DE19535313A1 - Method of producing haploid plant - Google Patents

Abstract

The nucleus either of an egg cell or of a spermatide is inactivated. The egg cell and the spermatide are fused together. The egg cell and spermatide are present in isolated form and the fusion cell cultivated. For obtaining a double haploid plant, the haploid plant obtained is subjected to duplicating treatment. The plant used is gramineae of the genus Zea.

Description

The invention relates to a method for producing a haploid plant by in vitro mock fertilization.

 Recently, for a large number of fruits Developed F₁ hybrid varieties. The type of F₁-type is Crossing a genetically fixed paternal line and mother production line. It is known that F₁ varieties a Hybrid behavior, and they are in many cases, ver  similar to varieties of a purebred line or an inbred line never or self-sacrificed line, stronger and yield higher out booties. Furthermore, F₁ varieties have the advantage that they are very homogeneous because they produce between purebred lines become.

To breed a F₁ variety, it is necessary to gene their parents to fix the table. In conventional methods, a Pure breeding line by five to eight times repeated selfing produced; These procedures took a long time.

Due to the development of cell culture techniques, it has been recently possible in a number of ways, by culturing pollen or ovules to produce a haploid plant len. A haploid plant brings a great advantage since a genetically fixed line immediately by doubling the Chromosome number of a haploid using a Che mikalie etc. can be produced. Although the technique to manufacture a haploid or a double haploid line some fruit plants such as tobacco, Chinese cabbage and eggplant in The practice has been implemented is the application of this technique on plant breeding nevertheless in a large number of Fruits due to the problems associated with the frequency of their Occurrence and their reproducibility, ziem difficult. Under these circumstances is the evolution a new technique that is more effective sheep a haploid or a double haploid line made possible light.

Among the methods for producing a haploid or a double haploid line using an ionizing Radiation, such as X-rays, are for example an Ver Drive known in which pollen irradiated before fertilization and the seeds obtained are sown to a plant to obtain, and a procedure whereby the fertilization under Use of pollen is carried out with an ioni  irradiating radiation was irradiated, the obtained Ovaries or ovules are cultivated (the or pseudo-fertilization and fruit nodule culture methods). However, these treatments can only be limited Number of plant species to create a haploid type. Furthermore, even in the same species is often between different varieties a big difference in frequency to watch their appearance.

Kranz et al. were in vitro fertilization in 1993 of maize (Kranz, E. and Loerz, H., "In vitro fer tilization with isolated, single gametes results in zygotic embryogenesis and fertile maize plants ", The Plant Cell 5: 739-746 (1993)). This in vitro fertilization is a tech nik, to an egg isolated from an ovule with a spermatids isolated from a pollen by in vitro cell fu fertilize and cultivate the preserved zygote in order to thereby obtaining a plant. According to this technique is it is possible from the moment of fertilization to the cultural condition adjust in a test tube. It is about therefore a very useful technique; This method however, it is only in a limited number of species successful, as there are a number of technically difficult steps includes, for example, the isolation of an egg from a Ovule, the fusion of an egg and a spermatid and similar steps. In particular, in the isolation of an egg cell will, in most cases, although some successful were, the egg after the digestion of the ovule with a Enzyme disappear. For example, in cruciferous vegetables, in de The detection of an embryo sack or an egg apparatus the isolation of the egg is difficult, if the above be Enzyme treatment is performed, it is schwie be able to distinguish an egg from other cells and therefore the isolation of an ovum is practically impossible Lich. Furthermore, the conditions that are required vary are to keep an isolated egg alive, and they  are dependent on the plant species. That's why the one in vi tro-fertilization in plants such as the cruciferous still more difficult in which no live egg has been isolated could be.

It was an object of the present invention to provide a method to provide these known from the prior art Avoids disadvantages and provides one technique to one haploid or a double haploid line efficiently len.

This object is achieved in that a Process for the preparation of a haploid plant bereitge The nucleus is either an ovum or a Spermatide is inactivated, the egg and the spermatids in isolated form are fused and the obtained fusio Nested cell is cultivated.

The term "isolated" in this context means that oocyte and spermatids from plant tissues or organs to which they belong.

To solve the above object have been made by the inventors extensive and time-consuming and costly research guided. It has been found according to the invention that it is possible is efficient, a haploid or a double haploid line in that an in vitro fertilization according to In activation of the nucleus of either an ovum or sperm matide is performed.

The present invention further relates to a method for Preparation of a double haploid plant, according to haploid plant obtained by the method described above is subjected to a doubling treatment to a diploid To get plant.  

Fig. 1 shows a preparation process for isolating egg egg in rape egg.

Hereinafter, the invention with reference to Ausfüh Examples described in more detail. The procedure according to the invention ren is applicable to any plant.

The inventive method for producing a haploid Plant is for example executable as follows. Be first the nuclei of either oocytes or spermatids inacti fourth. The inactivation of the nuclei may be by irradiation with an ionizing radiation such as ultraviolet rays, Röntgen beams and y-beams are performed. The dose of X-rays are preferably 50 kR - 200 kR, these being depending on the plant used, the target of Inactivation (oocyte nuclei or spermatid nuclei) etc. vari ated. If the kernels are inactivated by maize spermatids, the dose is preferably 80 kR - 120 kR.

After inactivation of the nuclei, the oocytes and Sper matids isolated from the plant bodies. To isolate the Eggs are different techniques in plants like Corn is used in which an embryo sack is recognizable from the outside is, and in such plants as Kreuzblütlern, in which one Embryo sac is not recognizable from the outside. For the first ge called plants are removed from a plant body treated with an enzyme to lyse the cell walls delt, and then the embryo sacks are removed. Of these Embryo sacs are used to isolate the oocytes. As an egg in is accompanied by an embryo sac of Synergiden is the egg le differ from other cells due to these indicators bar. For the latter plants, where an embryo sack is not recognizable from the outside, the ovule is without prepared the above enzyme treatment, and the egg is selected from a number of separate cells and isolated.  

The type of preparation of an ovule depends on the struc the ovule in the plant to be treated. In the case From rape, the preparation of an ovule can thus be carried out be as in the preferred embodiments of the above underlying invention is described. Although during the pre paration of an ovule a large number of cells abge can be separated, the selection and isolation of the egg based solely on the following three indicators be performed. These are:

• 1) The cell is taking a certain osmotic pressure either a spherical shape or not spherical Shape (an egg cell takes a spherical shape a certain osmotic pressure, as their cell wall not fully formed);
• 2) the cell diameter (in rapeseed the diameter of the Egg cell about 10 μm); and
• 3) The cell wall contains a large nucleolus or not (an egg contains a nucleolus).

It is necessary to keep an isolated egg cell alive to halve the osmotic pressure at an appropriate value This condition depends on the type of Plant off; Preferably, the osmotic pressure is in a Be rich of 400-900 mosmol / kg H₂O. For example, corn is the range at 450-700 mosmol / kg H₂O and rapeseed in the range of 650-850 mosmol / kg H₂O.

Isolation of spermatids can be achieved by breaking up the pole in a solution with a low osmotic pressure and Select only the spermatids from the released Spermatids and vegetative nuclei are performed. The Isolation of spermatids can also be achieved by other methods be performed. For example, a method is used bar, where the pollen is crushed. Because spermatids and vegetative nuclei are different in size  Spermatids selectable due to this difference. Because Sper They have a shorter survival time compared to oocytes , the isolation of the spermatids is preferred after Completed the isolation of the oocytes. A procedure Example is given below in the description, taking oocytes and spermatids after inactivation of the nuclei the spermatids are isolated. The inactivation of the nuclei However, after isolation of these cells is feasible.

Thus, an isolated egg and an isolated sperm matide then fused when removed from the plant tissues or organs to which they belong. The place This merger is arbitrary, as long as the merger not taking place in the living organism. Preferably, the Fusion carried out in a liquid drop formed in oil leads, as in the preferred embodiments of the above underlying invention is described. Such a liquid drop is preferred because the osmotic pressure is maintained at a certain level in it no water evaporates in it. The cell fusion is under use Polyethylene glycol, high concentration calcium etc. feasible. The cell fusion is preferably used performed electrical impulses, as in the before zugte embodiments of the present invention beschrie ben will. When cell fusion using electrical Impulse is carried out, the conditions, for example example, the voltage of the pulses, the number of applied Impulses etc. depending on the plant used, the Salt concentration in the liquid drop, etc. selected who the.

After completion of the cell fusion, the resulting fusion cells cultured with shaking in a liquid medium. The used medium can be arbitrary, as long as it is here a medium commonly used in plant cell cultures its about. Examples include MS medium, B5 medium,  N6 medium and similar media. Preferably, the osmotic Pressure in the medium to about 600 mosmol / kg H₂0 by adding Glucose or an agent for adjusting the osmotic Pressure set. As a plant hormone can contribute to the medium auxines are added by the way. In particular, be 2,4-D-naphthaleneacetic acid and similar hormones used. The Concentration in the case of 2,4-D is about 2 mg / l. In the Cultivation of the fusion cells are preferably feeder Cells added to the medium. If the fusion cells among the cultivated above conditions form in about 7 days Calli, although this time by the nature of the plant depends.

About 8 to 12 days after the start of the culture, the Calli to spherical formations with a diameter of 380-700 μm, and in parts of this an embryo formation begins. 10 to 12 days after the beginning of the culture calli, in which the Embryo formation progresses, transferred into MS medium containing a combination of plant hormones such as 2,4-D, NAA and Ben cyladenine (BA) or hormone-free MS medium is supplemented, where the calli are further cultivated. About 1 to 3 months after the beginning of culture of the fusion cells, the plants develop zenkörper.

The fusion cell obtained as described above differentiates usually via a callus to plant bodies, though at some plant species, the fusion cell directly and not over a callus grows to a plant body. The last ge called cases where a fusion cell does not have a Callus grows to the plant body, become of the invention also included.

Furthermore, by conventional duplication treatment of obtained haploid plant with colchicine or another Fabric a double haploid plant available.  

Subsequently, the present invention in detail under Reference to embodiments described. The invention however, is not limited to these examples.

example 1 Induction of Calli in maize by in vitro pseudo-fertilization technique 1) Plant material

It was grown in a greenhouse corn variety "A188" used. This plant was grown under additional light when the weather was not right.

2) Procedure for isolating oocytes

Female ears were in a suitable pollination state collected from plant bodies. The developmental stages of a Female spikes may be due to the length of their corn silks be estimated. Such female ears were used det, whose silks had a length of 15-20 cm. The collected female ears were sprayed with 70% Ethanol sterilized. Other sterilization procedures were unnecessary. After spraying the alcohol on the ears were using rubber gloves by hand husks and removed similar parts in a clean work bench, and the ears were used with a knife for easier handling in 3 to 4 Cut pieces. At this time, the lower part and the top (tip) of each ear removed. embryo Sacks with reproductive tissue parts were detached from the ears remove and immediately in an aqueous Mannitollösung with a osmotic pressure of 540 mosmol / kg H₂O watered or rinsed.

By the above procedures were 20 to 30 fruitkno tengewebefragmente in the aqueous Mannitollösung. Then became the ovary wall of each ovary tissue fragment  ment removed using tweezers. The so get The ovarian tissue fragments were placed in a 3.5 cm laboratory dish with 1 ml aqueous Mannitollösung with an osmotic Pressure of 540 mosmol / kg H₂O transferred.

To this laboratory dish was added 0.5 ml of an enzyme solution (the composition is shown in Table 1 below) adjusted with mannitol to an osmotic pressure of 570 mosmol / kg H₂O and a pH of 4.9-5.0 was. Then the laboratory dish was allowed to stand for 10 to 20 minutes at room temperature. Then, the isolation of the ova was carried out under a reverse microscope using glass needles.

Table 1. Composition of the enzyme solution

enzymes concentration Pectinase (Serva) | 1.5% Pectriase Y23 (Seishin) 0.5% Hemicellulase (Sigma) 1.0% Cellulase Onozuka RS (Yakult) 1.0%

If the number of bowls was too large, the insulation to start at once, the shells were in a cooling stored until the beginning of the isolation. As it is possible was the localization of an embryo sack in a semen Gengewebefragment due to the use of a microscope too confirm, the process was carried out so that the Gewe was fixed with a glass needle while the embryo sac stripped or scraped off with the other glass needle.

Since an egg cell is accompanied by two synergies, she was able to distinguished from other cells due to this indicator  become. The size of the oocytes obtained was about 60-70 μm.

The isolated oocytes were in a mannitol fluid transferred to a coverslip, wherein the cell fusion using a micropump (dispenser / thinner, Micro lab-M, Hamilton) under computer control.

3) Inactivation of spermatid nuclei by X-rays

Dust bags were ge before the bursting of plant bodies collected and with X-rays of 100 kR or 200 kR be shine. After the irradiation the dust bags were rocked net, and the pollen was taken out. Fertilization of greenhouse plants was irradiated using the Pollens performed to confirm that no Sa received, d. H. inactivates the nuclei of the spermatids were.

4) Process for isolating spermatids

Pollen for use in this experiment was from Staubbeu collected and transferred into a laboratory dish. It was no sterilization treatment performed. Because the isolated Spermatids have a fairly short survival time, be The isolation of the spermatids started just before the onset the cell fusion after the isolation process for the ovum was completed.

Upon addition of an aqueous Mannitollösung with an osmotic Pressure of 540-660 mosmol / kg H₂O to a pollen containing Laboratory dishes were picked up by the osmotic shock broken pollen particles spermatids released. vegetative Cores (large size) were also released from pollen particles set; Spermatids (small size) could due to their Size can be easily distinguished. The released spermati  it was transferred to a drop of liquid where the cell Fusion was performed using a micropump.

5) cell fusion

The liquid drop, the ova and spermatids to Ver was to take up the cell fusion, was as follows manufactured. The edges of a thick coverslip were placed in Re pel-silane (Pharmacia; dimethyldichlorosilane solution 20 g / l in 1,1,1-trichloroethane) immersed about 3 mm deep, and the deck glass was dried for 10 minutes. These processes were carried out in a chamber with air flow. The purpose of the This treatment consisted of the possible overflow of a on the coverslip to prevent oil to be given. Subsequently The coverslip was combined with its container with UV light Sterilized for 15 minutes.

Subsequently, about 0.3 ml of paraffin oil was applied to the coverslip 2 μl of a liquid were added to this paraffin oil dropping an aqueous Mannitollösung with an osmoti pressure of 540-660 mosmol / kg H₂O using a Micropump formed. In this liquid drop were the oocytes obtained under 2) and 3) as described above and spermatids introduced.

Electrodes used to carry out the electrofusion were platinum used. The platinum electrodes were under the condenser fixed by a microscope. The distance between these electric was turned on by the up and down movement of the microscope stage provides. After adjusting the localization of the liquid drop by applying an alternating current of 1 MHz 71 V / cm was the cell fusion by one to three times Anle gene of a DC pulse of 50 microseconds and 0.9-1.0 kV / cm carried out. The fusion efficiency was about 80%.

6) cultivation

The fusion cells were shaken on one side of a 12 mm Millicell placed semipermeable membrane in one Plastic laboratory dish with a diameter of 3.5 cm kulti fourth. The medium used was MS medium, which was mixed with 2,4-D supplemented with a rate of 2 mg / l and with glucose to one osmotic pressure of 600 mosmol / kg H₂O was set. to above 3.5 cm laboratory dish were 1.5 ml of MS medium and feeder calli from immature maize embryos added. For the Feeder calli are those with a diameter of about 3-5 mm suitable. 3 to 4 such calli were added to each laboratory dish added. The number of electrofusion out with X-ray irradiate irradiated spermatids and oocytes preserved Zygo in the first attempt was 7 and in the second attempt 22 These showed 6 zygotes or 1 zygote a division or Formation of microcalli.

Example 2 Isolation of oilseed rape 1) Plant material

It was grown in a greenhouse rapeseed "Topaz" used.

2) Collection of plant material and its sterilization

Buds or flowers just before blooming until shortly after Flourishing was collected. The petals, sepals etc. were removed from the collected buds and flowers to thereby to expose the ovaries. These ovaries were about Sterilization in 70% ethanol for 20 seconds surfaced. Then they were washed twice with sterile water. After the sterilization treatment these ovaries became until the beginning of the removal of the egg in sterile water held.  

3) Isolation of oocytes

The ovaries were placed in a 3.5 cm plastic laboratory dish with an aqueous Mannitollösung with an osmotic pressure of 750-850 mosmol / kg H₂O. The ovules were removed from the ovary in this laboratory dish with tweezers and a knife. It was confirmed that the osmotic pressure of the mannitol solution is not particularly limited, and that the oocytes can be kept under an osmotic pressure of 850 mosmol / kg H₂O or above. After removal of the ovules, the isolation of the oocytes was carried out using two glass needles. A seed plant was fixed with a glass needle. At this time, care was taken to ensure that the ge in Fig. 1 showed part A was damaged as little as possible, since the egg is located here. The ovule was cut with the other glass needle. Since the tissue B (see Fig. 1) representing the nucellus could be verified under a microscope, the ovule was cut on the line X (see Fig. 1) using the tissue B as indicator. This process occasionally produced nucellus, and was sometimes accompanied by haploid cells, such as an ovum and central cells. Subsequently, under Fixie tion of the point C, the line Y cut superficially (see, Fig. 1). Care had to be taken at this time not to injure the egg. Globular cells often appeared from the embryo sac by these procedures. When no spherical cells appeared by these methods, they were removed by scraping out the part A (see Fig. 1) with a glass needle.

It was assumed that one of the above-mentioned Ver drive preserved spherical cells with great probability the egg was an egg. Maybe removed, ballför mige cells include an egg cell, central cells, synergides, Antipodes and somatic cells. Since the Ver  However, if no enzyme is used, there is the possibility of one Isolation of somatic cells very low, and the differences Making an egg from other cells is less difficult. To distinguish an egg cell, the following In dicators used:

• (1) An ovum takes in an aqueous mannitol solution slightly spherical in shape because its cell wall is not is completely formed;
• (2) the diameter of an egg is about 10 μm; and
• (3) An ovum contains a large nucleolus.

The oocytes distinguished by these properties were transmitted by a micropump, and the inventive Treatment was performed.

According to the invention it is thus possible, the culture conditions in to enter a test tube from the moment of fertilization put. Accordingly, by the inventive method haploid plants or double haploid plants in comparison to produce conventional methods with higher frequency.

The present invention thus relates to a method for Production of a haploid plant, the nucleus either an ovum or spermatid of a plant inactivated , the egg and spermatids are fused together where both the ovum and the spermatids of the plant tissues or organs to which they belong iso The cultured fusion cell is then cultivated becomes. The invention further includes a method for producing position of a double haploid plant the above method obtained haploid plant of a Ver is subjected to a double-dip treatment to a diploid plant to obtain. Because of these methods are haploid plants or double haploid plants (diploids) with higher frequency available.

Claims (4)

1. A process for the preparation of a haploid plant, characterized in that the core is inactivated either an egg or a spermatid, the egg and the spermatids are fused mitein other, wherein both the egg and the spermatids are present in isolated form, after which the obtained Fusion cell is cultivated. 2. Method for producing a double haploid plant ze, characterized, that obtained by the process according to claim 1 haploid plant subjected to a duplication treatment is to get a diploid plant. 3. The method according to claim 1 or 2, characterized, that Gramineae be used as a plant. 4. The method according to claim 3, characterized, that as Gramineae uses a plant of the genus Zea becomes.