KR20080073388A - Mass production of bulblet via somatic embryogenic cell culture in lily - Google Patents

Abstract

A method for mass-producing bulblet of Lilium sp. industrially is provided to produce a massive amount of virus free bulblets economically within a short time through rapid proliferation of somatic embryogenic cells by applying a culturing technique of bulb scale segments of the Lilium sp. and a bioreactor culturing technique. A method for mass-producing bulblet of Lilium sp. using a bioreactor where an impeller is mounted and a plurality of air holes are formed at the bottom to inject sterile air comprises the steps of: (a) culturing bulb scale of the Lilium sp. in a solid culture medium where 0.1-10mg/liter of 4-amino-3,5,6-trichloropicolinic acid(PICLORAM) or 0.1-10mg/liter of 3,6-dichloro-O-anisic acid(DICAMBA) is added to Murashige Skoog(MS) culture medium under pH of 5.6-5.8 at a temperature of 25 deg.C in the dark state to induce somatic embryogenic cells therefrom and proliferate them; (b) culturing the proliferated somatic embryos in an MS culture medium excluding hormones at a temperature of 20-25 deg.C in the dark state to induce redifferentiation of bulblets; and (c) culturing the bulblets generated from the redifferentiation culture medium in a liquid MS culture medium at a temperature of 20-25 deg.C in the dark state to induce auxesis of the bulblets.

Description

Mass production of bulblet via Somatic Embryogenic Cell culture in Lily}

1 shows the conditions for rapid proliferation of Lilium somatic embryonic cells.

Figure 2 Lilium somatic embryo according to the present invention

Figure 3 is a rapid somatic embryo growth photos using the bioreactor according to the present invention

4 is a redifferentiated photograph of somatic embryo derived stem cells according to the present invention

The present invention relates to a method for industrially mass-producing a species of Lilium species, and more particularly, by applying the technique of human tissue culture and bioreactor culture technology of Lilium, somatic embryonic cells rapidly proliferate for a short time It is a method for mass production of economical disease-free species by mass production techniques.

Lilium sp. Is a vegetative propagation of human scales. The bulb acts as a reservoir for starch and other nutrients, and contains stem growth. On the other hand, bulbs are easily infected by viral diseases caused by phytopathogens, so the quantity and quality of the bulbs can not be used continuously for many years, so growers buy new bulbs every year.

In the Netherlands, the world's major producer of lily species, the scales of virus-tested hairballs are grown in a sterile state by tissue culture, followed by prolonged purifying and benign processes in the soil, producing and selling them to the world. Although several research institutes in Korea are studying mass production methods of lily seedlings using tissue culture methods, they rely on tissue culture methods using petri dishes or bottles for bacterial culture. As the ratio of labor costs to the production cost of large-scale production is high, the economic production of economic species is difficult. As a subsidiary project, some research institutes are conducting it, but the demand for farms is insufficient. Most farms depend on imports. In addition, the share of the seed cost of the cut flower production costs reaches 68%, and farmers producing lily for export use mostly imported seed ball due to the quality deterioration problem. It is true.

Therefore, the present inventors have provided a method of industrially mass-producing a species of Lilium species, as a result of the study, and as a result, using a somatic embryo cell culture technology that has a limitation on the production of species into tissue culture using conventional scalp culture When applied to bioreactor cultivation, not only continuous culture is possible, but also the growth rate is much faster when cultured somatic embryo cells, can be grown in a short period of time, and can be multi-varied with small facility investment by using small reactors. Mass production is possible, so harvested somatic embryonic cells can be stored for a long time (5 months) to control the production amount arbitrarily, and when somatic embryonic cells are re-differentiated, a small amount of somatic embryonic cells can be mass produced. And found that there is a large number of differentiated spouts per culture vessel, so that uniform mass production is possible. The invention was completed.

Accordingly, the present invention is to provide a method for economically mass production in the production of the species of Lilium by the purpose of domestic self-sufficiency of the species for export through the industrial species production.

In order to achieve the above object, the present inventors carried out tissue culture under various conditions in order to find a culture condition for somatic embryo induction, and carried out tissue culture under various conditions to find a culture condition suitable for rapid proliferation in bioreactor culture. The conditions for inducing regeneration of somatic embryonic cells and hypertrophy were performed. Species produced by culturing the bioreactor under the same conditions as in the tissue culture were transplanted into the general soil, and their purity was confirmed, and the survival rate was similar to that of the tissue culture.

That is, in order to achieve the above object, the method for producing a large number of lily lily species according to the present invention, inducing somatic embryonic cells from the scales and injecting the somatic embryonic cells into the reactor sterile air and many Rapidly proliferate using a bioreactor with air holes and induce somatic embryos into regeneration mediums to induce regeneration and add hypertrophy medium to mass-produce disease-free species in a short period of time. -4-amino-3,5,6-trichloropicolinic acid (hereinafter referred to as PICLORAM) in stuk medium (Murashige Skoog medium, hereinafter referred to as MS medium). Or in a solid medium to which 3,6-dichloro-O-Anisic acid (hereinafter referred to as DICAMBA) is added, at a pH of 5.6 to 5.8, at a temperature of 25 ° C. and in a dark condition. Cultured somatic embryonic cells Doha steps;

(2) rapidly proliferating somatic embryonic cells using a bioreactor in the same liquid medium;

(3) replacing somatic embryonic cells with a total amount of MS solid medium excluding hormones, incubating in a condition of pH 5.6 to 5.8, temperature 20 to 25 ° C., and blocking light to induce regeneration of magnetic domains;

(4) adding the liquid medium in which the salt concentration of the MS medium is reduced in half for the enlargement of the magnetic domains, thereby inducing the hypertrophy of the magnetic domains.

Example 1 Effect of Hormone Type and Concentration on Somatic Embryogenesis of Lilium

MS medium added with 3% sugar was treated with various growth hormones and concentrations, and then inoculated by cutting slices of Lilium, and the results of somatic embryos were investigated.

Table 1

kind Marco Polo 2,4-D One    0 3    0 6    0 9    0 Dicamba One    0 3   13 6    0 9    0 Picloram One   30 3   56 6   13 9    6 Casablanca 2,4-D One    0 3    0 6   10 9   10 Dicamba One    0 3    6 6    0 9    0 Picloram One   90 3  100 6   80 9    5 Sorbonne 2,4-D One    0 3    0 6    0 9    0 Dicamba One    0 3    0 6    0 9    0 Picloram One   52 3   89 6   97 9   92

      Effect of Hormone Types and Concentrations on Somatic Cell Formation in Lilium To induce somatic embryonic cells that are well re-differentiated in humans, the cultures were cultured in medium containing PICLORAM or DICAMBA, a growth regulator hormone. The amount of the hormone added is preferably in the range of 1.0 to 9.0 mg / l for PICLORAM and 1.0 to 6.0 mg / l for DIACMBA. Preferably, it is determined that it is desirable to add 3.0 mg / l for PICLORAM and 3.0 mg / l for DICAMBA.

  In the second stage culture, the somatic embryo cells derived in the first stage are rapidly grown in a short period of time in the same incubator in a large amount in a short period of time to produce a uniform device in induction during regeneration. Incubate with only MS medium added below.

Example 2 Somatic Embryonic Rapid Proliferation Condition

For rapid proliferation of somatic embryos, 50 ml of MS liquid medium (5% sucrose) was placed in a 250 ml Erlenmeyer flask using Oriental Nari “Marcopolo” somatic embryos and cultured by rotating shaking at 125 rpm. MS liquid medium (5% sucrose) was treated with various concentrations of picloram and zeatin at 0, 0.3, 1, 3mg / L, and examined the results of in vivo changes in somatic embryos depending on the treatment period. Shown in In the bioreactor culture for rapid somatic embryo proliferation, subcultured at intervals of 1 week in medium containing PICLORAM 1.0 mg / l may obtain the best results for rapid proliferation of somatic embryonic cells, as shown in FIG. 1.

  In step 3 culture, chopped somatic embryo cells rapidly expanded in step 2, excluding growth hormones, and submerged on MS solid medium containing 6% sugar and 0.5% medium solids (GELITE, Duchefa Co.). To induce.

Stage 4 culture is to enlarge the microspheres re-differentiated in step 3 and to increase the size of the autogenous medium by adding the autogenous hypertrophy medium through the ventilation hole of the culture vessel without additional subculture from the existing regeneration medium to the autogenous hypertrophy medium. The work efficiency can be increased by shortening the inefficient incubation time according to passage of the furnace.

 In stage 4 culture, it helps to develop the in-flight magnetic domains by using a container sealed with a breathable tape for the respiration of the re-differentiated magnetic domains. Simultaneous autogenous hypertrophy in the same culture vessel, rather than through culture, avoids the cumbersome work of subculture.

Through the rapid mass production culture of magnetic domains using these four-stage somatic embryo cells, a production system capable of producing large numbers of species with a small number of people is completed, thus reducing the burden on facility investment compared to the existing mass production system and effectively cultivating the system. It is possible to industrially supply large quantities of economically uniform quality seeds by constructing them, and the produced species are suitable for mass production of various varieties that meet the needs of farmers through precise cultivation in soil.

Hereinafter, the mass production of Lilium porcelain using rapid proliferation of somatic embryo cells through various embodiments is carried out in order to find suitable culture conditions for somatic embryo cell induction culture, tissue culture under various conditions, rapid proliferation conditions in bioreactor culture using the same, and differentiation. The culture of tissues under various conditions to find conditions and efficient culture system of in-flight magnetic domains will be described in detail. However, the present invention is not limited to these examples.

The growth rate of somatic embryogenic cells is much faster through the bioreactor culture according to the present invention. Since it can be supplied in large quantities, early bulk supply of new varieties can be applied to implementation.

Claims (4)

In the mass production method of lily lily species, an agitator (impeller) is installed in the middle of the reactor, and a bioreactor having many air holes formed in the bottom of the reactor to inject sterilized air, (1) 4-Amino-3,5,6-trichloropicolinic acid (Murashige Skoog medium, hereinafter referred to as MS medium) (4-Amino-3,5,6-Trichloropicolinic acid, hereinafter) PICLORAM) or 3,6-dichloro-O-anisic acid (hereinafter referred to as DICAMBA) in a solid medium, pH 5.6 ~ 5.8, temperature 25 ℃, Inducing and proliferating somatic embryonic cells in human cells by culturing in a cancerous condition; And (2) Proliferating somatic embryos are to replace the whole medium with a hormone-free MS medium, incubated in the temperature of 20 ~ 25 ℃, light-blocking cancer conditions to induce regeneration of the magnetic domains (3) The lily bulbs formed in the regeneration medium are put in a liquid MS medium for hypertrophy, incubated in a dark condition of the temperature of 20 ~ 25 ℃, light-blocked lily lilies comprising the step of inducing hypertrophy of the bulblets Method of mass production of species of species The method of claim 1, wherein in the first stage culture medium, the somatic embryo induction and proliferation of the lily lilies, characterized in that the PICLORAM is added in an amount of 0.1 ~ 10mg / l, DICAMBA is added in an amount of 0.1 ~ 10mg / l Method for mass production of somatic embryonic cells of a kind. The method for mass production of lily lilies spp. According to claim 1, wherein in a two-stage culture medium, 5-10% sugar and 0.5-0.7% GELITE are added. The method of claim 1, wherein the three-stage culture medium is a liquid medium in which hormones are excluded, salt concentration is reduced to 1/2, and sugar is added to 9%, and the medium is added every four weeks. Method of mass production of Lilium species.

Images