JP3889064B2 - Mass breeding method of ginseng seedlings - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a method for obtaining a large amount of ginseng seedlings that are medicinal carrots by tissue culture.
[0002]
[Prior art]
Panax ginseng CA Meyer is cultivated as an important medicinal plant and is usually propagated by seeds. It is a kind of herbal medicine that has been widely used as a traditional Chinese medicine and folk medicine since ancient times. Recently, it has been used for health foods, and the demand is increasing. However, medicinal carrots, which are used as raw materials for herbal medicines, usually take 4-7 years to harvest, are prone to growth depending on the weather, and are also useful for diseases (eg, Negusale disease, Tachigare disease, Hanten disease) and pests (Udokobu) It takes a lot of labor to cultivate because there is a lot of damage caused by weevil, weevil, etc. Therefore, stable storage and supply of excellent varieties were desired, but ginseng is self-fertile and requires at least 3 years to collect seeds, so it is difficult to obtain large numbers of excellent varieties. It was said.
[0003]
Therefore, in order to solve the above problem, callus is induced from tissue such as ginseng root, stem, leaf, etc. using a callus induction medium containing auxins and cytokinins, the callus is proliferated, and then the callus is A method of redifferentiation under light irradiation (Japanese Patent Laid-Open No. Sho 61-216619) has been proposed, but it takes a lot of work because it requires a process of inducing roots after inducing the above-ground part. There is also a problem with the survival in the field. Furthermore, the number of seedlings obtained by redifferentiation depends on the amount of callus, and since callus mutates at a significant rate when repeated, the amount of normal clonal seedlings obtained from a single plant is limited. There is a drawback that there is.
[0004]
On the other hand, in many plants such as edible carrots, many techniques for inducing seedlings by inducing somatic embryos that grow by taking the same morphological change process as seeds have been reported. A method of inducing callus from the cotyledon, cotyledon, etc., further inducing somatic embryos and redifferentiating them to obtain seedlings (WCChang et al; Theoretical and Applied Genetics, 57, 133 (1980), Japanese Patent Laid-Open No. 62-151117, Japanese Patent Laid-Open No. 63-248321) has been proposed. However, this method has a problem in the induction efficiency of somatic embryos, and has a drawback that it takes a long time from the start of culture to obtaining young seedlings.
[0005]
Therefore, in order to solve these drawbacks, a method for inducing somatic embryos using flower buds as a material (Japanese Patent Publication No. 2-51573) and a method for inducing shoots from somatic embryos and making them multiple shoots to proliferate (Japanese Patent Publication No. 3) -44725) and the like have been proposed. However, these methods also pass through the callus in the same way as the above-mentioned method of redifferentiating from calli by light irradiation, so the ability to form somatic embryos and redifferentiation is lost during repeated subcultures, and mutations occur. there's a possibility that.
[0006]
Therefore, a method has been proposed in which somatic embryo formation ability and redifferentiation ability are stably maintained and subculturing can be performed, and a somatic embryo is directly induced from a redifferentiated tissue by high-temperature treatment (Japanese Patent Laid-Open No. 4-370047).
[0007]
By the way, since somatic embryos usually differentiate gradually in various tissues, seedlings cannot be produced efficiently unless only mature somatic embryos are separated from other tissues. Therefore, in order to mass-produce seedlings from somatic embryos, it is necessary to obtain mature embryos as much as possible and synchronously within one culture cycle. Already for edible carrots, the method of arranging the cell mass size with a mesh (Hiro Kamada, Hiroshi Harada, Plant Cell Tissue Culture (Science and Technology) p.94 (1979)) and adjusting the density of the cell mass in the medium The method (Symposium of the 12th Annual Meeting of the Plant Tissue Culture Society, p.53 (1991)) has been proposed as a method for synchronously inducing somatic embryos. It has not been established yet.
[0008]
In edible carrots, it has been reported that somatic embryos can be induced by temporarily treating the shoot apex with a medium containing high concentrations of saccharose and mannitol (H. Kamada et al; In Vitro Cell Dev. Biol., 25, 1163 (1989)), but such methods and conditions were not known for medicinal carrots.
[0009]
Thus, a method has been proposed in which somatic embryos are continuously grown in large quantities by culturing somatic embryogenic tissues in a medium adjusted to a carbon source concentration of 4 to 20 g carbon source material / liter (Japanese Patent Laid-Open No. 5-168359). By these methods, high quality somatic embryos can be mass-produced.
[0010]
However, in order to mass-produce seedlings from somatic embryos, it is necessary to quickly germinate from somatic embryos and to efficiently carry out the acclimation process before transplanting to the field, but ginseng grows in a dry environment. The process took a long time.
[0011]
[Problems to be solved by the invention]
The present invention is intended to solve the above-mentioned problems, and an object of the present invention is to provide a method for efficiently germinating an adventitious embryo and further performing a habituation step before transplanting to a field.
[0012]
[Means for Solving the Problems]
The present invention relates to a germination process in which a somatic embryo derived from a ginseng tissue is cultured in a liquid culture and germinated to produce a germinated embryo, and a rooting process in which the germinated embryo is transplanted into a solid medium to root and extend to produce a rooted seedling. And a method for mass breeding ginseng seedlings having a acclimation step for acclimating rooted seedlings.
[0013]
An adventitious embryo as used in the present invention is a kind of embryo that arises from a somatic cell of a plant through the same morphological change process as a fertilized egg, and has the ability to develop into a complete plant body. It is also called an “embryoid” (see Biology Dictionary, 3rd edition (Iwanami Shoten), paragraph 1123 (1983)).
[0014]
The method for inducing somatic embryos as a material of the present invention can be achieved by any of the above-described methods known in the art, but from the aspect of somatic embryo growth efficiency and stable maintenance of somatic embryogenic tissues, For somatic embryo growth, it is preferable to use a method of continuous mass production by culturing in a medium adjusted to a carbon source concentration of 4 to 20 g carbon source material / liter.
[0015]
The liquid culture method used in the germination step of the present invention can be any of static culture, shaking culture, and tank culture, but from the aspect of preventing shear stress on somatic embryos, static culture In addition, tank culture by an air lift system is preferable. In addition, when performing static culture, it is preferable to supply air of 0.1 liter air / liter liquid medium / min or more to the liquid medium in order to supply oxygen to the tissue.
[0016]
In the germination step of the present invention, germination of somatic embryos can be induced under any light conditions, but culturing while irradiating light of 1000 lux or more for 8 hours or more per day improves germination efficiency. From the viewpoint of
[0017]
The support material for the solid medium used in the rooting step of the present invention can be any support material such as agar or gelling support material such as gellan gum, ceramic fiber, rock wool, or other horticultural planting materials. It is. It is not necessary to add plant hormones to the medium. However, if rooting is not good, it is possible to add auxin at a concentration of 5 ppm or less.
[0018]
In the acclimatization process of the present invention, any general horticultural planting material can be acclimatized, but sterilization by autoclave or dry heat sterilization is performed, or soil bacteria are removed in advance by disinfection sterilization with chemicals. It is necessary to keep.
[0019]
[Action]
Although the detailed interaction of the series of processes of the present invention on the improvement in productivity of ginseng seedlings is not clear, according to the observation of the tissue, by adding a germination step in a liquid medium, the nutrient supply to the embryo can be improved. As a result, the germination time is shortened and the yield of normal seedlings is improved. However, when the process proceeds from the liquid medium to the acclimation process as it is, it takes a long time for normal establishment in the soil due to the high moisture content of the seedlings. Therefore, by adding a rooting process with a solid medium, the moisture content of the seedling is reduced, and at the same time, the development of the hair root is promoted and can be quickly acclimated. Guessed.
[0020]
【Example】
5 years root of Panax ginseng, sucrose 30g / l, kinetin 1ppm added, Murashige-Skoog solid medium with 9g / l agar as support material, 20 ° C, 5000 lux, under light irradiation for 14 hours per day A shoot was induced.
[0021]
Two 100 ml flasks of this multiple shoot were subjected to high temperature treatment at 35 ° C. for 24 hours, and then cultured for 6 weeks under light irradiation at 20 ° C., 5000 lux, 14 hours per day to induce somatic embryos.
[0022]
The somatic embryogenic tissue treated at high temperature was placed in a 300 ml flask containing 100 ml of Murashige-Skoog solid medium with gellan gum as a supporting material, to which sucrose concentration was adjusted to 8 g / liter without adding plant hormones, and 20 ° C., 5000 lux. Culturing was continued for 6 weeks under light irradiation for 14 hours per day, and somatic embryos were mass-produced.
[0023]
Mass-produced somatic embryos are transplanted into a 2 liter bottle containing 500 ml of Murashige-Skoog liquid medium adjusted to a saccharose concentration of 3 g / liter without adding plant hormones, and 0.5-3 liter air / liter liquid medium About 100 germinated embryos were obtained by culturing for 3 weeks under aeration conditions per minute.
[0024]
Thirty of the germinated embryos were transplanted to Murashige-Skoog solid medium with 3 g / liter of gellan gum as a support, adjusted to a saccharose concentration of 30 g / liter without adding plant hormones, 20 ° C., 5000 lux, 1 Rooted seedlings were obtained by culturing for 3 weeks under conditions of 14 hours per day. When this seedling was transplanted to a pot containing cultivated soil for horticulture sterilized by an autoclave and dried on a petri dish lid to prevent drying, 28 seedlings were obtained.
[0025]
【The invention's effect】
According to the present invention, it has an excellent effect that it can efficiently produce a large amount of ginseng seedlings, and also has an exceptional effect that an established seedling can be obtained more quickly and efficiently than before. Further, in the liquid culture process, mechanization and automation are easy and labor saving is possible.

Claims (2)

The process of proliferating somatic embryos by culturing somatic embryogenic tissue of ginseng in a solid medium adjusted to a carbon source concentration of 4 to 20 g / L , and the somatic embryo in a liquid medium, 0.1 to 3 liter air / liter liquid medium Panax ginseng having a germination step of germinating by supplying air under the conditions of / min, a rooting step of transplanting the germinated embryo to a solid medium to elongate roots to generate a rooted seedling, and a conditioning step of acclimatizing the rooted seedling Seedling mass growth method.   The method for mass breeding ginseng seedlings according to claim 1, wherein in the germination step, culturing is performed while irradiating light of 1000 lux or more for 8 hours or more per day.