KR20200076167A - Method for haploid and doubled haploid plant production from embryos obtained by shed microspore culture of hot pepper - Google Patents

Abstract

The present invention relates to a method for producing a plant by increasing a regeneration rate through 2-step re-differentiation from embryos obtained by shed microspore culture of hot pepper. The method for producing a plant by 2-step re-differentiation according to the present invention has an effect of increasing the regeneration rate by reliably inducing unfolding of cotyledons and normal development of foliage leaves by 2-step re-differentiation, which is a method of culturing by transferring to a re-differentiation medium and then once again to a new re-differentiation medium. Accordingly, the present invention relates to a method for mass-producing microspore-derived haploid and doubled haploid.

Description

Method for haploid and doubled haploid plant production from embryos obtained by shed microspore culture of hot pepper

The present invention relates to a plant production method using pepper shed vesicle cultivation, specifically, to a method for producing vesicle-derived haploid or douploid haploid plants by increasing the re-differentiation rate by 2-step re-differentiation from pears obtained by pepper shed vesicle culture. Is about

Since the genes of haploid plants do not exist in pairs, the relationship between dominance and enthusiasm disappears and all traits appear, so selecting mutants or transformants from these haploid plants results in plants with useful recessive factors that do not appear in a dominant-recessive relationship. Can be selected.

Conventionally, as a method of acquiring haploids, mainly cultivation of medicine (anther culture) has been used, and each research institute and seedling company have established their own cultivation method, and commercial cultivation using medicinal cultivation is currently being performed, but in the case of red pepper, efficiency The biggest problem is that it is low and there are many diploid cells derived from somatic cells. Meanwhile, as it becomes possible to acquire a large amount of pears by culturing vesicles in rapeseed, studies on microspore culture in many plants have been conducted.

Embryos obtained from microspore culture are converted into plants by re-differentiation. The re-differentiation rate varies depending on the quality of the embryo, and during the process of re-differentiating the plants from the pears grown during pepper shed vesicle culture. There is a problem in that the aging phenomenon is repeated as the stomach becomes enlarged without going through a normal process of developing or developing the main leaf.

In the case of red pepper, it is possible to acquire a large number of pears and plants through shed-microspore culture, for example, as a prior art for a method for obtaining vesicles and plants derived from vesicles (Supena et al. 2006), Non-Patent Document 1 describes the improvement of shed-vesicle cultivation to increase the normal embryo production of red pepper, and Patent Document 1 shows the cultivation of vesicles of red pepper, after blending the medicine taken from the pepper bud After extracting the vesicles by texing, removing the somatic tissue debris from the extruded vesicles, centrifuging to harvest only the vesicles, and then pre-treating the harvested vesicles in pretreatment medium for 3 days at 32° C., and NLN the pretreated vesicles. One day after dentures at a density of 80,000 to 100,000 vesicles/ml in the medium, a normal pear production method of pepper was obtained by adding NLN medium and incubating for 3-4 weeks in a dark state at 27°C to obtain pears. It is disclosed.

In addition, Patent Document 2 is a method for producing a plant from a vesicle fold of pepper, comprising the step of culturing by transplanting a mature or immature cotyledonary embryo obtained from microspore culture of pepper into a re-differentiation medium, wherein the re-differentiation is performed. The medium contains 2% sucrose for mature cotyledon, 1/2MS or NN medium containing 0.4% phytagel, and 2% sucrose for immature cotyledon, 0.4% phytagel The added 1/2 MS medium is used, and the transplantation of immature cotyledon to the re-differentiation medium is performed by transplanting the 3-week old embryo after vesicle culture, the re-differentiation medium being included in the re-differentiation container, and the re-differentiation container has a diameter x height. Disclosed is a method for producing a plant from a vesicle vesicle of red pepper, characterized by using a 10x4 cm culture dish.

The inventor of the present invention solves the problem of repetition of the phenomenon that aging occurs as the pear grows larger and does not go through the normal process of developing the cotyledon or developing the main leaf during the process of re-differentiating the plant from the pear grown during cultivation of pepper shed vesicles. The rise was confirmed and the present invention was completed.

KR 10-0736150 B KR 10-0952103 B

E.D.J. Supena. Refinement of shed-microspore culture protocol to increase normal embryos production in hot pepper. Scientia HorticulturaeVolume 130, Issue 4, 31 October 2011, Pages 769-774

The problem to be solved in the present invention is to provide a method for producing a plant by cultivation of pepper shed vesicles, and more specifically, from the pear obtained by cultivation of pepper shed vesicles, the re-differentiation rate is increased by 2-step re-differentiation to derive the vesicles. It is an object to provide a method for producing haploid or embryonic haploid plants.

As a solution to the problem in the present invention, a plant production method by 2-step re-differentiation from a pear obtained by cultivation of pepper shed vesicles is performed by cultivating a drug collected from a red pepper bud to cultivate the exposed vesicle-derived pear in a re-differentiation medium (1). Then, it is again transferred to the re-differentiation medium (2) to produce a plant by 2-step re-differentiation, and consists of producing a haploid or douploid haploid plant derived therefrom.

In one embodiment according to the present invention, a method for producing plants by 2-step re-differentiation from a pear obtained by culturing pepper shed vesicles is a). The first step of re-differentiation and b) to obtain normal cotyledon embryos by transplanting pears derived from vesicles obtained by developing vesicles bursting out during the process of densifying and culturing the medicine collected from the pepper buds into the re-differentiation medium (1). It consists of producing a plant by a second step re-differentiation by transferring the culture obtained in the first step to the re-differentiation medium (2).

The method of producing plants by 2-step re-differentiation according to the present invention is compared to the conventional method of transferring to a re-differentiation medium after vesicle-derived embryos and cultivating without replacing the medium, and then transferring to a new re-differentiation medium and incubating again. The 2-step re-differentiation has the effect of reliably inducing the development of cotyledon and the normal development of the main lobe, thereby increasing the re-differentiation rate, from which it shows the effect of producing a large number of vesicle-derived haploid and douploid haploids.

1 is a video of the plant by the 2-step re-differentiation method of the present invention
Figure 2 is a two-step re-differentiation method of the present invention and the existing 1-step re-differentiation method of the plant image
3 is a video of the haploid and douploid haploid plants derived from red pepper vesicles

Hereinafter, the present invention will be described in more detail with reference to specific details, <Examples> and <Comparative Examples> for implementation, and the present invention is not limited by the details described below.

Method for producing a plant by 2-step re-differentiation from a pear obtained by cultivation of pepper shed vesicles according to the present invention described above a). The first step of re-differentiation and b) to obtain normal cotyledon embryos by transplanting pears derived from vesicles obtained by developing vesicles bursting out during the process of densifying and culturing the medicine collected from the pepper buds into the re-differentiation medium (1). It comprises a second stage re-differentiation to produce a plant by culturing the pear obtained in the first stage re-differentiation by transferring to the re-differentiation medium (2).

The first step re-differentiation according to the present invention is a method for culturing pepper shed vesicles (shed- microspore culture) to cultivate the medicine collected from the pepper buds and then cultivate the vesicles that have burst into the medium from the opened medicine for about 6 weeks. This is a re-differentiation step of culturing the embryo obtained by primary in the re-differentiation medium (1).

The shed vesicle cultivation method (shed-microspore culture) is a technique well known in the art to which the present invention belongs as a method of culturing a drug collected from a bud to extract vesicles and culturing the extracted vesicles in a medium for 6 weeks to obtain pears. Since it can be easily adopted, detailed description is omitted.

The primary differentiation step of culturing in the re-differentiation medium (1) of the present invention is a culture vessel of 100x20 mm in which the re-differentiation medium (1) was accommodated in the re-differentiation medium (1) obtained by incubating the drug for 6 weeks in a liquid medium. After transferring, the liquid medium is removed and cultured for 2 weeks, and the re-differentiation medium (1) according to the present invention is composed of 1/2MS+2% sucrose+0.4% Phatagel.

The shed-vesicle cultivation period is 6 weeks for the vesicle cultivation period, and since vesicles or vesicle-derived pears are immersed for a long time in a relatively long liquid medium, it may affect re-differentiation, so pears derived from shed-vesicle cultivation are normal. In order to re-differentiate into plants, a container with a size of 100x40 mm, which was previously used as a culture vessel, is not suitable.

In the present invention, the synergistic effect of the re-differentiation rate is achieved by culturing the vesicle embryos generated after 6 weeks of cultivation in a culture vessel having a size smaller than 100x20 mm, and using 2-step re-differentiation together.

The second stage re-differentiation, in which the pear obtained in the first stage re-differentiation according to the present invention is transferred to a re-differentiation medium (2) and cultured to produce a plant, is developed in the form of a heart-shaped or cotyledon in the first stage re-differentiation. If it changes, it is made to develop into a normal plant including selecting a standing pear and transferring it to a culture vessel of 100x40 mm containing the re-differentiation medium 2 for 6 weeks, and the re-differentiation medium 2 is 1/2 MS+2. % sucrose +0.4% Phatagel.

And in the pepper cultivation method according to the present invention, the plants developed by the second step re-differentiation are transplanted into sterilized topsoil, adapted to light conditions at 25° C. for 1 to 2 weeks, circulated air gradually and gradually opened while After growing and purifying for 6 weeks, the purified plants are transferred to pots with a diameter of 25 cm and grown in a greenhouse.

Hereinafter, a method for producing plants and a method for producing peppers by 2-step re-differentiation from pears obtained by culturing the pepper shed vesicles of the present invention through Examples and Comparative Examples will be described in more detail.

<Example 1> Shed-vesicle culture

-Bud collection and sterilization

In the late 1-nucleated vesicles, the condition of the pepper medicine containing the initial 2-nucleated pollen has a 1:1 ratio of calyx and petal. Open the buds of the HSM003 and HSM005 pepper varieties at the appropriate stage to check the coloration of the medicine, and collect the buds that have turned 50% purple and store the buds stored at 4°C for one day in sterile water of 2% NaOCl solution. After immersion for 10 minutes, the bud was sterilized by washing the bud three times with sterile water.

-Weak tooth and pretreatment

After dissecting the sterilized bud using 60x15㎜ petridish, peeling off the petals, and extracting the medicine, place it in NN solid medium containing 2% of maltose, and then dispense 1.5 ml of NN liquid medium. Subsequently, pretreatment is performed at 9°C for 1 week at low temperature (medium composition is shown in [Table 1] below).

-Drug culture

After the pretreatment, 1.5 ml of the liquid medium was added, transferred to a culture room, and cultured at 28° C. for 6 weeks.

Solid medium Liquid medium Autoclaving after adjusting to Nitsch medium+2% maltose+1% activated charcoal+0.4% Phatagel pH 5.8 Filter sterilization after adjustment to Nitsch medium + 2% maltose, pH 5.8

<Example 2> First and second stage re-differentiation

-1st stage re-differentiation

In <Example 1>, the medium obtained by culturing for 6 weeks was transferred to a culture vessel of 100x20 mm containing 25 ml of medium (medium 1) composed of 1/2MS+2% sucrose+0.4% Phatagel to remove the liquid medium. , Cancer culture for 2 weeks to develop into normal cotyledon.

-2nd stage re-differentiation

In the first stage re-differentiation, when the developmental form of the pear changes to a heart-shaped or cotyledonous form, the medium (medium 2) composed of 1/2MS+2% sucrose+ 0.4% Phatagel is cultured for 6 weeks with a 100x40mm medium container. After cultivation, the plants were developed into plants according to the second-stage re-differentiation, and the results of developing into normal plants using a 2-step re-differentiation method after shed vesicles of red pepper were shown as [FIG. 1].

<Comparative Example 1>

In order to compare the re-differentiation rate with respect to the 2-step re-differentiation method according to the present invention, HSM003 and HSM005 pepper varieties cultivated for 6 weeks were transplanted into a general re-differentiation culture vessel (100x40 mm) and re-differentiated by a 1-step method According to the method and the 2-step re-differentiation method described in <Example 2>, the results of re-differentiation and comparison of the re-differentiation rate are shown in [Table 2] and [Figure 2].

Genotype Redifferentiation
method No. of Embryo No. of Redifferentiated plantlet Redifferentiation rate(%) HSM003 1-step 26.7±4.9 1.0±1.7 3.3±5.8 2-step 117.0±36.8 72.0±39.6 59.1±15.3 HSM005 1-step 27.3±0.6 2.7±0.6 9.8±2.3 2-step 159.0±66.9 127.3±71.0 77.4±10.1

As shown in [Table 2], there is a difference between varieties when producing plants through HSM003 and HSM005, but it can be seen that the re-differentiation rate is very high for both HSM003 and HSM003 varieties. Compared to 3.3%, the re-differentiation rate according to the 2-step re-differentiation culture method is 59.1%, and HSM005 varieties have a re-differentiation rate according to the existing 1-step re-differentiation culture method, which is 9.8%, and re-differentiation according to the 2-step re-differentiation culture method. Since it can be seen that the rate is 77.4%, it can be confirmed that the re-differentiation efficiency by the 2-step re-differentiation method according to the present invention is greatly increased.

<Example 3> Pepper cultivation

After transplanting the plants according to <Example 2> into sterilized topsoil, after 1 to 2 weeks of acclimatization at 25°C, the air purification is gradually released and gradually opened to grow and purify for 6 weeks, and the finished plants are purified. It was transferred to a pot with a diameter of 25 cm and grown in a greenhouse.

Claims (4)

a). The first step of re-differentiation to obtain normal cotyledon by transplanting the vesicles derived from the vesicles obtained by developing vesicles bursting out during the process of denture and culturing the medicine collected from the pepper bud, into the re-differentiation medium (1),
b). Plants by 2-step re-differentiation from pears obtained by culturing pepper shed vesicles characterized in that it comprises a second-stage re-differentiation to produce a plant by transferring the pear obtained in the first step re-differentiation to a re-differentiation medium (2). Production method. The method according to claim 1, Medium (1) and medium (2), each of which is composed of 1/2MS+2% sucrose+0.4% Phatagel, a plant by 2-step re-differentiation from a pear obtained by pepper shed vesicle culture. Production method. The method according to claim 2, wherein the first step of re-differentiation, the culture medium of the culture medium (1) is 100x20㎜, the plant production method by 2-step re-differentiation from the pear obtained by pepper shed vesicle cultivation, characterized in that for two weeks. The method according to claim 3, wherein the second step re-differentiation is a plant production method by 2-step re-differentiation from pears obtained by shed vesicle cultivation of red pepper, characterized in that the culture vessel of the medium 2 is 100x40 mm and cultured for 6 weeks.

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