DK160392B - Process for the somatic hybridization of rape - Google Patents

Description

in

DK 160392 B

In particular, the present invention relates to a method of somatic hybridization, which enables the production of novel varieties of rapeseed.

An increase in rapeseed yield could constitute an element that made it possible to reduce the deficit of proteinaceous oil in the Community countries.

Although rapeseed, a species well adapted to the European climate, has already undergone important genetic improvements (introduction of disease resistance, improvement of oil quality and oilcakes), the varieties grown in France, 10, however, pure cultivated strains. Thus, one could hope for a significant increase in yield (of the order of 20%) if F1 hybrid varieties were produced. This preparation assumes that a cytoplasmic male sterility system is available, which allows for "genetic castration" of the strain which serves as the parent individual for these hybrids.

In the species of rapeseed (Brassica napus) no cytoplasmic male-sterile breeding plants have been found that could be exploited. In contrast, it has been possible to achieve stable male cytoplasmic sterility by transferring cytoplasm from radishes to rapeseed at appropriate junctions. Unfortunately, this transfer of cytoplasm has also caused the rapese a lack of heat-sensitive chlorophyll, rendering the male sterile strain obtained useless as such.

Namely, from 12 ° C, yellowing of the leaves can be observed, especially during the first growing period, which increases the sensitivity of the plant to cold during a critical growing period.

Applicants have been able to provide a method that allows rape to be produced with a male cytoplasmic sterility (chs) and with predetermined cytoplasmic traits carried by a fertile individual, especially a non-deficiency of chlorophyll in cold.

The invention relates to a method for producing male sterile rape with predetermined cytoplasmic features, characterized in that:

DK 160392 B

2 (a) somatic fusion of protoplasts from two rapeseed species, A and B, with A having male sterility and B having fertility, has said cytoplasmic characteristics, and 5 b) the fusion products are grown until seedlings are obtained.

The obtained cybrids (cytoplasmic hybr / d) are chs strains which, for example, do not exhibit yellow staining when the temperature drops below 12 ° C.

In particular, the A plant may be a Japanese rapeseed cytoplasm rapeseed or a rapeseed chs exhibiting low-temperature chlorophyll deficiency.

In particular, the B plant may be a fertile rapeseed without chlorophyll deficiency, which may have an interesting cytoplasmic characteristic, e.g., resistance to triazines (rapeseed with Canadian rapeseed cytoplasm resistant to triazines).

15 The latter characteristics make it possible to consider weed control in the rape using the triazines.

While it is possible to take into account various protoplast sources (a protoplast is a plant cell from which the pectocellulose wall has been mechanically or enzymatically degraded), preferably 20 protoplasts isolated from tissue from young leaves grown in vitro are used. .

The fusion of the protoplasts may be carried out according to known methods, especially according to the method described by Chupeau et al.

The fusion products are grown in a nutrient environment containing growth agents, especially auxin, especially α-naphthalene acetic acid (ANA), 2,4-dichlorophenoxyacetic acid (2,4-D) or other auxins in combination with cytokinins such as benzylaminopurine (BA) .

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The ratio of cytokinins to auxins is usually in the vicinity of 1, and the ratio of auxins to 2,4-D is between 0.1 and 1.

The weight ratio of these products can be: ANA: BA: 2.4 D = 5 0.5: 0.5: 0.5 and usually = 1: 1: 0.25.

In the first culture environment for the fusion products, glucose is preferably used as a carbon source, the osmotic agent being mannitol.

This first culture environment, of course, contains the various elements of a nutritional environment, in particular: (1) chemical elements (a) nitrogen derivatives, especially KNOg and (NH 2) SO 2, (b) phosphates such as NaHgPO 2, (c) calcium, CaCl 2 and (d) magnesium, MgSO 2) oligo elements, especially Fe in the form of ethylenediaminetetraacetate of Fe; 3) as well as vitamins and optionally amino acids.

These are known constituents in cultivation environments, as various details regarding the dosages and the exact nature of these constituents will be apparent from the examples below.

In order to ensure continuous culture of the fusion products, periodic dilutions of the initial environment (sometimes referred to as the callogenesis environment) should be made with fresh culture environments that accelerate the growth of newly formed tissue.

Instead of glucose, these nutrient environments preferably contain sucrose, and the content of osmotic agent (mannitol) is decreasing.

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Dilutions are preferably made after 12-14 days and 17-22 days of cultivation on the initial environment.

After 29-36 hours, the colonies are preferably placed on a solid nutrient environment of agar.

5 The developing sprouts are transferred to a fresh, solid environment for between 2 and 8 weeks.

When well-developed meristem tissue appears, the germs are propagated by cuttings in a nutritional environment.

The cuttings cause rapeseed plants from which the plants are selected which have the characteristic feature smc and one of the fertile individual's cytoplasmic markers.

More precise characteristics of the different environments are mentioned in the examples.

The invention is further illustrated by the following examples:

The examples cite various cultivation environments, which are denoted by letters and whose composition is shown in the table which is listed after the examples.

Example 1 20 Preparation and Fusion of Rapeseed Protoplasts The following individuals are used:

The first stem A is a spring radish with Japanese radish cytoplasm.

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It has the following characteristics:

Characteristics of the nucleus: a) flowers with large petals b) presence of erucic acid 5 Cytoplasmic characteristics: c) undeveloped dust buds - absence of pollen (male sterility) d) undeveloped nectaries - very little nectar e) the plants turn pale green to yellow at temperatures of below 12 ° C.

10 The second stem B is a spring rape of the Brutor variety with the following characteristics:

Characteristics of the nucleus: a) flowers with narrow petals b) absence of erucic acid 15 Cytoplastic genetic markers: c) well-developed dust buds - presence of pollen d) presence of nectaries - abundant nectar e) absence of chlorophyll in cold.

Furthermore, it is possible to distinguish between chloroplastic and mitochondrial DNA in these two individuals by electrophoresis of the restriction fragments.

These rapeseed plants are grown in the form of seedlings in vitro (2000 lux, white light, 24 ° C). They are obtained from aseptic sprouts (disinfecting the grains in calcium hypochlorite for 20 minutes and rinsing with sterile distilled water) on P environment.

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After 8 hours, the apex is isolated and grown on the same environment. Seedlings are then done at 4 week intervals. Only the apical bud is retained for subsequent planting.

The protoplasts are isolated from the leaves thus obtained. The 3 week old 5 leaves are used. They are cut into strips with a width of 2-3 mm and the main rib removed. The leaves are placed in flasks containing the following mixture:

Mixture A

Pectolyase Y23, 0.1% Cellulase R10, 0.2%

The flasks are shaken in the dark for 16 hours (a movement back and forth every two seconds, oscillation width 5 cm). Non-degraded leaf pieces are removed from the obtained protoplasts by filtration on a metal screen with a mesh size of 40 µm.

The liquid is centrifuged at 700 x g for 5 minutes. After the supernatant has been removed, the protoplasts are rinsed by two further centrifugations in an environment containing 2.5% KCl, 0.2% CaCl 2 h 2 O, pH 6.

The protoplasts are fused according to the method described by 20 Chupeau et al.

EXAMPLE 2

Cultivation of the merged protoplasts

The protoplasts obtained in Example 1 are grown in an amount of 50,000 / ml in the A environment and placed in the dark at 28 ° C. In particular, the A environment has the following characteristics:

DK 160392 B

7 a) It contains only glucose as a carbon source, b) it has mannitol as osmotic agent, c) it has a balance between BA: ANA: 2.4 D of 1: 1: 0.25 and d) the nitrogen supply originates mainly from KNO ^.

5 After 3 days, the cultures are exposed to light at the same temperature.

After 12-14 days of cultivation, an equal amount of B environment is added to the A environment and the suspensions are distributed in new culture dishes (Pe-tri plastic bowls with a diameter of 100 mm, each dish containing 10 ml of culture) .

The purpose of this fresh environment supply is to accelerate the growth rhythm of the newly formed tissue by decreasing the osmotic pressure and an energy supply in the form of sucrose.

After 17-22 days of cultivation, the cultures are diluted to a 1: 1 ratio with C environment and distributed in new Petri dishes.

After 29-36 days, the well-developed colonies are placed on a solid environment D in an amount of 20 colonies per day. bowl containing 25 ml of environment.

The sprouts that develop between the 2nd and the 8th week are isolated and grown on the F-environment. When new and well-developed meristem tissue is formed on these sprouts, they are isolated and propagated by cuttings in the F environment.

This trial protocol allows one colony per unit to be obtained. 100 growth-proto-plastics. One seedling is eventually obtained per 10 colonies planted on the D environment, ie. a total yield of one plant per plant. 1000 protoplasts.

The hybrids obtained have the following cytoplasmic characteristics.

25 c) absence of pollen d) presence of nectaries e) no lack of chlorophyll in cold.

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They have the chloroplastic genome for rape and a mitochondrial hybrid genome between rape and radish.

Their gendered descendants are homogeneous and retain these characteristics.

EXAMPLE 3

Proceed in the same manner as described in Examples 1 and 2, but using the following individuals:

The first seedling is still A.

The second seedling C is a spring rape (Tower) with cytoplasm of 10 a Canadian rape that is resistant to triazines.

It has the following characteristics in the nucleus: a) flowers with dense petals b) absence of erucic acid and the following cytoplasmic features: 15 c) presence of pollen d) presence of nectaries e) no deficiency of chlorophyll f) resistance to atrazine.

The chloroplastic and mitochondrial genomes are different from the A-20 stem plant and can be distinguished by restriction.

Hybrid plants are obtained which have the following cytoplastic characteristics: c) absence of pollen d) presence of nectaries e) absence of chlorophyll 25 f) resistance to atrazine.

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They have chloroplasts from canola.

Composition of the environments (mg / l)

P A B C D EF

5 NH4N03 1650 200 200 1650 1650 825 KN03 1900 2500 1250 1250 1900 1900 950 (NH4) 2S04 134 67 67

NaH2PO4 150 75 75 KH2PO4 170 35 35 170 170 85 10 CaCl2.2H20 440 750 525 525 440 440 220

MgSO4.7H20 370 250 250 250 370 370 185 H3B03 12.4 3 3 12.4 12.4 6.2 6.2

MnSO4.4H2 O 33.6 10 10 33.6 33.6 22.3 22.3

ZnSO4.7H20 21 2 2 21 21 8.6 8.6 15 K1 1.66 0.75 0.75 1.66 1.66 0.83 0.83

Na2MoO4.2H2O 0.5 0.25 0.25 0.5 0.5 0.25 0.25

CuS04.5H20 0.05 0.025 0.025 0.05 0.05 0.025 0.025

CoCl2.6H20 0.05 0.025 0.025 0.05 0.05 0.025 0.025

FeSO4.7H2O 22.24 27.8 27.8 27.8 27.8 27.8 22.24 Na2EDTA 29.84 37.3 37.3 37.3 37.3 37.3 29.84

Inositol 100 100 100 100 100 100 100

Nicotinic acid 0.5 111 0.5 1 0.5

Pyridoxine HCl 0.5 111 0.5 1 0.5

Thiamin 10 10 10 10 25 Glycine 2 2 2

Glucose 10000 20000 10000

Sucrose 10000 20000 20000 10000 10000

Mannitol 70000 40000 20000 ANA 1 0.2 1 0.1 0.01 30 BA 11 0.5

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Composition of the environments (mg / l) continued P A B C D EC

2.4 D 0.25 1

Adenine sulphate IPA 1 GA3 0.02

Tween®80 10

Agar (pH 5.8) 8000 8000 8000 8000 10 Gentamicin (sulfate) 10

Claims (7)

A method of producing male sterile rape with certain cytoplasmic characteristics, characterized in that (a) somatic fusion of protoplasts from two rape species, A and B, with A having male sterility, and B being fertile, is performed; have said cytoplasmic characteristics and b) the fusion products are grown until seedlings are obtained. Process according to claim 1, characterized in that the protoplasts are isolated from young leaf tissue grown in vitro. Process according to claim 1 or 2, characterized in that the culture environment for the first fusion products, or the callogenesis environment, is a culture environment which, in particular as a carbon source, contains glucose and as osmotic agent contains mannitol, the ratio of cytokinin to auxin being in the vicinity of 1, and the ratio of auxin to 2.4 D is between 0.1 and 1. Process according to claim 3, 20, characterized in that the primary callogenesis environment is diluted with environments in which the carbon source is sucrose and in which the concentration of mannitol is decreasing. Process according to any one of claims 1-4, characterized in that the colonies formed after dilution of the callogenesis environment are placed on a fixed agar culture culture. Method according to any one of claims 1-5, characterized in that the A-individual is a Japanese radish chs cytoplasm or a rapeseed chs. DK 160392 B Process according to any one of claims 1-6, characterized in that the B-individual is a fertile rape which has no chlorophyll deficiency and which may exhibit resistance to triazines.