NL9401447A - Organic compounds. - Google Patents

Description

ORGANIC CONNECTIONS

The present invention relates to obtaining Brassica oleracea plants exhibiting cytoplasmic male sterility (CMS). The invention particularly relates to the incorporation of CMS into commercially desirable plants of B. oleracea.

Male sterility is important in growing B. oleracea hybrid seed, because normal B. oleracea plants form flowers that are often self-pollinating. Male sterile plant lines do not form viable pollen and are not self-pollinating. Removing pollen from a parent plant at an intersection assures the plant grower of obtaining uniform quality hybrid seed. In the past, commercial hybrid seed producers have employed nuclear, self-incompatible systems to avoid self-pollination during seed formation; however, such systems can lead to impure hybrid seed portions. Further disadvantages of such systems are that they are time consuming and laborious.

Growers have turned to other means of introducing male sterility into B. oleracea plants. A system is described in British Patent GB2,211,205, where B. oleracea protoplasts containing an inactivated core and Ogura CMS cytoplasm are fused with protoplasts of B. oleracea. with a functional core and cold tolerant chloroplasts. Allogeneic cells thus obtained were regenerated into plants, which could then be used to prepare other cytoplasmic, male, sterile B. oleracea plants with a B. oleracea core, B. oleracea chloroplasts and mitochondria of the CMS ogura cytoplasma, using in vitro and / or crossing techniques. Thus, cytoplasmic male sterility was successfully transferred to commercial varieties. However, there are drawbacks to this method. Plants obtained according to the invention of GB 2,211,205 show some irregular color patterns in the leaves, which have to be grown out using other cultivation steps. In addition, the honey glands of plants according to GB 2,211,205 are generally not as well developed as those of non-ogura CMS containing B. oleracea plants and this underdevelopment can lead to less than optimal seed formation in such plants.

There is a need for an alternative method for CMS B. oleracea plants without the aforementioned drawback occurring.

A source of CMS is that of CMS Brassica iuncea (CMSj), Brown Mustard, an amphidiploid product of mono genomic parents, B. camoestris and B. niora. The source of CMS has been known for several years. The genome of B. naprus has also been reported to be transferred into the cytoplasm of B.iuncea (Mathias R. (1985) Z. Pflanzenzuchtig 95: 371374, to form CMSj B. napus plants. CMSj B. napus plants are however, hitherto not used in the commercial production of hybrid seed of B. napus, because the formation of hybrid seed is counteracted by the partial allogamic nature of that crop, such plants are not known to have been hitherto used to obtain B. oleracea plants, both for experimental and commercial purposes.

It has now been found that CMS B. oleracea plants containing mitochondria of the CMSj cytoplasm do not have the drawbacks found in the CMS plants with ogura cytoplasm.

An object of the present invention is to provide CMS B. oleracea plants containing mitochondria of the CMSj cytoplasm.

By the term "plant" is meant both the plant as a whole and a part or parts thereof. The part (s) can be either cultivated, for example a cutting, or an edible part. Edible parts include those typically sold on the fresh produce shelves of supermarkets, etc., or offered to the canning and / or inlay industries. Edible parts thus include, but are not limited to, parts of plants such as Brussels sprouts, broccoli heads or stems and / or shoots, cauliflower heads, cauliflower stems, cabbages, cabbage leaves.

The designation "CMSj" used hereafter denotes cytoplasm derived from Brassica iuncea, which contains Brassica iuncea mitochondria and, when present in Brassica oleracea plants, confers male sterility to it.

The term CMSj Brassica oleracea plant or plant material used hereinafter refers to a Brassica oleracea plant or plant material containing CMSj cytoplasm. Such CMSj cytoplasm will of course also contain Brassica iuncea chloroplasts.

The term "hydride seed" used herein refers to F1 hybrid seed and seed derived from plant material derived therefrom.

The present invention is particularly suitable for obtaining CMS in the following Brassica oleracea plants.

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. gemmifera DC (Brussels sprouts), 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 (swede), 8. Brassica oleracea L. convar. botrytis (L.) Alef. var. ithalica (broccoli).

and, in particular, in cauliflower, white cabbage, Brussels sprouts and broccoli, as mentioned above. It will be understood that Brassica types, listed above under 1-8, refer to Brassica types in a general sense. For example, under 8 Brassica oleracea L. convar. botrytis (L.) Alef. var. ithalica refers to broccoli plants, whether they are of purple sprouting, green or any other broccoli type.

According to another embodiment of the invention, a method of obtaining B. oleracea plants containing at least the characteristic of CMSj is provided, the method comprising: (i) performing a first cross between a CMSj Brassica plant and a B. oleracea plant, (ii) taking embryo material from this cross and developing plants from it, and (iii) selecting regenerated plants and performing repeated backcrosses on a selected progeny, until B. oleracea plants form normal seed and exhibits phenotypic uniformity to at least the male sterility property.

The CMSj-containing Brassica plant species used at the first crossing can be any plant species from the Brassicaceae family capable of associating with a B. oleracea plant. That is, a CMSj-containing plant, when crossed with an oleracea plant, will give rise to a viable progeny from which selections can be made for plants that are in a culture program to ultimately obtain CMSj-containing B ±. oleracea plants can be used. Examples of Brassica plants containing CMSj are B. iuncea and CMSj B. napus. The CMSj Brassica plant is the female fertile, male sterile (i.e. female parent) plant at the first crossing. The B. oleracea plant to be crossed is the female fertile, male fertile (i.e. male parent) plant. The crossing Ek. oleracea plant partner can be any plant selected from an agronomically important plant type, such as cauliflower, broccoli, cabbage (red, white or green) swede, Brussels sprouts, etc.

The embryo material may comprise all or part of the embryo, or material derived therefrom (e.g., callus material, as long as it is capable of being grown and regenerated in a B. oleracea plant, using from the state Methods Known in the Art The embryo material is typically a complete embryo separated from the seed coat, using widely known methods of embryo recovery known in the art An example of embryo recovery and regeneration methods of B. oleracea plants is illustrated herein (Example 1).

Selected progeny in step iii) with desirable traits for the breeder is subjected to repeated backcrosses with B. oleracea plants showing similar or other traits of interest to the breeder until at least the male sterility trait is uniform in the progeny of B oleracea plants is included. Thus, the plants obtained are phenotypically uniform, at least in terms of the male sterility property. Typically, the uniform inheritance of at least the property of cytoplasmic male sterility requires at least two backcrosses. The grower generally performs about 4 backcrosses, preferably at least 6, depending on the B. oleracea plant type of interest. The grower will also look for normal seed formed in the obtained B. oleracea plants, demonstrating that the obtained plants meet the phenotype typical of B. oleracea. Normally shaped seed means seed corresponding to the shaped seed of the parent backcross partner, that is, the female fertile, male fertile parent. The grower will also look at other traits for cultivation in the obtained male sterile B. oleracea plants and this may require a longer backcross period for cultivating such stable traits in the B. oleracea plants of the invention. Among such properties can be, for example, the quality of the head or of cauliflower florets and / or self-protection in cauliflower, the internal quality and whether the color in red cabbage, the resilience in Brussels sprouts, resistance to diseases, plant architecture, the absence of ears, etc., include.

Here is a general description of how B. oleracea plants according to the invention can be obtained.

B. oleracea, as a male parent and performing embryo recovery techniques on developing embryos, regenerating plants from the selected embryos, followed by back-crossing selected plants containing desirable properties with B. oleracea plants containing corresponding and / or desirable, additional properties until a seed is formed which is typical of a B. oleracea plant, i.e. indicates a ploidy value typical of B. oleracea. Preferably, the B. oleracea plant is diploid, i.e. 2n = 2x = 18. Such B. oleracea plants containing normal seed are repeatedly backcrossed with B. oleracea plants, which have corresponding and / or desirable additional properties, at least after the second backcross, preferably to at least the sixth backcross. Once the trait of male sterility has been evenly incorporated into a B. oleracea plant, this plant or the plant material from it can be further used, for example when backcrossing with plants of the same type or can be crossed with different plant types of B. oleracea , introducing the male sterility trait into other B. oleracea plant types. For example, a third backcross of male sterile broccoli or subsequent backcross can be crossed with a cauliflower, and by using repeated backcross with cauliflower, cytoplasmic male sterility can be incorporated into cauliflower. CMSj cabbage varieties (red, white or green), Brussels sprouts, swede, kale, etc. can be obtained in a corresponding manner. Methods for collecting embryos and crossing methods are known to the experienced grower.

Thus, the offspring of the first crossing can follow a number of possible avenues, including those described above and summarized below: 1

She can be crossed with the B. oleracea parent. Such a cross is a back cross and will have the effect of enriching the properties of the B. oleracea parent in the next generation.

2) She can be crossed with a plant of the same species or subspecies as the B. olereacea parent, either to introduce new traits or to confirm the inheritance of certain traits of that group.

3) She can be crossed with a plant of a species or subspecies that differs from the species or subspecies of the B. oleracea parent. Such a cross will have the effect of introducing other new properties.

4.) It can be crossed with a plant that itself is the product of one or more crosses.

Such breeding programs are cumulative and span several years with careful selection at each stage of the program. It will be understood that such breeding programs should be selected to obtain B. oleracea containing the chloroplasts and mitochondria of B. juncea and B. oleracea plant nuclei.

It will, of course, be apparent to the grower that embryo material from the first cross can be obtained via embryo recovery methods and regenerated into plants, or that plants can be obtained from seed obtained after the first cross. Preferably, the product plants of the first cross are obtained from embryo material, since the number of potential plants from which choices are made depends greatly on the number of selections that can be made from plants that can be obtained through conventional seed planting and plant selection methods.

The foregoing description illustrates the present invention. A more complete understanding of the invention can be made by the following examples. It is to be understood that the examples here are provided only to illustrate the invention and are not intended to limit the scope of the invention in any way.

Example 1; Embryo recovery method.

12 to 15 days after crossing a CMSj B. napus (female parent) with a B. oleracea (male parent), ovaries show signs of growth, that is, the fruit begins to swell and harden and has a greenish color, harvested and disinfected by immersing in 70% alcohol for 1 minute, then incubating the fruiting principles in 1 (wt.)% NaOCl solution for 10 minutes. It is then washed twice with sterilized water for 5 minutes per rinse. The developing seed is then removed under an analysis microscope (low magnification) in a space with a sterile, laminar air flow, and the embryo is removed from the seed coating.

Individual fractions of A 50-310 embryos were then incubated in contact with 25 ml of liquid White A medium in 75 ml Erlenmeyer flasks for 7 days at approximately 15 ° C with 16 h light / days. The White A medium contained per liter: 1 liter of filtered or distilled water, 0.94 g of White's basal salt mixture (commercially available from Sigma Chemicals, USA), 80 g of sucrose, 400 mg of casein hydrolyzate (commercially available from Sigma Chemicals , USA), adjusting the pH to 5.8 ± 0.2 before autoclaving and adding to the Erkenmeyer flasks.

When the embryos were ± 1.5 mm long, after 7-20 days, the embryos of liquid White A medium were transferred to 25 ml solid White B medium in 75 ml flasks, 1 embryo per flask, for 14-21 days , at 22 ° C with transfer to 25 ml of solid White B medium in 75 ml flasks, 1 embryo per flask, for 14-21 days, at 22 ° C with 16 hours of light-day cycles, i.e., to callus formation. The White B solid medium contained 1 liter of filtered or distilled water per liter, 0.94 g of White's basal salt mixture (Sigma Chemicals, USA), 20 g of sucrose, adjusting the pH prior to the addition of 8 g of agar (commercially available from Merck , NL), was adjusted to 5.8 ± 0.2 and then autoclaved in the flasks for the addition of embryos.

Once root-shooting callus appeared, the root-forming calli of solid White B medium were transferred to solid O medium (1 per opening) and kept at 22 ° C (16 hour light / day cycles) for 3 weeks. Every 3 weeks, O medium was replaced with fresh 0 medium. Further new plant material appeared after 28-60 days.

Solid O medium contained per liter: 4.42 g / 1 MS Macro elements (commercially available from Duchefa BV, NL) 1.00 ml MS Micro elements form a solution of 1 1, containing:

CoCl2. 6H20 0.025 mg / 1

CuSO 4. 5H20 0.025 mg / 1 H3BO3 6.20 mg / 1 KI 0.83 mg / 1

MnS04. H2 O 16.90 mg / 1

Na2Mo04. 2H20 0.25 mg / 1

ZnS04. 7H20 8.60 mg / 1 1 ml MS vitamins (commercially available from Duchefa BV) 1 ml FeNaEDTA (36.7 mg / ml) (FeNaEDTA commercially available from Sigma Chemicals, USA) 30 g sucrose

Developing shoots were separated from callus and placed on O medium, 4-5 shoots per pot and left for a period of time. After 10 days, roots with a length between 1 and 15 mm were observed. Once sufficient roots were formed, the plantlets were transferred to soil and kept in a greenhouse under a plastic film at a high relative humidity (RH: 90-100%). After a week, the plastic film was gradually removed and the plantlets exposed to a lower RH of about 40-80%. The greenhouse temperature is not critical and can vary between 15 and 25 ° C.

The average survival rate, that is, development into plants from the embryos, is between about 5 and about 10%.

Example 2: The formation of B. oleracea plants (broccoli), uniform in the property of CMSj.

Reference is made to culture schedule 1.

CMSj B. napus (2n = 2x = 38 female chromosome parent), obtained from The Institute of Agronomy and Plant Breeding, Georg August University, Gottingen, Germany was crossed with B. oleracea var. ithalica (2n = 2x = 18 chromosomes-male parent), (cv Zaadunie BV). 12 F1 plants were obtained via embryo recovery methods, as described in example 1.

An F1 plant with the designation No. 7 was assigned (2n = 2x = 36 chromosomes), was selected and backcrossed with a B. oleracea var. ithalica (2n = 2x = 18), after which seed was allowed to form. This is the first backcross (BC1). 9 plants were obtained (2n = 2x = 26-27 chromosomes). 1 Plant from BC1, which was designated 7-3 indoors, was selected and backcrossed with B. oleracea var. ithalica, giving the second backcross (BC2). Seeds from BC2 were grown and the selected plants backcrossed with B. Oleracea var. ithalica, forming the third backcross (BC3). Plants of BC3 show normal seed formation (2n = 2x = 18 chromosomes) for B. oleracea plants. Selected BC3 plants were further backcrossed with B. oleracea var. ithalica, to form BC4, from which selected progeny were backcrossed, to form BC5, and to select the progeny thereof further back, to yield BC6 plants. Plants of BC3-BC6 exhibit uniformity in the property of cytoplasmic male sterility. They show normal seed formation for B. oleracea var. ithalica (broccoli), which indicates that they are true B. oleracea plants with a normal diploid chromosome number (2n = 2x = 18).

Example 3: Production of B. oleracea var. botrytis (cauliflower) uniform in the property of CMSj.

Reference is made to culture schedule 2.

CMSj B. oleracea var. ithalica (broccoli; 2n = 2x = 18 chromosomes-female parent) obtained according to Example 2 above, was crossed with B. oleracea var. botrytis (cauliflower; 2n = 2x = 18 chromosomes-male parent). A Fl plant designated 920111 was selected and backcrossed with a B. oleracea var. botrytis (2n = 2x = 18) and allowed to form seeds. This is the first backcross (BC1). A plant from this BC1 population, which was assigned the designation 930131 indoors, was selected and backcrossed with B. oleracea var. botrytis, yielding the second backcrossing (BC2) population. B. oleracea var. botrytis plants of BC2 show normal seed formation (2n = 2x = 18 chromosomes) for B. oleracea var. botrytis plants and have a normal cauliflower shape and color.

Grow schedule 1

Grow schedule 2

Claims (12)

1. CMS B. oleracea plants or parts thereof containing mitochondria of the CMSj cytoplasm. Plants or parts thereof according to claim 1, wherein the CMSj cytoplasm is obtained with a CMSj B. napus plant. Plants or parts thereof according to claim 1 or 2, which are diploid. The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. acephela (DC.) Alef. var. botrytis L. (cauliflower). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. capitata (L.) Alef. var. alba. DC. (white cabbage). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. gemmifera DC. (Brussels sprouts). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. acephala (DC.) Alef. var. sabellica L. (kale). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. capitata (L.) Alef. var. sabauda L. (savoy cabbage). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. capitata (L.) Alef. var. rubra. DC (red cabbage). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. acephala (DC.) Alef. var. gongylodes (swede). The CMS Brassica oleracea plant or parts thereof according to claim 1, which are Brassica oleracea L. convar. botrytis (L.) Alef. var. ithalica (broccoli). Seeds of plants according to any one of claims 1-11. 13. Method for obtaining CMSj B. oleracea plants, which i) performing a first cross between a CMSj Brassica plant and a B. oleracea plant, ii) taking out embryo material obtained at the first crossing and forming plants from it and iii) selecting regenerated plants obtained from II and performing repeated backcrosses on their selected progeny, until B. oleracea plants obtaining normal seed and phenotypic uniformity in at least the male sterility property be obtained.