JPS63313579A - Preparation of callus originated from protoplast of leguminous plant - Google Patents

Abstract

PURPOSE:To surely and quickly obtain the titled callus useful for the development of new variety, etc., by separating a protoplast from a leguminous plant, culturing the protoplast in a specific liquid medium under irradiation of light, solidifying the culture product by the addition of a medium containing a solidifying agent and culturing in a liquid medium having low osmotic pressure. CONSTITUTION:A protoplast separated from a body of a leguminous plant (e.g. cotyledon of young soybean) is cultured under irradiation of light in a liquid medium free from ammonium ion and having a calcium ion concentration adjusted to a prescribed concentration of >=5mM. The produced culture product is solidified by the addition of a conventional medium containing a solidifying agent. The solidified product is cultured under irradiation of light in a liquid medium having lowered osmotic pressure and a callus is collected from the cultured product to obtain a callus originated from the protoplast of a leguminous plant.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for efficiently (reliably and quickly inducing callus) from mesophyll protoplasts of legumes such as soybeans, and for plant body regeneration from protoplasts. In addition to being used for this technology, it has the potential to be used for the breeding and development of new varieties by applying biotechnology such as genetic recombination of plant cells and cell fusion.

[Conventional technology]

The technology to regenerate plants from protoplasts is based on Solanaceae,
Although it has been perfected for many varieties such as Umbelliferae and Poaceae, it has not yet been perfected for soybean, which is a major legume.

There are several reports on the isolation and culture method of mesophyll protoplasts of legumes such as soybean, but in all of them, despite obtaining a high initial division frequency, it has not been possible to continue this method, and it has not been possible to maintain an efficient method. , and did not fail to induce callus with high cell content density.

[Problem that the invention seeks to solve]

Various methods have been reported regarding the isolation and culture of mesophyll protoplasts of legumes such as soybean, but despite obtaining a high initial division frequency of protoplasts, a large amount of NH,ゝ is present in the initial culture medium. As a result, this process could not be continued, and it was extremely difficult to induce callus with a high intracellular density.

[Means for solving problems]

In view of the above-mentioned current situation, the present inventors have conducted extensive studies regarding the cultivation of protoplasts isolated from legume plants such as soybeans, and as a result, the present inventors have found that protoplasts isolated from legume plants (N) in a culture medium I4'' and C
The present invention was completed based on the knowledge that callus with a high intracellular density can be formed efficiently in a short period of time by culturing a411 in a liquid medium containing a411 at a desired concentration under light irradiation.

That is, the present invention cultivates protoplasts isolated from leguminous plants under light irradiation in a liquid medium in which NH4'' has been substantially removed and Ca- has been adjusted to the required concentration. It is characterized by solidifying the product by adding a medium containing a normal solidifying agent, culturing the solidified product in a liquid medium with reduced osmotic pressure under irradiation with light, and then collecting callus from the culture. This is a method for producing callus derived from legume protoplasts.

Examples of the legumes include soybeans, peanuts, cowpeas, green beans, and peas, with soybeans being particularly preferred.

The legume plant body is preferably the leaf part of a young legume plant, and when the legume is soybean, the cotyledon part thereof is particularly suitable.

Isolation of protoplasts from the plant body of a legume can be carried out by a conventional method. Preferably, protoplasts can be isolated by a conventional method from the leaves of seedlings of seedlings grown by sowing legume seeds by a conventional method. be separated.

The seedlings of the legumes mentioned above are obtained by sowing the seeds of the legumes in the soil by a conventional method, and under light irradiation (about 16 hours day length).
At about 20-30℃ for 5-30 days, preferably 7-21 days
Obtained by growing for several days.

As a method for isolating mesophyll protoplasts from the leaves of the obtained seedlings, the leaves are injected with a sterilizing solution used in a normal sterilization method, such as a 70% ethanol solution, and/or a hypodermic solution containing a surfactant. It is desirable to sterilize by adding a sodium chlorate solution or the like.

Next, the leaves after sterilization are immersed in distilled water or a neutral solution containing cytokinin and incubated in a dark place at about 3 to 10°C.
It is desirable to treat the leaves at a low temperature for about 1 to 24 hours.The leaves after the low temperature treatment are cut into thin pieces of an appropriate thickness, and then mixed with a solution containing an osmotic pressure regulator such as mannitol (pH 5.5-6).
．． 5) at room temperature for about 30 to 180 minutes.

The soaked product is mixed with a pectin degrading enzyme and a cell wall lytic enzyme in a solution with adjusted osmotic pressure (for example, a mannitol-containing solution, or a solution to which a reducing agent such as mercaptoethanol, a cell membrane protecting agent such as dextran potassium sulfate, etc. is added as necessary). ) and add it to the enzyme solution dissolved in
Let the enzyme react for about an hour.

As the pectin degrading enzyme, maceration enzymes such as pectolyase Y-23 and Macerozyme R-10 are preferably used, and as cell wall lytic enzymes, for example, cellulase y-c and cellulase Onozuka R are preferably used.
-10, Cellulase Onozuka RS, Driselase, etc. are preferably used.

As a means for isolating protoplasts from the above-mentioned enzyme reaction solution, mesophyll protoplasts are isolated by, for example, filtration with Miracloth (manufactured by Calbiochem Behring, USA), nylon mesh, etc., followed by centrifugation.

Next, the protoplasts were removed from NHr and a+
In a liquid medium enriched with *, under light irradiation, liquid static culture is performed for 5 days or more, preferably 7 to 10 days.

Note that as a liquid medium in which the above-mentioned NH4'' is substantially removed and Ca4+ is adjusted to the required concentration, for example, MS medium (Murashige & Skoog, Physi
ol, Plant,.

15:473-479.1962) and KM medium (Kao
etc., Planta 126+105~110.1975
), etc. Among the inorganic component compositions of a normal protoplast culture medium, NH4'' is about 111M or less, and Ca- such as calcium chloride and calcium nitrate is 5mM or more, preferably 8~
Culture media adjusted to about 25mM are used. Furthermore, 2.4-D (2゜4-dichlorophenoxyacetic acid), NAA (naphthaleneacetic acid), BA (
benzyladenine), Zeatin.

Plant hormones such as Ga4 (gibberellic acid), vitamins such as myo-inositol, nicotinic acid, pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, folic acid, ascorbic acid, sugars such as glucose, shakerose, ribose, xylose, arginine, asparagine ,
A suitable protoplast culture medium contains amino acids such as glutamine, an osmotic pressure regulator such as mannitol, etc., and is adjusted to a pH of about 5.5 to 6.0.

Next, a medium containing a normal solidifying agent is added to the culture after the liquid static culture to solidify it. V
), or solidify the culture by adding alginate and suspending it and contacting it with a calcium chloride solution to obtain a solidified product in which protoplast-derived cells are fixed.

Next, the obtained solidified product is divided into several pieces as necessary, and then placed in a suitable culture container and heated under light irradiation, preferably for 7 to 70 minutes.
Suspension shaking culture is carried out in a liquid medium for one month or more while the osmotic pressure is continuously or intermittently lowered by gradually decreasing the osmotic pressure regulator such as mannitol at 15-day intervals.

The liquid medium with lowered osmotic pressure may be obtained by gradually decreasing the osmotic pressure adjusting agent such as mannitol from the protoplast culture medium mentioned above by about 1 to 2 to 1 to 10 at intervals of 7 to 15 days. A particularly preferred example is a medium that is finally substantially free of the osmotic pressure regulator.

In addition, as the above-mentioned floating shaking culture means, for example, a culture container such as a Petri dish containing the above-mentioned culture solidified product is placed on the rotary plate of a normal circular shaking culture machine, and the culture is subjected to floating shaking culture while rotating. do.

By the above operation, callus derived from protoplasts of legumes is obtained.

〔Example〕

Seeds of soybean (variety: Oyachi No. 2) were sown in soil,
±2℃, 30001ux, 10 days (16 hour photoperiod)
The cotyledons of the grown soybeans are sterilized for 20 minutes with a sterilizing solution prepared by adding a few drops of Tween 20 to a sodium hypochlorite solution containing 1% available chlorine, and then washed with sterile water. After sterilizing the cotyledons, the cotyledons were subjected to a low temperature treatment in sterilized water at 5°C for 1 hour in a dark place, and then cut into strips with a thickness of about 0.5 mm or less in a sterile room. Immerse in a mixture of 6M mannitol and 1mM mercaptoethanol for 1 hour at room temperature.

Enzyme solution [0.1% (W/V) Pectolyase Y-2
3 (manufactured by Seiun Pharmaceutical Co., Ltd.), 0.5% (revised ■) Macerozyme R-10 (manufactured by Yakult Co., Ltd.), 0.5% (-c) Cellulase VC (manufactured by Seiun Pharmaceutical Co., Ltd.) and 0 .5% (-c) Driselase (manufactured by Kyowa Hakko Co., Ltd.) was dissolved in a mixture of 0.6M mannitol, 0.1% (W/V) dextran sulfate potassium, and 1mM mercaptoethanol, and the pH
5, 7) in the dark at 25°C.
Shake at 40 r, p, m for 4.5 hours to allow enzyme reaction.

The enzyme reaction solution was filtered through Miracloth (Calbiochem Behring, USA), and then filtered through a 40 micron sieve.
Separate protoplasts through (nylon mesh) and then incubate at 700 r, p, i using a mixture of 0.64 mannitol and ld mercaptoethanol.

Repeat centrifugation and washing three times for 3 minutes to obtain protoplasts.

The number of protoplasts obtained was 1X10'/d.

Culture at 25°C under 10001 ux light irradiation in the liquid culture medium described above [a medium having the following composition (hereinafter referred to as B5-1 medium) filtered with a 0.2 μm sterile filter at pH 5.75]. do.

Composition of B5-1 medium Inorganic salts: Same composition as inorganic salts in MS medium, plant hormones: 2.4-D, NAA, BA, Zeatin+
G A 3 each 0.5 wg/A! , vitamins;
Myo-inositol 100■/j! , nicotinic acid 1■/I
t, pyridoxine hydrochloride 1w/Jt, thiamine hydrochloride 1O■
/l.

Calcium pantothenate 1■/It, folic acid 0.4■/L
, ascorbic acid 2/2. Sugar; glucose 30g/
j! , Shake Rose 30g #I, Ribose 0.5g
/1. Xylose 0.3 g/l, amino acids;
Arginine 10■/! , asparagine 0.3 g/(1° glutamine 0.3 g/j!, osmotic pressure regulator; mannitol 45 g/l.

At that time, the inorganic salt (N11.") of the above medium was used.
NO3 and (CaC1g.2HzO and Ca(NO3)z.4■1□0 as:a$4) were set in the experimental categories as shown in Table 1.

Table 1 shows the initial division frequency after liquid stationary culture for 7 days in a medium modified with the inorganic salt shown in Table 1.

(Margins below this page) Table 1 (Margins below this page) From Table 1, all of the classifications of the present invention have a significantly superior division frequency compared to the control classification.

Next, agarose is added to the culture of the present invention-2 described above and solidified to obtain a culture solidified product having an agarose concentration of 0.7% (-8). After cutting the solidified product into several blocks with sterilized microspertil, this was placed in a plastic petri dish (inner diameter 63), and a medium was prepared by removing ammonium nitrate from the B5-1 medium and adding calcium chloride and calcium nitrate. (NH4NOx: Q “Town CaC1g
・2HzO; 9.1mM, Ca(NO3).

A medium (hereinafter referred to as B5-2 medium) in which the amount of mannitol in the medium has been reduced by 174 from 4H 0; 7.0++M) is placed in the Petri dish and then sealed with Parafilm.

Next, the above-mentioned petri dish was subjected to continuous shaking culture of suspended liquid at 25° C. and 6 Or, p, e on a circular shaking incubator under light irradiation of 10,001 ux.

After culturing this culture for 1 week, the agarose blocks were transferred from B5-2 medium to a liquid medium in which the amount of mannitol was reduced to 172, and incubated at 25°C for 2 weeks at 1200 °C.
Floating shaking culture is performed under 1ux light irradiation. Furthermore, the blocks cultured for 2 weeks were transferred to a liquid medium in which mannitol had been completely removed from the B5-2 medium, and incubated at 25°C for 1 hour.
Callus derived from soybean protoplasts with a diameter of 1 ml or more was obtained by floating shaking culture for 2 weeks or more under light irradiation of 2001 ux [callus formation rate]. As a result of carrying out the test according to the method, almost no callus formation was observed (callus formation rate was 0.1% or less).

〔Effect of the invention〕

According to the present invention, protoplast-derived callus can be obtained more efficiently and reliably than leguminous plants, and in a shorter period of time.

Claims (1)

[Scope of Claims] 1. Protoplasts isolated from leguminous plants are cultured under light irradiation in a liquid medium in which NH_4 is substantially removed and Ca^+^+ is adjusted to the required concentration. The resulting culture is solidified by adding a normal solidifying agent-containing medium, and the solidified product is cultured in a liquid medium with reduced osmotic pressure under light irradiation,
A method for producing callus derived from protoplasts of a leguminous plant, which comprises then collecting callus from the culture. 2. The method for producing callus derived from protoplasts of a legume according to claim 1, wherein the legume plant is the mesophyll of a leaf of a young legume. 3. The method for producing callus derived from topoplasts of a legume according to claim 2, wherein the leaf of the legume seedling is a cotyledon of a soybean seedling. 4. The liquid medium for culturing protoplasts is NH_4^+
is removed to a concentration of about 1mM or less, and Ca^
4. The method for producing callus derived from topoplasts of leguminous plants according to any one of claims 1 to 3, which is a liquid medium in which +^+ is adjusted to a concentration of 5 mM or more. 5. The protoplast-derived leguminous plant according to any one of claims 1 to 4, wherein the first-stage culture is a static culture, and the second-stage culture is a floating shaking culture. Callus production method. 6. Claims 1 to 6, characterized in that when culturing the solidified product under light irradiation in a liquid medium with reduced osmotic pressure, the culturing is performed while lowering the osmotic pressure in stages. A method for producing callus derived from protoplasts of leguminous plants according to any one of Item 5.