JP4523769B2 - Mass propagation method of fine tree of Para rubber tree by tissue culture - Google Patents

Description

  The present invention belongs to the technical field of mass growth of para rubber tree.

  Natural rubber is manufactured from latex called rubber latex. As rubber tree cultivated for the purpose of industrial rubber raw material, the rubber tree of the family Euphorbiaceae, Havea brasiliensis, is particularly important, but as rubber tree that produces latex, there are other rubber tree (Manihot glaziovii), mulberry plant Indian rubber tree (Ficus elastica).

  It takes 5 to 7 years from planting rubber tree seedlings to growing latex to the extent that it can be collected, and the collection period is approximately 20 to 30 years.

  In addition, about 35% of the rubber component in the latex produced by Para rubber tree, a number of useful substances such as protein, inositol, saccharides such as ketebraquitol, a kind of vitamin E and tocotrienol, which is also effective as a natural anti-aging agent It is included.

  By the way, para rubber trees are difficult to grow by cutting, and as a method of clonal propagation of specific varieties of para rubber trees, seedlings are grown by sowing, and are used as rootstocks at a certain stage of growth, and buds obtained from cloned seedlings are used. There is a method of growing by using a sprout method that sproutes the rootstock onto this rootstock (Non-patent Document 1). However, such a sprout method makes it difficult to obtain true clone seedlings due to the heterogeneity of rootstock seedlings, and there is a limit to their growth.

Menara Perkebunan 1998 Vol.66, No.1 p.1-8

  In order to increase the production of useful substances derived from latex or other rubber tree, and to improve the environmental adaptability of rubber tree, it is desired to further improve the characteristics and characteristics of rubber tree. Recently, breeding of excellent varieties by genetic engineering breeding has been attempted.

  By the way, as for para rubber tree, even if excellent varieties have been developed through breeding, at present, para rubber cannot be propagated by grafting. Therefore, seedlings are nurtured by sowing, and are grown as rootstocks when they are grown to some extent, and the shoots obtained from the cloned seedlings are proliferated by sprouted onto the rootstocks. However, there is a limit to the number of shoots obtained from cloned seedlings, and in order to rapidly disseminate excellent varieties, it is desired to develop a technique for mass propagation of excellent varieties of cloned seedlings.

  The inventors of the present invention collected a tissue containing the top buds or side buds of Para rubber tree clone seedlings, shot them with a shoot elongation induction medium, cut the shoots, or repeated divisional growth and transplanted the shoots to the root induction medium. The rooted shoots were found to be complete plants and can be used as cloned seedlings.

  On the other hand, the tissue from which the shoot is removed is returned to the shoot elongation induction medium again, the shoot is elongated, and the shoot portion is cut out again. It has been found that by repeating this process as many times as possible, it is possible to produce a large amount of clone seedlings of excellent varieties, and the present invention has been completed.

  According to the present invention, a tissue containing the top buds or side buds of Para rubber tree clone seedlings is collected. By collecting shoots from the tissue as many times as desired, excellent varieties of cloned seedlings can be produced in large quantities.

  The present invention can be used for sprouts by introducing a shoot that has been induced to elongate from a tissue containing rubber tree apical buds or side buds, and then excising the shoots and rooting them with a root induction medium. In addition, the tissue from which the shoot part has been cut can be re-extended with the shoot elongation induction medium again, and the sprout for sprout can be collected a suitable number of times.

Particularly preferred examples of the rubber tree used in the present invention include para rubber tree, but besides these, there are ceara rubber tree, indian rubber tree, panama rubber tree, guayule rubber tree, rubber dandelion, lagos rubber tree, gutta pergano tree, parata rubber tree, cis-chickle. Can be mentioned.
Hereinafter, para rubber trees will be described as an example, but other rubber trees can also be implemented by the same method.

1. Tissue to be used (step of collecting tissue containing apical or side buds)
As a material used for propagation, cloned seedlings grown in a place isolated from the outside can be used to prevent environmental pollution. A tissue containing the top buds or side buds of the grown clonal seedling can be collected and cut into an appropriate size for use.

  First, the surface of the arachis clone seedling is washed. Next, the tissue including the top buds or side buds of the collected clone seedlings is sterilized or sterilized. Sterilization or sterilization can be performed using a known disinfectant / sterilant, but ethanol, benzaconium hydrochloride, and sodium hypochlorite aqueous solution are preferable, and particularly preferably, the surface is first cleaned with ethanol. Then sterilize with diluted sodium hypochlorite solution with stirring.

2. Stretching step of the shoot The tissue containing the sterilized or sterilized apical buds or side buds is cut at the cut end in order to remove the influence of the sterilizing agent, and the portion containing the shoots is inserted into the solid medium to extend the shoot. As the medium for shoot elongation, BAP (benzylaminopurine) can be added to a suitable solid medium for plant growth as a plant hormone so as to be 0.5 to 3.0 ppm. Examples of the medium include White medium, Heller medium, SH medium (Schenk and Hildebrandt medium), MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), Gamborg, B5, etc. MS medium or its modified or MS modified medium is preferred. Moreover, as a carbon source, it is suitable to add and use sucrose so that it may become 20-40 g / l. As the solidifying agent, gellite or powder agar can be used. 5 wt. % Coconut water can also be added.

More preferably, TDZ (thidiazuron) is added in an amount of 1.0 to 5.0 ppm, or cultivated in a combination of 1.0 to 5.0 ppm of TDZ (thiazurone) and 1 ppm of NAA (naphthalene acetic acid) to obtain a multi-bud. it can.
The culture for shoot elongation is preferably performed under light irradiation conditions of 27 ° C. and 16 hours per day, and if necessary, it is cultured about once a month.

3. Rooting step and transplanting step In the above-mentioned medium, culture is performed for about 2 to 3 months, and shoots grown to an appropriate size of about 2 to 3 cm are collected. The collected shoots are rooted with a rooting induction medium.

  The rooting induction medium can be used by adding a plant hormone to an appropriate plant growth medium. Examples of the medium include White medium, Heller medium, SH medium (Schenk and Hildebrandt medium), MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), Gamborg, B5, etc. MS medium or its modified or MS modified medium is preferred. As the carbon source, it is preferable to add sucrose so as to be 30 g / l.

  BAP (benzylaminopurine), 2iP (isopentinylaminopurine), kinetin (6-furfurylaminopurine), zeatin can be used as the cytokinin plant hormone.

  Auxin plant hormones include pCPA (chlorophenoxyacetic acid), 2,4-D (dichlorophenoxyacetic acid), IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), NOA (naphthoxyacetic acid), It can be used at about 1 ppm.

Preferably, as the plant hormone to be added, IBA (indolebutyric acid) 3 to 10 ppm and NAA (naphthalene acetic acid) 0 to 5 ppm are used in combination. As the solidifying agent, gellite or powder agar can be used. 5 wt. % Coconut water can also be added.
When roots are induced in 1 to several months and become complete plants, they are transplanted from the rooting induction medium to the culture soil.

  Process of changing humidity and temperature conditions (acclimation process) to adapt to the external environment in order to shift from culture under controlled conditions (humidity 100%, temperature 28-30 ° C) to growth in the external environment ), A healthy seedling can be obtained.

[Example 1]
(1) The surface of a Para rubber tree clone seedling (grown in a managed field) is washed. Next, the tissue containing the top buds or side buds of the collected clone seedlings is first surface-washed with ethanol, and then sterilized with stirring with a diluted sodium hypochlorite solution.
(2) The tissue containing sterilized apical buds or side buds is cut at the cut end, and the portion containing the buds is inserted into a solid medium and stretched. As a medium for this shoot extension, MS (Murashige scoog) mixed medium is basically added as a plant hormone, BAP (benzylaminopurine) is 2.0 ppm, and as a carbon source, sucrose is added at 30 g / l, As a solidifying agent, gellite is used and 5 wt. Add% coconut water.
(3) In order to obtain multi-buds, as a plant hormone, NAA (naphthalene acetic acid) 1 ppm instead of BAP (benzylaminopurine) and TDZ (thiazuron) are cultured in a combination of 3 ppm.
(4) Culture for shoot elongation is carried out approximately every month under light irradiation conditions of 27 ° C. and 16 hours per day to extend shoots. Elongated shoots can be divided into two or three parts, and can be proliferated by transplanting each. (FIG. 1).
(5) Collect shoots that were subcultured and grew to an average size of 2-3 cm. The collected shoots are rooted with a rooting induction medium. As rooting induction medium, sucrose is added to MS medium (Murashige and Skoog mixed medium) to 30 g / l, and plant hormones are combined with 5 ppm of IBA (indolebutyric acid) and 3 ppm of NAA (naphthaleneacetic acid). Use. As a solidifying agent, gellite is used, and 5 wt. Add% coconut water. (FIG. 2).
(6) A root is induced in one month to several months, and a complete plant body is obtained. Therefore, the rooting induction medium is transplanted to the culture soil. In order to shift from a controlled culture room (humidity 100%, temperature 28-30 ° C.) to growth in the external environment, acclimatize to 80% humidity for 2 weeks and 60% weekly in order to adapt to the external environment. Get healthy seedlings grown from cloned seedlings.

  The method of the present invention can be used for large-scale propagation and breeding of an elite tree of rubber tree.

(A) A shoot elongated in a shoot elongation medium, (B) A shoot transplanted in two. Plants that have been regenerated through transplanting the elongated shoots to the rooting medium, rooting, and root elongation.

Claims (7)

After the step of obtaining a tissue containing the apical or Wakime from clone seedlings Hevea, the step of stretching the chute chute extension medium supplemented with BAP (benzylaminopurine) from the tissue, and the chute taken, the tissue the comprises the step of obtaining the multiple shoots with multiple shoots elongation medium supplemented with TDZ (thidiazuron) added or TDZ and NAA (the naphthalene acetic acid), mass propagation method clone seedlings Hevea. After harvesting or after harvesting and divided growing shoots, further mass propagation method clone seedlings Hevea of claim 1 including the step of rooting in root inducing medium. Rooted shoots were transplanted into culture soil, after acclimatization, mass propagation method clone seedlings Hevea according to claim 2, further comprising the step of a complete seedlings. Mass propagation method clone seedlings Hevea of claims 1 to 3 any one of claims containing solid medium further sucrose used in the stretch-induced shoot. Root inducing medium, IBA (indole butyric acid) 3～10Ppm containing the claim 2-4 mass propagation method clone seedlings Hevea according to any one. The method for mass-proliferating a cloned seedling of Para rubber tree according to any one of claims 2 to 5, wherein the rooting induction medium contains IBA and NAA. The method for mass-growing para rubber tree clone seedlings according to any one of claims 1 to 6, comprising dividing the collected shoots and further planting them.

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