KR20190025236A - Method of embryogenic tissue induction from immature zygotic embryo in Japanese larch - Google Patents

Abstract

The present invention relates to an induction method of an embryogenic tissue from an immature embryo in a Larix kaempferi seed, which can maximize various lines of the embryogenic tissues. The induction method comprises the steps of: isolating and surface-sterilizing an immature seed contained in squama of immature strobilus of collected Larix kaempferi; removing testa of the surface-sterilized immature seed, cutting the testa-removed immature seed in longitudinal section and bisecting the same; and culturing and denticulating the bisected section to be in contact with embryogenic tissue-inducing media.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inducing embryogenic tissue from an immature embryo,

The present invention relates to a method for inducing embryogenic tissue from a seedless immature embryo of a Japanese brownwood, and more particularly, to a method for isolating and sterilizing an immature seed contained in a human scrotum A step of cutting off the seeds of the sterilized immature seeds and then cutting the seeds of the immature seeds from which the seeds have been removed into a longitudinal section and bisecting the seeds; and culturing the divided sections so as to contact the embryogenic tissue- The present invention relates to a method for inducing embryogenic tissue from an immature embryo of a seed tree.

Japanese larch ( Larix kaempferi ) is a deciduous needle tree. In Korea, seeds were introduced from Japan for the first time in 1904 and planted in various parts of the country. Currently, it is one of the main planting species and is currently being planted in a large area.

The characteristics of Japanese mulberry trees are juvenile growth, and when grown for 30 years, it is 259 m ³ / ha The production of seedlings of Japanese larch (larch) largely depends on the production of seedlings, but the production of seeds is very different in 5 ~ 6 year cycle. It is impossible to produce regular seeds every year.

As an alternative, there is a method of cutting, but this also shows a large difference in the rooting rate depending on the degree of tissue maturation of the cutting, and the produced seedlings have a disadvantage that they show a slanting growth for several years. In addition, because of the diverse genetic characteristics of the various parameters, there is a large difference in the rooting rate between the clones, and it is not possible to continuously produce a large number of rootstocks.

In order to overcome these disadvantages, mass propagation of high quality individuals through tissue culture technology has been used, among which plant propagation using somatic embryo induction method is the most widely used method in advanced forestry countries (Dong JZ, Dunstan DI 2000. Molecular 20, Tree Physiol., 20: 20-21, 1981. In particular, it has been found that, in the case of the somatic embryogenesis, 921-928).

In addition, biochemical techniques have been applied to the development of new varieties by inserting genes resistant to pests, diseases, and environmental stresses into cells using somatic embryos as samples. However, despite the breakthrough technology that can induce somatic embryogenesis and germinate into whole plants, there are still many problems to be solved. Among them, the most difficult thing is to induce embryogenic tissue which is the material from immature material.

The extent of the embryogenic tissue induction is known to be largely dependent on the degree of zygotic embryogenesis in the immature seed and the genotype of the embryo, among which the developmental stage of the zygotic embryo is the most influential factor (Becwar , 1990, Can. J. For Res. 20: 810-817), and many research reports focus on this.

The study of somatic embryos of conifer species was done by Hakman et al., 1985, J. Plant Physiol. 121: 149-158)Picea abies), The somatic embryo was first induced from the immature seed embryo,LarixThe somatic embryo induction of the genus was performed by Nagmani and Bonga (Can. J. For. Res. 15: 1088-1091 (1985)) European larch (L. decidua) Were induced from the female gametophyte tissue for the first time.

In addition, L. laricina (Klimaszewska et al., 1997, Can. J. For Res. 27: 538-550), L. x leptoeuropaea (.. Lelu etc., 1994, Plant Cell Tiss Org Cult 36:. 117-127), L. occidentalis (Thompson and von Aderkas, 1992, Plant Cell Rep. 11: 379-385).

In the case of Japanese brownwood, the somatic embryo development from immature embryo (Kim et al., 1999, Plant Cell Tiss. Org. Cult. 55: 95-101) , Plant Cell Tiss. Org. Cult. 88: 241-245), and the effect of addition of antioxins such as TIBA (Yong-Wook Kim, Hong-Gyu Hong, 2009, Line effects (Kim, Yong-Wook, 2010, Korean Journal of Aging 99: 633-637).

However, since only one embryogenic tissue line is used for inducing somatic embryo transfer as described above, there is no genetic diversity of the clone seedlings produced therefrom.

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems of the prior art, and an object of the present invention is to provide a method of producing microbial seeds, And the correlation between the embryogenic tissue induction rate according to the culturing technique of the zygotic embryos was clarified and it was confirmed that the seedless immature embryo of Japanese brownwood, To provide a method for inducing embryogenic tissue.

(A) isolating and sterilizing the seeds immersed in the scallops of the germinated Japanese mulberry leaves, (b) sterilizing the surface seeds of the surface sterilized seeds, A step of cutting the immature seed from which the seed coat has been removed and cutting the seed into a longitudinal section and then bisecting the seed; and (c) cultivating the endogenous seed to contact the embryogenic tissue- Provides a method of inducing embryogenic tissue from a stomach.

According to the induction method of the embryogenic tissue from the seedless immature embryo of the Japanese Lepticromaster of the present invention, it is possible to induce a embryogenic tissue of a high induction ratio according to the step of embedding the embryo and the method of culturing the embryo, Somatic embryo induction and plant production are also possible from the developing tissues, and new variety breeding of various Japanese brownwood trees becomes ultimately possible.

In addition, advanced biotechnology techniques such as genetic engineering can be applied to forestry, and it can contribute greatly to the forest resources such as the improvement of the productivity of the forest through the breeding of the forest species.

Fig. 1 shows the effect of embryogenic tissue induction rate on the seedless immature embryo harvesting time of Japanese brownwood.
Fig. 2 shows the effect of embryogenic tissue induction rate according to the technique of seedless immature embryo culture of Japanese brownwood.
Fig. 3 is a photograph showing an embryogenic tissue occurring from a seedless immature embryo of a Japanese brownwood.

(A) isolating and sterilizing the seeds immersed in the scallops of the collected immature seeds of Japanese japonicus, (b) removing the seeds of the surface sterilized seeds, A step of cutting the seed to a longitudinal section and bisecting the seed; and (c) culturing the seed to contact the embryogenic tissue induction medium for cultivation. .

The success of the development of clone seedling propagation technology through the somatic embryo induction technique of Japanese myrtle tree depends on the success or failure of inducing embryogenic tissue which is the original material of somatic embryo induction. This is because the more numerous embryogenic tissues with different genotypes, the more clonal seedlings can be produced from them.

Among the conifer species, Picea species are reported to induce 60% or more of embryogenic tissues, and even induce embryogenic tissues from mature seeds stored in refrigerated storage. On the other hand, in Pinus species, the induction rate of embryogenic tissue is generally low, and the induction rate is remarkably different according to the characteristics of these species.

Although various plant mediums and growth regulators have been added to improve the induction rate of low embryonic tissues, the most important factor is the development stage of zygotic embryo This is largely dependent on the fact that the embryogenic tissue of the conifers begins with cell division from the zygote or suspensor attached to the zygotic embryo, at which time the zygote embryo enters the maturation stage and, from then on, Because it is hard to expect.

In the method for inducing embryogenic tissue from the seedless immature embryo of the Japanese Lepticromaster of the present invention, the harvesting time of the immature embryo may be the first week of June or the second week of June.

In the method for inducing embryogenic tissue from the seedless immature embryo of the Japanese Lepticromaster of the present invention, the harvesting time of the immature embryo may be from June 3 to June 9, preferably June 9.

In the method for inducing embryogenic tissue from the seedless immature embryo of the Japanese Lepticromaster of the present invention, the seed embryo of the immature seed may be a proembryo or a globular embryo.

In the method for inducing embryogenic tissue from the seedless immature embryo of the present invention, the harvesting time of the immature seed is in the first week in June when the seeds of the immature seed are in the centrifuge, , And the time of collection of the immature oocyte is the second week of June.

In the method for inducing embryogenic tissue from the seedless immature embryo of the present invention, the surface of the immature seed is washed with ethanol for 0.5 to 5 minutes, preferably 1 to 2 Minute, and sterilized in a sodium hypochlorite solution for 1 to 5 minutes, preferably 2 to 3 minutes.

In the induction method of the embryogenic tissue from the seedless immature embryo of the present invention, the embryogenic tissue induction medium is selected from the group consisting of L-glutamine 1000 mg / L, sucrose 30 g / L , 2.0 mg / L of 2,4-dichlorophenoxyacetic acid, 1.0 mg / L of 6-benzylaminopurine and 0.2% (w / v) of gellan gum.

In the method for inducing embryogenic tissue from the seedless immature embryo of the present invention, the culture is cultivated for 7 to 9 weeks, preferably 8 weeks, at 24 to 26 캜, preferably at 25 캜, Lt; / RTI >

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited to these examples.

< Example 1> Plants  Collection of materials

For the extraction of the immature oocyte, it was collected at the National Forest Cultivation Center (Chungbuk, Chungbuk, Korea) from June 3 to 30 at Jaejongwon, Korea. The immature oocytes were refrigerated at 4 ° C until they were cultured.

< Example 2> Culture  Sterilization of plant materials for

For the preparation of samples for induction of embryogenesis, first, the immature seeds contained in the scallop pieces of the immature saliva were separated using a surgical knife (No. 21, Feather), and then immersed in 70% ethanol for 1 minute , Surface sterilized in 2% NaClO solution with a few drops of Triton X-100 for 2.5 minutes, and then washed several times with sterilized distilled water.

Samples for investigation of developmental stages of seed embryos were prepared by immersing immature seeds in a collection fixative, cutting them into fine flakes and observing them under a microscope.

< Example 3> Embryogenesis  Preparation and preparation of medium for induction of tissue

The culture medium for inducing embryogenic tissue from the immature embryos was the P6 (Teasdale et al. 1986) medium of Table 1 as the basic medium. L-glutamine (Sigma), 30 g / L sucrose, 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg / L 6-benzyladenine (BA) was added.

The pH of the medium was adjusted to 5.7 and 0.2% (w / v) gellan gum (Phytagel, Sigma) was added to the solid medium.

After heat sterilization of the medium, L-glutamine was separately added when the temperature of the medium was cooled to about 50 ° C., and the medium was dispensed into a plastic Petri dish (SPL) of 87 × 15 mm in an amount of about 15 ml, For one night.

< Example 4> Embryogenesis  Induction and proliferation of tissue

Prior to culturing the immature seeds, the seeds of the sterilized seeds were first removed, and then impregnated in parallel with the surface of the medium. The seeds were cultured for 8 weeks at 25 ° C. under a culture condition of the dark without changing the culture medium.

The proliferation of the induced embryogenic tissues was examined in a medium supplemented with 20 g / L sucrose, 2.0 mg / L 2,4-D, 1.0 mg / L BA, 1,000 mg / L L-glutamine and 0.4% The cells were grown every 2 weeks with subculture.

< Example  5> Depending on the harvesting time of immature seeds Zygotic sperm  Identification of developmental stages

In order to confirm the developmental stage of the zygotic embryo according to the harvesting time of the immature seeds, the immature seeds in the bract were separated from the unripe embryo using a dissection knife (No. 21, Feather).

After that, the seeds were held with tweezers and cut using the No. 11 dissection knife to bisect exactly half the length of the seeds. The cleaved sites were placed under a dissecting microscope (Nikon) and the degree of development of the zygotic embryos in the seeds at each sampling time was examined. The number of irradiations was observed to about 20 per harvesting period.

< Example  6> Fine In the micro-section  by Zygotic  Identification of developmental stages

First, a preprocessing process is required. Ten to fifteen seeds were removed from each seed stage and stored in phosphate buffer solution (0.05M, pH 6.8) supplemented with glutaraldehyde (1.5%) and paraformaldehyde (1.6% It was immersed in the refrigerator for about 3 months.

The dehydration process consisted of an ethanol series of different concentrations, and when dehydration was complete, it was permeated into the tissues with the Historesin (Technovit 7100, Kluzer, Germany). The microtomes were cut into 3-μm-thick tissue sections with tungsten-carbide microtomes and stained with Periodic acid-Schiff's (PAS) (0.1%) and Toluidine blue O (0.05% Photographs were taken.

< Experimental Example  1> Seeds Immature  By collection time Embryogenesis  Tissue induction rate

Fig. 1 shows the results of correlation observation of embryogenic tissue induction rate by immature seed harvesting time at the interval of one week from June 3 to June 30, 2017.

As shown in Fig. 1, the maximum induction rate of the embryonic tissue, 10.9%, was observed in the immature seeds collected on June 9, and then dropped to 2.6% in the samples on June 19, It can be seen that induction of embryogenic tissue was not performed at all.

The development of seed embryos according to harvesting time was confirmed only in proembryo which was the earliest type in the samples of June 3 (7.2% of embryogenic tissue induction rate), and in the samples of June 9 (10.9% (Globular embryo) stage zygotes were observed only.

Subsequently, on June 19 (2.6%), the electronic cottage (precotyledonary embryo) was observed, and only the cotyledonary embryo was observed in the subsequent period, and after June 23 It can be seen that the development of the seed embryo is almost complete.

Therefore, the most suitable seeding period for inducing embryogenic tissue of Japanese brownwood was June 9, and the stage of development at that time proved to be most appropriate for the stage of development.

< Example 7> Seeds Immature  Depending on the culture technique Embryogenesis  Tissue induction rate

In order to obtain the maximum embryogenic tissue induction rate, four kinds of culturing methods were performed as shown in FIG.

The method a in Fig. 2 is a method in which only the seed coat is removed and then cultured in an equilibrium state on the medium. In method b, the seeds are sieved on both sides of the seeds peeled off the seed coat and then immersed in a P6 liquid medium for 24 hours and then cultured in a solid medium The method c) is a method in which the cotyledon of the seed is cut to about 30%, the seed is dipped in a P6 liquid medium for 24 hours and then cultured in a solid medium. Finally, the d method is a method in which the seed is removed And then the cut surface is brought into contact with the medium.

As a result of the above four methods of a to d, the highest tissue induction rate was 0.56% when cultured with the d method, whereas none of the other three methods showed induction of embryogenic tissue at all.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. The scope of the invention is defined by the appended claims. It will be apparent that many modifications are possible by those skilled in the art.

According to the induction method of the embryogenic tissue from the seedless immature embryo of the Japanese Lepticromaster of the present invention, it is possible to induce a embryogenic tissue of a high induction ratio according to the step of embedding the embryo and the method of culturing the embryo, Somatic embryo induction and plant production are also possible from the developing tissues, and new variety breeding of various Japanese brownwood can be ultimately made possible, so that the present invention can be effectively applied to the technical field to which the present invention belongs.

Claims (7)

(a). Isolating and sterilizing the immature seeds contained in the scallops of the Japanese myrtle tree;
(b). Cutting off the seeds of the surface-sterilized seeds after cutting off the seeds of the seeds, removing the seeds from the seeds, And
(c). And culturing the two divided sections so as to contact the embryogenic tissue-inducing medium. The method of inducing embryogenic tissue from a seedless immature embryo of a Japanese brownwood. [Claim 2] The method according to claim 1, wherein the harvesting time of the immature oocyte is the first week of June to the second week of June. The method according to claim 1, wherein the harvesting time of the immature oocyte is from June 3 to June 9. The method according to claim 1, wherein the seeds of the immature seeds are proembryo or globular embryos. 2. The method according to claim 1, wherein the seeds of the immature seeds are proembryo or globular embryos. The method according to claim 1, wherein the surface sterilization is performed by washing the surface of the separated immature seed, then immersing in ethanol for 0.5 to 5 minutes, and then sterilizing in a sodium hypochlorite solution for 1 to 5 minutes. Induction method of embryogenic tissue from seedless immature embryo of. The embryogenic tissue induction medium according to claim 1, wherein the embryogenic tissue induction medium is selected from the group consisting of L-glutamine 1000 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid 2.0 mg / A method for inducing embryogenic tissue from a seedless immature embryo of Japanese Leptocaroni, characterized in that 6-benzylaminopurine 1.0 mg / L and 0.2% (w / v) gellan gum are added.
[Table 1]

The method according to claim 1, wherein the culturing is carried out for 7 to 9 weeks under culturing conditions of 24 to 26 占 폚 and darkness.

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