JPH0799855A - Method for culturing woody plant protoplasts - Google Patents

Abstract

PURPOSE:To improve the colony forming activity of an arboreous plant by culturing a protoplast of the arboreous plant in the coexistence of a protoplast of another arboreous plant having a higher division potential under a specified condition. CONSTITUTION:A protoplast of an arboreous plant is culture in the coexistence of a protoplast of another arboreous plant having a higher division potential in a liquid medium prepared by adding one kind of auxin, plural kinds of cytokinins, a carbon source and an osmotic pressure adjusting agent to a basal medium for plant tissue culture. The obtained colony of the arboreous plant is cultured under irradiation with light according to the vertical roller tube culture method to form a seedling germ and a plant body can be regenerated therefrom.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing woody plant protoplasts. More specifically, it is one of the commonly used plant tissue culture media for inducing colonies from woody plant protoplasts. A method for efficiently forming a colony by culturing a combination of auxins and multiple types of cytokinins, thereby enabling regeneration of a woody plant through a shoot disc. It is a thing.
Therefore, the present invention relates to a method for efficiently obtaining a new and useful woody plant from a protoplast of a woody plant obtained by gene recombination or cell fusion, which is an important technique for producing mutants.

[0002]

2. Description of the Related Art Since protoplasts produced from plant cells have no cell wall, cell fusion or introduction of a useful foreign gene or the like is possible. If it becomes possible to efficiently form a colony from such a protoplast and further regenerate a plant body, it becomes easy to produce a completely new plant that has never existed before. For example, in the case of crops, it is possible to produce varieties or plant species that are high in yield, have a good taste, and can withstand pest damage and weather damage.

A medium for culturing protoplasts is a basal medium (MS: Murashige & Skoog, Physiol. Plant. Plant. Combined with various inorganic salts and organic salts as shown in Table 1.
15: 473-497, 1962, B5: Gamborg et al., Exp.Cell Re
s.50: 151-158,1968, WPM: Lloyd & McCown, Proc.Int.Pl
ant Prop.Soc.30: 421-427,1980), and various plant growth hormones and carbon sources (sucrose, etc.) added to it.
And osmotic pressure regulators (mannitol, glucose) and the like. So far, techniques for regenerating plants from protoplasts through colonies using such a medium have been established in tobacco, petunia and many other herbaceous plants.

However, regarding woody plants, Trovita orange (Kobayashi Shozo "Breeding magazine" 34 volumes (2), 32-33, 1984), Kozo (Narumi Oka, Katsuo Oyama "Breeding magazine" 34 volumes) (Another 2) 26-27, 198
4), poplar (Kazuya Ito et al., JP 63-301784, Russel
l, JAand BHMcCown, Plant Sci.46: 133-142,198
6), Eucalyptus (Yasuyasu Tachido et al., JP Sho 63-301784), Sa
ndal wood (Rao, PSandP.Ozias-Akina Protoplasma
, 124: 80-86, 1985), Elm (Sticklen, MB et
al. 47: 29-34, 1986) and so on.

[0005] The cause thereof is inhibition by polyphenolic substances excreted from the culture medium into the medium during the protoplast culturing process, or medium components, plant growth hormones, etc. cause protoplast division and colony growth due to the long culture period. -It has a great relation to formation. For this reason, callus or seedling hypocotyls or cotyledons with high differentiation potential have been used as materials recently, and after protoplasts are isolated from them, solid media are used instead of liquid media to avoid cell aggregation. , A beet type culture method in which protoplasts are embedded in agar and suspended in a liquid medium for culture, and a feeder layer method in which protoplasts inactivated by X-rays or ultraviolet rays are embedded in agar and then cultured Has been done.

[0006] In Eucalyptus, which is a woody plant, the present inventors have proposed a method of regenerating a plant from a protoplast through colonies by co-culturing with a protoplast of kenaf, which is a herbaceous plant. (JP-A-2-28631). Further improve the colony formation rate by changing the basic medium for plant tissue culture during protoplast culture, and then, when inducing shoot primordium from the obtained colony, change to a different basic medium from the one used for colony production. By doing so, a method of improving the reproduction rate is proposed (Japanese Patent Application No. 5174631).

However, even when these novel and effective methods are used to produce a new plant by the gene recombination or cell fusion, which is a technique for producing a mutant, the rate of colony formation or the regeneration efficiency of the plant is improved. I was not completely satisfied with the points. The present inventors have solved these problems and easily divide the protoplasts of woody plants to form colonies, produce shoot primordia from them, and then carry out research on methods for regenerating plant bodies. As a result, the present invention has been completed.

[0008]

The object of the present invention is to provide an effective culture method for protoplasts.

[0009]

Means for Solving the Problems The present invention provides protoplasts of woody plants and protoplasts of herbaceous plants with high mitotic activity, and one auxin and several types of cytokinins in a basic medium for plant tissue culture. This is a method for producing colonies of woody plants by co-culturing in a liquid medium containing a combined plant hormone, carbon source, and osmotic pressure regulator.

The woody plant colonies thus obtained are prepared by changing the basal medium to a basal medium different from the basal medium for producing the colonies, and further adding a plant hormone, a carbon source and an osmotic pressure regulator. It is possible to produce shoot primordia by further transplanting to a liquid medium and performing vertical rotary culture under light irradiation, which is a conventional method, and further regenerate them into plants.

The type of plant used in the present invention is not particularly limited. Examples of herbaceous plants having a high division ability to be subjected to co-cultivation include kenaf, tobacco, petunia and other herbaceous plants. On the other hand, woody plants for the purpose of regeneration include eucalyptus, acacia, mangrove, Hevea brasiliensis, evergreen broad-leaved trees such as coffee, poplar, thorn tree,
Quercus, kunugi, deciduous broad-leaved trees such as sumac, pine, cedar, cypress, spruce, fir, larch and other coniferous trees, citrus fruits, grapes, almonds, mangos and other fruit trees, roses,
Examples include flowering trees such as plums, sakura, camellias and oleanders.

[0012] Hereinafter, the callus used for isolating the protoplasts used in the present invention, aseptic seedlings, shoot primordia and shoot production methods, protoplast isolation / conditioning methods, protoplast culture methods, shoot origin from colonies. A method for producing a base, a method for regenerating a plant, and the like will be described in detail.

(Callus, aseptic plant, method for producing shoot primordia) A woody plant intended to be regenerated, and a callus produced from a herbaceous plant used for co-culturing with it, and woody nature As a material used for producing a sterile plant body of a plant, one obtained by sterilizing seeds and then placing it on a tissue culture medium of a plant, for example, an agar medium such as B5 medium or MS medium and germinated is used.

Next, the length including the grown shoot tips is 0.5 to 2 cm.
The stems are cut off and plant hormones such as naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) or indoleacetic acid (IAA) and benzyladenine are added to B5 medium or MS medium. (6BA), kinetin, 1- (2-chloro-4-pyridyl) -3-phenylurea (4-PU), zeatin, etc. are placed or cut on an agar medium containing cytokinins. 20 this
Cultivate at a temperature of ~ 30 ° C and an intensity of 2,000 ~ 5,000 lux to obtain callus or shoots. The shoot primordia can be produced by the method already proposed by the present inventors (JP-A-62-55020 and JP-A-63-7720).

(Protoplast Isolation / Preparation Method) Using the callus, aseptic plant or shoot primordia of the woody plant and herbaceous plant obtained by the above-mentioned method as a material, pectin which is an intercellular substance is decomposed. Pectin degrading enzyme,
Cellulose-degrading enzyme, which decomposes the cell wall, and an enzyme solution containing mannitol and polyvinylpyrrolidone (PVP) were added, and the protoplasts were subjected to shaking treatment at a shaking speed of 20 to 30 rpm at a temperature of 20 to 30 ° C for 6 to 24 hours. Let go. In this case, it is desirable to perform a washing treatment with 0.1 to 1% of a surfactant (Tween) as a pretreatment for isolating protoplasts from a sterile plant. Next, the enzyme solution is removed and the protoplasts are washed and centrifuged to adjust the protoplasts of each plant.

(Mixing of protoplasts) After the enzyme treatment, the protoplasts of the washed woody plants and herbaceous plants are adjusted to a concentration of 10 ⁴ to 10 ⁵ cells / ml, respectively. Then, both protoplasts are mixed at a mixing ratio of 1: 5 to 5: 1, and a plant tissue culture medium such as WPM, B5, or MS medium, preferably WPM, containing plant hormones such as NAA, 2,
One of auxins such as 4-D, IAA and 6BA, 4-
It is cultured in a liquid medium containing a plurality of types of cytokinins such as PU, kinetin, zeatin, and thidiazuron, or a medium containing a support agent. As the support agent, agar, agarose or gellan gum (Kelco division, Me
rck) is used. At this time, the support agent can be contained in a low concentration.

(Protoplast Cultivation Method) Regarding the culture conditions, it is preferable that the culture is carried out in a dark place in the initial stage and gradually brightened as the protoplasts grow. The temperature during the culture period is preferably 20 ° C to 30 ° C, and particularly preferably 25 ° C to 28 ° C.

(Method for forming shoot primordia by vertical rotary culture) A liquid obtained by changing the basic medium from WPM medium to B5 medium or MS medium, and further adding auxin, cytokinin, carbon source and osmotic pressure adjusting agent to this. The obtained colonies are transplanted into a medium and subjected to vertical rotary culture. At that time, a B5 basal medium is preferable. Culture conditions are 1-10rp
Rotation speed of m, illuminance up to 2,000-20,000 lux, 20-
At a temperature of 30 ° C, the culture is continued by subculturing it in a fresh medium every 14 to 40 days. In this way, shoot primordia can be obtained 40 to 120 days after the start of vertical rotary culture.

(Method of Regenerating Plant Body) A shoot primordium obtained by culturing by spin culture is used as a plant hormone in a medium for regenerating shoots, such as B5 medium and MS medium, as a plant hormone, for example.
Culture in a medium supplemented with auxins such as NAA, 2,4-D and IAA, cytokinins such as 6BA, 4-PU, kinetin, zeatin and thidiazuron, and a carbon source. Culture is 20 ~
The shoots are regenerated at a temperature of 30 ° C and an illuminance of 2,000 to 3,000 lux for about 60 days, and further rooted to regenerate a complete plant.

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 (Test plant) Eucalyptus camaldulensis (Eucalyptus camaldulensis) as a woody plant, and kenaf (Hibiscus cann) as a herbaceous plant co-cultured with it
abinus) was used.

(Method for Producing Aseptic Eucalyptus Plant) Eucalyptus seeds were sterilized in a bacterial state with antiformin diluted 10-fold for 1 hour, and aseptically added to ethanol at a concentration of 70% for 15 seconds, then It was sterilized by dipping it in 5 times diluted antiformin for 20 minutes. This was germinated aseptically on an agar medium containing 3% sucrose in B5 medium, which is a tissue culture medium for plants. One month after germination, the stalks of aseptic plants were cut to a length of about 1 cm including the shoot apex, and NAA 0.5 mg / l and BA 0.1 mg / l as plant hormones were added to B5 medium to adjust the pH.
The seedlings were cut into agar medium adjusted to 5.6 (containing 0.6% agar). As a result of culturing this at a temperature of 28 ° C. and an illuminance of 3,000 lux, seedlings that formed roots were formed about 60 days after culturing. This was used as a material for the isolation of eucalyptus protoplasts.

(Method for inducing kenaf callus) Kenaf seeds were sterilized with 70% ethanol for 30 seconds and then with 10 times diluted antiformin solution for 10 minutes, and then aseptically B5.
The medium was germinated on an agar medium containing 3% sucrose. About 30
The shoot stem obtained by culturing for a day was cut from the tip to a length of about 1 cm, and this was used as a plant hormone in B5 medium as 2,4-D
2 mg / l, 6BA 0.01 mg / l added agar medium (sucrose 3%,
It was placed on agar (0.6%). This was cultured at a temperature of 28 ° C. and an illuminance of 3,000 lux. About 40 days after the start of culture, yellowish-white callus was obtained, and this was subcultured every month to a fresh medium having the same composition, and used as a material for isolation and preparation of protoplasts.

(Method for isolating / preparing protoplasts) The shoots of eucalyptus obtained by the above method were treated with 0.1% surfactant.
Washed with (Tween) and chopped with a knife for 0.1 g, and for kenaf 1 g callus 20 ml of enzyme solution (1
% Cellulase Onozuka RS, 0.1% Pectolyase Y-23,
13% mannitol, pH 5.6) was added and the enzyme treatment was carried out at a temperature of 28 ° C. and a shaking frequency of 30 rpm for 6 hours to isolate protoplasts of both plants separately. The resulting protoplast suspension was filtered through a nylon mesh (225 mesh) to remove undigested cell mass and the like, and further centrifuged at 750 rpm for 3 minutes to separate the enzyme solution and the protoplast, and the washing solution (13 % Mannitol) and washed twice before culturing.

(Cultivation of protoplasts) The culture medium of protoplasts contained WPM containing NAA, which is an auxin, at 5 mg / l.
Cytokinins 4-PU 1 mg / l, and 6BA 0.2 mg / l
Was added in combination. The WPM in this case is the WPM of the basal medium plus 1% sucrose and 9% mannitol. Eucalyptus and kenaf protoplasts were added to this medium at a ratio of 3: 1 (3 eucalyptus, 1 kenaf) and 5 × 10 5.
^The mixture was mixed to a concentration of ⁴ cells / ml and plated on a petri dish having a diameter of 6 cm. The plate liquid volume was 3 ml per dish. The plated protoplasts were cultured under a dark condition at a temperature of 28 ° C. As a result, the colony formation rate was 1.07% (Table 2).

(Method of Regenerating Plant) Next, shoot primordia were produced from the obtained protoplast-derived colonies by a conventional technique, and the plant was further regenerated. That is, the colony after 2 months of culture was changed from WPM to B5 as the basal medium, and the medium containing NAA 0.02 mg / l, 4-PU 0.2 mg / l, sucrose 1%, and mannitol 3% was added. As a result of inoculating a colony at a ratio of 1 individual to one test tube and performing vertical rotary culture, shoot primordia were obtained after 1 month of culture. (Table-
4)

Furthermore, this shoot primordia was added to B5 medium with NAA 0.02.
The cells were transplanted to a solid medium supplemented with mg / l, BA 0.2 mg / l, sucrose 1%, and gellan gum 0.2%. As a result, shoot regeneration was recognized one month after transplantation. NA of the obtained shoots in B5 medium
When transplanted to a rooting medium supplemented with 0.01 mg / A of A, 1% of sucrose and 0.15% of gellan gum, rooting was observed after 1 month and the plant became a complete plant.

Comparative Example 1 As a culture medium for protoplasts, WPM was added with NAA 5 mg / l, sucrose 1%, and mannitol 9% with 4-PU 1 mg / l, a cytokinin, and 6BA 0.2 mg / l. Protoplasts were cultured by the method described in Example 1 using each of the added media. As a result, the colony formation rate was lower than that in the practical example 1. (Table-2)

Example 2 (Plant) Eucalyptus camaldulensis (Eucalyptus camaldulensis) was used as a woody plant, and kenaf (Hibiscus cann) was used as a herbaceous plant cocultured with it.
abinus) was used.

(Method for producing Eucalyptus shoot primordia) The shoot apex of a eucalyptus aseptic seedling one month after germination was cut out,
NAA 0.02mg / l as a plant hormone in B5 basic medium
And 4-PU were added at a ratio of 0.2 mg / l, and the mixture was inoculated in a liquid medium whose pH was adjusted to 5.6. The culture conditions were 2 rpm, a temperature of 27 ° C. and an illuminance of 1,000 to 10,000 lux, and a shoot primordium was induced using a rigid rotary culture device. As a result, shoot primordia were produced after 30 days of culture. It was used as a material for culturing protoplasts while subcultured at 1-month intervals.

(Protoplast Isolation / Preparation Method) The eucalyptus shoot primordium obtained by the above method was loosened with tweezers, and 20 ml of enzyme solution was added to 1 g of the sample.
Protoplasts were isolated by performing enzyme treatment for 12 hours at a temperature of 28 ° C. and a shaking frequency of 30 rpm. The obtained protoplasts were filtered and washed by the method described in Example 1 and then subjected to culture. In addition, kenaf protoplasts were also obtained and adjusted in the same manner as Example 1.

(Protoplast culture and plant regeneration method) The culture medium for protoplasts is WPM (basic medium WPM).
And NAA 5 mg / l, sucrose 1%, and mannitol 9%), combined with 4-PU 0.8 mg / l and 6BA 0.2 mg / l. Eucalyptus and kenaf protoplasts were mixed with this medium at a ratio of 3: 1 (Eucalyptus 3, kenaf 1) at a concentration of 5 × 10 ⁴ cells / ml and plated on a Petri dish having a diameter of 6 cm. The plate liquid volume was 3 ml per dish. The plated protoplasts were incubated at a temperature of 28 ° C. in the dark. 60 after the start of culture
Colonies formed after day were investigated. Results The colony formation rate was 4-PU 0.8 mg / l and 6BA 0.2 m for cytokinins.
It was 0.11% in the hormonal condition in which g / l was combined. (Table-3). As a result of inducing shoot primordia using these colonies and further attempting to regenerate the plant body, shoot primordia were efficiently induced in the same manner as in the culture of protoplasts from shoots described in Example 1, and , Regeneration of the plant body was observed.

Comparative Example 2 In the culture of protoplasts, cytokinins were treated with 4-
As a result of forming colonies in the same manner as in the practical example 2, except that PU 0.8 mg / l and 6BA 0.2 mg / l were individually added, the colony forming rate was lower than that in the practical example 2.
(Table-3)

[0033]

[Table 1]

[0034]

[Table 2]

[0035]

[Table 3]

[0036]

As described above, when culturing protoplasts, a single auxin and a plurality of cytokinins were used in combination to improve the colony forming rate.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor, Kazuya Ito 24-9, Nobinocho, Kameyama City, Mie Prefecture Kamiyama Laboratory, Forest Tree Breeding Research Institute, Oji Paper Co., Ltd. (72) Inventor, Masaru Shibata 24-9 Koinomachi, Kameyama Laboratory, Forest Tree Breeding Research Institute, Oji Paper Co., Ltd.

Claims (1)

[Claims] 1. A protoplast of a woody plant and a protoplast of a herbaceous plant having a high division ability are cultured in a liquid medium in which a basal medium for plant tissue culture contains a plant hormone, a carbon source and an osmotic pressure adjusting agent. A method for cultivating a protoplast of a woody plant that produces a colony of a woody plant, wherein the plant hormone is one auxin and one or more types of cytokinins.