KR100838699B1 - Technique of Cryopreservation Using Somatic Embryo in Japanese Larch Larix leptolepis and Induction of Embryogenic Culture - Google Patents

Abstract

The present invention relates to a cryogenic storage technology using larch somatic embryos. More specifically, the induced somatic embryos are dehydrated and stored and thawed in liquid nitrogen, and the thawed somatic cells are absorbed by distilled water, and the embryos are incubated in embryogenic tissue-derived media to reinduce embryonic tissues. It is composed.

The present invention is a cryogenic storage technology that is simple to pretreatment than the conventional liquid embryonic cell method, and can be stored for a long time in liquid nitrogen even without expensive cell freezing. In addition, since cryogenic storage does not use DMSO (dimethylsulphoxide), the somatic embryo mutation is relatively less likely to occur. And by utilizing the cryogenic storage technology using the larch somatic embryo of the present invention, it is possible to apply and apply biotechnology such as somatic embryo through the cryogenic storage of valuable germplasm of coniferous species in the future. It is expected to be possible.

Larch, Somatic Embryos, Cryogenic Storage, Embryonic Tissue Induction

Description

Technology for Cryopreservation Using Somatic Embryo in Japanese Larch (Larix leptolepis) and Induction of Embryogenic Culture}

1 is a graph showing the effect of embryogenic tissue induction according to the medium concentration and rehydration treatment period.

Figure 2 is a graph showing the effect of embryogenic tissue induction according to the medium concentration and somatic embryo type

Figure 3 is a graph showing the effect of embryogenic tissue induction according to the type of Solute

4 is a graph showing the effect of embryogenic tissue induction according to dehydration temperature and duration.

5 is a graph showing the effect of induction of embryonic tissue according to storage temperature and storage period.

Figure 6 is a comparative photograph illustrating the technical difference between the cryogenic storage method using the existing embryonic development cells and somatic embryos.

(a: using existing embryogenic cells, b: ultra-low temperature storage using somatic embryos)

Figure 7 is a photograph of larch somatic embryos to be used in the present invention in one place

FIG. 8 is a photograph of the somatic embryo in 24-well for dehydration of the somatic embryo.

Figure 9 is a photograph stored in the liquid nitrogen tank for cryogenic storage of somatic embryos

10 is a photograph of rapid thawing of frozen somatic embryos in lukewarm water at about 35 ° C. after cryogenic storage

FIG. 11 is a photograph of a thawed somatic embryo on a paper filter moistened with sterile distilled water for rehydration.

FIG. 12 is a photograph showing regeneration of embryonic tissue in a medium to which 2,4-D (2,4-Dichlorophenoxyacetic acid) and BA (Benzyladenine) are added from somatic embryos derived from cryogenic storage.

FIG. 13 is a close-up photograph of the developing embryonic tissue of FIG. 12. FIG.

The present invention relates to a cryogenic storage technology using larch somatic embryos. More specifically, dehydrated somatic embryos are stored and thawed in liquid nitrogen and absorbed distilled water into thawed somatic cells to induce embryonic tissues by incubating them in embryonic tissue-derived medium. do.

When a tree derived from somatic embryos is found to be a plant with excellent traits by field test, etc., when mass growth is needed, the tissues of the target parameters are already mature and no longer exist by conventional growth methods such as grafting and cutting. It cannot be easily propagated. Therefore, long-term storage of vital cells without any loss of vitality until the end of the transfection is essential by storing cryogenic embryonic cells of the target parameters in liquid nitrogen at the same time as the field assay (Chen and Kartha 1987; Gupta et al. 1987). At this time, the most commonly used material for cryogenic storage is embryogenic cells or tissues. Somatic embryo development and somatic embryo induction technology research from these cells is currently attracting much attention. Therefore, in recent years coniferous species have been able to cryogenic storage using embryogenic tissue, and clone propagation (Charest and Klimaszewska 1995a, b) has become possible.

Gupta et al. (1987) succeeded in regenerating the embryogenic cells of Picea abies and Pinus taeda into vigorous tissues after cryogenic storage in liquid nitrogen at -196 ° C for 10 minutes.

Kartha et al. (1988) reported that Picea glauca embryonic embryonic cells were cryogenically stored in liquid nitrogen for one year and then regenerated to report somatic embryo induction and plant regeneration from them, thus confirming that long-term storage of embryogenic tissues is possible through cryogenic storage. It was.

Recently, Bercetche et al. (1990) have regenerated the embryonic cells of Picea abies into tissues after cryogenic storage for about 3 months, and incubated in medium supplemented with ABA (Abscisic acid) in a faster time than the cells without cryogenic storage. It was possible to induce somatic embryos, which reported that non-embryogenic cells mixed with embryogenic cells in cryogenic storage were killed, and only embryonic cells rich in cytoplasm survived, so that only pure embryonic tissues were propagated and cryogenic storage was effective for inducing somatic embryos. have.

Galerne et al. (1992) report that Picea glauca promoted the induction of more somatic embryos after the cryogenic storage of embryonic cells, which is caused by the selective culling of non-embryogenic cells during cryogenic storage. Therefore, the cryogenic storage of embryonic cells is a storage method made in liquid nitrogen, which is much labor-saving and stable than low-temperature storage methods such as 4 ℃, and thus is widely used as a long-term preservation technology of germplasm. Cell viability after cryogenic storage has also been reported to be high, with 97% of 357 genotypes survived without somatic inheritance in Picea glauca engelmannii species (Cyr et al. 1994), and Park et al. (1994) from 551 clones of Picea glauca. About 83% of the induced embryogenic tissues reported cell viability after cryogenic storage. It is reported that the cell viability varies according to genotype.

The cryogenic storage method using the liquid nitrogen of germplasm or embryogenic cells of the conifer species mentioned above is to first dehydrate the embryonic cells with the cryogenic preservative solution and then slowly dehydrate the cells to -40 ℃ with a freezing rate of 0.33 ℃ / min. Immediately after freezing, it is placed into liquid nitrogen and preserved. This method is very toxic to DMSO (dimethyl sulphoxide), which is added as cryopreservative during cryogenic storage, and is highly likely to cause somatic lesions after recovery from liquid nitrogen preservation to room temperature. There is a disadvantage of requiring a freezing device. And while the cryogenically stored cells are thawed in hot water, hot water flows in from the outside of the storage tube due to the pressure difference in the outside of the storage tube, and all the cells in the tube are highly contaminated. On the other hand, the present invention relates to the development of technology for preserving liquid nitrogen directly into a cryogenic storage tube after dehydration using somatic embryos instead of liquid embryonic cells.

The feature of this technology is that the pretreatment process is simpler than the above-mentioned method using liquid embryogenic cells, and does not require expensive equipment such as a freezer for freezing up to -40 ° C and does not require the addition of DMSO during cryogenic storage. As a result, somatic embryo mutations are much less likely. However, in order to utilize this technology, the development of technology capable of inducing embryonic tissues from somatic embryos after cryogenic storage remains a prerequisite problem. Therefore, the present invention includes the development of technology capable of inducing embryonic tissue from somatic embryos.

Cryogenic storage using somatic embryos has been reported in a few hardwood species (Bajai 1995). However, coniferous species use embryogenic cells (Ford et al. 2000; Touchell et al. 2002). On the other hand, cryogenic storage using dehydrated somatic embryos was able to preserve dehydrated (or dried) somatic embryos of dehydrated (or dried) cells at room temperature for 1 year without any loss of vitality (Senaratna et al. 1990). Pears are also reported to be susceptible to cold storage for 8 months at 4 ° C after sucrose treatment and relative humidity of 45% (Lecouteux et al. 1991). In coniferous species, Attree et al. (1995) reported high re-differentiation rate (76-84%) after 1 year of cold storage at -20 ° C after dehydration of somatic embryos of Picea glauca. It is not the result of cryogenic storage. However, Bomal and Tremblay (2000) report the possibility of plant regeneration and embryogenic tissue reinduction after cryogenic storage of two kinds of Picea species (P. mariana, P. glauca) in liquid nitrogen. It can be said that it laid the foundation for the cryogenic storage research.

On the other hand, as a conventional technology related to the present invention, cryogenic storage technology for long-term storage of plant germplasm in -196 ℃ liquid nitrogen is the safest and cost-saving storage technology from bacterial contamination and DNA mutations. In such cryogenic storage, all cell division and metabolic processes are stopped, so cryogenically stored cells can be stored indefinitely without any modification or change, and can be stored in very small amounts (volumes). It is a very efficient long term storage technology that is free and requires minimal maintenance.

In the case of coniferous species, the development of a protocol for cryogenic storage of cell germplasm, which is a material for producing genetically engineered plants or trees required for large-scale afforestation, and the use of this technology are emphasized. Cryogenic storage is particularly important for forestry sarcoma applications using biotechnology such as somatic embryo induction. In particular, embryogenic tissues used to induce somatic embryos are known to be well maintained compared to other tissues in a stable state without DNA mutations for many years, but the growth or somatic cells of embryonic tissues are frequently generated by frequent subculturing for several months to several years. It is also very likely that the ship's induction capacity will slow down or be completely lost. Therefore, there is a particular need for a technique capable of safely and long-term storage of embryogenic tissue from such an adverse environment. This storage technology can also be used for tissue selection, such as cryo-selection, which involves the removal of non-embryogenic tissues or unwanted tissues or cells mixed in embryogenic tissues in liquid nitrogen. As a result, more pure embryogenic tissue can be selected. Therefore, it can be applied to re-ghosting embryogenic tissue (Engelman, 2004).

Ultra-low temperature storage technology using embryogenic tissue of coniferous species was first developed by Kartha et al. (1988) and is still widely used in the coniferous species of the genus Picea or Pinus. Most of the cryogenic storage technologies currently used include processes such as dehydration of materials to be stored, cell freezing up to -40 ° C, storage in liquid nitrogen, and thawing of frozen plant cells. At present, such coniferous species that are capable of cryogenic storage include P. taeda (Gupta et al. 1987), P. caribaea (Laine et al. 1992), P. radiata (Hargreaves and Smith 1992; Hargreavesb et al. 2002), P. pinaster (Bercetche and Paques 1995), P. sylvestris (Haggman et al., 1998), P. patula (Ford et al. 2000) and P. roxburghii (Mathur et al. 2003).

To date, very few studies have reported on cryogenic storage using somatic embryos of coniferous species, and only one report by Kim et al. (1999) on cryogenic storage using embryogenic tissue of larch, Studies on dedifferentiation and cryogenic storage into embryonic tissues using somatic embryos have not yet been conducted.

Cryogenic storage technology can be divided into two types, classic and new. First, the classical method includes dehydration process, while the new technology includes vitrification of materials. This term refers to the transition from a liquid state to an amorphous or glass state. This process prevents crystalline ice formation and prevents damage or death of organelles from freezing damage caused by cryogenic temperatures. give. In addition, because it freezes the ice crystallization process (because no freezing device is used), the process is simpler and more widely applicable.

The success of cryogenic storage with liquid nitrogen is in the pretreatment of the plant material to be stored, (1) encapsulation-dehydration method, (2) vitrification, (3) encapsulation-vitrification, (4) Dehydration, (5) pre-growth, (6) pre-growth-dehydration, and (7) droplet freezing. However, no matter what method is applied, the key is to obtain a high tissue survival rate when the liquid nitrogen is returned to room temperature after storage. Therefore, in the present invention, the dehydration treatment method was selected among various pretreatments, and the optimal condition related thereto was finally revealed. Finally, after the dehydration process of somatic embryos, the contents of embryonic tissue reinduction effect according to the storage time of the liquid nitrogen are described. It is the main gist of the invention.

The object of the present invention is to dehydrate the somatic embryos of larch larch instead of the embryonic cells, which are widely used in the prior art, and then, after cryogenic storage using liquid nitrogen, the somatic embryos are removed from the liquid nitrogen and rapidly thawed, and then the new embryonic tissues are re-induced. It is to enable the acquisition of rejuvenated organizations. In addition, it is economical because it can be stored for a long time in liquid nitrogen without an expensive freezer, and is simpler than the cryogenic storage method of embryonic cells using conventional cell freezers, and does not require the addition of a poisonous cryogenic protective agent such as DMSO added during storage. The chance of DNA mutations in recovered embryogenic cells is very low.

The present invention clarifies the optimal dehydration conditions of somatic embryos, which are pretreatment for cryogenic storage, and also stores embryogenic tissue after 4 hours, -20 ℃ and -80 ℃ including storage to -196 ℃ liquid nitrogen. By re-differentiation, the practical feasibility of cryogenic and cryogenic storage was confirmed. Therefore, the technical problem to be solved by the present invention is ① embryogenic tissue induction effect according to the medium salt concentration and rehydration treatment period, ② embryogenic tissue induction effect according to the type of medium and somatic embryo, ③ Embryonic tissue induction effect according to the type of dehydration solvent (solute), ④ Embryonic tissue induction effect according to dehydration temperature and treatment time, ⑤ Storage temperature (4, -20, -80 and -196 ℃) The purpose of this study is to identify the effect of inducing tissues.

The present invention includes the steps of dehydrating the induced somatic embryos, storing and thawing at an extremely low temperature, and rehydrating the thawed somatic cells to induce embryonic tissues by incubating the embryonic tissues in the induced medium to induce embryonic tissues. It shows the cryogenic storage technology and embryogenic tissue induction method using larch somatic embryo.

The induction of somatic embryos is derived from the growth medium to recover the embryonic tissue, put the liquid medium and pulverize the cell suspension obtained by filter vacuum culture method to separate the cells on the somatic embryo induction medium and 23 ~ 26 ℃, in the cow It can be obtained by incubating for about 6-8 weeks.

In the dehydration treatment of the somatic embryo, the recovered somatic embryo can be treated for 1 to 7 days in the dark at 4 to 25 ° C.

The dehydrated somatic embryo was placed in a cryogenic preservation tube, stored in a liquid nitrogen tank for 1 week, then taken out and thawed for 5 to 15 minutes in hot water at 30 to 40 ° C.

The rehydrated somatic cells were thawed for 12 to 36 hours on a filter in which somatic embryos were absorbed in distilled water, and then sufficiently rehydrated. Can be cultured.

When the derived somatic embryos are dehydrated and stored at very low temperatures, cold storage can be stored in -20 ° C. to -196 ° C. in a nitrogen container.

The present invention can be broadly divided into two parts; first; Induction of embryogenic tissue according to medium type, rehydration, somatic embryo type, dehydration solvent type, dehydration temperature and treatment time using induced somatic embryos; It consists of embryonic tissue induction stage according to storage temperature (4, -20, -80 and -196 ℃) and storage period.

Hereinafter, the specific configuration and operation of the present invention have been described through specific experimental examples and examples. However, the technical configuration and the scope of rights related to ultra low temperature storage of larch species similar to the disclosed materials of the present invention are limited only to the embodiments described below. no.

Example 1 Dehydration and Somatic Regeneration of Somatic Embryos

① Somatic Embryogenesis

Somatic embryo induction of larch used in the present invention was carried out in the growth medium as follows. Recovered embryonic tissues were collected and put into a 200 mL Erlenmeyer flask at a rate of 35 mg per mL of liquid medium and the corresponding liquid medium was added. It was. Then, the fine tissue suspension was prepared by crushing the tissue finely by repeating suction and discharge with a disposable sterile plastic pipette. Somatic embryo induction was performed using a filter vacuum culture method. First, a Buchner funnel was firmly fixed on the top of a 1 L vacuum eggplant flask to prevent air leakage, and a 5.5 cm paper filter (Whatman No. 2) was placed in the center of the funnel and 3.25 mL ( Extract the cell suspension (including 95 mg total tissue) and spread it evenly over the paper filter. After that, the vacuum pump was operated to create a vacuum state for 4 to 5 seconds, and the liquid in the paper filter was drained down through the funnel, leaving only the cells on the filter, and the filter was soaked on the somatic embryo induction medium. Embryo induction began. The culture environment was incubated for about 7 weeks in cows at 24 ° C., and no subculture was performed with fresh medium.

② Dehydration of somatic embryos

To recover the induced somatic embryos, first pick them out with tweezers (FIG. 7). Dehydration of somatic embryos was carried out using a multi-well plate of 24 wells, each of which puts about 6-7 somatic embryos, floats one space, and adds 1.0 mL of distilled water (Figure 8). It is then sealed with a wrap film and dehydrated at 4 or 24 ° C for 1-7 days.

Example 2 Storage with Liquid Nitrogen, Rapid Thawing and Reduction of Embryonic Tissue

① Storage as liquid nitrogen

Carefully pick up the dehydrated somatic embryos with tweezers and place 5-6 in each cryogenic preservation tube and close the lid tightly. It is then inserted into the tube support and immediately into the liquid nitrogen tank (Figure 9).

② Recovery and rapid thawing from liquid nitrogen

Recovery from the liquid nitrogen immediately removes the cryogenic preservation tube stored in the liquid nitrogen and inserts it into hot water at about 35 ° C. and rapidly thaws for 10 minutes. Rapid thawing inhibits the formation of crystalline ice, which can occur during thawing, thereby increasing the survival rate of somatic embryos.

③ Somatic Embryogenesis Rehydration and Embryonic Tissue Induction

The thawed somatic embryo has a very low moisture content because the cryogenic storage is carried out through dehydration. Therefore, it is necessary to examine the effects of increasing the water content of somatic embryos on the induction of embryonic tissues. First, the somatic embryo was removed from the tube and placed on a paper filter soaked in distilled water and placed in a cow for 24 hours to allow sufficient water absorption (Fig. 11). The rehydrated somatic embryos are then densified in embryogenic tissue-derived media and cultured in cows at 23-25 ° C.

<Example 3> Embryonic tissue induction effect according to the type of medium and rehydration treatment period

In this experiment, the somatic embryos were separated from the somatic embryo induction medium, and then transferred to the induction medium to which 2,4-D and BA plant hormones were added. The result of rehydration according to (0-7 days) was compared to the effect of embryogenic tissue induction. As shown in Fig. 1, embryogenic tissue induction showed the highest 88.2% in the control group that was not rehydrated, but the induction rate of embryogenic tissue tended to decrease with longer rehydration period, and 22.2% on the 7th day of rehydration treatment. Was the lowest. Somatic embryos induced at high sucrose and gelrite concentrations had low water content in tissues, but suggest that rehydration is not required for reinduction as embryogenic tissues. In comparison with the salt concentration of culture media used for induction of embryonic tissue, all treatments except for 7 days of rehydration showed higher induction rate in LMDB media that did not reduce salt concentration in half compared to 1 / 2LMDB. The medium was shown to be more effective (FIG. 1).

<Example 4> Embryonic tissue induction effect according to the media salt concentration and somatic embryo type

  This study was conducted to investigate the effects of embryonic tissue induction according to the media salt concentration and the type of hematopoietic embryos (normal and abnormal). The maximum embryogenic tissue induction treatments were applied to the full-strength LM medium. 61.4% when hemorrhaged (Fig. 2). In addition, abnormal growth of somatic embryos in full-strength LM medium was 61.7%, indicating that embryogenic tissue induction was more affected by salinity of hemorrhagic media than in somatic embryos. On the other hand, the lowest induction rate was obtained by culturing abnormal (25%) somatic embryos in 1 / 2LM medium.

<Example 5> Embryonic tissue induction effect according to the type of dehydrated solvent (solute)

Dehydration of somatic embryos has been shown to be associated with the growth of broad-leaved trees, conifers, and plant development. In particular, this treatment allows long-term storage of several somatic embryos, including black spruce, in a manner that stops metabolism of cells. It has also been used as a means (Bomal and Tremblay 2000; Shiota et al. 1999). And dehydration rate during dehydration process plays an important role in triggering the mechanisms that plant organs can withstand dehydration treatment, but rapid dehydration treatment can modify or damage plant cells, especially membrane components and structures. The method for dehydration of In particular, the most important factor that enables long-term storage of somatic embryos in liquid nitrogen is to reduce the water content in the somatic embryos to a certain level. If the ultra low temperature is carried out with high moisture content in the somatic embryo without dehydration, the moisture in the tissue is frozen, which causes severe damage to the entire tissue, and thus greatly affects the survival rate.

  Figure 4 shows the types of solute used during dehydration (distilled water (100% relative humidity), Na2HPO4 (relative humidity 97%), Na2CO3 (relative humidity 88%), (NH4) 2SO4 (79% relative humidity), NH4NO3 ( Embryonic tissue induction rate according to 63% relative humidity) and K2CO3 (43% relative humidity). The maximum induction rate of embryogenic tissue was 43.5% when the dehydration treatment was performed with (NH4) 2SO4 (79% relative humidity) saturated solution. Next were treatments of Na 2 HPO 4 (generating 97% relative humidity) and Na 2 CO 3 (88%) with induction rates of 34.6 and 34.2%, the lowest in distilled water with 19.6%. Taken together, the optimal dehydration humidity environment for somatic embryos should be between 79 and 97%, and above or below humidity is not favorable for embryogenic tissue regeneration. However, the relative humidity conditions are not always constant, and the physiological conditions and water content of somatic embryos, and tissue lines are widely different between species, so it is necessary to find the optimum dehydration conditions for cryogenic storage.

Example 6 Embryonic tissue induction effect according to dehydration temperature and treatment time

Figure 5 relates to the embryogenic tissue induction effect according to the dehydration temperature (4 ℃ or 25 ℃) and the dehydration period (1-9 days) during the dehydration treatment of somatic embryos stored for 1 week in liquid nitrogen and then re-embedded embryonic tissue The result is derived. The highest induction rate was 56.8% for 2 days dehydration treatment at 25 ° C., and 56.3% and 56.6% for 2 and 3 days dehydration at 4 ° C., which was very effective. However, dehydration for more than 2 days at 25 ℃ showed excessive dehydration of plant tissues, indicating that embryogenic tissue induction rate decreased. And even at 4 ℃ dehydration tissue induction rate began to gradually decrease even after 3 days or more dehydration treatment, the deduction rate of about 30% was very low on the 9th day, the dehydration treatment period of 2 or 3 days was appropriate. On the other hand, in the case of no treatment without dehydration, the tissue induction rate after cryogenic storage was 15.1% (Fig. 4), so that the dehydration process of somatic embryos was essential for obtaining a high embryogenic tissue induction rate for cryogenic storage using liquid nitrogen. Shows that

<Example 7> Embryonic tissue induction effect according to storage temperature and storage period

Figure 5 shows the embryonic tissues according to the storage temperature (4 ℃, -20 ℃, -80 ℃ and -196 ℃) and storage period (1 day, 7 days, 28 days and 84 days) of the somatic embryo after dehydration treatment The induction effect was investigated. In the storage period of 1 day, the embryonic tissues were induced the highest by the storage of liquid nitrogen (-196 ℃) (66.9%). As the storage period increased, the tissue induction rate of liquid nitrogen gradually decreased, which was the lowest at 19.4% at 84 days, and also decreased at the other treatment temperature. In fact, cryogenic storage in liquid nitrogen should show almost constant embryogenic tissue induction rate regardless of storage period, but the optimal somatic embryo dehydration condition is not achieved yet, so the induction rate is estimated to decrease gradually with storage period.

In developed countries such as Canada and New Zealand, many practical researches have been made in the past to develop asexual growth technology applying biotechnology for the selection, breeding and mass production of high value added conifers. Is up to the actual stage of dissemination. In order to achieve the practical use using the somatic embryo induction technology, it is impossible to cultivate the seeds of superior coniferous tree first, and the embryogenic tissue is not preceded by the induction of a specific tissue. Subsequently, such tissue is used as a material, followed by the production of somatic embryos, followed by production to plants. Various induced embryonic tissue lines are usually expanded and maintained through passage in fresh medium. However, there are many problems such as the disposal of biocontamination caused by mold and bacteria during the subculture, and the increase in the occurrence of DNA mutations caused by the proliferation using a medium containing plant hormones such as 2,4-D. . In addition, after a certain period of time after planting the test trees produced from various tissue lines on the test forge, excellent trees are selected among them, which requires a long time requiring several years or more. In this case, the embryogenic tissue needs to be cryogenically stored during outdoor selection. If the field is later determined to be excellent wood, only the embryonic tissue lines can be removed from the liquid nitrogen and thawed, and then the material can be mass-produced using somatic embryos.

Gonadal storage of coniferous species in the past decade has been mostly done by cryogenic storage, which is stored in liquid nitrogen using embryogenic tissue. This method is very likely to generate somatic lesions under the influence of the cryopreservative DMSO added during tissue pretreatment, and has the disadvantage of requiring expensive freezing equipment, which requires slow freezing up to -40 ° C. On the other hand, the present invention relates to the development of technology to preserve the liquid nitrogen after the dehydration process by inducing somatic embryos instead of cryogenic storage using liquid embryogenic cells. The advantage of the present invention is that the pretreatment step is simpler than the method using embryogenic cells, expensive equipment such as a freezer is not required, and the possibility of somatic embryogenesis is significantly lower because DMSO is not added during cryogenic storage. However, in order to use this technology successfully, technology development (dehydration treatment, etc.) that can be regenerated from somatic embryos into embryonic tissues must be made.

The effect of the present invention is to use the result of 'ultra low temperature storage technology using lactic somatic embryos', and induce organ induction and somatic embryo through ultra low temperature storage of valuable germplasm (embryogenic tissue, pollen, shoots, etc.) of conifers in the future. It is expected to be able to apply to biotechnologies such as insect repellents for mass growth and dissemination of superior conifers and ultimately contribute to the establishment of clonal forestry, tree planting and forest resources. do.

Claims (5)

Sealing the induced somatic embryos and dehydrated in a cow at 4-25 ° C. for 1-7 days to store and thaw at ultra low temperatures; Ultra-low-temperature storage technology and embryonic tissue induction method using larch somatic embryos comprising re-hydrating the thawed somatic cells to induce embryonic tissue by inducing the embryonic tissue from the induced medium. delete 2. The larch somatic cell embryo according to claim 1, wherein the dehydrated somatic embryo is placed in a cryogenic preservation tube and stored in a liquid nitrogen tank for 1 week, and then taken out and thawed for 5 to 15 minutes in warm water at 30 to 40 ° C. Cryogenic Storage Technology and Embryonic Tissue Induction Method Using The thawed somatic cells are rehydrated for 12 to 36 hours on a filter in which somatic embryos are absorbed in distilled water. Cryogenic storage technology and embryogenic tissue induction method using larch somatic embryos characterized in that the cow culture at ℃. The method of claim 1, wherein the cryogenic storage is carried out in -20 ℃ to -196 ℃ in a nitrogen container ultra-low temperature storage technology and embryogenic tissue induction method using larch somatic embryos.

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