JPS6258934A - Mass propagation of potato by tissue culture - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION B) Industrial application field
Or Dloscorea batatas DEC
(7) Relating to a method for mass propagation of tissue culture.

More specifically, a section of the stem containing the axillary and apical buds is collected from the vine of the plant, sterilized and cultured aseptically to induce the formation of many buds from one bud, and then the individual buds are divided. By obtaining seedlings through stem and leaf regeneration and rooting,
Concerning a method for mass propagating Chinese potatoes. With this method,
It becomes possible to propagate large quantities of superior strains, such as disease-free strains from which viruses have been removed, in a genetically uniform state within a short period of time.

(b) Prior Art and Problems Like many other plants of the Dioscoreaceae family, Japanese yam is a vegetatively propagated plant that does not reproduce by seeds. In normal cultivation, reproduction is attempted by dividing the underground root-bearing bodies (so-called potatoes) or by using the vines formed in the leaf sap of the vines, but the multiplication rate is extremely low, ranging from several tens to a hundred times more per year. .

;There have been many attempts at propagation using methods such as shoot apex (growing point) culture and callus culture, but the growth of this plant species on medium is generally slow and complete individual regeneration is not possible. In addition to requiring a long period of time, it has been difficult to achieve sufficient efficiency for practical propagation. Furthermore, these callus-based methods tend to cause genetic variations in regenerated plants due to chromosomal mutations during culturing, and there are many problems as a means of obtaining uniform lawn plants in large quantities.

For Japanese cultivars such as Nagaimo, Ichoimo, Tsukuneimo, and Yamatoimo, multiple buds are formed on the medium from the axillary buds, and then these are divided and each is regenerated as an individual. There is no known method for obtaining a large number of seedlings.

(/→ Purpose and composition of the invention As a result of intensive studies on the conditions for multi-bud formation and the conditions for regeneration of seedlings from divided buds of the Dioscoreaceae family, the inventors found that when cultured in a medium containing 10 M or more of cytokinin, The present invention has been completed based on the discovery that a high rate of multi-bud formation can be obtained and that the split buds can be efficiently regenerated into seedlings by culturing in a medium that does not contain plant growth hormone or has a low concentration of plant growth hormone.

The present invention involves forming multiple buds by aseptically culturing the axillary buds or apical buds of Japanese potato on a medium, and then dividing these multiple buds and transplanting the individual buds onto the medium. Provided are a method for producing young plants of Dioscoreaceae, a plant belonging to the Dioscoreaceae family, which is characterized by obtaining young plants, and a method for propagating the young plants, which is characterized by growing these young plants.

Regarding the method of carrying out the present invention, (1) growing plants to be used as materials, (2) collecting and sterilizing axillary buds and apical buds, (3) inducing formation of multiple buds, (4) dividing/transplanting multiple buds, ( 5) Acclimation of young plants and planting, which are divided into the above five stages, will be described in detail below in order.

(1) Cultivation of plants used as materials Examples of the Dioscoreaceae plant to which the present invention is applied include long yam, ginkgo yam, Tsukune yam, and yam yam. These seed potatoes are cut, sterilized, and sprouted using conventional methods, and then planted in pots or fields.

Of course, it is also possible to use Mukago.

In the case of normal cultivation, the size of the stamp section is 100 to 250y.
However, since the purpose of this method is to collect vines, 50 to 100 y per piece is sufficient. In the case of Nagaimo, the size of the seed potato varies depending on the variety, but in general, the size of the seed potato is 1000f or more for Nagaimo, and 5000F for Tsukuneimo.
It has some weight. Therefore, a 10 to 20-fold multiplication is achieved by cutting seed potatoes using the conventional method (first stage multiplication).

It is desirable to disinfect the cut end of the stamp section immediately after cutting. As a disinfectant, benlate, thiuram, cabtan, or oatside is used, and hydrating agent immersion treatment or powder coating treatment is performed, but as a simple method, slaked lime powder coating is also effective. After disinfection, store the cut sections at room temperature and in the dark.
It is desirable to heal the cut end and encourage germination. Usually, in about a month, sprouts the size of adzuki beans are formed on the surface of the cuttings, and these are planted in pots or fields. In the case of this method,
Is the nutrients in the cut section sufficient? - Because the vines elongate,
Just use ordinary soil. After sprouting above ground,
By erecting a support and attracting vines, it is possible to promote the elongation of the vines and the flourishing of the stems and leaves. When the vine droops, the growing points of the leaf sap tend to form bulrushes, and such axillary buds are not suitable for this method, so care must be taken in cultivation management.

(2) Collection and sterilization of axillary buds and apical buds Cut the vines that have reached a height of 1 to 2 m and collect the axillary buds and apical buds. In general, apical dominance is observed in Dioscoreaceae plants,
While the apical bud is healthy, the axillary or lateral buds do not elongate and only remain in the leaf sap region. When cutting the gong, rather than cutting off the entire above-ground part, by leaving some of the axillary buds, the vines will grow from that part after cutting, and the axillary buds can be harvested again.

This makes it possible to usually collect 200 to 300 axillary buds from one potato section (second stage multiplication).

The collected vines are separated into axillary buds in the laboratory. The most desirable form is to cut the axillary buds leaving about 51 cm above and below the stem, and also leave about 5 cm of the petiole. Such a plant has a suitable size and is easy to handle as an explant. It is desirable to sterilize the collected axillary buds before culturing. The sterilization method usually involves immersing the product in approximately 1% sodium hypochlorite solution for 5 to 10 minutes, followed by washing with sterile water 8 to 5 times under aseptic conditions. By this treatment, the contamination rate of various bacteria can be suppressed to 5% or less per number of axillary buds placed on the bed.

(3) Induction of multiple bud formation The sterilized axillary buds are placed on a suitable plant tissue culture medium under sterile conditions. The medium was L8 (Linsmaier &
8koog 1964) or MS (Murash
A commercially available plant tissue culture medium, such as G. ige & Skoog 1962), is sufficient, containing sucrose at a concentration of 1-5%, preferably 2-4% and 0.5-1.5%, preferably 0.8%. Agar added with a concentration of ~1.2% is used. The most important factor for forming multiple buds is the addition of cytokinins, which are plant growth hormones. As a cytokinin, kinetin 6
Commercially available products such as -benzylaminopurine (BA) or 2-isopentenyladenine (2iP) can be used. The added concentration of cytokinins effective for multi-bud formation is 10 M or more, preferably 10 to 10 M.
M @ degree is suitable. The incubator can be of any shape, such as a test tube or Erlenmeyer flask, but the incubator must have a diameter of 60 mm.
A trowel is also suitable for work efficiency, such as a plastic petri dish with a size of about 10 cm and a culture medium of about 10 mm. As for the culture conditions, the temperature is 20 to 27 degrees Celsius, preferably 23 to 27 degrees Celsius.
is suitable, and the photoperiod is 1000-10000lux,
Preferably, conditions of 2000 to 5000 lux and 12 to 24 hours are suitable.

When cultured under the above conditions for 3 to 4 weeks, the growing points of the axillary buds or apical buds placed on the bed become enlarged, and it is observed that many new growing points are formed at the base of each leaf primordium. After 7 to 8 weeks of culture, each growing point grows as an independent bud with several pairs of leaf primordia, and appears as multiple buds formed in a single leaf sap region. The number of buds formed per leaf sap is usually 10.
~40 pieces. (Third stage proliferation).

(4) Division and transplantation of multiple buds The axillary buds on which multiple buds have been formed are removed from the medium, and individual buds are divided using a scalpel or the like under sterile conditions. As long as each bud has one growing point and at least 2 to 3 pairs of leaf primordia, there will be no problem with subsequent growth. These buds are transplanted onto an agar medium to develop stems and leaves and root. As in (3), a commercially available plant tissue culture medium is sufficient as the medium, but in this case no plant growth hormones are added, or cytokinins and auxins are added at a low concentration of 10 M or less.

As auxin, IAA (β-indole acetic acid)
, NAA (α-naphthalene i[) or 2.4-D (
2,4-dichlorophenoxyacetic acid), etc. Culture conditions are the same as described above.

Approximately half of these divided buds can be cultured on the same medium for 1 to 2 months to regenerate as seedlings each with 1 to 2 expanded leaves and 1 to 2 roots. It is possible.

(5) Acclimatization and planting of seedlings The seedlings with regenerated stems, leaves and roots are transferred to germ-free conditions, acclimated to the outside environment, and grown as healthy seedlings. Once at least one adult leaf has developed and one root with a length of 1 to 2 leaps has been regenerated, the plant can be transferred to the outside environment as a young plant. Acclimation is
It is advisable to thoroughly wash the agar off the roots of the seedlings and transplant them into a suitable potting soil with good drainage, such as vermiculite, peat moss, or perlite, and keep it moist. Plants take root.

The seedlings that have taken root on the soil will develop new stems and leaves in about two weeks and can be transplanted into the soil.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 The axillary buds of Nagaimo cultivar Gankumijika produced in Stomori Prefecture were collected, sterilized by immersion in a 1% sodium hypochlorite solution for 10 minutes, washed eight times with sterile water, and cultured according to the following conditions.

The medium contains 8% sila sugar and 1% agar I) H5,8
The MS medium adjusted to 100 ml was dispensed into mayonnaise bottles with a diameter of 40 days and a height of 30 m. Plant growth hormone contains BA as cytokinin without additives and 1
A total of 2 x 8 = 6 concentration combinations were provided: 2 groups with 0 M addition, and 8 groups with no NAA addition, 10 -6 M addition, and 10 Mm addition as auxin. Two culture bottles and six axillary buds were placed in each hormone concentration zone, 20"C, 2
The cells were cultured for 2 months under 000lux 12 hour illumination.

Table 1 Shape of axillary buds of Nagaimo cultivar Gankumijika after 2 months of culture 0 Stem and leaf elongation/rooting Rooting
Callus formation IO multi-bud formation Multi-bud formation Callus formation As seen in Table 1, stem elongation and rooting from axillary buds occurred easily in the hormone-free medium, but in
Apical dominance was maintained even under vitro culture conditions, with only one stem elongating. When BA, a cytokinin, was added to the medium, the elongation of the apical bud within this axillary bud was suppressed, and new growth points were formed at the base of the leaf primordium. Addition of auxin NAA had no effect, and at high concentrations, callus formation was observed, browning of the medium was also significant, and growth was inhibited.

Example 2 Axillary buds were collected from two Japanese potato cultivars Gankumijika and Torakuri produced in Aomori Prefecture, sterilized by immersion in a 1% sodium hypochlorite solution for 10 minutes, washed three times with sterile water, and cultured according to the following conditions. The medium is 396 sulfur sugar and 1% agar.
60m5 in diameter containing MS medium adjusted to pH 5.
．． The sample was dispensed into a plastic Petri dish with a height of 10 m and used. As for the plant growth hormone, 10 M BA was added as cytokinin. Three to four axillary buds were placed in each petri dish and cultured for 2 months under 25"C, 2000 lux, 24 hour continuous lighting.

Table 2 Nagaimo varieties Gankumijika and Trakuri variety name Number of buds placed in bed 0-1010-2020-308()-
Cut-average bud formation number Ganku water deer 112 34
42 30 6 16 Totsukuri
84 18 10 9 2
15 As seen in Table 2, multiple bud formation was easy, high, and stable. In addition, the cultivar's opening size is not very large, and it is possible to divide each axillary bud into an average of 15 to 16 independent buds after two months of culture.

Example 8 Eight axillary buds of the Japanese potato cultivar Gankumijika were sterilized and cultured according to Example 2 to obtain multiple buds.

The formed buds were divided and each was transplanted into a test tube with a diameter of 18 nods and a length of 130 days, in which 10-M8 hormone-free agar medium was dispensed in the form of a slant. I cultivated my strength for months. The seedlings that showed regeneration of stems and leaves and rooting were taken out from the test tubes and transplanted into plastic cups with a diameter of 55 cm and a height of 60 cm containing about 100 ml of culture soil, and then placed at 25"C.

Acclimatization was performed for one month under continuous lighting for 24 hours.

Table 3 Multiplication of Nagaimo cultivar Gankumijika by multi-bud division $1 40 19
18$2 27 14
12 As shown in Table 3, the multiple buds formed from the axillary buds can be divided into 30 to 40 pieces, and about half of them can be regenerated as seedlings, and the growth after acclimatization is was also going well.

2) Effect of the invention The multiplication rate of Japanese potato by this method is 3rd stage multiplication rate 10
It is possible to achieve a cumulative growth rate of at least 20,000 times, with a second stage growth rate of 200 times and a third stage growth rate of 10 times. In addition, the propagation process by this method uses only the plant's inherent ability to form growing points, and unlike methods that use callus-derived somatic embryos, it is a method that can minimize genetic mutations. be. Therefore, by this method, it is possible to propagate a large amount of specific individuals or strains in a genetically uniform state in a short period of time, such as virus-free disease-free or selected superior strains.

Claims (8)

[Claims] (1) After forming multiple buds by aseptically culturing the axillary buds or apical buds of Dioscorea yam on a medium,
A method for producing young plants of Dioscoreaceae, a plant belonging to the family Dioscoreaceae, which comprises dividing these multiple buds and cultivating the individual buds in a culture medium to obtain a large number of young plants. (2) Cytokinin from 10^-^4 to 10^-^6M
2. The method according to claim 1, which comprises culturing using the added medium to form multiple buds. (3) 20-30℃, photoperiod 1000-10000lux
3. The method according to claim 2, wherein the method is cultured for 12 to 24 hours to form multiple buds. (4) Claims 2 or 3, characterized in that seedlings are obtained by culturing using a medium without the addition of plant hormones or with addition of cytokinin and auxin at a concentration of 10^-^7M or less. the method of. (5) After forming multiple buds by aseptically culturing the axillary buds or apical buds of Dioscorea yam on a medium,
A method for propagating yam, a plant belonging to the Dioscoreaceae family, characterized by dividing these multiple buds and culturing individual buds in a medium to obtain a large number of seedlings, which are then grown and regenerated as complete plants. . (6) Cytokinin from 10^-^4 to 10^-^6M
6. The method according to claim 5, which comprises culturing using the added medium to form multiple buds. (7) 20-30℃, photoperiod 1000-10000lux
7. The method according to claim 6, wherein the method is cultured for 12 to 24 hours to form multiple buds. (8) Claims 6 or 7, characterized in that seedlings are obtained by culturing using a medium without the addition of plant hormones or with addition of cytokinin and auxin at a concentration of 10^-^7M or less. the method of.