JP2005130813A - Method for producing latex by tissue culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce latex by a tissue culture method independent of the growth of a gum tree and stably produce natural rubber in high efficiency. <P>SOLUTION: An adventitious root or a sterile root derived from the tissue culture product of a gum tree is mass-cultured in a liquid to achieve the mass-culture of latex. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention belongs to the field of substance production technology by plant tissue culture technology, in particular, rubber tree tissue culture.

  Natural rubber is manufactured from latex called rubber latex. As rubber tree cultivated for the purpose of industrial rubber raw material, Euphorbiaceae para rubber tree, Havea brasiliensis, is particularly important. Indian rubber tree (Ficus elastica).

  Latex is present in the ducts within the bark, and the ducts develop several layers a year outside the formation layer. If a groove is scratched with a knife, milky latex will flow out. At present, a groove is manually formed by a knife so that the trunk of the rubber tree bark is half-turned diagonally, and a latex collector is provided at the bottom of the groove to collect the latex.

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

  Up to now, there have been attempts to produce latex by cell culture, but none of them can be used industrially (Patent Document 1).

  In addition, about 35% of the rubber component in the latex produced by Para rubber tree, there are many 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.

JP-A-60-27392.

  Conventional production of natural rubber began with the cultivation of rubber trees, and since it was grown, latex was collected by hand. (1) After making the production plan, there was a period of time until latex could actually be produced. Too much (2) Latex (rubber) collection must be done manually, but in Southeast Asia, the main rubber cultivation area, labor shortage is expected due to industrialization. Development of a new production method for stable and efficient rubber is desired.

  Therefore, the present invention has an object to produce latex in a tissue culture method that is not influenced by the growth of rubber tree, and to achieve an efficient and stable production of natural rubber.

  Furthermore, it is also an object to produce a useful material such as rubber, protein, sugar, and tocotrienol in latex by mass-producing para rubber tree tissue in a relatively short time by producing latex in tissue culture.

  The present inventors have found the conditions for producing adventitious roots after tissue culture of rubber tree, and established the basis of a large-scale culture method of latex by culturing adventitious roots in a large amount in liquid. Furthermore, the present inventors have repeated trial and error, succeeded in culturing immature embryos and inducing sterile roots.

  According to the present invention, adventitious roots or aseptic roots can be prepared systematically in a large amount and in a short period of time in a systematic manner. Thereby, conventionally, it has become possible to extract latex, which has been collected from rubber trees, manually from adventitious or aseptic roots cultured in large quantities. Furthermore, useful substances such as proteins, sugars, and tocotrienols can be selectively and efficiently obtained by separating rubber components from latex produced by tissue culture by a technique such as centrifugation.

  The present invention relates to a method for producing latex by tissue culture of rubber tree, generating adventitious roots, and culturing adventitious roots in large quantities. Furthermore, the present invention relates to a method for producing latex by tissue-culturing immature rubber tree embryos, inducing sterile roots, and culturing a large amount of sterile roots.

  Particularly preferred examples of the rubber tree to be subjected to tissue culture include para rubber tree. Chicle.

  Hereinafter, para rubber trees will be described as an example, but other rubber trees can also be implemented by the same method.

  As materials used for tissue culture, various parts such as para rubber tree roots, stems, leaves or flowers can be cut into appropriate sizes, but preferably buds, immature fruits or stems are used. The cocoon taken out from the bud, the immature seed taken out from the immature fruit, or the formed layer taken out from the stem can be used as the material.

  First, the surface of the para rubber tree material is washed. When using the inside of a plant as a material, for example, it may be washed with a scouring powder, but it can also be washed with water containing about 0.1% of a surfactant. When using leaves, wash the surface with a soft sponge.

  The material is then sterilized or sterilized. Sterilization or sterilization can be performed using a well-known disinfectant / sterilant, and ethanol, benzaconium hydrochloride, and sodium hypochlorite aqueous solution are preferable.

  Callus can be derived from material taken from para rubber tree, adventitious roots can be derived from callus, or sterile roots can be derived directly from material collected from para rubber.

I. Method for Inducing Callus and Inducing Adventitious Roots from Callus Preferably, cocoons, immature seeds or formed layers are used as the material. To induce callus, cytokinin plant hormones and / or auxin plant hormones are added to the medium at a low concentration. Medium includes White medium, Heller medium, SH medium (Schenk and Hildebrandt medium), MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), Gamborg, B5, MB medium, etc. However, an MS medium or an MS modified medium obtained by changing the composition thereof is preferable.

  As the cytokinin plant hormone, BAP (benzylaminopurine), 2iP (isopentinylaminopurine), kinetin (6-furfurylaminopurine), and zeatin can be used. Preferably, BAP (benzylaminopurine) is used. Purine) or KIN (Kinetin) can be used at 1 to 3 ppm.

  As auxin plant hormones, pCPA (chlorophenoxyacetic acid), 2,4-D (dichlorophenoxyacetic acid), IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), NOA (naphthoxyacetic acid), suitable For example, 2,4-D (dichlorophenoxyacetic acid) can be used at 0.3 to 1.0 ppm, IAA (indoleacetic acid) at about 1 ppm, and NAA (naphthaleneacetic acid) at about 1 ppm.

  More preferably, a cytokinin plant hormone and an auxin plant hormone can be used in combination. For example, BAP or KIN, 2,4-D, and IAA or NAA can be used in combination.

  Furthermore, in order to promote callus induction, sucrose may be added at a high concentration of 6 to 7 wt%, and coconut water may be added. Preferably, the pH is adjusted to 5.6 to 5.8, the culture temperature is 26 to 28 ° C., and callus is induced in the dark. As the callus support, gellite or agar can be used.

  The induced callus is cultured in a dark medium at the same composition for 1 to 2 months at a culture temperature of 26 to 28 ° C. to grow the callus. Adventitious roots are induced from the grown callus. For the induction of adventitious roots, a medium in which 3 to 10 ppm of IBA (indolebutyric acid) is added to the MS basic medium is preferably used. More preferably, 6-7 wt% of sucrose is added. Auxins such as IAA and NAA may or may not be added.

2. Induction of sterile roots from immature embryos Preferably, an immature green fruit at a stage prior to hardening of the pericarp is utilized. The surface of the immature fruit is sterilized with well-known sterilizing agents such as ethanol, benzaconium hydrochloride, sodium hypochlorite, preferably ethanol. After sterilization, the seeds inside the fruit are removed. The part that grows into an immature seed embryo is removed.

  The extracted immature embryo is grown in a medium. Examples of the medium include a White medium, a Heller medium, an SH medium, an MS medium, an LS medium, and the like, and an MS medium or an MS modified medium obtained by changing the composition thereof is preferable.

  By adding sucrose from a relatively grown immature embryo based on the MS basic medium, roots can be induced without being significantly affected by the hormone species.

3. Mass growth of roots Or 2. The adventitious or sterile roots induced in step 1 are cut into small pieces having an appropriate size, preferably 2 to 3 cm square. An appropriate medium, for example, White medium, Heller medium, SH medium, MS medium, LS medium, or MB medium can be used. Preferably, MS medium is used, and 3 to 10 ppm of IBA is added to the medium. , The root can be propagated by culturing at 26 to 28 degrees. The medium may be solid or liquid, but preferably shake tow culture is performed using the liquid medium. A jar fermenter can also be used for liquid culture. By culturing in liquid, more efficient growth is possible and the transplantation operation is facilitated.

4). Confirmation and Extraction of Latex from Roots When a root culture that has undergone rapid growth is cut, latex is precipitated and can be visually confirmed.

  Furthermore, the rubber component was confirmed by analyzing a small amount of latex by means of pyrolysis GC / MS or FT-IR.

  Further, by extracting latex from a large amount of grown roots, and further separating the rubber component by a technique such as centrifugation, proteins, sugars (sucrose, kebraquitol that can be used as a humectant, myo-inositol, etc.), Useful substances such as tocotrienols and amino acids can be selectively and efficiently obtained.

Method for producing latex by tissue culture (1) <Method for obtaining adventitious roots from calli, immature seeds, and formation layers via callus>
Male flower buds used as explants were collected from para rubber trees, disinfected with diluted sodium hypochlorite solution, opened and removed. Immature seeds were collected from soft fruits with a green outer skin. In the formation layer, young branches were collected, and the surface was washed with ethanol and then sampled.

  For callus induction, add 1 to 3 ppm of BAP or KIN, 0.3 to 1 ppm of 2,4-D, and 1 ppm of NAA or IAA to the MS basic medium, and then add 6 to 7% sucrose and about 5% coconut. The medium to which water was added was prepared. The pH was adjusted to 5.6 to 5.8, and gellite or agar powder was used as the support.

Growth of callus was confirmed by culturing for 1-2 months in the dark at 27-28 ° C.
The grown callus was transplanted to a medium having the same composition as the callus induction, in which only the hormone composition was changed to 3 to 10 ppm. Auxins such as IAA and NAA may or may not be added. Adventitious roots could be induced by adding IBA and culturing for 1 to several months in an environment of 27 to 28 ° C. irradiated with light for 16 hours. The formed root is shown in FIG.

(2) <Method for inducing sterile roots from immature embryos>
Sampling relatively rooted immature embryos from seeds and cultivating them in a medium of MS basal medium with 6-7% sucrose to induce sterile roots without being significantly affected by hormonal species I was able to.

(3) <Root mass growth>
The roots obtained by the above cultures (1) and (2) were cut into small pieces of 2 to 3 cm and the roots were grown on a medium supplemented with 3 to 10 ppm of IBA. Good results were obtained with either solid or liquid medium. (See Figure 2.)

(4) <Confirmation of latex in cultured roots>
Latex leaching was visually confirmed in the culture of roots that had been proliferated by cutting during transplantation. In order to further analyze the composition, Soxhlet extraction was performed on the cultured roots with an organic solvent (such as chloroform), and the extracted portion was analyzed by techniques such as pyrolysis GC / MS, FT-IR, and NMR.

  For example, when CDC13 was measured by NMR using a solvent as a solvent, high peaks were observed at positions of 135.24 ppm, 125.08 ppm, 32.25 ppm, 26.44 ppm, and 23.44 ppm, and in addition, 128.0 ppm, 120 Low peaks were observed at positions of .4 ppm, 29.7 ppm, 22.7 ppm and 14.1 ppm.

  As a result, it was confirmed that cis polyisoprene, which is a main component of natural rubber, was present in large quantities, and useful components such as sugars, amino acids, proteins, and higher fatty acids were identified. (See Fig. 3 NMR spectrum)

Callus rooting in IBA-containing medium Elongated cultured root 13C-NMR spectrum of an extract extracted from cultured roots with chloroform

Claims (10)

  A method for producing latex, comprising culturing adventitious or sterile roots derived from a rubber tree tissue culture.   The method according to claim 1, wherein the adventitious root derived from a rubber tree tissue culture is an adventitious root from callus derived from an anther, an immature seed or a formation layer of the rubber tree.   The method according to claim 1, wherein sterile roots are derived from a tissue culture of immature embryos of rubber tree.   The method according to any one of claims 1 to 3, wherein IBA (indolebutyric acid) is used to induce adventitious or sterile roots.   The method according to claim 4, wherein the concentration of IBA (indolebutyric acid) added is 3 to 10 ppm.   The method according to any one of claims 1 to 3, wherein sucrose is used to induce adventitious or sterile roots.   The method according to claim 6, wherein the addition concentration of sucrose is 6 to 7 wt%.   The method according to any one of claims 1 to 7, wherein the rubber tree is a para rubber tree.   A method for producing useful components contained in latex, comprising collecting useful components from latex produced by culturing adventitious or sterile roots derived from a tissue culture of rubber tree. Adventitious or aseptic root cultures derived from rubber tree tissue cultures.

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