KR100961161B1 - Method of high frequency plant regeneration of Perilla frutescens - Google Patents

Abstract

The present invention relates to a regeneration method of perilla seedlings, and more specifically, (1) sterilizing germinated perilla seed; (2) inducing somatic cells using hypocotyl derived from the germinated seeds as explants; (3) culturing for 3-4 weeks in MS medium to which BA (6-benzylaminopurine) is added to shoots generated from the induced somatic cells; And (4) culturing the culture of (3) in MS medium to produce roots.

Perilla, hypocotyl, buds, recurrence rate, seedlings

Description

Method of regeneration of perilla seedlings {Method of high frequency plant regeneration of Perilla frutescens}

The present invention relates to a method for regenerating perilla seedlings.

Perilla frutescens [L.], a broad-leaved vegetable crop that produces oil, has been grown in Southeastern and East Asian countries, including Korea. It belongs to the Lamiaceae family, a self-pollinating annual plant. It is divided into two perilla species according to their shape and use. One is Perilla frutescens (L.) var. Perilla , which is frutescens, and edible as herbs or vegetables frutescens (L.) var. crispa (Lee et al ., 2002. Crop Sci 42: 2161-2166). Both species are essential additives in the cooking of South and East Asians (Li, HL. 1969. Econ. Bot. 23: 253-260). To date, Perilla frutescens (L.) var. frutescens has been widely grown and used in Korea (Nitta, M. 2001. Ph.D. Thesis, Kyoto Univ., Kyoto, Japan). These perilla species are widely used for medicinal, oil producing and broadleaf vegetable crops (Honda et al., 1990. Jpn. J. Breed. 40: 469-474). However, the perilla plant breeding method and the method of introducing the cellular cytoplasm have not been widely used, and cultivation plants that can be used as oil production or vegetable crops alone have not been developed in Korea (Lee et. al ., 2002. Crop Sci 42: 2161-2166). In the last 20 centuries, the striking progress of this effort has begun to yield genetically improved perilla, and that effort continues. Anther (Lee et al., 1994. Korean J. Breed. 26: 345-352), leaves and cotyledon (Kim et al., 1993 Korean J. Plant Tissue Cult. 20: 47-50), hypocotyl (Kim et al. al., 2004. Plant Cell Rep. 23: 386-390). Indirect organogenesis through callus from ex-plants of cotyledon, leaf and hypocotyl is already easy (Lee et al., 2003 Korean J. Breed. 35 (4): 237-240).

A study using Yeopsil, Daesil and Dasil, Korean perilla cultivars, confirmed that high quality oil production was possible. On the other hand, the broad-leaved vegetable perilla, which is widely used as a side dish or as a side dish in the Korean diet, has yet to be reported on genetic regeneration through a plant biotechnological approach.

Recently, the highest rate of shoot induction using cotyledon (cotyledons) was reported to be 27.3% (Kim et al., 2007. J. Plant Biotechnol 34 (1): 69-73). In vitro regeneration systems with improved reoccurrence by developing tissue explants that can regenerate in perilla are essential for increasing qualitative value under molecular breeding programs.

The present invention has been made in accordance with the above-described needs, and the present invention establishes an in-flight culture system of perilla through a plant bioengineering approach, thereby establishing a mass growth system capable of reviving the increasing demand of perilla in Korean and global diets. To establish.

In order to solve the above problems, the present invention

(1) sterilizing the germinated perilla seed;

(2) inducing somatic cells using hypocotyl derived from the germinated seeds as explants;

(3) culturing for 3-4 weeks in MS medium to which BA (6-benzylaminopurine) is added to shoots generated from the induced somatic cells; And

(4) it provides a regeneration method of perilla seedlings comprising the step of culturing the culture of the above (3) in MS medium to produce a root.

The present invention can provide a method for regenerating perilla seedlings having a high regeneration rate using a reproducible, reliable and convenient in vitro regeneration system.

In order to achieve the object of the present invention, the present invention

(1) sterilizing the germinated perilla seed;

(2) inducing somatic cells using hypocotyl derived from the germinated seeds as explants;

(3) culturing for 3-4 weeks in MS medium to which BA (6-benzylaminopurine) is added to shoots generated from the induced somatic cells; And

(4) it provides a regeneration method of perilla seedlings comprising the step of culturing the culture of the above (3) in MS medium to produce a root.

The ventral axis of the present invention grows into a part forming the central axis in the pear of the higher plant, and becomes a stem, the upper part of which is a cotyledon and young eyes, and the lower part of a young root.

In the method according to an embodiment of the present invention, the sterilization step of step (1) may be performed by soaking in 70% ethanol for 1 minute and then soaking in 4% sodium hypochlorite for 20 minutes. Can be.

Seed sterilization kills germs that adhere to seeds, and there are chemical methods of using drugs and physical methods of using heat or ultraviolet rays. Chemical method is a method of treating Tri Sodium Phosphate (TSP), Tsunami, Nomadin, Solid Matrix Priming (SMP), Gibberellic acid (GA) or KNO ₃ for a certain period of time, and physical methods include pre-cooling of seeds, hot water treatment, Dry heat treatment, mechanical treatment, and the like, and these may also be included within the scope of the present invention.

In the method according to the embodiment of the present invention, the explant of the second step may be an apical bud or a top section of the embryonic axis. Mean reoccurrence of plants from explants showed 64.3%, 15.66%, 4%, and 1.33%, respectively. Preferably, the explants of the hypocotyls are preferably a sperm or a top, more preferably sperm.

In the method according to an embodiment of the present invention, the embryonic axis of step (2) is characterized in that 9-12 days old seedlings after seed germination, preferably 10 days old seedlings.

In the method according to an embodiment of the present invention, the concentration of BA in the third step (3) is characterized in that 2-6ppm, preferably 2ppm.

In a method according to an embodiment of the present invention, the step (4) is characterized in that the root spontaneously (spontaneously) in the hormone-free MS medium.

The method of the present invention may further comprise the step of culturing the rooted shoots in distilled water for 3-4 days and transferring the culture to the soil.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Provision of explants

Perilla Perilla ( Perilla) frutescens [L.] Britton "Manbaeg" was provided as a source of explants of the present invention. Seeds of the cultivars were obtained from the National Crop Experimental Station, Rural Development Administration (Suwon, Korea).

2. Sterilization of seeds

For sterilization of seeds Two different treatment procedures were performed. One placed 2 mg of dried mature seeds in a 50 ml Falcon tube, soaked in 70% ethanol for 1 minute and washed several times with distilled water. Second, as shown in Table 1, the seeds were treated with different concentrations of HgCl ₂ and / or commercial bleach (YUHAN COLOREX, 4% sodium hypochlorite). As soon as the second treatment was finished, the sterile bodies were washed 3-4 times with distilled water in a Laminar Air Flow Cabinet. The treated seeds were washed 3-4 times at 1 hour intervals while maintaining sterile conditions. Immerse in sterile water overnight at room temperature.

3. In vitro germination of seeds

The treated seeds were pressed with a filter paper and placed in basal half-strength MS (Murashize T. and F. Skoog 1962. Physiol Plant 15: 473-497) medium containing 1.5% (w / v) sucrose without plant hormone. Germinated aseptically. The pH of the medium was titrated with NaOH or 1N HCl with pH5.7 ± 0.1 (using “istek” pH meter, Model 720P), and 0.4% (w / v) of Plant Agar (Duchefa Biochemie, The Netherlands) was added. The medium was sterilized with autoclave (Hanback Scientific Co .; HB-506-6) at 121 ° C. for 20 minutes at 1.5 kgcm ⁻² . In order to obtain seedlings with well-grown hypocotyl, approximately 22-25 seeds were plated in plant culture dishes containing the medium. The plated seeds were subjected to 16 / 8-h (day / night) photoperiod with a 40 W blue-white fluorescent lamp with a photon flux density of 45 μMol photon m ^-2 S ^-1 in a 25 ± 2 ° C culture chamber. Incubated with. After incubation for 8-10 days under such conditions, the germinated seeds were prepared as initial samples of the present invention.

4. Regeneration of whole plants

The direct somatic organogenic method was used to induce embryonic development directly from the embryonic axis without the development of an intermediate callus.

a. Explant

Leaf fragments and cotyledon (Kim et al., 1993. Korean J. Plant Tissue Cult. 20: 47-50), anther (Lee et al., 1994. Korean J. Breed. 26: 345-352 to regenerate perilla Limited research has been conducted with However, the comparative efficiency of regenerating perilla plants using hypocotyl segments has not been reported yet. In the present invention, the explants were selected as the explants, the common explants reported earlier in other perilla species. Dividing the ventral axis into individual pieces, each 5-6mm long (except the apical bud) explants are defined as basal, middle, and top sections (Figure 1). The relative reoccurrence capacity under different conditions was investigated. Regeneration rates were investigated with the same explants originating from the hypocotyls of different ages of seedlings (10, 12 and 14 days). Sections were cut from the base to the top of the ventral shaft by cutting on a sterile paper tray with a sharp surgical knife and tweezers. Special care must be taken to avoid damaging the cells at the end of the cut.

b. Outbreak

The organ genetic capacity of embryonic sections (5-6 mm long) was observed by incubating in solid MS basal medium at pH 5.7 ± 0.1 with varying concentrations of BA (6-benzylaminopurine). Temperature and light conditions are the same as the in vitro germination conditions of the seed. Initially tested in 15 treatments with a combination of five concentrations of BA 0.0 ppm, 2.0 ppm, 4.0 ppm, 6.0 ppm and 8.0 ppm and three hypocotyl segments (basal, middle, and top) It was. Thirty random pieces of isolated hypocotyl sections were treated individually, and this experiment was repeated three times and rearranged into a completely random design. Based on these attempts, we investigated the addition of apical buds to compare the regeneration rate of perilla. Explants containing the explants were subcultured from 2 weeks to 2 months. In fresh medium by subculture, shoot regenerators were cut at approximately 2/3 ^rd position from the tip of shoot shoot.

c. Shoot elongation and root induction of shoots

Shoots grown on the cutting plane of explant explants were transferred to MS basal medium solidified with hormone-free 0.8% plant agar for elongation and rooting of shoots. Plants with well-developed roots separated the roots from the medium, and the medium on the roots was washed with water. Rooted plants were allowed to acclimate in water for 3 days and in small plastic pots filled with sterile soil (compost: vermiculite = 1: 1). Well-adapted plants were transferred to larger pots of soil, planted, and transferred to greenhouses to investigate their development and growth.

<Example 1. Sterilization method of seed and seedling germination>

In order to maximize the number of well-developed embryos for use as explants in vitro, different sterile drugs were treated with perilla seeds under several conditions. When the sterilant consisting of HgCl ₂ (0.3%) and commercial liquid bleach (100%) was treated sequentially for 15 and 20 minutes respectively, no contamination of the seeds was observed. In this way, germinated seeds (25%) grew to 4% seeding (Table 1). In contrast, when only commercial bleach on seeds was treated at this time, no contamination was observed in the medium, and seeds germinated (95%) in sterile cultures grew to 20% seedlings. Seed treatment time also affects the cleaning effect from microbial contamination of the seed in the culture medium. When seeds were soaked in 100% (v / v) commercial bleach for 15 and 20 minutes respectively, longer treatment (20 minutes) was free of contamination while short treatment (15 minutes) resulted in contamination. Short treatments (15 minutes) showed 3% greater seedling growth than long treatments (20 minutes) (Table 1). Seedling germination is not affected by these conditions. Although the same amount of contamination was found in the culture medium, there was no difference in the germination rate (%) and the number of seedlings after 15 minutes of 100 and 50% (v / v) commercial bleach. Similarly, 0.15% (v / v) seedlings in different ways yet be natjiman shown that pollution is frequently little, as well as the germination rate used is the HgCl ₂ and HgCl ₂ 100% (v / v) doubled compared to method (A) with commercial bleach. Due to the low efficiency in obtaining seedlings compared to other methods, HgCl ₂ treatment It was not used as a method of sterilization in the present invention. However, long treatment of commercial bleach (4% sodium hypochlorite) for 20 minutes compared to all methods was selected as the best method for obtaining the most seedling water at 100% sterilization conditions.

TABLE 1Increase rate of in vitro seeding by seed sterilization method

<Example 2. Influence of seedling age and shoot induction>

Based on the different growth rates of seedlings generated through the sterilization method (data not shown), the age of seedlings suitable as explants for shoot induction was optimized. After plating the sterilized seeds in germination medium, it usually takes 9-10 days to obtain explants for shoot induction. In special circumstances, seedlings aged 15-16 days are suitable for use of the axilla as an explant. However, the 10-day-old seedlings, one of the three types of seedlings, appear to be the best material for excretion as an explant for induction of shoots. These results explain that the continual increase in seedling age shows a gradual decrease in percent shoot induction.

Example 3. Generation of Shoots

In the preliminary experiments, the recurrence ability of the top, middle, and basal sections, different segments of the hypocotyl, and the response to BA show various variations (FIG. 3). The top section of the embryonic axis appears to be the best explant for spontaneous shoot regeneration with acceleration compared to the middle and basal sections in MS medium supplemented with 2 ppm BA. Even with the best conditions in the top and middle sections with 2 ppm BA added, the lower part of the hypocotyl appeared to increase regenerating shoots up to 6 ppm BA. In addition, it was found that calli masses that did not reoccur were induced into segments of the embryonic axis. In this case, BA values strongly influenced this kind of callus induction, and it was found that the occurrence of shoots increased in all segments of the hypocotyl designated by the increase in BA concentration (FIG. 4). However, based on this fact, further research is needed to investigate the organogenic response as to whether the use of apical buds as an alternative to explants for in vitro regeneration of perilla. Thus, in addition to the hypocotyl section, sperm was plated in regenerating medium to which 2 ppm BA (the best amount of cytokinin in the previous results) was added as described above. Among the explants tested in this manner, shoot regeneration rate and total induced shoots were higher in sperm (FIG. 5). The second highest value for the same variable was recorded as the upper section, followed by the interruption of the axis and the section derived from the lower section. As such, regenerating media comprising sperm induced increased regenerating plant numbers over other explant types. The average reoccurrence rate of vegetation from sperm showed 64.33% higher than the upper end (15.66%), discontinuation (4%) and lower end (1.33%). Thus, quinoa can be used as the best explant of broadleaf perilla in vitro (FIG. 5).

Example 4 Plants Adapted to Root Induction

Induced shoots perform a spontaneous response that takes root in MS medium without the addition of hormones. Usually, plated shoots induce roots in the aforementioned medium through a 7-10 day incubation period. In some cases, fast-growing shoots fail to take root. Longer shoots also fail to acclimate in the root dish. Fast growing shoots are rooted by incubating 1-2 weeks with distilled water in a 100 ml flask at room temperature (FIG. 6). However, regenerated shootlets with well-developed roots survive in greenhouse soils. Plants surviving in the soil (data not shown) were enhanced by planting in small plastic pots filled with sterile soil (vermiculite: compost = 1: 1) and growing with distilled water for 3-4 days. The acclimated plants were broad-leaved perilla, showed their general form, grew and blossomed, and developed well to make seeds. The reoccurrence process of broadleaf perilla is shown in FIG. 7.

1 is the location of explant explants including sperm in the broad axis of broadleaf perilla.

Figure 2 shows the shoot induction rate (%) of embryonic explants according to seedling age in broad leaf perilla. The data represents the mean value repeated three times.

FIG. 3 shows the effect of hypocotyl segments (top, middle, basal) and BA on shoot regeneration rate of broadleaf perilla. Vertical bars represent standard deviation of 40 explants.

4 shows the effect of BA on unrecurred calli mass induced from the hypocotyl explant sections of broadleaf perilla. Vertical bars represent standard deviation of 40 explants.

5 is a result showing the recurrence rate from the lower end of the hypocotyl explant to the bud in the reoccurrence variable of broadleaf perilla. Vertical bars represent standard deviation of 40 explants.

6 shows the rooting of shoots grown in water. (A) Elongated shoots before taking root in water, (B) Bones after taking root in water.

7 shows the regeneration of broad leaf perilla plants using hypocotyl as explants. (A) Axle slices of 9-10 day age seedlings, (B) Explants in growth medium: top-axle upper slice, bottom left-axle interrupted slice, bottom right-axle lower slice, (C) callus removed Explants of plants such as (B), (D) shoots derived from rooting medium, (E) plants regenerated in small pot soil, (F) perilla plants grown in greenhouses.

Claims (6)

(1) sterilizing the germinated perilla seed; (2) inducing somatic cells using hypocotyl derived from the germinated seeds as explants; (3) culturing for 3-4 weeks in MS medium to which BA (6-benzylaminopurine) is added to shoots generated from the induced somatic cells; And (4) a method for regenerating perilla seedlings comprising culturing the culture of (3) in MS medium to generate roots. The method according to claim 1, wherein the sterilization of step (1) is performed by immersing the seeds in 70% ethanol for 1 minute and then soaking in 4% sodium hypochlorite for 20 minutes. How to. The method of claim 1, wherein the explant of the second step is an apical bud or a top section of the embryonic axis. The method of claim 1, wherein the embryonic axis of step (2) is 9-12 days old seedlings after seed germination. The method of claim 1, wherein the concentration of BA in step (3) is 2-6 ppm. The method of claim 1, wherein the step (4) is characterized in that the spontaneously roots in the hormone-free MS medium.

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