KR101137819B1 - Proliferation method of pro-embryogenic in yellow poplar - Google Patents

Abstract

The present invention relates to a method for propagating embryonic tissue of a lily tree, and more specifically, collecting seed pears from a lily immature seed after pollination; Inducing the embryogenic tissue of the lily tree by culturing the seed embryo of the collected lily tree in a medium; And it relates to a method for growing embryonic tissue of the lily, characterized in that it comprises the step of culturing the induced lily tree embryogenic tissue in the medium.
The present invention examines the degree of developmental stage of seed pear according to the time of harvesting the yellow-poplar seed, and histologically confirms it by providing important information for determining the optimal sampling time when inducing embryonic tissue of the yellow-lily. Embryonic tissues can be generated by high embryogenic tissue induction rate by checking the optimal harvesting time of immature seeds, and somatic embryos can be induced, proliferated and plant production from embryonic tissues of various lines. Will contribute to production.

Description

Proliferation method of pro-embryogenic in yellow poplar}

The present invention relates to a method for propagating embryonic tissue of a lily tree, and more specifically, collecting seed pears from a lily immature seed after pollination; Inducing the embryogenic tissue of the lily tree by culturing the seed embryo of the collected lily tree in a medium; And it relates to a method for growing embryonic tissue of the lily, characterized in that it comprises the step of culturing the induced lily tree embryogenic tissue in the medium.

Yellow poplar is a North American ornamental and timber tree belonging to Magnoliales Magnoliaceae, also known as yellow poplar and whitewood. It is distributed in mixed deciduous forests in eastern North America and grows larger than other deciduous trees in the region, sometimes over 2m in diameter and 60m in height. Bright green leaf with long petiole is bilaterally divided into 2 or 4 branches, and its end is straight or broadly V-shaped. The leaves turn golden in autumn and have large chin leaves at the bottom of petiole. The large yellow-green flowers look like tulips, with six petals with yellow undersides and three bright green calyx leaves. In addition, the winged fruit clusters like a conifer at the end of the stem, the fragrant and purplish brown branches have winter snow resembling a duck's beak, and a long oval shape on the straight stem It is characterized by the formation of water crowns. It will not fully grow until about 200 years. The yellow wood is light yellow to yellowish green and is used to make furniture, plywood panels, paper, woodwork, boxes and crates and is relatively resistant to pests. In places where trees are suitable for growth, they are useful as large green trees.

It is no exaggeration to say that the completion of the somatic embryo induction system of the broad-leaved deciduous tree, including the lily tree, depends on how many inductions (tissue lines) of embryogenic tissue, which are essential materials for somatic embryo development, are induced. This is because even if a variety of embryogenic tissues are induced, not all of them can induce a large amount of somatic embryos, and in fact, only a few tissue lines can be used for mass production of clone seedlings. In addition, embryonic tissue induction is very limited in time because it is possible to collect immature seeds once a year. Therefore, information on the timing of sampling (seed) and developmental patterns of immature embryos in seeds is very important for inducing successful embryogenic tissue.

Methods for inducing embryonic tissue include various factors such as timing of proper sampling, determination of plant medium and growth regulators and concentration, sterilization of sample, change of medium composition according to seed culture stage, and culture method of sample. .

In the case of woody plants, somatic embryos have been studied in many woody plants since Hakman first induced somatic embryos (or artificial seed embryos) from immature seedlings of the Picea abies in 1985 (Hakman I., von Arnold). S. 1985. Plantlet regeneration through somatic embryogenesis in Picea abies (Norway spruce) .J. Plant Physiol. 121: 149-158.

The recent somatic embryo induction technology is a kind of plant cloning technology that is recognized as a very useful tissue culture technology capable of producing a large number of clones in a short time and can be applied to research such as gene insertion and transformation. Do.

The first case of a lily somatic embryo development study reported plant regeneration after Merkle and Sommer induced embryogenic tissue from immature seed embryos in 1986 and developed somatic embryos (Merkle SA, Sommer HE. 1986. Somatic embryogenesis in tissue cultures of Liriodendron tulipifera.Can.J . For.Res. 16: 420-422.). Since then, research on the development of several somatic embryo induction techniques has been conducted.

A recent study on the development of the somatic embryos of the lily tree was carried out in 2001 by Dai, Liriodendron. Tulipifera × L. chinense ) (Dai J., Vendrame WA, Merkle SA 2001. Enhancing the productivity of hybrid yellow-poplar and hybrid sweetgum embryogenic cultures.In Vitro Cell.Dev.Biol. -Plant 40: 376-383.), But there is no detail on embryogenic tissue induction.

There are few reports of embryonic tissue induction and proliferation related to the present invention. Among them, Sotak et al. (1991) reported that 28% of seed embryos were cultured at 8 weeks after post-pollination. Sotak RJ, Sommer HE, Merkle SA 1991.Relation of the developmental stage of zygotic embryos of yellow-poplar to their somatic embryogenic potential.Plant Cell Rep. 10: 175-178 The seed embryo at that time was found to be in the globular stage of development. A domestic study of this technique was conducted by Lee et al. (2003) on the induction of embryonic tissues according to the harvest timing of immature seeds (Lee JS, Moon HK, Kim YW 2003. Mass propagation of Liriodendron tulipifera L. via somatic embryogenesis. J. Plant Biotech. 30: 359-363.), And Son et al. (2005) reported results related to parameter genotypes for embryogenic tissue generation and the correlation of light associated with culture (Son SG, Moon HK). , Kim YW, Kim JA. 2005.Effect of mother trees and dark culture condition affecting on somatic embryogenesis of Liriodendron tulipifera LJ Kor.For . Soc. 94: 39-44. There are very few reports.

Therefore, in the present invention, the seed pears are collected from the immature seeds of pollen after pollination, and the seed pears are cultured in a medium to induce embryogenic tissue of the lily tree, and then the lily pear embryonic tissues are cultured in the medium. The purpose of the present invention is to provide a method for proliferating embryonic tissue of a lily tree that is grown.

An object of the present invention is to provide a method for growing embryonic tissue of a lily tree.

Another object of the present invention is to provide an embryogenic tissue of a lily tree grown by a method of growing embryonic tissue of a lily tree.

The present invention comprises the steps of harvesting the seed pears from the yellow immature seeds after pollination; Inducing the embryogenic tissue of the lily tree by culturing the seed embryo of the collected lily tree in a medium; And it can provide a method for growing embryonic tissue of a lily tree comprising the step of culturing the induced lily tree embryogenic tissue in the medium.

The present invention can provide an embryogenic tissue of a lily tree grown by the embryogenic tissue propagation method of the above-mentioned lily tree.

Seed fruit of lily tree is very low in seed fertility and inevitably imports large amount of seeds from foreign countries for production and distribution of seedlings. Fortunately, the development of plant regeneration technology through somatic embryo development in tissue culture technology has led to the successful mass production and commercialization of useful coniferous species such as German spruce and Teda pine, especially in forested countries such as Canada and New Zealand. Therefore, in the case of lily tree, it is possible to achieve the commercialization of mass production of clone seedlings by applying somatic embryogenesis technology. First, it is necessary to secure various embryonic tissue tissue lines capable of mass production of somatic embryos. Mass production of cloned seedlings through induction systems.

The prerequisite for applying this mass production system to the actual forest breeding is a very important step of inducing many embryonic tissue lines and successfully growing them. The reason is that it is very important to produce cloned seedlings with various genotypes.

Therefore, the effect of the present invention is to first determine the developmental stage of the seed embryo according to the timing of harvesting the immature seeds, and to provide an important information in determining the optimal sampling time when inducing embryogenic tissue of the lily tree by confirming histologically anatomical Embryonic tissues can be generated by high embryogenic tissue induction rate by confirming the optimal harvesting time of unripe seedlings, and somatic embryos can be induced, proliferated and plant produced from various embryogenic tissues in the future. It could contribute to the mass production of wood.

1 is a photograph showing the seed development according to the seed collection time after pollination of the lily tree.
Figure 2 is a graph showing the embryogenic tissue induction rate according to the seed collection time after pollination of the lily tree.
Figure 3 is a photograph showing the appearance of embryonic tissues induced from the lily immature seeds after pollination of the lilies.

The object of the present invention is a method of inducing embryogenic tissue of a lily tree.

The present invention comprises the steps of harvesting seed pears from pollen immature seeds after pollination; Inducing the embryogenic tissue of the lily tree by culturing the seed embryo of the collected lily tree in a medium; It relates to a method for growing embryonic tissue of a lily tree comprising the step of culturing the induced lily tree embryogenic tissue in the medium.

Since the seed pears of immature seeds of the lily tree appear clearly after 10 weeks after the pollination of the lilies, it is better to collect the seed pears from the immature seeds of the lily tree after 10 weeks (weeks). .

Since the seed embryos of the immature seeds of the lilies begin to develop rapidly after 6 weeks after pollination of the lilies, they are generated to the size necessary for induction of embryogenic tissue. Harvest seed pears from tree immature seeds.

Seed embryos harvested from the lily tree may be cultured in a medium to induce embryonic tissue of the lily tree.

Seed pears collected from the lily tree in the PEM medium, and after primary culture in 23 ~ 25 ℃, cow for 4 to 5 weeks (week), to obtain the endosperm except the seed of the seed, the endosperm in the PEM medium Embryos can be induced in 23 ~ 25 ℃, the second culture in the cow for 4 to 5 weeks (week).

The seed pears collected from the lily tree are put in PEM medium at 25 ° C., after primary culture for 4 weeks in cows, to obtain endosperm except seed seed, and then the embryos are placed in PEM medium at 25 ° C., Secondary cultures in the cow for 4 weeks can induce embryonic tissue of the lily tree.

Embryonic embryogenic tissue derived from the above cultured in PRO medium at 23-25 ° C. for 4-5 weeks (cow) can be grown in embryogenic tissue.

Embryonic embryogenic tissue induced above may be cultured in PRO medium at 25 ° C. for 4 weeks in the cow to propagate embryogenic tissue.

After cultivating the above-derived lily tree embryogenic tissue in PRO medium, the embryonic tissue can be grown after observing the growth level of embryogenic tissue, and then passaged at 2 weeks intervals in PRO medium.

In the PEM medium, 100 mg / L casein hydroiysate, 3.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.25 mg / L benzyl adenine BA), 40 g / L sucrose and 0.2% gelite.

The RPO medium is 250mg / L casamino acid, 250mg / L L-glutamine, 2.0mg / L 2,4-dichlorophenoxyacetic acid in 1 / 2LM basal medium (2, 4-D), 0.25 mg / L benzyl adenine (BA), 20 g / L sucrose (sucrose) and 0.4% gelite (gelite) is included.

When the seed pears are harvested from the pollen immature seeds after pollination, the immature seeds of the lilies may be sterilized. The sterilization step in this sterilization step is sufficient if it can be carried out using a process for sterilizing the surface of the conventional seed. As an example, put an immature seedling tree in a triangular fresco and shake it with Triton X-100, wash it thoroughly with tap water, and then (1) 70% ethanol in the aseptic culture pad. Immerse the yellow-poplar seed and shake it continuously for 3 minutes (2) Sterilize for 10 minutes by adding 2% NaClO solution with TritonX-100 (3) Sterilize for 5 minutes with 0.5% HgCl ₂ solution (4 Surface sterilization can be performed by washing 3 ~ 4 times with sterilized distilled water.

The method of inducing embryogenic tissue of the lily tree of the present invention was carried out under various conditions. In order to achieve the object of the present invention, it is preferable to provide a method of inducing embryonic tissue of the lily tree under the above-mentioned conditions.

The present invention includes the embryogenic tissue of the lily tree grown by the embryogenic tissue propagation method of the above-mentioned lily tree.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

<Experimental Example 1> Seed dissection anatomy according to the passage of time after pollination of lilies

1 is a photograph comparing the seed cutting dissection cutting the seeds as the seal elapsed after the water pollen. As shown in Fig. 1, the result of collecting the immature seeds of the lily tree did not form the endosperm in the seed from 3 weeks (May 27) to 7 weeks (June 17) after pollination and after 7 weeks (June 24) It can be seen that this is started.

After 7 weeks of pollination, the development of endosperm in the immature seedlings was rapidly developed, and a clear, mucus endosperm was observed, but after 11 weeks of pollination (July 22), white opaque endosperm was formed. . The emergence of an immature seedling embryo was not observed until 6 weeks (June 17) after a few minutes, but after 7 weeks. The morphology consisted of an embryonal head, hypocotyl and suspensor and was translucent. It is juicy mucous ecology.

Since then, it has continued to develop, and after 10 minutes (July 15), the seed pear has changed into opaque pale yellow color. Since then, it has continued to develop and seed until 12 weeks (July 29). It was confirmed that development continued.

In particular, until 10 weeks after pollination of the yellow-poplar tree, the yellow-poplar seed pear had an amorphous form containing a lot of water, but since then, the shape of the seed pear is gradually inclined. Therefore, it is appropriate to collect seed cultivation material from unripe seedlings after 10 weeks (July 15) after the pollination of the seedlings. It is considered to be.

<Experimental Example 2> Seed fidelity according to the sampling period

Based on the anatomical data on the development of seed embryos after pollination in Fig. 1, seed seeding was performed three times from July 20 to July 27, which is a time when the seed embryos appeared distinctly. As shown, the rate of fidelity varies widely.

In general, the rate of improvement varies from 4.7% to 14.4% depending on the harvesting date and individual subjects.

Comparison of Fidelity Rates of Lily Tree Seeds According to Seed Harvesting Times sign Seed Collection Date (Mon./Sun) Object Fidelity rate (%) 1-1 7.20 One 14.4 1-2 7.20 2 - 1-3 7.20 3 - 1-4 7.20 4 12.4 2-1 7.24 One 4.3 2-2 7.24 2 11.9 2-3 7.24 3 - 2-4 7.24 4 12.1 3-1 7.27 One 4.7 3-2 7.27 2 - 3-3 7.27 3 - 3-4 7.27 4 11.9

Experimental Example 3 Comparison of Embryonic Tissue Induction Ratio According to Sampling Time (After Minutes)

Figure 2 is a graph measuring the rate of induction of embryogenic tissue from seeds over time after pollination of lilies.

In 3 to 5 weeks after pollination, no embryonic embryos were induced, and embryonic tissues were not induced at 6 weeks (0.89%) after pollination. Since then, embryogenic tissues continued to induce and were high at 9 weeks (1.19%) after pollination, and the highest value was 2.78% at 11 weeks after pollination.

As shown in FIG. 2, the change in the rate of induction of embryonic tissue according to the harvesting time after pollination of the lily tree does not show a constant tendency, but the seed embryos develop rapidly from 6 weeks after the pollination of the lily tree, indicating that the embryonic tissue is formed to the size necessary for induction of the embryogenic tissue. Could.

Example 1 Induction and Growth of Lily Tree Embryonic Tissue

(1) sample collection

Immature seeds were first collected to induce embryonic tissue from the immature seeds of the yellow-poplar tree. The samples were collected in the middle of May ~ July 2004, in the Forest Resources Development Department of the National Forest Research Institute, Suwon, ② June 2006, Gangwon. The samples were collected from mature trees in the Chuncheon City Lily Tree Group. Dozens of seeds were harvested at each harvest time, with no distinction between individuals. The harvested seeds were refrigerated at 4 ° C. until use.

(2) sterilization of samples

In the conifer of one lily tree, a large number of immature seeds are united with each other and are thickly wrapped with pulp, so that they cannot be easily separated. First, they are separated into approximately one seed with a surgical knife (No. 21), and then surface sterilization is performed.

Seed sterilization was performed by first separating the seed into a triangular flask and shaking by adding a few drops of Triton X-100, then rinsing thoroughly with tap water, and then immersing the immature seeds treated above in 70% ethanol in a sterile culture pad. The solution was continuously shaken for 3 minutes, sterilized for 10 minutes by adding 2% NaClO solution added with TritonX-100, sterilized for 5 minutes with 0.5% HgCl ₂ solution, and washed 4 times with sterile distilled water to perform surface sterilization. It was.

(3) induction and proliferation

One lily seed was picked up with tweezers, stood vertically, and the seed was accurately bisected using a scalpel. Each nutrient seed was incubated for 1 week at 25 ° C. in the dark by placing the seed cut surface in contact with the medium on the PEM medium.

 After 4 weeks, the embryos except for the seed of each nutrient seed cultured were scraped off with a scalpel and placed on the PEM medium. Occurrence was observed.

After induction of light yellow embryonic tissues, if grown to a size of 0.5 cm or more, the original culture explants and embryogenic tissues were separated with tweezers and dispersed over PRO medium, followed by incubation for 4 weeks in 25 ° C and cows. The lily germ tissue was expanded.

Four weeks after the second culture of the embryonic seeds, the pale yellow embryonic tissue begins to be derived from the seed explants, and the tissue is composed of a mass of small globular somatic embryos. In addition, depending on the tissue line, the type and tissue properties (powder type or mucus) forming the tissues were different (see FIG. 3).

The composition for the PEM medium and PRO medium used for embryogenic tissue induction and proliferation is shown in Table 2 below.

Medium composition of each step of induction Medium component (mg / L) PEM Badge PRO badge Salts NH ₄ NO ₃ 1650.0 825.0 MgSO ₄ 180.54 90.27 CaCl ₂ 332.02 166.01 KH2PO ₄ 170.0 85 KNO ₃ 1900 950 FeNaEDTA 36.70 18.35 MnSO ₄ ? H ₂ O 16.9 8.45 ZnSO ₄ ? 7H ₂ O 8.6 4.3 KI 0.83 0.415 H ₃ HO ₃ 6.2 3.1 Na ₂ MoO ₄ ? 2H ₂ O 0.25 0.125 CoCl _2? 6H ₂ O 0.025 0.0125 CuSO ₄ ˜5H ₂ O 0.025 0.0125 Vitamines myo-inositol 100.0 100.0 Glycine 2.0 2.0 Nicotinic acid 0.50 0.50 Pyridoxine®HCl 0.10 0.10 Thiamine? HCl 0.10 0.10 additive Casein hydrolysate 1000.0 - Casamino acid - 250.0 L-glutamine - 250.0 2,4-D 3.0 2.0 BA 0.25 0.25 Sucrose 40,000.0 20,000.0 Gelrite 0.2% 0.4% pH 5.7 5.7

* "-" In Table 2 means not used.

<Example 2>

In Example 1, if grown to a size of 0.5 cm or more after induction of light yellow embryonic tissues, the original culture explants and embryogenic tissues were separated with tweezers and dispersed over PRO medium at 25 ° C. for 4 weeks in cows ( When the growth was observed after incubation for a week), the embryonic embryonic tissue was further proliferated and subcultured at intervals of 2 weeks in the PRO medium to obtain the lily tree embryogenic tissue.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. It will be understood that the present invention can be changed.

The present invention examines the degree of developmental stage of seed pear according to the time of harvesting the yellow-poplar seed, and histologically confirms it by providing important information for determining the optimal sampling time when inducing embryonic tissue of the yellow-lily. It is possible to generate embryonic tissues with high embryogenic tissue induction rate by confirming the optimum harvesting time of immature seeds, and to induce somatic embryos and plant production from embryonic tissues of various lines in the future. There is industrial applicability as it will be able to contribute.

Claims (6)

Harvesting seed pears from yellow-poplar immature seeds after pollination;
The seed pears of the above-obtained lilies were placed in 100 mg / L casein hydroiysate, 3.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.25 mg in an LM base medium. / L benzyl adenine (BA), 40g / L sucrose (sucrose) and 0.2% gelite (gelite) containing in the PEM medium and seeded after primary culture for 4 to 5 weeks (23 ~ 25 ℃, cow) Obtaining embryos except for the endothelial of the embryos and incubating the embryos in the PEM medium at 23-25 ° C. for 2 to 4 weeks in the cow to induce embryonic tissue of a lily tree;
The induced lily tree embryogenic tissue was placed in a 1 / 2LM medium for 250 mg / L casamino acid, 250 mg / L L-glutamine, 2.0 mg / L 2,4-dichlorophenoxy. 23 to 25 ° C in RPO medium containing acetic acid (2,4-D), 0.25 mg / L benzyl adenine (BA), 20 g / L sucrose and 0.4% gelite, 4 to 4 Embryonic tissue proliferation method comprising the step of culturing for 5 weeks (growth) embryogenic tissue. The method of claim 1,
A method of growing embryonic tissue of a lily tree, characterized by harvesting seed pears from an immature seed of the lily tree after 10 weeks of pollination. The method of claim 1,
A method of growing embryonic tissue of a lily tree, characterized by harvesting seed pears from an immature seed of a lily tree 6 weeks after pollination. delete delete 5. Embryonic tissue of a lily tree grown by the method of any one of claims 1 to 3.

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