JPH0198480A - Method for cultivating tissue of hybrid plant body belonging to genus panax - Google Patents

Abstract

PURPOSE:To obtain young seedling belonging to the genus Panax in a large amount, by forming shoots of a hybrid plant body belonging to the genus Panax and subjecting the above-mentioned shoots to multiple shooting. CONSTITUTION:Sections of roots, stems, flower buds, etc., of hybrids, etc., between Panax ginseng C.A. Meyer and Panax japonicus C.A. Meyer, Panax ginseng and Panax quinquefolium L and Panax japonicus C.A. Meyer and Panax ginseng quinquefolium L. are cultivated in a culture medium, such as Murashige.Skoog culture medium, containing cytokinins, etc., added thereto to form calluses and induce adventitious embryos. Mature embryos of the above-mentioned adventitious embryos are then shot in a redifferentiation culture medium or the immature embryos are subjected to maturity promotion thereof, formation and propagation of secondary embryos and subsequently shot in a redifferentiation culture medium. The resultant shoots are then cultivated in a culture medium containing benzylaminopurine (BAP)-gibberellin (GA) or naphthaleneacetic acid (NAA)-benzylaminopurine (BAP) added thereto while being irradiated with light to form multiple shoots, which are subsequently cultivated in a rooting culture medium containing auxins while being irradiated with light to root and afford the aimed young seedlings.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for obtaining a large amount of young seedlings of hybrids of the genus Panax using tissue culture techniques.

With traditional techniques. Panax ginseng C, A
, Meyer), Panax japan (Panax ja
ponicium C, A, Meyer) and American ginseng (Panax quinquefolium
Members of the genus Panax, such as L., are cultivated as important medicinal plants and are usually propagated by seeds.

Recently, attempts have been made to create hybrids of the genus Panax by crossbreeding these carrots with the aim of introducing new traits such as disease resistance and hybrid vigor, and it is known that they exhibit excellent hybrid vigor. There is. However, these hybrids are highly sterile and cannot produce progeny, and the above-mentioned Panax ginseng, Chikusetsu ginseng, and American ginseng are
Because it is monofertile, it is difficult to collect hybrid seeds efficiently and in large quantities, and hybrids were thought to have no practical use.

On the other hand, attempts have been made to propagate Panax ginseng by tissue culture, and this method involves inducing callus from plant tissues such as roots, stems, and leaves of Panax ginseng using a callus induction medium containing auxins and cytokinins. , a method of proliferating the callus and then redifferentiating the callus under light irradiation (JP-A-61-216619), a method of inducing and propagating somatic embryos from the callus and then redifferentiating (W, C, Chang, Y, 1.11
sing: Theoretical and Applied Genetics
Genetics) 57, 133 (1980)) etc.
Proposed.

There are many reports on the method of multiplying shoots derived from the shoot apex by multiple shooting (the so-called Noricron method), for example, for Gerbera plants [for example, T, Mu
Rashige et, al.: HortScience (H
ortscience) 9.175 (1974)), but there are no reports on tissue culture of hybrids of the genus Panax.

By the way, in the above-mentioned method of direct sprouting and rooting from the callus of Panax ginseng, the number of young seedlings obtained by redifferentiation depends on the amount of callus, and in general, callus is
Since repeated subculturing results in a considerable rate of mutation, there is a problem in that there is a limit to the amount of normal or lawn seedlings that can be obtained from an individual plant.

In addition, with the method of inducing and propagating somatic embryos from callus, the ability to redifferentiate from somatic embryos is lost through repeated subculturing, and as mentioned above, there is a limit to the amount of normal lawn seedlings that can be obtained from a single plant. There is a problem that there is.

Problems that the present inventor is trying to solve every day As a result of intensive research in order to solve these problems, the present inventor has discovered that hybrids of the genus Panax (shoo)
t) into multiple shoots (multiple 5h
oot).

Therefore, the present invention aims to produce cloned seedlings of Panax genus hybrids in large quantities and efficiently by forming living tissues of Panax genus hybrid plants into shoots using tissue culture and converting the shoots into multiple shoots. The challenge is to provide a method that can be produced efficiently.

The present invention will be explained in detail below.

The main feature of the present invention is to form shoots of a hybrid plant of the genus Panax, and to convert the shoots into multiple shoots (
The goal is to multiply by multiple shooting (multiple shooting).

The term "Panax hybrid" as used herein means a hybrid plant that is a combination of six species, including reverse crosses between Panax ginseng and Panax ginseng, Panax ginseng and American ginseng, and Panax ginseng and American ginseng.

The shoots used in the present invention may be derived from somatic embryos, shoot tips, somatic embryos, etc., but those derived from somatic embryos are preferred for ease of culturing.

In the present invention, in order to form a shoot, first, a part of the living tissue such as roots, stems, leaves, and flower buds of a hybrid plant of the genus Panax is cut off, and then a portion of the living tissue such as roots, stems, leaves, flower buds, etc. Alternatively, after sterilization with a sterilizing solution such as ethanol, it is preferable to culture in a callus induction and somatic embryo induction medium to obtain a callus, and then to induce somatic embryos from the callus. In this case, in particular, when flower buds are used, somatic embryos can be efficiently induced from callus in a short period of time.

As callus induction and somatic embryo induction medium, auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D), indole acetic acid (IAA), naphthalene acid fII (NAA), or benzylaminopurine (BAP), Murashige-Skoog (MS), White, Linsmeyer-Skoog, added with cytokinins such as kinetin,
Mediums such as Gauslet and Hella can be used.

The living tissue after sterilization is cultured in the dark for about 12 weeks in this callus formation and somatic embryo induction medium, and then the living tissue is transformed into a callus and then somatic embryos are induced.

The mature embryos among the somatic embryos thus induced will germinate in about 4 weeks by culturing in a regeneration medium containing cytokinins, gibberellin (GA), etc. under light irradiation, and shoots will form. can get.

On the other hand, immature embryos are subcultured in the dark in a medium containing cytokinins, auxins, etc. to promote embryo maturation and formation and proliferation of secondary embryos, which are then transformed into shoots in the above-mentioned redifferentiation medium. It's good to let it happen.

The above-mentioned shoot obtained in this way is BAP-GA
Alternatively, when cultured under light irradiation in a medium supplemented with NAA-BAP, multiple shoots are formed.

These multiple shoots consist of 2 to 10 normal shoots with about 2 leaves, and can be propagated by repeating a series of operations in which they are divided and further cultured in the above-mentioned multiple shoot formation medium. Can be done.

When the shoots obtained in this way are cultured under light irradiation in a rooting medium containing auxins such as NAA, indole acetic acid (IAA), and indole butyric acid (IBA),
The seeds will germinate in about a week and young seedlings will be obtained.

By transplanting these seedlings into soil, hybrid plants of the genus Panax can be cultivated.

The present invention will be specifically explained below using Examples.

Example 1 About 2 weeks after germination, 51 flower bud sections from the hybrid side of Panax ginseng x Panax ginseng, with the mouth flower buds and flower sepals removed, root sections of about 3 mm square with the root epidermis removed, and stems with the thickness A stem section cut into a disk shape of approximately 211IO1 and a leaf section cut into a leaf section of approximately 1-1 IO1 were each cut into Tween 80.
The sample was sterilized for 10 minutes with a 3% by weight aqueous sodium hypochlorite solution containing 0.1% by weight of , and then for 30 seconds with a 70% by volume ethanol solution, and then washed twice with sterile purified water.

Each section after sterilization was transplanted into MS medium supplemented with tppm of 2.4-D, and kept in the dark at a temperature of 25 ± 1°C for 12 hours.
Cultured for a week. As a result, more than 80% of the various explants turned into callus, and somatic embryo formation was observed in 65% of the callus sections derived from flower buds. On the other hand, none of the root, stem, and leaf sections formed somatic embryos at 12 weeks, but after 9 months, 1%, 3%, and 11%, respectively, formed somatic embryos.

Mature embryos were taken out of these somatic embryos, the MS medium composition was diluted 2 times, and BAP and GA were added to this with 0% each.
．． 5 ppm each, and sucrose (sucrose)
The cells were transplanted onto an agar medium prepared by adding 1.5% or 3% by weight of e) and cultured for 4 weeks at a temperature of 22±1° C. under illumination for 16 hours/day. As a result, 1 sucrose
．． In the medium supplemented with 5 wt%, 70% of the embryos and 3
In weight% medium, normal shoots were obtained from 40% of the embryos.

Next, this shoot was diluted twice with the MS medium composition solution,
At a temperature of 22 ± 1°C on an agar medium supplemented with the amounts of GA, BAP and sucrose shown in Table 1,
The cells were cultured for 8 weeks under 16 hours/day light. As a result, multiple chutes having the numbers shown in the table were obtained.

After dividing and propagating the shoots obtained above under the same culture conditions as above, NAA, IBA, and IAA were each grown in lp.
NAA
30% of the shoots were rooted in the medium supplemented with iBA and 30% of the shoots were rooted in the medium supplemented with 75% iBA, and clone seedlings were obtained.

These young seedlings were transplanted into soil and are growing well.

Example 2 The same method as in Example 1 was carried out using flower buds from the hybrid side of Panax ginseng x American ginseng, which were about two weeks old after germination.
The cells were cultured in a medium, formed into a callus, and formed into somatic embryos. As a result, more than 80% of the area became callus, and 10pp of 2.4-D was added.
In the medium supplemented with m, 85% formed somatic embryos within 12 weeks.

Mature embryos were removed from these somatic embryos and cultured in a medium using the same method as in Example 1. As a result, sucrose was found to be 1.
Normal shoots were obtained from 70% of the embryos in the medium containing 5% by weight, and from 34% of the embryos in the medium containing 3% by weight.

Next, this shoot was diluted twice with the MS medium composition solution,
This was cultured in the same manner as in Example 1 on an agar medium supplemented with GA, BAP, and sucrose in the amounts shown in Table 2, and as a result, multiple shoots having the numbers shown in the table were obtained.

The shoots obtained above were cultured using the same method and medium as in Example 1. As a result, 60% of IB
In the medium supplemented with A, 30% of the shoots were rooted and cloned seedlings were obtained.

These young seedlings were transplanted into soil and are growing well.

Example 3 The same method as in Example 1 was carried out using flower buds about 2 weeks after germination from hybrid buds of American ginseng.
The cells were cultured in a medium, formed into a callus, and formed into somatic embryos. As a result, more than 80% of the embryos turned into callus, and 63% formed somatic embryos within 12 weeks.

Mature embryos were removed from these somatic embryos and cultured in a medium using the same method as in Example 1. As a result, sucrose was found to be 1.
Normal shoots were obtained from 85% of the embryos in the medium containing 5% by weight, and from 60% of the embryos in the medium containing 3% by weight.

Next, this shoot was diluted twice with the MS medium composition solution,
This was cultured in the same manner as in Example 1 on an agar medium supplemented with the amounts of GA, BAP and sucrose shown in Table 3, and as a result, multiple shoots having the numbers shown in the table were obtained.

The shoots obtained above were cultured using the same method and medium as in Example 1. As a result, 60% of IB
In the medium supplemented with A, 30% of the shoots were rooted and cloned seedlings were obtained.

These young seedlings were transplanted into soil and are growing well.

As described above, the present invention forms shoots of a hybrid plant of the genus Panax and multiplies the shoots by multiple shooting. This method has a special effect in that clone seedlings of hybrid plants of the genus Panax can be obtained.

Claims (1)

[Claims] Forms shoots of hybrid plants of the genus Panax,
A method for tissue culturing a hybrid plant of the genus Panax, which is characterized in that the shoots are multiplied by multiple shooting.