JPH0256051B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to American ginseng (Panax).
quinquefolium L.) using tissue culture techniques. Conventional techniques and problems American ginseng is cultivated as a medicinal plant and is usually propagated by seeds.
Attempts have been made to propagate Panax ginseng, which is of the same genus as American ginseng, through tissue culture. For this method, Panax ginseng root,
A method of inducing callus from plant tissues such as stems and leaves using a callus induction medium containing auxins and cytokinins, propagating the callus, and then redifferentiating the callus under light irradiation 61−
216619) Also, a method for inducing and propagating somatic embryos from callus and then redifferentiating [WCChang,
YIHsing: Theoretical and Applied Senetics
Genetics) 57 , 133 (1980)], etc. have been proposed. In this case, roots, petioles, leaves, flower stalks, etc. have been tried as explants for tissue culture of Panax ginseng [RG Butenko et.al.
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, there are examples of using flower buds as explants in plants such as Freesia [RLMPierik et.
al.: Netherlands Journal of Agricultural Science
Journal of Agricultural Science) 23 , 334
(1975)], but this example was aimed at inducing somatic embryos, and there is no description of somatic embryos. By the way, the method of inducing and redifferentiating callus from roots, stems, and leaves of Panax ginseng, or the method of inducing somatic embryos from this callus to proliferate and redifferentiate, requires a long time to induce somatic embryos from callus. In addition, there was a problem that the induction rate of somatic embryos was low. Problems to be Solved by the Invention The present invention was devised in view of the above-mentioned problems, and involves culturing somatic embryos from callus in an extremely short period of time by performing tissue culture using flower buds of American ginseng as explants. It is an object of the present invention to provide a method for efficiently producing seedlings by inducing somatic embryos and proliferating somatic embryos at a high induction rate. The present invention will be explained in detail below. Structure of the Invention The main feature of the present invention is to produce seedlings by performing tissue culture using flower buds of American ginseng as explants. Furthermore, the present invention is characterized in that the flower buds are tissue cultured to form calluses, somatic embryos are induced and propagated from the callus, and then redifferentiated to produce seedlings. Means for Solving the Problems In the present invention, flower buds of American ginseng used as explants are not particularly limited, but are obtained from plants immediately after flower bud differentiation and immediately before flowering, and the pedicels and sepals are removed. Preferably, it is cut into sections 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. For tissue culture, first, flower bud sections sterilized as described above were treated with 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid).
4-D), Murashige-Skoog (MS), White, Linsmeyer-Skoog with added auxins such as indole acetic acid (IAA) and naphthalene acetic acid (NAA), or cytokinins such as benzylaminopurine (BAP) and kinetin. ,
This is carried out by culturing in the dark in a callus and somatic embryo induction medium such as Gauslett or Heller for about 12 weeks. Through this culture, the flower buds turn into calluses, and then somatic embryos are induced. In the present invention, mature embryos among the somatic embryos thus induced are cultured in a redifferentiation medium containing cytokinins, gibberellin (GA), etc. under light irradiation, so that they germinate in about 4 weeks. and forms shoots. On the other hand, the immature embryos among the above-mentioned somatic embryos are subcultured in a medium containing cytokinins, auxins, etc. in the dark to promote embryo maturation and form and multiply secondary embryos. It is best to redifferentiate in a medium. The shoots formed by the redifferentiation form multiple shoots when cultured under light irradiation in a medium supplemented with BAP-GA or NAA-BAP. Then, the obtained multiple shots are
Seedlings can be obtained by culturing and rooting for about 7 weeks under light irradiation in a rooting medium containing auxins such as NAA and indolebutyric acid (IBA). American ginseng can be cultivated by transplanting the seedlings thus obtained into soil. As mentioned above, in accordance with the present invention, by performing tissue culture using flower buds of American ginseng as explants, somatic embryo induction from callus formation can be achieved in a very short period of about 12 weeks, and moreover, as described below. As seen in the Examples, somatic embryos are induced at a high induction rate, making it possible to produce American ginseng seedlings very efficiently in a mass production manner. Incidentally, when performing tissue culture using roots, stems, leaves, etc. of American ginseng as explants, it takes a long period of 6 to 9 months for somatic embryo formation, as seen in the comparative example below. Moreover, there are extremely few cases that lead to somatic embryonic differentiation. EXAMPLES The present invention and its effects will be specifically explained below with reference to Examples. Example: Approximately 5 mm flower bud sections from American carrot buds grown in Nagano Prefecture, 2 weeks after germination, with the peduncles and sepals removed, were prepared in a 3 wt% sodium hypochlorite aqueous solution to which 0.1 wt% Tween 80 was added. After sterilization for 10 minutes with a 70% volume ethanol solution for 30 seconds, the tube was washed twice with sterile purified water. The sterilized flower bud sections were transplanted into MS medium supplemented with 2,4-D at the concentrations shown in Table 1, and cultured in the dark at a temperature of 25±1° C. for 12 hours. As a result, 80
More than % of the embryos turned into calluses, and somatic embryo formation was observed in the proportions shown in Table 1.

[Table] Take out the mature embryo from these somatic embryos, dilute the MS medium composition 2 times, add 0.5 ppm each of BAP and GA, and add 1.5% or 3% by weight of sucrose. Transfer to an agar medium prepared by
Cultured for 4 weeks under 16 hours/day light. As a result,
In the medium supplemented with 1.5% by weight of seuucrose,
Normal shoots were obtained from 70% of the embryos and from 40% of the embryos with 3% by weight medium. 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 cultured under the same conditions in a medium supplemented with 1.5% by weight of sucrose, and as a result, normal shoots were obtained as described above. Next, dilute the MS medium composition solution twice with this shoot, and add 0.5 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 7.7 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 30% 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 culture as in the example using roots, stems, and leaves from the same American ginseng tissues from which the flower buds in the example were obtained.
In this case, the root is cut into a disc-shaped section with a thickness of about 2 mm and a diameter of about 5 mm, excluding the epidermis, and the stem is cut into a disc with a thickness of about 2 mm.
The leaves were used as disk-shaped sections of about 1 cm square. As a result, more than 80% of root, stem, and leaf sections turned into callus, but none reached somatic embryo formation at 12 weeks, and somatic embryo differentiation did not occur even after 6 to 9 months. The percentage of cases that reached this level was about 1 to 10%. Effects of the Invention The present invention uses flower buds of American ginseng as explants to perform tissue culture, so somatic embryos of American 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 American carrot seedlings, which comprises producing young seedlings by performing tissue culture using flower buds of American carrot as explants. 2 By performing the above tissue culture, flower buds are formed into calluses, somatic embryos are induced to proliferate from the calluses, and then redifferentiated to produce young seedlings.Claim 1
Method for mass production of young American ginseng seedlings as described in Section 1. 3 The above redifferentiation involves the development of shoots from somatic embryos.
3. The method for mass production of young American carrot seedlings according to claim 2, wherein the method is carried out after forming and propagating the shoots. 4. When the somatic embryo is induced to proliferate, when the somatic embryo is an immature embryo, the immature embryo is matured, a secondary embryo is formed and multiplied, and then redifferentiated. Claim 2
Method for mass production of young American carrot seedlings as described in section.