JPH06104062B2 - Method for producing useful substances using hairy roots - Google Patents

Description

Detailed Description of the Invention

[0001]

The present invention relates to Agrobacterium
The present invention relates to a method for producing a pigment using a hairy root induced by Rhizogenes.

[0002]

2. Description of the Related Art When plants are used to produce useful substances such as pharmaceuticals, ordinary plants are usually used. Recently, however, they have a significantly faster growth rate than those plants (hence,
The movement to utilize hairy roots, which has a high production rate of substances, is active.

As a method for industrially producing a secondary metabolite of a plant by a plant cell / tissue culture method, the plant has a root bacterium, Agrobacterium rhizogenes.
It is known to inoculate the hairy roots and culture the growing hairy roots. This method is based on the following principle. That is, Agrobacterium rhizogenes
When inoculated on leaves and roots, roots called hairy roots develop from the infected site. This root is generated when a part of the gene of the giant plasmid (R _i plasmid) existing in Rhizogenes is integrated into the plant gene. Then, by establishing hairy root clones and selecting high-producing strains, efficient production of secondary metabolites becomes possible. Therefore, by utilizing this property, Agrobacterium rhizogenes is inoculated into a plant containing a useful substance in the root, the generated hairy root is cut out and cultured in a device such as a tank,
The useful hairy substance can be taken out by destroying the grown hairy root.

[0004]

However, in this method, useful substances are often stored in the tissues and cells of hairy roots, and the hairy roots after culturing are separated and collected from the culture solution to obtain organic, It is necessary to repeat the extraction with an inorganic solvent.

Therefore, the operation of separating and extracting the useful substance from the cells of the hairy root is complicated and the manufacturing cost is increased. Further, it is difficult to continuously remove the growing hairy roots from the culture tank, and only batch production can be performed. Therefore, the hairy root once subjected to the extraction operation cannot be used for the culture again, and it is necessary to select the hairy root clone again.

An object of the present invention is to provide a method for producing a useful substance using hairy roots, which enables continuous and efficient separation and extraction of pigments from hairy root cells derived from edible beets. That is.

[0007]

According to the present invention, an edible beet plant is transformed with an R _i plasmid carried by Agrobacterium rhizogenes, and the hairy roots produced by this transformation are cultured in a liquid medium. Then, the oxygen concentration in the liquid medium is reduced to release the betanin pigment and the burgaxanthin pigment from the hairy root into the liquid medium, which relates to a method for producing a useful substance using the hairy root. .

[0008]

The present inventor has reached the present invention by conducting extensive research on the culture of hairy roots induced by the R _i plasmid carried by Agrobacterium rhizogenes.

That is, the present inventors have found that the dissolved oxygen concentration in the liquid medium has a very close relationship with the state of hairy roots. Specifically, when hairy roots are grown in a liquid medium and cultured, when the dissolved oxygen concentration in the liquid medium is lowered, useful substances are released from the hairy root cells into the liquid medium, and the dissolved oxygen concentration is again detected. It was found that the hairy roots started to grow again when they were raised. Therefore, the useful substance can be easily extracted by removing the liquid medium after the useful substance is released. Further, an extraction operation for releasing useful substances from the hairy roots into the liquid medium by sequentially increasing and decreasing the dissolved oxygen concentration in the liquid medium, and a culture operation for culturing the hairy roots in the liquid medium. It can be performed sequentially and continuously. Therefore, the useful substance can be continuously produced by repeating the method of performing the extraction operation, replacing the liquid medium, and performing the culture operation.

[0010]

Examples The following are examples of combinations of useful substances obtained from plant bodies to be transformed with the R _i plasmid carried by Agrobacterium rhizogenes and hairy roots derived therefrom.

Plant: Edible beet (Detroit dark red) Useful substances: Betanin, Bulgaxanthin

Examples of Agrobacterium rhizogenes include the following. Agrobacterium rhizogenes 25818 (ATCC 25818) Agrobacterium rhizogenes 15834 (ATCC 15834) Agrobacterium rhizogenes 8196 Agrobacterium rhizogenes A4 (ATCC 43057) Bacteria in which a partial T-DNA is introduced can also be used.

When a plant is treated with Agrobacterium rhizogenes, a part of the Ri plasmid (T-DNA) in Rhizogenes is introduced (transformed) into the nuclear DNA of the plant cell.

As a method for obtaining transformed hairy roots by introducing Ri plasmid T-DNA into the stems, roots, leaves, etc. of plants, the following method can be mentioned, for example.

1. Direct inoculation method for plant individuals 2. Leaf disk method using leaf pieces (RB Horsch et a
L., SCIENCE 227. 1229 (1985)) 3. Co-culture method using plant protoplasts (Z.
M. Wei et al., Plant Cell Rep., 5; 93-96 (198)
6)) 4. Protoplasts of plants and spheroplast method of Agrobacterium rhizogenes (R. Hain et al., Plan
t Cell Rep., 3, 60 (1984)) 5. A method of directly injecting Ri plasmid of Agrobacterium rhizogenes or a part of T-DNA thereof by a method such as microinjection.

When the Ri plasmid is introduced by the above-mentioned methods 1 to 4, it is necessary to eradicate the Agrobacterium rhizogenes bacterium thereafter, and the method includes the following. 〇High temperature treatment (40 ℃) 〇Antibiotic treatment 〇Spring transfer of hairy root tip in fast cycle

In the present invention, for example, a liquid medium conventionally used for tissue culture of plants, that is, an inorganic component and a carbon source are essential components, and at least one selected from plant growth regulators, vitamins and amino acids. It is possible to use a liquid medium to which one or more components are added and other components are added if necessary.

The inorganic components in the liquid medium include nitrogen, zinc, iron, copper, molybdenum, boron, phosphorus, cobalt, potassium, calcium, magnesium, sulfur, manganese, chlorine, sodium, iodine, and the like. Is
Ammonium nitrate, ammonium dihydrogen phosphate, ammonium sulfate, ammonium chloride, sodium nitrate, calcium nitrate, potassium nitrate, zinc sulfate, ferrous sulfate, ferric sulfate, 1-sodium ethylenediaminetetraacetic acid secondary sodium
Iron, copper sulfate, molybdic acid, sodium molybdate,
Examples include boric acid, phosphoric acid, 1 sodium phosphate, 1 potassium phosphate, 2 sodium phosphate, 3 sodium phosphate, cobalt chloride, potassium chloride, calcium chloride, magnesium sulfate, sodium sulfate, manganese sulfate and potassium iodide. it can.

Examples of carbon sources include sucrose, other carbohydrates, their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol.

As plant growth regulators, indoleacetic acid (IAA), naphthaleneacetic acid (NAA), p-chlorophenoxyisobutyric acid, 2,4-dichlorophenoxyacetic acid (2,4-D)
And auxins, kinetin, zeatin, dihydrozeatin, and other cytokinins.

Examples of vitamins include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, ascorbic acid (vitamin C), inositol and nicotinic acid.

As amino acids, glycine, alanine,
Examples thereof include glutamine and cysteine. The concentrations of the components in the liquid medium can be varied within wide limits. Usually, about 0.1 μM to about 1000 mM of inorganic components, about 1 g / l to about 120 g / l of carbon sources, about 0.01 μM to about 10 μM of plant growth regulators, vitamins and amino acids, respectively. It can be about 0.1 mg / l to about 100 mg / l.

In the present invention, the initial planting amount of hairy roots in the liquid medium can be varied within a wide range. Usually, it is desirable to inoculate about 10 mg to about 1 g (fresh weight) of hairy roots to 50 ml of liquid medium.

Light is not always necessary in the culture of hairy roots. The culture temperature is about 10 ° C to about 35 ° C, especially about 23 ° C to about 28 ° C.
C is preferred. That is, if the temperature is lower than about 10 ° C, the growth rate of hairy roots is low, and if the temperature is higher than about 35 ° C, the growth rate of hairy roots is also low.

The following method can be considered for maintaining a relatively high dissolved oxygen concentration in the liquid medium during the culture operation. -Rotating the liquid culture medium. -Bubbling the medium with an O ₂ -containing gas such as air.

The dissolved oxygen concentration in the liquid medium during the extraction operation is preferably 3 ppm or less. The following methods are available to reduce the dissolved oxygen concentration in the liquid medium during the extraction operation. -Stop the rotary shaking of the liquid medium and stop the renewal of the gas-liquid interface to lower the dissolved oxygen concentration.・ Reduce the O ₂ concentration in the gas by replacing it with N ₂ gas.・ Reduce the O ₂ concentration in the bubbling gas.・ Reduce the amount of bubbling gas.

After the extraction operation, it is preferable that the liquid medium after the useful substance is released is partially or wholly replaced with a new liquid medium.

Hereinafter, more specific experimental examples will be described. (Induction of Hairy Roots by Leaf Disc Method) The leaves of edible beets (Beta vulgaris) were immersed in a 2% aqueous solution of sodium hypochlorite for sterilization. The leaves were cut with a cork borer to have a diameter of 10 mmφ and immersed in an Agrobacterium riogenes suspension to infect Agrobacterium riogenes bacteria.

The leaves after infection were then transplanted onto Murashige-Skoog (MS) agar solid medium containing 20 g / l sucrose. Hairy roots took root from the leaves after 1-2 weeks.

From a large number of obtained transformants, adventitious root cells were selected which were excellent in terms of betanin pigment, bulgaxanthin pigment content and growth characteristics. From the results of obain analysis by filter paper electrophoresis, it was confirmed that the adventitious root cells were hairy roots.

(Culture Operation and Extraction Operation) 300 cc of hormone-free Murashige and Skoog (MS) liquid medium containing 20 g / l sucrose was prepared. Volume of these 500c
It was put in the Erlenmeyer flask of c and capped with a cotton plug. 25 ° C in a dark room
Then, the Erlenmeyer flask was rotatably shaken at a rotation speed of 100 rpm. At this time, oxygen is smoothly supplied into the liquid medium by updating the gas-liquid interface. This dissolved oxygen concentration is 8ppm
It is a degree.

In the embodiment of the present invention, as shown in FIG.
, After culturing as described above for 9 days, the rotary shaking was stopped for 2 days, and the flask was rotary shaken again for 9 days.
Stop the rotary shaking for 2 days, and rotate for 11 days,
The rotary shaking was stopped for 2 days and repeated culture was performed. And
The cell mass (g / l) was measured after 9, 11, 17, 20, 22, 28, 33, and 35 days from the first day, and expressed as a line graph (stopped). On the other hand, also in the example in which the flask was continuously shaken by shaking from the first day, the cell amount was measured after 11, 17, 22, 28, and 35 days and represented as a line graph (without stop). In the examples of the present invention, the liquid medium was replaced after the completion of one cycle of the rotary shaking of the flask and the stop of the rotary shaking.

Further, in both the examples of the present invention and the comparative examples, the production amounts of betanin (BT) and burgaxanthin (VX) dyes were determined by HPLC method (colorimetric analysis) 11, 22 and 35 days after the first day. It was quantified and expressed as a bar graph.

As can be seen from the results in FIG. 1, by stopping the rotary shaking of the flask for 2 days, a significant amount of the dye is released to the outside of the cells. This is due to the decrease in dissolved oxygen concentration in the liquid medium (3pp
It is considered that the hairy root cells relax and the pigment is released. It is also understood that the hairy roots start to grow again when the rotary shaking is stopped for 48 hours and then the flask is again rotary shaken to supply oxygen into the liquid medium.

In the next experiment, hairy roots were first cultured in MS medium for 10 days in the same manner as described above. Immediately before stopping the rotary shaking, the amount of intracellular pigment in hairy roots was measured, and it was found that betanin (BT) was 3.69 mg / l-broth and bulgaxanthin (VX) was 12.9 mg / l-broth. It was

Then, 12 hours after stopping the rotary shaking,
After 24 hours, 36 hours, and 48 hours, the amount of extracellular production of BT and VX, that is, the amount released into the liquid medium was measured, and the results are shown in FIG. As can be seen from this, the amount of dye released rapidly increases, especially after about 24 hours from the stop of rotation and shaking.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between culture time and the amount of extracellular production of dye and the amount of cells for each of the examples and comparative examples of the present invention.

FIG. 2 is a graph showing the change over time in the extracellular production amount (release amount into the liquid medium) of each dye after the rotation and shaking were stopped.

[Explanation of symbols]

 BT Betanin VX Bulgaxanthin

Claims (2)

[Claims] 1. A edible beat plants transformed with R _i plasmids Agrobacterium rhizogenes holds, the hairy root caused by the transformation was cultured in a liquid medium, and then oxygen in the liquid medium A method for producing a useful substance using a hairy root, which comprises lowering the concentration to release a betanin pigment and a burgaxanthin pigment from the hairy root into the liquid medium. 2. Extraction for lowering the oxygen concentration in the liquid medium relative to the oxygen concentration in the liquid medium during the culture of the hairy root to release a useful substance from the hairy root into the liquid medium. The production of a useful substance using a hairy root according to claim 1, wherein an operation and a culture operation of culturing the hairy root in the liquid medium by increasing the oxygen concentration in the liquid medium are alternately repeated. Method.