JPH05115229A - Vegetable cell hybrid plant of agrostis and its production - Google Patents

Abstract

PURPOSE:To obtain a vegetable cell hybrid plant of Agrostis stolonitere having excellent properties by subjecting a protoplast derived from Agrostis stolonitere to cell fusion with a protoplast of other plant belonging to the genus Agrostis by an electric method CONSTITUTION:A cultured cell or plant body derived from Agrostis stolonitere is treated in an enzyme containing cellulase RS, driserase, etc., to form a protoplast and the protoplast is subjected to electric cell fusion treatment with a protoplast of other plant belonging to the genus Agrostis to provide the objective regetable cell hybrid plant.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a somatic cell hybrid plant using a cell fusion method and a method for producing the somatic cell hybrid plant. Particularly, a somatic cell hybrid plant suitable for improving a variety of the Helicoptera patens within the genus Nucabo and a method for producing the same. Regarding

[0002]

2. Description of the Related Art Conventionally, plant breeding has been carried out by artificial crossing or mutation. Artificial mating yielded only hybrid cells of closely related species, and the mutations were poorly reproducible, despite long-term testing. However, in recent years, cell fusion technology has advanced and it has become possible to obtain hybrids over a fairly wide range. Such a technique has been used mainly for rice to improve the variety of plants used for food.

[0003]

The plant of the genus Nucabo is used in golf course greens and the like, and the main plant thereof is a North American native creeping vent ( Agrostis stolonifera ).

[0004] Conventional breeding of the hemlock squirrel is carried out by artificial crossing mainly in North America. By such a method, varieties such as pen cloth, pen links, and pen eagle have appeared.

However, in breeding by artificial crossing, it is difficult to give many effective genetic traits because crossing is only possible between closely related species. In particular, there is a need for a Helicoverpa armorum that has properties such as disease resistance, cold resistance, heat resistance, insect resistance, trampling resistance, and dwarfing.

The present invention has been made in view of the above problems, and provides a somatic hybrid plant composed of Helicoptera punctata introduced with the trait of other plants in the genus Nucabo and a method for producing the same. The purpose is.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a somatic hybrid plant of the genus Hydrangea and Nucabo using the cell fusion method, and a method for producing the same. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to produce Helicoverpa armigera having useful traits such as disease resistance, cold resistance, heat resistance, and insect resistance. For example, it is possible to produce a plant more suitable for Japan by obtaining a hybrid between an exotic plant, Helicoptera japonica, and a plant native to Japan in the genus Nucabo.

[0008] In the present invention, somatic hybrids are produced by cell fusion of protoplasts derived from Rhizopus and protoplasts of other plants derived from the genus Nucus.

[0009] and Agrostis genus referred to in the present invention, high creeping bentgrass (Agrostis stolonifera), creeping bentgrass (Agrostis alba), Himenukabo (Agrostis canina), Colonial Bent (Agrostis tenuis), Agrostis (Ag
rotis clavata ), Yamanukabo ( Agr
ostis clavata ), Ezonukabo ( Agro
stis scabra ), Black squirrel ( Agro
stis valvata ), Miyamakubo ( Agro
stis flaccida ), Komiyamanukabo ( Ag
rotis mertensii ).

The cultured cells having a redifferentiation ability used for cell fusion are callus composed of tissues such as mature and immature seeds of the above-mentioned plant, growth points of runners, roots, etc., and cultured by shaking in a liquid medium. Can be obtained. For example, after sterilizing the above materials by a conventional method, these plant pieces are aseptically placed on an LS or MS agar medium containing a plant hormone, and then placed at 25 to 28 ° C., 0 to 3000 lux, 12 to 20.
Callus is induced under the condition of photoperiod.

The plant hormones mentioned here are dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NA).
A), indole acetic acid (IAA), 4-amino-3,
4,6-Trichloropicolinic acid, 2,6-Dichloro-0
-Anisic acid, kinetin, benzyladenine (B
A), abscisic acid (ABA), gibberellin and the like.

[0012] After substituting the induced callus in the same medium, a callus having a redifferentiation ability was selected, and N6, AA,
Liquid culture in R2 medium or the like. As a result, suspension cells having redifferentiation ability can be obtained. The suspension cells are pre-cultured in MS medium containing sugar and auxin such as 2,4D for several days to isolate protoplasts.

To obtain protoplasts from cultured cells and plants, cellulase RS, cellulase R-10, macerozyme R-10, dolicerase, pectriase Y
15 to 1 of these tissue pieces in an enzyme solution containing -23 etc.
The treatment is performed at 6 ° C. for 0 to 50 rpm for 3 to 18 hours.

Particularly, the plant body is shredded into 0.5 to 1 mm square pieces, shaken with a hypertonic solution (containing 0.2 to 0.8 M mannitol), and further shaken with an enzyme solution. It can facilitate the isolation of protoplasts.

After the enzyme treatment, the undigested tissue is removed through a 30-100 μm mesh, and the protoplasts are washed with a washing solution. Cleaning solution is 0.3-0.8
M mannitol, 0.1-0.8 mM calcium chloride 2
It is an aqueous solution containing hydrous salt. For washing, the filtrate and the washing solution are added to the centrifuge tube, the mixture is centrifuged at 500 to 2000 rpm for 3 to 15 minutes, the supernatant is removed, and the washing solution is newly added to wash the attached protoplasts with a hippet or the like. Centrifuge under the same conditions. Do this operation 2 to 4
Completely remove the enzyme solution by repeating the procedure.

When undigested substances and the like are present in the protoplast solution, the protoplast solution is placed in a 15-25% sucrose aqueous solution, and only the protoplasts are separated by floating.

The obtained protoplasts were mixed with 0.3 to 0.
The cells are suspended in a fusion solution containing 8M mannitol and subjected to electric cell fusion treatment. As the protoplast used here, it is preferable to use one having redifferentiation ability in at least one side.

That is, for example, protoplasts have a density of 0.
1-0.8 M mannitol, 0.1-10 mM ME
S, mixed with a fusion solution containing 0.1 to 10 mM calcium chloride dihydrate and suspended. Furthermore, the number of protoplasts is 10
Adjust the concentration with the fusion solution so that it becomes ⁵ to 10 ⁸ cells / ml. An alternating current is applied to the protoplast solution to form a state in which the protoplasts are arranged in a line between the electrodes (pearl chain). The AC voltage at this time is preferably a frequency of 0.1 to 1 MHz, a voltage of 50 to 300 V / cm, and an application time of 2 to 60 seconds.

Next, a DC pulse of 1-8 KV / cm
Giving 1 to 5 times every 10 seconds for 10 to 100 microseconds,
Combine protoplasts.

Further, the above AC voltage is applied for 0 to 120 seconds to restore the fused protoplasts. After that, the power is turned off, the electrode is removed, and the protoplast solution is left to stand.

The fused protoplasts were mixed with 5 to 15
% Glucose, R2 containing 0-5 mg / 12,4D,
Resuspend in MS, 8P, B5, KM medium. This suspension is mixed with a medium containing low melting point agarose in an equivalent amount and solidified in a petri dish. The concentration of agarose at this time is
0.5 to 1.5%. Further, the number of protoplasts to be redifferentiated is adjusted to 10 ² to 10 ⁷ cells / ml. If the protoplasts are difficult to grow, the agarose gel containing the solidified protoplasts is cut and cultured in a liquid medium containing the same components as the solid medium.

At this time, cultured cells such as rice may be co-cultured in the liquid medium. Culture conditions are 10
Dark conditions are set at ˜80 rpm and 25 to 30 ° C. By culturing the fused protoplasts for 2-3 weeks,
It grows even if it does not coexist in the growth medium. Furthermore, by culturing from a liquid medium to a solid medium,
After 10 weeks, colonies are formed and then callus is formed.

MS containing 3% of this sucrose
By placing on a redifferentiation medium such as a solid medium and culturing, a milky white adventitious embryo is formed to become a plant.

By gradually decreasing the concentrations of plant hormones (cytokinins) and sugars in this medium,
Acclimation can be made efficient.

[0025]

EXAMPLE 1 (cell fusion of high creeping bentgrass and stolonifera) (1) Induction of callus and suspension of creating high creeping bentgrass (Agrostis Stolonif
era ) seeds were sterilized by a conventional method, placed in an MS medium containing 3% sucrose, 2 mg / l 2,4D, and 2 g / l casein hydrolyzate, and callus was induced at 26 ° C. in the dark.

White and compact callus having a redifferentiation ability in this callus was selected and 3 mg / l 2,4
D shaking culture in AA liquid medium containing 3% sucrose and 3% sorbitol (100 rpm, 26 ° C., dark condition)
Then, suspension cells were obtained. A part of the callus grown here was placed on a hormone-free MS solid medium for redifferentiation, and the redifferentiation ability of the callus was confirmed.

2 mg / l 2,4 of suspension cells
D, protoplasts were isolated by preculture (100 rpm, 26 ° C., dark condition) for 2-3 days in MS medium containing 3% sorbitol.

(2) Isolation of Protoplasts Suspension cells of Helicobacter palustris were treated with 2% cellulase RS, 2% macerozyme R-10, 0.1% pectolyase Y-23, 0.5 M mannitol, 7 mM calcium chloride dihydrate, 7 mM. The enzyme solution containing MES and 1 / 2MS medium was treated at 26 ° C., 40 rpm for 4 hours. At this time, 40 ml of enzyme solution was used for 1 g of callus.

[0029] On the other hand creeping bentgrass (Agrostis al
ba ) is obtained by sterilizing new leaves or larvae cultivated under illumination of 100 to 500 lux according to a conventional method, and 0.5 to 1.
Shred into 0 mm square, 0.5 M mannitol, 7 mM M
The cells were permeated with a hypertonic solution containing ES and 1/2 MS medium. This hypertonic solution was shaken at 100 rpm and 26 ° C. for 1 hour. After removing the hypertonic solution, 3% cellulase RS, 2% macerozyme R-10, 0.1% pectolyase Y-23, 0.5.
M mannitol, 7 mM calcium chloride dihydrate, 7 mM
40 rpm with an enzyme solution containing MES and 1 / 2MS medium,
It was treated at 26 ° C. for 4 hours.

After the enzyme treatment, the oxygen solution was filtered to obtain 0.5M.
Mannitol, 0.3 mM calcium chloride dihydrate, 0.
A washing solution containing 3 mM MES was added, the mixture was centrifuged, and the precipitated protoplasts were washed twice with the washing solution.

(3) Cell fusion
Each was suspended in the washing solution at 1.0 × 10 ⁷ cells / ml and mixed so that the protoplasts were uniform.
To this suspension, an alternating current of 1 MHz and 250 V / cm is applied.
It took about 15 seconds to form a pearl chain. After that, the protoplasts were fused by applying a DC pulse of 5 KV / cm three times for 100 μs.

(4) Culture of fused cells The fusion-treated protoplasts were mixed with 11% glucose and 2%.
Suspended in 8P medium containing mg / l 2,4D, the diameter is 3
It was uniformly spread on a 5 mm petri dish and solidified with a low melting point agarose. At this time, the culture density of protoplasts having a proliferation ability was about 10 ⁶ cells / ml.

The solidified agarose was cut out, suspended in a petri dish having a diameter of 6 cm and filled with 8P medium containing 11% glucose, 2 mg / l 2,4D, and shake culture was carried out. The conditions at this time were 30 rpm, 26 ° C., and dark conditions. After 2 weeks of culture, M without plant hormones
The agarose was shake-cultured in the S medium. After 2-3 weeks, the grown pieces were treated with 3% sucrose, 3% sorbitol,
Regeneration was performed in MS medium containing 0.7% agarose. This resulted in the formation of somatic embryos, followed by shoots and rooting, resulting in hybrid plants.

Example 2 (of the rainbow trout and Yamanukabo)
Cell fusion) (1) Callus induction and preparation of suspension By the method described in the paragraph (1) of the example, a suspension of Helicoverpa armigera was obtained.

(2) Adjustment of protoplasts [ Agrostis stolonif]
era ) protoplasts were prepared according to the method described in Example 1 (2).
It was obtained by the method described in the section.

In addition, Yamanukabo ( Agrostis cl
Avata ) protoplasts were obtained from sprouts and young sprouts by the method described in item (2) of Example 1. After the enzyme treatment, the enzyme solution was filtered, and 0.5 M mannitol, 0.
A washing solution containing 3 mM calcium chloride dihydrate and 0.4 mM MES was added and centrifuged, and the precipitated protoplasts were further washed twice with the washing solution.

Since the undigested material remained, 21% sucrose was put in the bottom of the centrifuge tube, and the protoplast solution was gently put in the upper layer of the centrifuge tube, and normal protoplasts suspended by centrifugation were used. did.

(3) Electric cell fusion According to the method described in paragraph (2) of Example 2, the protoplasts obtained were suspended in a washing solution at 1.0 × 10 ⁷ cells / ml, and gently suspended with a pipette. It became turbid and made into a fusion liquid.

An alternating current of 1 MHz and 200 V / cm was applied to the fusion solution for 10 to 15 seconds to form a pearl chain, and then a DC pulse of 5 KV / cm was applied twice for 50 μm seconds to fuse the protoplasts. ..

(4) Culture of fused cells The fusion-treated protoplasts were mixed with 11% glucose and 2%.
The cells were suspended in 8P medium containing mg / l 2,4D, spread on a petri dish, and solidified. At this time, the density of cells having a proliferation ability was set to about 10 ⁶ cells / ml.

The solidified agarose gel was cut out, and 11%
It was floated on 8P medium containing glucose, 2 mg / l2, 4D and shaken. Shaking was performed at 30 rpm and 26 ° C. under dark conditions. As a result, cell proliferation is observed,
Colonies formed after 3 weeks.

After that, shaking culture was carried out in the same manner in MS medium from which plant hormones were removed. After 2-3 weeks, the grown pieces were transplanted to MS medium containing 3% sucrose, 3% sorbitol and 0.7% agarose. By culture, formation of milky white somatic embryos was observed from the colonies, and then shoots and rooting were observed by applying light conditions, and hybrid plants were obtained.

Example 3 (Hyconukusa and Colonial Beetle)
By the method of cement cell fusion) (1) Callus induction and suspension of the creation of Example 1 (1) above, wherein, to obtain a high creeping bentgrass and colonial vent callus, created the suspension.

(2) Preparation of Protoplasts The protoplasts of Rhizopus and colonial vents were prepared from the suspension by the method described in item (2) of Example 1. After the enzyme treatment, the enzyme solution was filtered through a mesh, and the protoplasts were washed by the method described in item (2) of Example 1.

(3) Iodoacetamide treatment The concentration of the protoplasts of the colonial vent obtained by the above method was adjusted to 10 ⁶ cells / ml, and after suspension, the iodoacetamide solution was adjusted to 25 mM. In addition, the mixture was treated at 5 ° C for 15 minutes. After the treatment, it was washed twice with a washing solution and adjusted to 1.0 × 10 ⁷ pieces / ml.

(4) Cell fusion The thus obtained protoplasts of Rhizopus and the iodoacetamide-treated colonial vent protoplasts were mixed to prepare a fusion solution.

In this fusion liquid, 1 MHz, 200 V / cm
Was applied for 5 to 10 seconds to form a pearl chain, and then a DC pulse of 3 KV / cm was applied for 30 µ seconds to fuse the protoplasts. After this, 1 MH
An alternating current of z, 200 V / cm was applied while being attenuated for 30 seconds.

(5) Culture of fused cells The fusion-treated protoplasts were mixed with 11% glucose and 2%.
The cells were suspended in B5 medium containing mg / l 2,4D and fixed in a petri dish with agarose. At this time, the density of cells having a proliferation ability was about 10 ⁶ cells / ml.

The solidified gel was floated in a B5 medium containing 11% glucose and 2 mg / l 2,4D in a size of about 5 × 20 mm and shake-cultured. Shake 30 rp
m, 26 ° C. under dark conditions.

After this, cells started to grow in the gel, and colonies formed after about 20 days. After the colonies grew to some extent, they were transferred to a growth medium and grown. The growth medium mentioned here is 12 mM proline, 100 pp.
Hydrolyzed m-casein, 2% sucrose, 4 mg / l
MS liquid medium containing 2,4D and 3% mannitol.

The further grown callus was used to form somatic embryos in a regeneration medium. The redifferentiation medium referred to here is 20
0ppm casein hydrolyzate water, 2% sucrose, 6%
Sorbitol, 2 mg / l 2,4D, 5 mM MES
MS solid medium containing. Milky white somatic embryos were seen after 4-6 weeks on the regeneration medium.

This somatic embryo was placed on a seedling medium to obtain a hybrid plant. This seedling medium was an MS solidified medium containing 3% sucrose, and the concentration of sugar was gradually reduced to acclimatize.

[0053]

INDUSTRIAL APPLICABILITY As described above, the present invention relates to a somatic hybrid plant obtained by a cell fusion method of protoplasts of Helicoptera japonicus and other plants of the genus Nucabo and a method for producing the same. According to such a somatic cell hybrid plant and a method for producing the same, disease resistance, which is a competent genetic trait possessed by other plants in the genus Nucabo for Helicoptera,
It is possible to have properties such as cold resistance, heat resistance, insect resistance, trampling resistance, and dwarfing. Moreover, it becomes possible to arbitrarily control these properties by selecting the type of other plants of the genus Nucabo that are fused with Helicoptera japonicus, so that it is possible to provide somatic hybrid plants of Helicoptera serrata with excellent traits. become.

Claims (2)

[Claims] 1. A high creeping bentgrass (Agrostis s
a somatic hybrid plant obtained by cell fusion of a protoplast derived from Tolonifera ) and a protoplast of another plant in the genus Nucabo. 2. A production method for producing a somatic hybrid plant by cell-fusion of protoplasts obtained from cultured cells or plants of Helicoptera chinensis and protoplasts obtained from cultured cells or plants of the genus Nucabo.