JPH0251573B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to the use of Panax ginseng (Panax ginseng).
This paper relates to a method for producing large quantities of young seedlings by tissue culture (CAMeyer). [Prior Art] Panax ginseng (commonly known as Panax ginseng) is cultivated as a medicinal plant and is usually propagated by seeds, but recently attempts have been made to propagate Panax ginseng by tissue culture. In this method, callus is induced from plant tissues such as roots, stems, and leaves of Panax ginseng using a callus induction medium containing auxins and cytokinins, the callus is multiplied, and then the callus is exposed to light. Method of redifferentiation (Japanese Patent Application Laid-open No. 61-216619) Also, a method of inducing and propagating somatic embryos from callus and then redifferentiating them {WCChang, YIHsing: Theoretical and Applied Genetics 57 , 133
(1980)} have been proposed. Incidentally, roots, petioles, leaves, flower stalks, etc. have been tried as explants for tissue culture of Panax ginseng {RGButenko et.al.: Botanicheskii Zhurnal)
7, 906 (1968)}, there are no examples of flower buds being used. Furthermore, the above-mentioned document states that no culture was obtained when flower stalks were used. On the other hand, an example of using flower buds as explants is Freesia {RLMPierik et.al.: Netherlands Journal of Agricultural Science.
Science) 23 , 334 (1975)}, but this example is aimed at inducing adventitious buds, and there is no description of somatic embryos. [Problems to be Solved by the Invention] The method for inducing and redifferentiating callus from the roots, stems, and leaves of Panax ginseng, or the method for inducing and propagating somatic embryos from such callus and redifferentiating, There were problems in that long-term culture was required to induce somatic embryos, and the induction rate of somatic embryos was also low. As a result of intensive research in order to solve these problems, the present inventor has found that somatic embryos can be induced from callus in an extremely short period of time by using flower buds of Panax ginseng as explants, and that the induction rate is also low. shoots formed from somatic embryos can be multiplied into multiple shoots; furthermore, immature somatic embryos can be subcultured to proliferate through secondary embryo formation; We discovered that it is possible to redifferentiate. The present invention was made based on this knowledge, and an object of the present invention is to provide a method for efficiently obtaining a large amount of cloned seedlings of Panax ginseng in a short period of time. [Means for Solving the Problems] The present invention consists of culturing the flower buds of Panax ginseng as explants to produce young seedlings. Particularly preferably, the production of young seedlings by the tissue culture is The method comprises inducing and propagating a somatic embryo from a callus and then redifferentiating the somatic embryo.Furthermore, this redifferentiation is carried out by forming a shoot from the somatic embryo, propagating the shoot, and then rooting the somatic embryo. This is a method for mass production of Panax ginseng seedlings, which involves maturing the immature embryo, forming and multiplying the secondary embryo, and then redifferentiating it. The flower buds of Panax ginseng used in the present invention are
Although there are no particular restrictions, it is preferable to use a piece obtained from a plant within about 6 months immediately after flower bud differentiation, with the peduncle and calyx removed, and cut into pieces with a size of 1 to 10 mm. The flower bud sections are sterilized with a sterilizing solution such as a sodium hypochlorite aqueous solution containing Tween 80 or ethanol, and then subjected to tissue culture. In the present invention, first, a section of this flower bud is
2,4-dichlorophenoxyacetic acid (2,4-D),
Murashige Skoog (MS), white, which contains auxins such as indole acetic acid and naphthalene acetic acid (NAA), or cytokinins such as benzylaminopurine (BAP) and kinetin;
The cells are cultured in the dark in a callus and somatic embryo induction medium such as Linsmeyer-Skoog, Gauslett, Heller, etc. for about 12 weeks. As a result, the flower bud turns into a callus, and then somatic embryos are induced. Among the somatic embryos induced in this way, mature embryos are cultured in a regeneration medium containing cytokinins, gibberellin (GA), etc. under light irradiation, and germinate in about 4 weeks and produce shoots. is obtained. On the other hand, immature embryos are subcultured in a medium containing cytokinins, auxins, etc. under light irradiation to promote embryo maturation and formation and proliferation of secondary embryos, and then re-cultured in the above-mentioned redifferentiation medium. It is good to differentiate. The above shot is BAP-GA or NAA-BAP
When cultured under light irradiation in a medium supplemented with , multiple shoots are formed. The shots obtained in this way are NAA,
When cultivated under light irradiation in a rooting medium containing auxins such as indolebutyric acid (IBA), roots will develop in about 7 weeks and seedlings can be obtained. Panax ginseng can be cultivated by transplanting these seedlings into soil. [Example] Example A 5 mm flower bud section obtained by removing the pedicel and sepal from a bud of Panax ginseng approximately 2 weeks after germination was prepared using 3 wt % sodium hypochlorite to which 0.1 wt % Tween 80 was added. After sterilization with an aqueous solution for 10 minutes and a further 30 seconds with a 70 volume % ethanol solution, it was washed twice with sterile purified water. The sterilized flower bud sections were transplanted to MS medium supplemented with 1 ppm of 2,4-D and cultured in the dark at a temperature of 25±1° C. for 12 weeks. As a result, 100% callus formation was observed, and somatic embryo induction was observed in 80% of the calluses. A mature embryo was taken out of the somatic embryos, the MS culture composition was diluted 2 times, and BAP and GA were added at 0.5 ppm each, and sucrose was added at 1.5% or 3% by weight. The cells were transplanted onto an agar medium and cultured for 4 weeks at a temperature of 22±1° C. under illumination for 16 hours/day. As a result, normal shoots were obtained from 70% of the embryos in the medium containing 1.5% by weight of sucrose, and from 45% of the embryos in the medium containing 3% by weight. Regarding immature embryos among the above somatic embryos,
As a result of subculturing on MS agar medium supplemented with 2 ppm NAA and 2.5 ppm BAP, all immature embryos matured.
The proliferation of somatic embryos through secondary embryogenesis occurred actively. This mature embryo was sucrose 1.5% by weight above.
As a result of culturing under the same conditions using an additional medium, normal shoots were obtained as described above. Next, dilute the MS medium composition solution twice with this shoot, and add 5 ppm each of GA and BAP to this.
The cells were cultured for 8 weeks on an agar medium supplemented with 1.5% sucrose at a temperature of 22±1° C. and under light for 16 hours/day. As a result, multiple shots with an average number of 5.9 shots were obtained from all shots. The shoots obtained above were placed on MS agar medium supplemented with 1 ppm each of NAA and IBA for 20±
As a result of culturing at a temperature of 1° C. for 7 weeks under light for 16 hours/day, 75% of the shoots were rooted in the medium supplemented with NAA and 50% in the medium supplemented with IBA, and clone seedlings were obtained. These young seedlings were transplanted into soil and are growing smoothly. Comparative Example Callus and somatic embryos were induced in the same medium as in the example using roots, stems, and leaves from the same Panax ginseng tissues from which the flower buds in the example were obtained. still,
In this case, the roots are cut into disc-shaped pieces with a thickness of about 2 mm and a diameter of about 5 mm, excluding the epidermis, the stems are cut into disc-shaped pieces with a thickness of about 2 mm, and the leaves are cut into pieces with a leaf blade of about 1 cm square. there was. As a result, 100% of the root, stem, and leaf sections turned into callus, but none of them reached somatic embryo formation at 12 weeks, and some of them showed somatic embryo differentiation even after 6 to 9 months. was about 1 to 10%. [Effects of the Invention] The present invention uses flower buds of Panax ginseng as explants to perform tissue culture. Therefore, somatic embryos of Panax ginseng can be efficiently induced in a short period of time, and cloned seedlings can be obtained in large quantities. This has a special effect.

Claims (1)

[Scope of Claims] 1. A method for mass production of young Panax ginseng, which comprises culturing flower buds of Panax ginseng to produce young seedlings. 2. The method for mass production of Panax ginseng seedlings according to claim 1, wherein the production of seedlings by tissue culture involves inducing and propagating somatic embryos from callus and then redifferentiating them. 3. The method for mass production of young Panax ginseng seedlings according to claim 2, wherein the redifferentiation is performed by forming shoots from somatic embryos, multiplying the shoots, and then rooting the shoots. 4. When the somatic embryo is an immature embryo, the embryo is matured, a secondary embryo is formed and multiplied, and then the embryo is redifferentiated, according to claims 2 and 3. A method for mass production of young Panax ginseng seedlings is described.