FR2740656A1 - Compositae crop plants with cytoplasmic male sterility - Google Patents

Abstract

Crop plants of the family Compositae whose cytoplasm contains mitochondria contg. DNA which is at least partly derived from another species of Compositae and is a carrier of cytoplasmic male sterility (CMS) are new. Also claimed are seeds and plant parts derived from such plants. The donor species is a Helianthus, Cirsium, Chrysanthemum, Senecio, Centaurea, Sonchus, Hieracium, Tagetes, Dahlia or Aster sp., esp. Helianthus annuus, which provides petiolaris-type CMS. The recipient has a normal species-specific chloroplast and nuclear genome and is rendered phenotypically male-sterile.

Description

PLANT HAVING MALE CYTOPLASMIC STERILITY OF THE
COMPOSITAE FAMILY AND ALSO PROCESS FOR OBTAINING
THIS PLANT
The invention relates to a plant having male cytoplasmic sterility of the Compositae family according to the main claim, that is to say, relates to new mother lines of this plant of said family. The invention further relates to a process for obtaining the above-mentioned plant having male cytoplasmic sterility, as well as the hybrid seed obtained from the new mother lines of the Comnositae family.

 In the world of plant breeders it is usual to combine the hereditary properties of two mother lines of the hybrid by crossing. If both mother lines are direct pollinated, direct pollination is prevented, for example by removal of the anthers (called emasculation), to ensure that the desired hereditary properties of both parents are present in the hybrid. This hybrid is also called F1 hybrid, and is of great importance in the world of plant breeders. F1 hybrids are usually characterized by a very high degree of uniformity (color, taste, size and the like), high vigor and high yield from plant parts, such as fruit and the like, which can be marketed. of an F1 hybrid in particular for companies specializing in phytogenetics is above all the fact that it is not possible to cultivate products of the same quality by the line coming from the seed obtained from it (greater heterogeneity and lower yield), so that the company is guaranteed an inherent protection of its F1 hybrid, as it was.With the plants of the family ComDositae the development of mother lines and the commercial production of pure hybrid seed F1 is very problematic, because these plants are in general with direct pollination and because the measures of direct anti-pollination that i have been used so far not only require a lot of time and labor, but have mostly proven to be insufficiently precise.

As a result, this latter fact results in seed lots that are not of uniform quality.

 For example, with cabbage (Brassica oleracea) it is currently not possible to produce 100% pure F1 hybrids using modern techniques. The fact is that with the radish (RaPhanus sativus), which like cabbage belongs to the Cruciferae family, a cytoplasm causing male sterility has been found, which cytoplasm is also known as the CMS Ogura cytoplasm. The mitochondria of the Ogura CMS cytoplasm are introduced into Brassica oleracea plants by fusion of protoplasts, where it follows that the nucleus and chloroplasts (chlorophyll granules) all come from normally fertile Brassica oleracea plants. Thus Brassica oleracea plants with CMS are obtained. Currently no F1 hybrid of important plants of the Composite family is yet available, despite the high demand on the market. The above-mentioned CMS Ogura cytoplasm cannot be used with plants of the Compositae family, due to the genetic incompatibility between the species of said families.

 The object of the invention is to introduce male cytoplasmic sterility into a plant of the Comositae family, and for this purpose the cytoplasm of this plant is provided with mitochondria comprising DNA which originates at least partially from another species of the Compositae family and which carries male cytoplasmic sterility (CMS). Surprisingly, research has shown that despite the strong genetic incompatibility of plant species of the Compositae family, the introduction of CMS into a plant of the Composite family from a plant of another species of the same family, by Protoplast fusion is very possible. Preferably the plant is phenotypically a plant with male cytoplasmic sterility, i.e. male sterility (generally defined as the inability of a plant to produce fertile pollen seeds) manifested itself in the plant. Due to the cytoplasmic inheritance of CMS the entire line of this sterile male plant has this property unchanged. It will be evident that the foregoing form of hereditary transmission of genotypic CMS is of great importance for the efficient development of mother lines of plant species and for the subsequent production of F1 hybrids. It should be noted that the plant does not necessarily have to phenotypically possess male cytoplasmic sterility, but that it is also possible for the nucleus to possess what are called "repairing genes", which phenotypically annihilate male sterility. to say prevent it from manifesting itself in the plant despite the presence of DNA in the cytoplasm, which is a carrier of CMS. In this case it will be necessary to maintain the crossing until a plant with phenotypic CMS is obtained.

 In a first embodiment in accordance with the invention, the plant cytoplasm further comprises a nuclear genome and a chloroplastid genome specific for the species which are normal for the plant in question. Due to the fact that a nucleus and chloroplasts from normal fertile plants are present in the unmodified state (in the case of the nucleus it is usually a situation of diploidy), the product is a 100% pure product.

In a preferred embodiment of the invention, the other species of the Compositae family was chosen from the group consisting of
Helianthus spp and Circium spp. These species have been shown to contain the
CMS. It was chosen more particularly Helianthus annuus (L.) (sunflower) with CMS of the petiolaris type. Surprisingly, the introduction of CMS from these plants genotypically possessing male cytoplasmic sterility can be done effectively by fusion of the protoplasts, despite the genetic incompatibility. In this regard, it should be noted that the transmission of CMS from the aforementioned plants to plants belonging to the same family cannot be carried out using conventional methods, due to crossing barriers.

According to the invention, the other species introducing CMS (the "donor"), which belongs to the family Compositae, can also be a species which does not contain CMS, but which introduces what is called an "alloplasmic" form. "of CMS in plants by fusion of protoplasts. In accordance with the invention, a species of this type is preferably chosen from the group consisting of Chrvsanthenum,
Senecio, Centaurea, Sonchus, Hieracium, Taaetes, Dahlia and Aster. Good results have been obtained according to the invention, in particular with Chrvsanthenum, more particularly with Sonchus.

 In another embodiment in accordance with the invention, the plant was chosen from the group consisting of Cichorium intvbus (L.) var.

foliosum (Heai) (chicory), Cichorium endivia (L.) (endive), Lactuca sativa (L.) (lettuce), Scorzonera hisoanica (L.) (black salsify), Cvnara scolvmus (L.) (artichoke) and Taraxacum officinale (L.) (dandelion). Extensive experiments have shown that in particular Lactuca sativa, but particularly Cichorium intybus (L.) var. foliosum and Cichorium endivia possessed the important advantage that no other property is lost which is not characteristic of the plant following the introduction of the
CMS.

 The invention further relates to the seeds or plant parts from the plant according to the invention.

 The general steps of the process for obtaining plants, seeds or plant parts having CMS and belonging to the ComDositae family according to the invention include: - the isolation of protoplasts (plant cells from which the cell membrane has been removed) tissues of various plants belonging to the ComDositae family, hereinafter called "acceptor", and tissues of another species of the ComDositae family, for example Helianthus marigolds, below called "donor" (see Example 1); - optional treatment of protoplasts, for example by irradiation, cytoplastization or with specific chemicals, which in addition in the process results in an increase in yield (see Example 2); - fusion of protoplasts (in vitro fusion of cells), for example under the influence of chemical substances, or using electricity (see example 3); - the subsequent and optional realization of one or more processes, the result of which is a certain form of selection of hybrid cells; cells resulting from the fusion of one or more acceptor protoplasts with one or more donor protoplasts (see Example 4); - the subsequent culture of cells and their regeneration to give complete plants (see example 5); the selection of individuals from the regenerant collection thus obtained by analysis of mitochondrial, chloroplastidial and genomic DNA, followed by the phenotypic evaluation of the properties of the plant (see example 6); - specific crossbreeding of previous individuals, or their lineage, with other genotypes of the same species and other species (see example 7); - large-scale seed production.

 The invention will be explained in more detail below with reference to the examples (which have already been briefly referred to above) discussed below, which constitute all the preferred embodiments of the process according to the invention. In order not to complicate these examples unnecessarily, the examples are based on Helianthus annuus (L.) who is the donor. As already explained above, the invention relates generally to a donor from the ComDositae family of a species other than the plant and is therefore not limited to the donor cited in reference in the examples.

 In all the examples below, the operations must be carried out aseptically and all the solutions, media (the compositions of which will be described in a later step) and instruments to be used must be sterile. The petri dishes are further sealed with a film layer, such as Fuji, Nesco or Parafilm.

Example 1:
Culture of acceptor material; chicory, endive and lettuce leaf in vitro
Wash the seeds with 70% ethanol for 10 seconds, then sterilize for 10 minutes in 1% Na-hypochlorite, then wash thoroughly with sterile water. Seed aseptically in a disposable tissue culture container with approximately 75ml of MS medium (approximately 8 seeds / box). Cultivate at 25 "C in the light (16 hours of illumination; approximately 2000 lux / 8 hours of darkness).

After about two weeks the young seedlings are placed in a box with a fresh medium of DM (1 seedling / box). One month after sowing the plants are ready to be used as starting material.

Isolation of protoplasts from sheet of acceptor material; chicory, endive and lettuce
Cut the sheet material in vitro (+ 95cl2) with a scalpel in a Petri dish (TC, diameter 9 cm) with about 10ml of PPO.

Replace the solution after cutting with 10ml of maceration solution. Incubation: still, dark, 25 "C, about 16 hours.

Filtration through a 45pm nylon sieve, final washing and dilution of the suspension with approximately 3 volumes of washing solution. Centrifuge the protoplast suspension in 15ml centrifuge tubes for 5 min at 600 rpm (= 63 x g). Wash the deposit twice with 15 ml of washing solution, then centrifuge for 5 minutes at 600 revolutions per minute. The protoplasts (pps) are now ready for fusion or for culture, now an inactivation step, preferably treatment with iodine acetate, can be performed, if desired.

Culture of the donor material; sunflower hypocotyls
The etiolated hypocotyls are chosen as the starting material of the donor from which the protoplasts are isolated. In comparison to the leaf, which is chosen as the "acceptor" tissue, this plant material contains a much greater amount of mitochondria. In addition, the hypocotyls contain no chloroplasts (they certainly contain a number of proplastides), unlike the leaf cells. This tissue selection leads to a greater chance of obtaining the correct organelle composition of a fusion product. In addition, this tissue selection allows optical selection following fusion; the fusion products of the donor and acceptor protoplasts are recognizable.

 Sterilize sunflower seeds in 1% Na-hypochlorite for 1 Omn, then wash thoroughly with sterile water.

Inoculate in replication dishes with MS medium. Dark, 25 "C.

Isolation of protoplasts from the donor material; sunflower
Cut the upper half of the hypocotyls in PPO 15 after 5 days, cut the hypocotyl parts very finely in a 9cm TC box with 10ml of PPO, renew the PPO solution after cutting and leave to stand for 1 hour, dark (ambient temperature).

 Replace the PPO solution with a maceration solution.

Incubation: still, dark, 25 "C, 16 to 18 hours (possible staining with FITC / FDA; 2 drops of saturated stock solution, in acetone, in 1 Oml of maceration solution).

 Filtration in a 45pm nylon sieve, final washing with the washing solution. Centrifuge for 8 minutes at 600 rpm.

Wash the deposit again with the washing solution; centrifuge for 8 minutes at 600 rpm. The protoplasts (pps) are now ready for fusion. Now a step of inactivation / elimination of the nucleus, for example irradiation or cytoplastization, can be carried out, if desired.

 The cultivation conditions for chicory / endive or lettuce are however such that the sunflower chloroplasts actually form parts or calluses, but that there is no regeneration of the shoots.

Example 2:
Inactivation of the acceptor cytoplasm; iodine acetamide treatment
Like iodine acetate, iodine acetamide is a biochemical inhibitor oriented towards the cytoplasm. It is used here to inactivate the mitochondria of acceptor protoplasts, as a result of which these cells can barely survive, if at all possible. This gives the mitochondria of the donor in a fusion product a selective advantage over those of the acceptor, while in addition the continuous growth of the fusion products is supported in preference to that of the protoplasts of the acceptor using this chemical substance. The treatment of protoplasts with iodine acetamide will for example be as follows:
Stock solution of 60mM, use 1 to 5mM (preferably 2.5mM) in the washing solution with a cell density between 104 and 106 (preferably 10), incubate for 5 to 30 minutes. (preferably 15 minutes) at a temperature between 2 and 22 "C., more particularly at 4" C. Then wash the treated protoplasts completely with the washing solution. Then introduce the protoplasts into the fusion solution.

Inactivation of the donor nucleus; irradiation
The aim of irradiation by X-rays, gamma rays or ultraviolet rays is to fragment the chromosomal DNA of the donor protoplasts, as a result of which these cells can no longer divide. In addition, as a result of this the donor nucleus in a fusion product will have a selective disadvantage in comparison with the acceptor. The advantage of irradiation is that the protoplastid membranes are not too damaged, which is important because the fusion can have a disastrous effect. The disadvantage is, however, that the nucleus remains present, even in the fragmented state, with the corollary the risk of at least partial transmission of chromosomal DNA from the donor to the acceptor.

 X-ray irradiation will for example be as follows: place the pps in the suspended state in a pps culture medium at a density of approximately 2.5 × 10 6 pps / ml in a TC petri dish 6cm (2ml) under an X-ray source (eg Baltobloc CE 100) at an appropriate distance, so that the dose will be 10rad per minute, for 3 to 20 minutes (preferably 5 minutes). Then wash the cells twice with the washing solution.

 Gamma irradiation is usually done using a source of Cobalt 60. Similarly, the protoplasts are introduced into pps culture medium. An appropriate dose is 2 to 30 krad. Followed by washing twice in a washing solution.

Elimination of the donor nucleus; cytoplastization
During cytoplastization the nucleus is removed from the protoplast, so that the only thing left is a cytoplast which contains mitochondria and protoplastids. There are several ways to achieve this. For example, the difference in specific mass between the nucleus and the cytoplast can be used. The nucleus is literally driven out of the cell by high speed centrifugation and a discontinuous gradient (percolle / sucrose), with the possibility of the presence of cytochalacin-B. A milder different method is to perform a strong plasmolysis during the enzymatic incubation, using a maceration solution having a relatively high osmolarity (for example 0.7M Mannitol), in this state the cytoplasts are formed spontaneously; up to 40% of cells. These cytoplasts can then be separated from protoplasts using the difference in specific mass between protoplasts and cytoplasts. The pps / cps mixture is loaded onto a percollelmannitol gradient and refracted by centrifugation at low speed (160 × g; 10 minutes). The success of these operations depends to a large extent on the selection of the starting material. A combination of the methods described above is possible, if necessary. Of course the cytoplasts are washed (washing solution) after purification, and are introduced into the fusion solution. An essential advantage of cytoplasts is that the nucleus is eliminated, which eliminates the possibility of transmission of chromosomal DNA from the donor to the acceptor. A disadvantage of PEG fusion is that cytoplasts have a low specific gravity, as a result of which they sum up.

Example 3:
PEG fusion of mesophylic chicoreelendivellaitue protoplasts with protopasts of sunflower hypotocyles
Give the 100cul drops (1 or 2 for chicory, 4 to 8 for endive and lettuce) with a suspension of pps a density of 106, from which it follows that the partners of the fusion are mixed at the right 1: 1, to remain undisturbed in a fusion solution in cans
Uncoated petri dish (9cm in diameter for chicory, 6cm for endive and lettuce) for 15 minutes. For each drop of pps solution two small drops (about 20pl) of PEG1 are loaded; after 2 to 10 minutes, depending on the fragility of the pps (most often 4 minutes), the box is kept in an inclined position and the liquid can be aspirated at the edge by means of a pasteur pipette, while that the protoplasts remain stuck to the bottom where they have established themselves.

A few drops of PEG2 are carefully loaded and after 3 to 15 minutes (most often 7 minutes) they are removed in the same way as the PEG1 solution. Do the same for PEG3, add and remove after 5 to 30 minutes (most often 10 minutes).

Then fill again with the protoplast culture medium (10 ml of WA medium for chicory, 2 ml of AA medium for endive or 2 ml of SA medium for lettuce), and cultivate as described above. PEG fusion is a very suitable process for fusing large quantities of protoplasts at one time.

Electrofusion of mesophylic chicoreelendivellaitue protoplasts with hypotocylic sunflower protoplasts
Electrofusion should be preferred to PEG fusion when the protoplasts of the two parents of the fusion have, for example, quite different specific masses. This is, for example, the case when the cytoplasts (see example 2 above) are used as donor material; as a result of the absence of a nucleus, the cells are relatively light, which makes them float, which makes PEG fusion impossible. An advantage is that the electrofusion process makes it possible to work with great precision and with great mastery; even the fusion of only two protoplasts is possible. For this, an apparatus intended for electrofusion and a fusion chamber are necessary. The procedure can be as follows: the mixture of donor and acceptor protoplasts is suspended in an isotonic solution (0.5M Mannitol + 0.2mM CaCl2) , and is loaded into the melting chamber.

 Then an electric field generated by a high frequency alternating current (oscillation between 0.3 and 1.5 MHz) is applied, which means that the protoplasts arrange themselves in chains; duration (10 seconds to 10 minutes). The density of pps (104 to 2 x 106) and the intensity of the field (1 to 150V RMSlcm) determine the length of the chains and must constantly be optimized. Then the protoplasts are exposed to one or more strong and short current spikes, which can cause the membranes which are in contact with each other to fuse. The amplitude of the pulse or pulses (40 to 3000 V / cm) as well that the duration (10 microseconds to 1 milliseconds), the shape (block), the number (1 to 10) and the width of the pulse (s) have an influence on the fusion frequency and the survival of the cells. The field generated by the alternating current will be maintained for some time, after which the cells can be introduced into the pps culture medium.

Example 4:
Sort cells
It is possible to separate the desired fusion products, preferably 5 to 10% of the total number of protoplasts, from the fusion mixture by purification directly after the fusion. It goes without saying that this will benefit the efficiency of the whole procedure. For this, techniques which are known per se can be used, such as micromanipulation, sorting by flow or other techniques. It is not, however, necessary to use cell sorting, in particular if inactivation (for example with iodine acetamide), irradiation and cytoplasts are used. In addition, sunflowers will not be regenerated under the conditions described for chicory, endive or lettuce.

EXAMPLE 5
Cultivation of chicory protoplasts
Inoculate in WA medium, the density being 5 x 103 pps / ml, 10 ml in a 9 cm TC petri dish.

 Culture at 25 "C, in the dark.

 The dilution with WB medium is done in 10 days after isolation, in order to prevent browning and staining of the cells. Dilute 1/10 in 9cm TC Petri dishes (10ml per dish). Keep the boxes again at 25 "C, but now in soft light (16 hours in the light; about 500 lux / 8 hours in the dark).

 When the calluses have a reasonable diameter (from 2mm), approximately 1 to 2 months after the isolation of the protoplasts, they must be transferred to a medium for regenerating the shoots; WC medium, approximately 50 calluses per Petri dish (diameter 9cm) with approximately 20ml of medium. 25 "C, in the light (16 hours in the light; about 1000 lux / 8 hours in the dark).

 After about a month, shoots can be cut from the calluses and transferred to disposable tissue culture containers (6 to 8 per box) with about 75 ml of medium D for root development, in the light (16 hours in the light; about 1000 lux 18 hours in the dark) at 25 "C.

Culture of endive protoplasts
Inoculate in AA medium, density from 104 to 105 pps / ml, 2 ml in a 6 cm TC petri dish.

 Culture at 25 "C, in the dark.

 When there is a division and at least a part of the protoplasts has reached a 4 cell stage, the dilution must be carried out with WB medium, for example 1/2. Keep the boxes again at 25 "C, but now in soft light (16 hours in the light; about 500 lux 18 hours in the dark).

 When the calluses have a reasonable diameter (from 2mm) approximately 2 to 4 months after the isolation of the protoplasts, they must be transferred to the shoot regeneration medium; AC, approximately 50 calluses per Petri dish (diameter 9cm) with approximately 20ml of medium. 25 "C, in the light (16 hours of lighting; about 1000 lux / 8 hours in the dark).

 After 2 to 4 months shoots can be cut from the calluses and transferred to the disposable tissue culture containers (6 to 8 per box) with approximately 75ml of medium D for root development, in the light (16 hours of illumination; about 1000 lux / 8 hours in the dark) at 25 "C.

Culture of lettuce protoplasts
Inoculate in SA medium, density 2.5 x 104 pps / ml, 2 ml in a 6 cm TC petri dish.

 Culture at 25 "C, in the dark.

 One week after isolating the pps, the first dilution with SB medium will be made, for example in a ratio of 1: 1 and then every 5 days, in order to prevent browning and staining of the cells.

 When the calluses have a reasonable diameter (from 2mm), approximately 1 month after the isolation of the protoplasts, they must be transferred to the regeneration medium for the shoots; SC medium, approximately 50 calluses per Petri dish (diameter 9 cm) with approximately 20 ml of medium. 25 "C, in the light (16 hours of lighting; about 1000 lux / 8 hours in the dark).

 After two months shoots can be cut from the calluses and transferred to the disposable tissue culture containers (6 to 8 per box) with approximately 75 ml of medium D for root development, in the light (16 hours illumination; about 1000 lux / 8 hours in the dark) 25 "C.

Example 6:
Analysis of mitochondrial and chloroplastid DNA
Total DNA is isolated from the leaf material of the donor, acceptor, fusion and line products using the Dellaporte method (Plant Molecular Biology reporter, vol. 1, number 4 (1983)). Total DNA includes genomic, mitochondrial and chloroplastid DNA. Total DNA is digested into smaller fragments by restriction enzymes. The digested DNA is then separated according to the size of the fragments by electrophoresis in an agarose gel. After separation, single strand DNA fragments are transferred to a positively charged membrane by vacuum blotting, the so-called "Southem blotting principle" (Journal Mol.

Biol. 98, 503-517 (1975)). The various membranes comprising the single-stranded DNA products are hybridized with: - mitochondrial DNA sequences, codes: pEZMT22 and pEZMTA2; - the chloroplastid DNA sequence, code: pEZCP64.

 For labeling, hybridization and detection, the "direct detection and labeling system for nucleic acids ECL n RPN3001" (Amersham) is used.

 Any modification of the mitochondrial DNA of the regenerants which is detected can on the one hand be attributed to the appearance of specific rearrangements during the fusion and regeneration procedures which followed. The fusion product and the line may similarly contain, at least partially, the donor mitochondrial DNA. FIG. 1 shows a specific fusion product and the line thereof containing mitochondrial DNA, which is the result of specific recombination events between the mitochondrial DNA of the donor (Helianthus) and that of the acceptor (cichorum).

Figure 1:
Hybridization with pEZMTA2, bglll restriction enzyme. The figure represents Helianthus (the donor), Cichorium (the acceptor) and various fusion products (A to J). The figure clearly shows that the specific Helianthus bands are present in a number of fusion products (Cichorium phenotype). Due to the procedures that were followed the mitochondrial genomes of the donor and the acceptor were merged.

Figure 2:
Hybridization with pEZMT22, EcoRI restriction enzyme. The figure represents Helianthus (the donor), Cichorium (the acceptor) and 1 CMS fusion product. The figure also shows 5 plants which are backcrosses of the fusion products (the mother) and the
Cichorium / acceptor (the pollinator). From the mitochondrial DNA profile of the fusion product and the line (backcrosses) it appears that the mitochondrial DNA has changed. The mitochondrial DNA thus modified is stably transmitted to the line.

Figure 3:
Hybridization with pEZCP64, restriction enzyme Dral. The figure shows that the CMS fusion product and its line contain the acceptor chloroplast type (Cichorium).

Figure 4:
Determination of the ploidy of sunflower-chicory fusion products (CMS). This is done by means of relative measurements of the quantity of nuclear DNA by flow cytometry (Ulrich & Ulrich, Protoplasm, 1991, 165: 212-215). Measures relating to chicory, sunflower and a fusion product at CMS of chicory. This comparison clearly shows that the fusion product is no different from chicory with regard to the level of ploidy.

Figure 5:
Chicory flower with normal fertility, with anthers and pollen clearly discernible.

Figure 6:
Chicory flower with male cytoplasmic sterility, clearly showing that the anthers are not developed and that no pollen is formed, while the rest of the flower is normal.

Figure 7:
Detail of two radiant chicory flowers (showing a flower with normal fertility on the left and a flower with male cytoplasmic sterility on the right), the anthers and pollen being present in the fertile flower and missing in the sterile flower.

Example 7:
Crossings
It is naturally necessary to multiply the fusion products and to adapt the genotype to the latest requirements again by means of current methods of plant breeders. The seed must be produced for this purpose. This can be done on a large scale in cages using bees or flies, or especially by manual crossing. In this case the pollens of an appropriate pollinator are placed on the pistils of the fusion product (or of its line) 1 after which the seeds are formed. As a result of maternal inheritance, all the properties encoded by the cytoplasm, such as CMS, will be present throughout the line. In this way it is possible to make combinations that are both inter-specific and intra-specific. A good example of a simple intra-specific crossing is the combination of chicory and endive; no special technique, such as the rescue of the embryo, is necessary to achieve this crossing.

Media and solutions
PPO (solution-preplasmolysis) 54.64 gl of sorbitol 7.35 gl of CaCl2.H2O 0.59 g / l MES pH 5.8, autoclave (20 minutes, 115 C)
Maceration solution
Dissolve enzymes in WA medium
Cellulase R-10 0.0667%
Driselase 0.033% Macero @ yme R-10 0.013%
0.2% Cellulysin
0.03% macerase
MES 0.2 g / l 2.4D 0.33 mg / l
BAP 0.16 mg / l pH 5.6 sterile filter (0.22 M)
Washing solution
CaCl2.2H2O 0.2% Kd 2.5%
MES 0.06% pH 5.8 autoclave
Fusion solution
Sorbitol 0.1 SM CaCl2.2H2O 0.03M
KCI 0.075M
Tris-HCI 0.05M pH 7.2 autoclave
PEG1
PEG1500 40%
Glucose 0.3M CaCI2.2H20 50mM
Filter sterilization (0.22 M)
PEG2
PEG1500 13.3%
0.1M glucose
CaCl2.2H2O 0.067M
Sorbitol 0.067M
Filter sterilization (0.22 M)
PEG3
PEG1500 6.7%
Glucose 0.05M CaCl2.2H2O 0.083M
Sorbitol 0.083M
Filter sterilization (0.22 M)
Abbreviations used
PEG polyethylene glycol
NAA a-naphthalene acetic acid 2,4D 2,4dichlorophenoxy acetic acid
BAP 6-benzyl amino purine
IAA indole-3-acetic acid
IBA indole3-butyric acid
MES (2 (N-morpholino) ethanesulfonic acid
FITC fluorescein isothio cyanate
FDA fluorescein diacetate
TC tissue culture
Composition of media (mg / l)
MS AA WA WB WC D SA SB SC AC
KNO, 1900 1900 -. 1900 950 1900 1900 1900 1900
NH4NO3 1650 600 - - 1650 825 80 80 80 80
MgSO4.7H2O 370 300 123 123 370 185 370 370 370 370
KH2PO4 170 170 68 es 170 85 170 170 170 170
CaCl22H2O 440 600 440 440 440 220 440 440 440 440
KCI - 300 750 750 - -
Kl 0.83 0.75 0.75 0.75 0.83 0.42 0.83 0.83 0.83 0.83
MnSO4.4H2O 22.3 10 10 10 22.3 11.15 22.3 22.3 22.3 22.3
H2BO2 6.2 3 3 3 rd, 2 3.1 rd, 2 nd, 2 nd, 2 nd, 2
ZnSO4.2H2O 8.6 2 2 2 8.6 4.3 8.6 8.6 8.6 8.6 8.6
NaMoO4.2H2O 0.25 0.25 0.25 0.25 0.25 0.125 0.25 0.25 0.25 0.25
CuSO4.5H2O 0.025 0.025 0.025 0.025 0.025 0.013 0.025 0.025 0.025 0.025
CoCl2.6H2O 0.025 0.025 0.025 0.025 0.025 0.013 0.025 0.025 0.025 0.025
Fe-EDTA 43 43 43 43 43 21.5 43 43 43 43
Thiamine-HCL 0.1 10 10 10 0.1 0.05 0.5 0.5 0.5 0.5
pyridcodine-HCl 0.5 1 1 1 0.5 0.25 0.5 0.5 0.5 0.5
Acid, nlcothnic 0.5 1 1 1 0.5 0.25 0.5 0.5 0.5 0.5
Ascorbic acid - 2 2 2 - - - - -
Sodium pyruvate - 20 20 20
Citric acid - 40 40 40
Acide ma @ que 40 40 40
Fumaric acid - 40 40 40 - - - -
Wisteria 2 - - - 2 1 2 2 2 2
Fructose - 250 - - -
Ribose 250
Xylose 250
Mannose 250
Rhamnose 250
Cellobiose 250
Sorbitol 250 - -
Mannitol - 250 80,000 60,000 - - 91,000 - -
inositol 100 100 100 100 100 50 100 100 100 100
Sucrose 10,000 250 20,000 20,000 10,000 10,000 10,000 10,000 20,000 10,000
Glucose - 68400 - - - - - - -
Casamine acid 250
Coconut milk ml / l - 20 - - -
AGAR 8000 - - 8000 8000 8000 8000
1WEEN-20 - 8 - - - - -
Glutamine 750 750
Soluble starch - - - - 10,000 - -
NAA 0.1 0.1 - 0.1 0.5 0.5
2,4-D 0.25 - - - -
IAA 1.0
Zeatine 1.0 1.0
BAP 0.1 - 1.0 0.5 0.5 0.5 1.0 0.1
IBA - - - - - - - - - 0.01 pH 5.7 5.6 5.8 5.8 5.7 5.7 5.8 5.8 5.8 5.8 5.8
NB Autoclave medium with AGAR: 20 minutes, 115 C
Filter sterilization medium without AGAR / 0.22 M

Claims (9)

 CLAIMS 1. Plant belonging to the Compositae family, whose cytoplasm is provided with mitochondria comprising DNA which at least partially originates from another species of the Comoositae family. and who is the carrier of male cytoplasmic sterility (CMS). 2. Plant according to claim 1, the cells of which comprise a nudary genome and a chloroplastid genome specific for the species which are normal for the plant in question. 3. Plant according to claim 1 or 2, wherein said plant phenotypically has male cytoplasmic sterility. 4. Plant according to claim 1, 2 or 3, wherein the other species of the family Compositae was chosen from the group consisting of Helianthus sop. and Cirsium spp. 5. Plant according to claim 4, wherein the other species of the family Compositae is Helianthus annuus (L.) with CMS of the petiolaris type. 6. Plant according to claim 1, 2 or 3, wherein the other species of the family Compositae was chosen from the group consisting of Chrvsanthemum. Senecio, Centaurea, Sonchus, Hieracium. Taaetes. Dahlia and Aster. 7. Plant according to any one of the preceding claims I to 6, in which the plant was chosen from the group consisting of Cichorium intvbus (L.) var. Foliosum (Heai), Cichorium endivia (L.). Lactuca sativa (L.), Scorzonera hispanica (L.), Canara scolvmus (L.) and Taraxacum officinale (L.). 8. Seeds or plant parts from a plant according to any one of the preceding claims. 9. Method for preparing a plant according to any one of claims I to 7, characterized in that it consists in:  - isolate protoplasts from tissues of various plants belonging to the ComPositae family. so-called "acceptors" and tissues from another species of the Comoositae family. called "donor";  - possibly treat protoplasts, in particular by irradiation, cytoplastization or with specific chemicals;  - fusing protoplasts, in particular under the influence of chemical substances or using electricity;  - possibly select certain hybrid cells;  - then cultivate the cells and regenerate them to give complete plants; ;  - select individuals from the regenerant collection thus obtained by analysis of mitochondrial, chloroplastidial and genomic DNA, followed by phenotypic evaluation of the plant's properties;  - specifically cross previous individuals or their lineage with other genotypes of the same species and other species;  - produce seeds on a large scale.