JPH02299595A - Production of brassinosteroid by somatic adventitious embryo - Google Patents

Abstract

PURPOSE:To enable safe production of a safe natural substance without generation of isomers as by-products by inducing an adventitious embryo from a seed or a plant tissue of a leguminous crop, a Cruciferae crop or an Umbelliflorae crop. CONSTITUTION:A plant tissue of an immature seed, a full-ripe seed, etc., is arranged and cut to a suitable size to prepare a slice for using as an explant. The resultant slice is planted in an MS medium or a White medium respectively containing an auxin such as 2,4-D and cultured at 23-27 deg.C to form a callus. The obtained callus is then transplanted into an auxin-free MS medium and cultured at 23-27 deg.C to induce an adventitious embryo. The resultant adventitous embryo is crushed, extracted using a solvent such as ethanol and subjected to separation and collection, thus obtaining the objective brassinosteroid. By the above mentioned method, a safe substance can be safely produced and readily isolated and the above-mentioned extract, etc., can be used without a special treatment in some situations.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention involves culturing plant tissue pieces, inducing somatic embryos (hereinafter referred to as somatic embryos), and extracting prasinosteroids from the obtained somatic embryos. This is a method of producing prasinosteroids by separating and collecting prasinosteroids. The prasinosteroid according to the present invention is agriculturally useful as a plant growth regulator in crop production.

[Prior Art] In recent years, research on production methods, usage methods, etc. of brassinolide has been vigorously advanced for use in agriculture. Although inexpensive synthetic methods are being considered for its practical use, it has been relatively difficult to supply it at a low cost because it has a complex chemical structure with stereoisomers. For this reason, attempts have been made to produce it using microorganisms.

Brassinolide is ubiquitous in higher plants, and is particularly abundant in tissues during reproductive growth such as pollen and immature seeds, but only in trace amounts. Therefore, research has recently been carried out on the production of brassinolide by plant cells, and it has been reported that crown gall cells of Catharanthus periwinkle and callus Scutellaria barbata produce prasinosteroids, suggesting the possibility of brassinolide production using cultured plant cells.

[Problem to be solved by the invention] If this brassinolide can be produced by a plant cell culture method, it will not use dangerous reagents (and will not produce isomers as a by-product) compared to synthetic methods. Therefore, it becomes possible to safely produce safe natural products.
Through years of research on tissue culture of plant tissue pieces, we have learned that prasinosteroids are produced in extremely small amounts in callus that has dedifferentiated from plant tissues.

[Means for Solving the Problems] The present inventors focused on the fact that among plant tissues, brassinolide is particularly abundant in tissues in the reproductive growth period such as pollen and immature seeds. The result of intensive research aimed at finding clues in somatic embryos and safely producing brassinolide using somatic embryos derived from plant cells without using expensive or dangerous reagents used in chemical synthesis. ,
He completed his invention by discovering for the first time that somatic embryos produce relatively large amounts of prasinosteroids.

That is, induction of somatic embryos for leguminous crops (dice, green beans, Fuji beans, etc.), cruciferous crops (rapeseed rape, canola, Chinese cabbage, etc.), and umbelliferous crops carrots, which are known to contain brassinolides. They conducted research on this and discovered that the somatic embryos produced produced prasinosteroids.

The present invention involves inducing somatic embryos from seeds or plant tissue pieces of leguminous crops, cruciferous crops, or umbelliferous crops, and separating and collecting prasinosteroids from the obtained somatic embryos. This is a method of producing steroids.

Furthermore, the present invention specifically relates to culturing immature or ripe seeds or plant tissue pieces of leguminous, cruciferous, or umbelliferous crops in a medium containing 2.4-D, and then culturing them in a liquid medium containing no auxin. This is a method for producing prasinosteroids, which is characterized by transplanting, inducing and developing somatic embryos, and separating and collecting prasinosteroids from the obtained somatic embryos.

Note that the prasinosteroids of the present invention refer to those that exhibit brassinolide-like activity, such as brassinolide and castasterone.

In carrying out the present invention, pieces of plant tissue such as immature seeds, ripe seeds, or sprouts are prepared, and after sterilizing the tissue pieces according to a conventional method, the tissue pieces are cut into appropriate sizes to form explants. Prepare sections to be used as samples. In the case of immature seeds or ripe seeds, after removing the seed coat, cut the cotyledons of the seeds to make sections. This section is planted in a medium to form a callus. As a medium, as auxin, IAA
, NAA or 2.4-D, preferably MS medium or white medium supplemented with 2.4-D can be used. The culture temperature is preferably 23-27°C, and 12-1
It can be cultured with a 6-hour photoperiod and 10 mouths of 0 to 20,001 ux.

The formed callus is transplanted into an auxin-free MS liquid medium and cultured to induce somatic embryos. Culture is 8
0~12Orpm, 23-27 degrees, 1000-20
It can be cultured under conditions of 0.001ux. The somatic embryos thus obtained are treated with a conventional method for separating plant bodies using prasinosteroids. That is, if the somatic embryo is crushed, extracted and separated using ethanol, ethyl acetate, or methanol, and an organic solvent such as chloroform or benzene, then the embryo is collected.
Prasinosteroids can be obtained.

As mentioned above, somatic embryos that produce prasinosteroids can be easily obtained by dividing the embryo into two phases: the induction period and the developmental period.

MS or White medium is suitable as a callus-forming medium during the induction period of somatic embryos, but it is not limited thereto. As a carbon source, it is preferable to use 1% sucrose, but it is not necessary to use it. There are various auxins to be used, such as IAA, NAA, 2.4-D, or their salts, but 2.4-D is preferred.

The concentration of auxin used may be any concentration suitable for inducing somatic embryos and is usually 10-6 to 10-'M. The preferred auxin used is 2.4-D of 0.1
-2.5 mg/l, preferably 1.0-2.0 mg/l.

During the developmental stage of somatic embryos, somatic embryos are induced when the formed callus is transferred to MS medium with high salt concentration containing reduced nitrogen, and the presence of high concentrations of nitrate ions and reduced forms such as nitrate and ammonium ions induce somatic embryos. The coexistence of nitrogen promotes the induction of somatic embryos.

The effect of reduced nitrogen is small during the induction period, but the effect of reduced nitrogen is observed during the developmental period.If the medium contains reduced nitrogen such as nitrate ions and ammonium ions, it will It is not limited.

During the developmental stage of somatic embryos, the presence of auxin is not desirable, so an auxin-free liquid medium is used.
It is possible to promote the induction of somatic embryos by adding small amounts of gibberellin, cytokinin, and abscisic acid as plant hormones other than auxin.

Culture differs depending on the plant, but it can be carried out under normal plant tissue culture conditions6. Explants for somatic embryo induction can be collected from the outside of the cotyledons of immature seeds and mature seeds, and from the embryos of budding seeds. Callus is formed from the axes, leaves, apical buds, and roots, and the formed callus is transplanted into an auxin-free liquid medium and cultured to induce somatic embryos. When young tissue pieces such as apical buds are used as explants, the formation of somatic embryos is particularly good.

Next, callus formation and somatic embryo induction will be explained in detail.

(2) Callus formation To induce somatic embryos, ripe seeds of dice (variety: Amsoy) or Fuji peas were soaked in 70% alcohol for 2 minutes, then placed in 3% sodium hypochlorite solution for 10 minutes and sterilized by stirring. After thoroughly washing the seeds with sterilized water, wiping off moisture thoroughly and removing the seed coat, the cotyledons in the seeds were cut into 5 x 5 mm square pieces as explants, and 2.4-D
Murashige Skoog containing 2mg/l
e & Skoog (MS) and white wh
ite (W) agar medium and cultured at 25°C, 16 hour photoperiod, 20001 ux to form callus.

■ Somatic embryo induction Next, the formed callus should be transplanted into MS and W liquid medium that does not contain 2.4-D.
g7l00, reciprocating shaking incubator, 115 rpm, 23
Cultured at ~27°C, 500-10001 ux.

Induction of somatic embryos for prasinosteroid production can be carried out in two phases.

As a result of various studies, as a preferable condition for somatic embryo induction and formation for prasinosteroid production, we found that Dice is cultured in a medium containing 2.4-D for 7 to 10 days to form a callus, and then, Transfer to auxin-free medium and culture for 7 to 10 days. In Chinese cabbage, 7 to l
After O days, the callus is transplanted and cultured for 14 to 21 days. In carrots, somatic embryos are cultured until just before the start of differentiation, then transplanted into a liquid culture medium and cultured for 21 to 28 days, at which time the growth amount and activity of somatic embryos reach their peak.

Prasinosteroids produced from the somatic embryos thus obtained can be extracted, separated, and purified with easy operations.

[Example] Results are shown below in Examples, but these are just examples,
It goes without saying that this does not limit the scope of the present invention.

Example A, Examination of callus transplantation period Fully ripe seeds of Dais (variety: Amsoy) and Fuji peas were sterilized by a conventional method, the seed coat was removed, the cotyledon part of the seeds was cut into 5 x 5 mm square pieces, and the seeds were placed on MS medium ( +2.4-D
2 mg/l) solid medium. 3.7.14.
21. After 28 days, the cells were transplanted into auxin-free MS medium and the induction of somatic embryos was observed, and the results shown in Table 1 were obtained.

Table 1 Effect of callus transplantation on somatic embryo induction from mature seeds and cotyledons of Fuji peas Transplantation time (days) 3 7 14
21 28 Am Soi − ++
＋ ＋ ＋ ＋Fuji beans
＋ ＋＋ ＋ ＋
-1: None +=5 or less ++: 5 to 10 +
++: 10 to 20 (Note 2: The number means the number of somatic embryos per colben.) The largest number of somatic embryos was induced when the callus was transplanted on the 7th day for both Amsoy and Fujimame.

Therefore, it is desirable to transplant at an early stage after callus formation.

Example B Creation of somatic embryos with good synchrony Use the 1st and 2nd petioles from core leaves of carrots 30 to 60 days after seeding as explant sites and sterilize them with 70% alcohol and 1% antiformin. After that, cut into 1-2mm pieces,
．． M added with 4-D (0.1-1.0 mg/l)
The cells were placed on S medium to induce callus. The formed callus is maintained and grown in the same medium, and somatic embryos are induced.

The callus induced with 2.4-D was once transplanted onto an auxin-free MS agar medium, and cultured as the cells proliferated until just before the start of somatic embryo differentiation. This callus is transplanted into an auxin-free MS liquid medium and stirred with a magnetic cooler to homogenize large cell clusters into small cell clusters, and the cell suspension is filtered through a nylon mesh with a pore size of 90 mμ. The filtered cell suspension is dispensed into Erlenmeyer flasks for culture and cultured with shaking to induce somatic embryos. As a result, when the callus was transplanted to a hormone-free MS agar medium and cultured for 3 weeks, and the cell suspension was observed under a microscope on day 1O0, many spherical embryos of uniform size were observed. .

When the culture is continued, torpedo-shaped somatic embryos can be seen with the naked eye, and many embryos with fully developed cotyledons, hypocotyls, and roots are obtained.

Example C Relationship between number of culture days, growth amount, and lamina joint activity Callus grown and formed on a solid medium (MS + 2.4-, D 2 mg/l + sucrose 1%) after 7 days was finely crushed with a bottle set, and 80 ml of Transplant into a 500 ml Erlenmeyer flask containing auxin-free MS liquid medium (transplant amount: around 1600 mg for Dai, around 200 mg for Chinese cabbage), and after culturing for a specified number of days, filter the amount in 3 flasks.
10 g of each fresh weight of the obtained somatic embryos was ground using a glass homogenizer, extracted and separated with ethanol and ethyl acetate in a conventional manner, and then the ethyl acetate was distilled off.

The residue was transferred to a Petri dish, air-dried, and then 10 ml of distilled water was added to prepare a test chemical solution.The lamina joint activity was examined, and the results shown in Table 2 were obtained.

Table 2 Relationship between number of days of culture, growth amount, and lamina joint activity after transplantation of Dais callus. On the 4th day after transplantation, the induction of somatic embryos is still at an early stage, and it is necessary to visually judge whether somatic embryos have been formed. Although it is difficult to do so, on the 7th day, many well-formed somatic embryos with an apical end and a basal end can be seen, and the amount of somatic embryos formed reaches its peak.

The formed dice callus was transferred to auxin-free MS medium and cultured for 4, 7, 10°14, 21 and 2.
The lamina joint activity of somatic embryos after 8 days was determined from 7 days after culture.
Somatic embryos after 10 days show relatively high activity.

Table 3 Relationship between culture days after Chinese cabbage callus transplantation, growth amount, and lamina joint activity In Chinese cabbage, the amount of somatic embryos obtained was higher than that of dice, and the number of somatic embryos increased from 7 days to 10 days, and on the 14th day. reach the peak.

The relationship between the number of culture days of Chinese cabbage and lamina joint activity shows a peak on days 14 to 21.

Table 4 Relationship between the number of culture days after carrot callus transplantation and lamina joint activity The relationship between the number of culture days and lamina joint activity in carrots peaks on the 21st day, and maintains a high value even on the 28th day.

Based on the obtained amount of somatic embryos and the results of lamina joint activity or lamina joint activity, it was found that somatic embryos of 7 to 10 days after transplantation were used for dais, somatic embryos of 14 to 21 days after transplantation were used for Chinese cabbage, and carrots were used for somatic embryos of 14 to 21 days after transplantation. 21 in
It has been discovered that brassinolide can be produced by culturing somatic embryos on day 28.

Test Example The mass culture of somatic embryos was studied under the above-mentioned conditions such as callus induction, transplantation amount, transplantation time, culture solution amount, culture days and growth amount, and 500m1 triangular culture was carried out for each of dice, Chinese cabbage and carrots. The embryos were cultured in 50 flasks, and fresh somatic embryos weighing 22.3 g of dice (Amsoy), 54.6 g of Chinese cabbage, and 32.5 g of carrots were obtained. The somatic embryos obtained were ground using a glass homogenizer in a conventional manner, extracted with ethanol and ethyl acetate,
Separated, each extract was spotted on a TLC silica gel plate 60F 264 and developed with ethyl acetate:ethanol (22+3). Silica gel with an Rf value of 10
The mixture was divided into equal parts, each fraction was extracted with acetone, and the acetone was distilled off to obtain a residue. A test solution diluted to express the activity of the activity test described below was prepared and subjected to the lamina joint test in which brassinolide specifically reacts, the rice seedling growth test, and the wheat leaf blade expansion test. As a result, all somatic embryo extracts showed brassinolide-like activity in the three biological tests mentioned above, as shown in the table below, and in each case, in the fraction with an Rf value of 0.2 to 0.3. The Rf of brassinolide, which is a standard product, shows strong activity.
It was confirmed that the active substance was brassinolide.

The amount of brassinolide contained in each somatic embryo extract was determined from the calibration curve in the lamina joint test, and the amount of brassinolide per fresh weight was 188 μg/Kg, Chinese cabbage 10
0 μg/Kg, 1; and carrot 207 μg/Kg.

In addition, the extract obtained by treating somatic embryos obtained from the same experiment using a conventional method was applied to a Merck thin-layer silica gel plate 60F.
254 was developed with ethyl acetate:ethanol (22:3) as a mobile phase, and detected by spraying vanillin sulfuric acid, the result matched the Rf value.

Table 5 Rf of each somatic embryo extract and standard brassinolide
Value Dice Chinese cabbage Carrot Brassinolide Rf0.28 0.26 0.28 0.26Developing solvent, ethyl acetate:ethanol (22:3) As a result,
The active substance contained in each extract can be identified as brassinolide.

The finding that these somatic embryos produce brassinolide has never been reported and is a completely new finding.

The results of the lamina joint test, rice seedling growth test, and wheat leaf blade expansion test, in which brassinolide specifically reacts, will be explained below.

fa) Lamina joint rice seeds are soaked in water at 30'C for germination and sown in vats. After cultivating at 30°C in the dark for 7 days, cut out the lamina joints. A rice section is floated in distilled water and kept in the dark for 24 hours. A section bent at about 30 degrees is selected from among the sections and floated in a sample solution. After 48 hours, the angle is measured.

Test substance tortuosity Curvature rate (%l = x 1o.

Curvature of control substance (bl) Rice seedling growth test This is an assay method that utilizes the fact that brassinolide twists the roots of rice seedlings.
Observe the degree of torsion of the roots.

(c) Wheat leaf blade expansion test Wheat seeds were germinated on moist cotton for 24 hours, and then sown in a vat containing vermiculite at 26°C.
C2 After cultivating for 6 days in the dark, 1.5-3 cm from the tip
Cut the part to a length of 1.5 cm and float it in the sample solution.
Measure the width of the leaf blade after 24 hours.

Table 6 Dice, Chinese cabbage, and carrot somatic embryos As described above, in the present invention, somatic embryos are induced, and brassinolide is separated from the somatic embryos using an organic solvent.
Can be collected.

C Effects of the Invention] Unlike chemical synthesis methods, the method of the present invention does not produce various isomers as by-products, and under conditions that can be artificially controlled.
Provided is a method for producing natural prasinosteroids using somatic embryos. Therefore, the present invention produces a safe substance by a safe method, can be easily separated, and since the material is a plant, depending on the situation, the extract or crude product can be used without special treatment. made it possible to do so.

Claims (1)

[Claims] Production of prasinosteroids, which is characterized by inducing somatic embryos from seeds or plant tissue pieces of leguminous crops, cruciferous crops, or umbelliferous crops, and separating and collecting prasinosteroids from the obtained somatic embryos. Method.