cell rescue method. After determining the minimum dose required to inactivate cell division, the protoplasts are subjected to different levels of X-rays at a minimum dose of 30 and 10 and 20 times the minimum dose and below. The protoplasts thus obtained are then used in the process of the present invention.

Generally, satisfactory nucleus inactivation can be achieved with gamma irradiation in the range of 3 to 30 cr.

The nucleation can also be accomplished by incubating the protoplasts in medium with high osmotic value to obtain cell-free alprotoplasts. 40

A suitable method for the ultracentrifugation of cell nuclei, which is suitable for the preparation of the cell-free protoplast excretion material of the present invention, is described in Spangenberg, EUROPEAN JOURNAL OF CELL BIOLOOY 45 39, 41-45 (1985). Preferably, Cytochalasine B is added to facilitate the release of nuclei from the cells.

Ogura CMS Brassica oleracea plants can also be obtained by conventional culture techniques from B. oleracea 50 and CMS Raphanus sativus (see introduction to this application).

'It should be noted that the Brassica plants produced by the process of the present invention may be used as starting materials for other Brassica oleracea strains having the desired mitochondrial matrix of Ogura CMS, with chloroplasts of normal fertilized B.oleracea and, if desired, further desirable characteristics; for their production in vitro and / or crossing! Techniques may be used. Such in vitro and crossover techniques are well known to those skilled in the art from the literature.

The following examples illustrate the process of the invention.

The solutions used in the experiments are as follows (% values are% by weight unless otherwise indicated):

a) TVL solution

0.3 mol / liter of sorbitol

0.05 mol / L CaCl ₂ · 2H ₂ O pH 5.6-5.8

b) Enzyme solution

0.6-1% cellulizine

0.1% macerase

Dissolved in BRl but with double sucrose without agarose and tenfold concentration

With 2,4-D pH-5,4-5,8

c) Solution W5 (1 L) of 18.4 gCaCl ₂ 2H ₂ O 9.0 g NaCl

1.0 g of glucose

0.8gKCl pH-5.6-5.8

d) CPW16s (l liter)

16% Sucrose 0.0272 g KH ₂ PO ₄ 0.1010 gKNO 1.4800 g CaCl ₂ 2H ₂ O 0.2460 MgSO ₄ 2H ₂ O 0.00016 g KI

0.000025 g of CuSO ₄ '5H ₂ O pH 5.5-5.8

e) Fusion Solution 1 (FS-1)

0.015 M CaCl ₂ · 2H ₂ O 0.075 mol / IKCl

0.05 mol / l tris (hydroxymethyl) -ano-methane hydrochloride pH-7.2

f) Fusion Solution 2 (FS-2)

30-40% PEG (molecular weight: 1500) 0.3 mol / L glucose 50 mM CaCl ₂ 2H ₂ O

g) Fusion Solution 3 (FS-3)

13.3% PEG (molecular weight: 1500) 0.1 mol / L glucose 0.067 mol / l sorbitol 0.067 mol / lCaCl _2? 2H ₂ O

h) Fusion Solution 4 (FS-4)

6.7% PEG (molecular weight: 1500) 0.05 mol / l glucose 0.083 mol / l sorbitol 0.083 mol / l CaCl ₂ * 2H ₂ O

EN 204561

Table 1

Composition of media (mg / l)

MS BC-1 BC-2 BR-1 BR-2 kno ₃ 1900 1900 1900 2500 2500 NH4NO3 1650 600 600 250 250 MgSO ₄ «2H ₂ O 370 300 300 250 250 KH2PO4 170 170 170 - - CaCl ₂ * 2H ₂ O 440 600 600 300 300 KC1 - 300 300 - - NaH ₂ PO ₄ «H ₂ O - - - 150 150 (NH ₄ ) ₂ SO ₄ - - - 134 134 WHO 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 NaMoO ₄ -2H ₂ O 0.25 0.25 0.25 0.25 0.25 CuSO ₄ -5H ₂ O 0,025 0,025 0,025 0,025 0,025 CoC1 ₂ 6H ₂ O 0,025 0,025 0,025 0,025 0,025 FeEDTA 43 43 43 43 43 Tiamin'HCl 0.1 10 10 10 10 Piridoxin'HCl 0.5 1 1 1 1 nicotinic acid 0.5 1 1 1 1 ascorbic acid - 2 2 - - Sodium pyruvate - 20 20 - Citric acid - 40 40 - - maleic - 40 40 - - fumaric - 40 40 - - glycine 2 - - - - fructose - 250 250 - - ribose - 250 250 250 , 250 xylose - 250 250 -  - mannose - 250 250 - - rhamnose - 250 250 - - cellobiose, - 250 250 - L sorbitol 250 250 - - mannitol - 250 250 - - inositol 100 100 100 100 100 saccharose ** 250 250 70,000 50,000 glucose - 68,400 68,400 - - casamino 250 250 - - Coconut Water * 20 20 - - agarose - - - 2000 4000 agar 6000 - - - - NAA - 0.1 0.1 0.1 - 2,4-D - 1 - 0.1 - IAA - - - - (0.1) zeatin - - - - 1 BAP - 0.5 0.5 . 0.5 0.5

* ml / 1 ** see examples

Examples Example 1

Magsterilization and germination

Brassica oleracea Delira type cauliflower, which

Raphanus sativus CMS has a CMS cytoplasm from Ogura (hereinafter referred to as B.oleracea called 61043) for a short time, dipped in 20% alcohol and in a 1% sodium hypochlorite solution for 20 minutes on a rotating shaker at 160 rpm 22 Sterilized at a temperature of ° C. Then rinse thoroughly with sterile distilled water. Seeds were placed on hormone-free medium containing 1% by weight of sucrose (see Table 1). To grow sterile green seedlings, the seeds are replicated5

HU 204561 is grown over a 16-hour illumination period at 3000 lux at 22 ° C. Sterile shoots were cultured under the same conditions in a plastic container.

To isolate protoplasts from white tissues, e.g. hypocotyls or seeds in Petri dishes are grown in the dark at 22 ° C.

Example 2

Analogously to the procedure of Example 1, Brassica oleracea SG 121 variant (cauliflower), deposited on December 8, 1987 at the American Type Culture Collection under deposit number ATCC 40399, is germinated by sterilization.

Example 3

Isolation of protoplasts

The four-week sterile shoots of the plant material of Example 1 were cut into small pieces and pre-plasmized in about 1 guillocraft in a dark solution.

The TVL-αdate was removed and the bacterial contamination of the plant material was checked by culturing a portion of the incubated TVL solution in culture medium at 22 ° C overnight.

An enzyme solution containing 0.6-1% of cellulose and 0.1% macerase was added to the material and the substance was incubated through 16 guards in the dark at 22 ° C.

The suspension was then filtered through a nylon mesh (70 µm) and washed with half a volume of CPW solution at centrifugation at 750 rpm for 7 minutes. This results in the floating of intact protoplasts. The protoplasts were collected and rinsed first with W5 solution and then washed with the fusion solution 1 at 500 rpm for 5 minutes.

Example 4

The 8-day hypocotypes of the plant material of Example 1 were isolated according to the procedure of Example 3, with the exception that 1 µg / ml fluorescein isothiocyanate was added during enzymatic treatment. In this way, painted protoplasts suitable for manual or machine selection are obtained.

Example 5

The seeds of Brassica oleracea SG 121 (cauliflower) are sterilized and germinated according to the procedure of Example 1 and treated for 4 weeks with the method of Example 3 to obtain protoplasts of B.oleracea SG 121.

Example 6

B.oleracea SG 121 (cauliflower) seeds were sterilized and germinated according to the procedure of Example 1, and treated for 8 days with hypocotyls according to the procedure of Example 3 with the addition of 1 µg / ml fluorescein isocyanate during enzymatic treatment. to obtain painted protoplasts suitable for manual or machine selection.

Example 7

Irradiation of Protoplasts The freshly isolated protoplasts of Example 3 were placed in 6 cm Petri dishes in W5 (2 mL). The protoplasts were irradiated with an X-ray source (Baltoblac CE 100) at a dose of 10 kr / min for 5-20 minutes. After irradiation, the inactivated protoplasts are diluted with the first fusion solution before being used for fusion experiments.

Example 8

Cytoplasmization by Ultracentrifugation A gradient of 10 ml of sucrose saturated water and 4 ml of 1.5 mol / l sorbitol and 0.5% of dimethylsulfoxide (DMSO) and 30 pg / ml of cytochalasine Bt contains 2 ml (= 140 ⁶ ). The protoplast prepared according to Example 3 is loaded in fusion solution 1. The material was subjected to a 15-minute centrifugation at 4000 g at 25 ° C to obtain B.oleracea A 61043 nuclear-free cytoplasm.

Example 9

Fusion procedure

The protoplasts of claims 5 and 7 are mixed in a sterile chamber at a 1: 1 sterile air flow to a final concentration of 540 ⁵ protoplasts (pps) / ml fusion solution 1.

200 μΐ droplets are placed in the uncovered

Allow the petri dish (5-7 drops / 6 cm petri dish) to settle for 15 minutes. (The sterile air flow is switched off to avoid disturbing the settling protoplasts.) A fusion solution (25-100 µl / droplet) is added to induce agglutination for 3-7 minutes.

The sterile airflow is again switched on and after 5 minutes, 1 ml of the solution is replaced by 1 ml of fusion solution 3 and then replaced in 5 minutes with 1 ml of fusion solution 4 for 5 minutes. Finally, the fusion solutions were replaced with 1.5 ml of te40 culture medium (BC-1).

10.példa

Selection and growth of fusion products 45 Fused cells, which can be visually recognized by the presence of double fluorescence, are taken up with a micromanipulator.

Hybrids or cibrides were cultured on Biopor filter membranes having a diameter of 1.1 cm or 3 cm 50 and a pore size of 0.45 to 3 µm; they contain 100-1G of ⁵ cells per ml of culture medium. The filters are placed in a Petri dish containing 2 to 2.5 ml of nutrient cell (IOVml) and incubated at 22 ° C in the dark.

When 20% of the cells are divided, culture medium 2 is added.

When a typical star-shaped microchip develops, it is transferred to a regenerating feed bed in the covered Petri dish (BR-1) and cultured at 22 ° C for 60,500 lux for 4-6 weeks.

EN 204 561

Example 11

The hypocotyl protoplasts stained according to the procedure of Example 4 were irradiated according to the procedure of Example 7. The inactivated stained protoplasts thus obtained were fused with the protoplasts of Example 5 and the fused protoplasts were selected in a cell sorting apparatus equipped with a mercury lamp (HB 100) for two parameter fluorescence grading.

The fluorescent excitation radius of the cell classifier is adjusted between 488 and 500 nm. Emission radiation from excited cells is broken down by "dichroic" mirrors on two light beams: one with a wavelength of between 560 and 610 nm, representing the autofluorescence of chloroplasts, and the other having a fluorescence of fluorescein isothiocyanate at 500 to 560 nm. Two photoconductors are used to measure these signals. The coating fluid (carrier fluid used in the cell classifier to dilute the protoplast sample in a continuous flow of fluid) contains autoclaved and degassed W5 medium.

The classifier is based on the principle that the fluorescence of non-fused parent cells exhibits only unique fluorescence (red fluorescence of leaf mesophilic protoplasts or yellow / green fluorescence of fluorescein isothiocyanate of dyed hypocotyl protoplasts), while fused cells exhibit double fluorescence (red and yellow / green). ).

The selected hybrids and cibrides were cultured in the same manner as used for micromanipulated cells.

Example 12

The total fusion mixture of Example 9 was held in a Petri dish in which the fusion was performed and cultured at 22 ° C in the dark. After 1-2 days, the cells are pipetted into a covered petri dish. After about 7 to 14 days, when 10% of the cells divide, two-fold culture medium (BC-2) was added. Ten days later, additional single volume BC-2 was added. Cells were further cultured in the dark at 22 ° C. After 2-4 weeks, when the cells form typical star-shaped microcells, the cells are transferred to regeneration medium 1. The microcells are then cultured at low light intensity (500 lux) for 16 hours at 22 ° C.

Example 13

plant regeneration

The callus according to claim 12, which has evolved to a diameter of 2 to 5 mm, are transferred to regeneration medium 2 (BR-2) and cultured at 22 ° C at 3000 lux for 16 hours.

Approximately 1 cm shoots are transferred to MS medium containing 1% sucrose and free of hormone and rooted.

Example 14

Molecular analysis of fusion products

a) Cell nuclear DNA composition

The characterization of the nucleus composition of the fusion products is performed using specific DNA test samples. A 0.9 kb KpnI / BamHI fragment of the beta tubulin gene of Arabidopsis thaliana hybridizes with different bands in the endonuclease digestion of B.oleracea A 61043 CMS donor nucleus DNA. This digestion pattern is specific for B.oleracea A 61043 and differs from the B.oleracea culture lines that are used as receptors for CMS characteristics. (Figure 1A)

b) Mitochondrial DNA composition

Characterization is performed with pBO 604 DNA; a clone containing a 1.5 kbp SacI fragment from the mitochondrial DNA of raphanus sativus Ogura CMS. The clone gives hybridizing signals to B. oleracea The mitochondrial DNA digestion of endothelial DNA from the cytoplasm of the 61043 Ogura CMS cytoplasm, but it does not signal mitochondrial DNA from the fertilized cytoplasm of B.oleracea breeding line.

c) Chloroplast DNA composition

DNA present in chloroplasts of chloroplasts susceptible to Ogura CMS cytoplasm is characterized by the lambda Bcp17 assay. This clone contains a 4.5 kbp XhoI / SacI fragment from Ogura CMS chloroplast DNA. The clone hybridizes with a 57.5 kbp band in the Ogura CMS-chloroplast DNA Saliva digestion and a 9.9 kbp band in the Sall digestion of chloroplast DNA from fertilized cytoplasm in B.oleracea (line 1) figure).

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

Claims (1)

Patent Claim Point CLAIMS 1. A method of producing Brassica oleracea plants having cytoplasmic male sterility comprising Ogura cytoplasmic male sterile cytoplasmic mitochondria and normal fertile Brassica oleracea chloroplasts, characterized in that a protoplast of B. The resulting allogeneic cells are regenerated.