JPH0430810B2 - - Google Patents

Description

[Detailed description of the invention]

[Field of Industrial Application] The present invention relates to a method for regenerating plants from protoplasts of woody plants. This invention relates to a method for regenerating plants from protoplasts. Therefore,
By applying the present invention, it becomes possible to develop mass production technology for producing embryoid bodies from single cells. Furthermore, it has become possible to create new plants through genetic recombination and cell fusion, which are important techniques for creating mutants. [Prior Art] Since protoplasts produced from plant cells do not have cell walls, they can be subjected to cell fusion treatment or the introduction of useful genes. If it becomes possible to regenerate plants from such protoplasts, we will not only be able to create completely new plants that have never existed before, but also produce crops that meet the purposes of human use, such as high-yield, delicious food. It will be possible to create new species that have good properties and can also withstand pests, diseases, and weather damage. Techniques for regenerating plants from protoplasts obtained by treating plant tissues such as leaves and stems with enzyme solutions are being established for tobacco, petiunia, and many other herbaceous plants. however,
Among woody plants, Trovita orange (Shozo Kobayashi et al., "Breeding Journal", Vol. 34 (Part 2), 32-33, 1984,
JP-A No. 61-192283), Kozo (Narumi Oka, Katsuo Oyama, “Breeding Journal”, Vol. 34 (Separate 2), 26-27,
1984), the present inventors used the poplar example (Kazunari Ito et al.,
Patent Application No. 61-64800, Russell, J, A. and BH
McCown, Plant Sci, 46, 133-142, 1986)
Sandalwood (Rao, PSand P. Ozias−Akins,
Protoplasma, 124, 80-86, 1985), Elm (Sticklen, MBet al. Plant sci, 47, 29-34,
There are only a few examples, such as 1986). The reason for this is that in the case of protoplasts obtained from plant tissue, substances such as polyphenols discharged into the medium during the culture process and aggregation of protoplasts act as an inhibitor to their survival and division, which inhibits plant regeneration. There are many things that could not be achieved. For this reason, recently, in order to avoid aggregation of protoplasts, solid media are used instead of liquid media, and immature embryos or immature seeds are used as materials to create callus with higher differentiation potential, and then protoplasts are have been isolated and cultured, but
Even so, as mentioned above, there are still only a very small number of cases in which complete plants have been obtained, especially in woody plants. Therefore, the present inventors have completed the present invention as a result of repeated research on a method for solving these problems and easily regenerating woody plants from protoplasts. [Problems to be Solved by the Invention] The present invention allows the protoplasts of a woody plant to coexist with the protoplasts of a herbaceous plant with a high division rate of protoplasts at the early stage of culture, so that the plant body cannot be recovered from a single protoplast. The purpose is to regenerate plants from protoplasts of woody plants that are difficult to regenerate. [Means for Solving the Problems] The present invention is a method for regenerating a plant from protoplasts of a woody plant, which is characterized by culturing protoplasts of a woody plant in the coexistence of protoplasts of a herbaceous plant and then differentiating them. It's a method. The types of plants used in the present invention are not particularly limited, but types of herbaceous plants include kenaf, tobacco, vetyunia, etc., while woody plants to be regenerated include eucalyptus, mangrove, acacia, etc. Evergreen broad-leaved trees such as Hevea brasiliensis and coffee, deciduous broad-leaved trees such as poplar, thorn, Quercus oak, Japanese oak, and sumac; useful coniferous trees such as pine, cedar, cypress, fir, spruce, and larch; tangerines, grapes, figs, and almonds; These include fruit trees such as mangoes, and flowering trees such as roses, plums, camellias, and cherry blossoms. Hereinafter, the method for producing callus and shoots, the method for isolating and adjusting protoplasts, the culture medium for protoplasts, culture conditions, etc. used in the present invention will be explained in detail. Method for creating callus and shoots After sterilizing the seeds of woody and herbaceous plants,
Plant tissue culture media, e.g. Gam
-borg) B5 medium or Murashige/Skoog's MS medium or other agar medium, and germinate. Next, cut off the grown leaves, stems, and roots.
B5 medium, MS medium, etc. contain plant hormones, such as auxins such as naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), or indole acetic acid (IAA), and benzyladenine ( BA), kinetin, KT-30, or cytokinins such as zeatin are added to agar medium. This is cultured at a temperature of 20 to 30°C and an illuminance of 2000 to 5000 lux to obtain callus or shoots. In the case of woody plants, it is also possible to obtain shoots by sterilizing the stems, subjecting them to circular peeling until they reach the cambium layer, and then cuttings into an agar base in the same manner as described above. Isolation and preparation method of protoplasts Using the callus or shoots obtained by the above method as material, pectolyase Y-25 is an enzyme that decomposes pectin, an intercellular substance, cellulase Onozuka RS is an enzyme that decomposes cell walls, and mannitol are added. The protoplasts are isolated by adding the containing enzyme solution and shaking at a temperature of 20 to 30° C. for 6 to 24 hours at a shaking speed of 20 to 30 rpm. Next, the enzyme solution is removed, the protoplasts are washed, and the protoplasts are centrifuged to prepare the protoplasts. Mixing of protoplasts 10 ⁴ protoplasts of woody plants to be regenerated, which were isolated and prepared after enzyme treatment, and protoplasts of herbaceous plants to be cultured in coexistence with these.
Adjust the concentration to 10 to ¹⁰⁵ particles/ml. Next, the mixing ratio of both protoplasts was 1:3~
plant tissue culture medium, mixed in a 3:1 ratio;
For example, after selecting Kanborg's B5 medium, Murashige-Skoog's MS medium, etc. according to the properties of protoplasts, plant hormones such as naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4 -D), auxins such as indole acetic acid (IAA) or benzyladenine (BA),
Culture in agar medium or liquid medium supplemented with cytokinins such as KT-30, kinetin, and zeatin. Method for culturing protoplasts Protoplasts are cultured using isolated and prepared protoplasts in the above-mentioned tissue culture medium. However, a more effective method is to add ``dierite'' as a support agent to support protoplasts in the culture vessel in a concentration range of 0.001 to 0.05% in addition to the above-mentioned tissue culture medium and plant hormones. be. As for the culture conditions, it is preferable to initially culture in the dark and gradually increase the light as the protoplasts grow. Also, the temperature during the culture period is preferably 20℃ or 30℃, but especially 25℃.
Temperatures between 28°C and 28°C are preferred. In the agar medium method, separately prepared protoplasts are added to a heated and molten medium, mixed, and then fixed and cultured in a floating state in the medium, which solidifies as the temperature decreases. It is. In this case, placing protoplasts in a heated culture medium has an adverse effect on the subsequent survival and growth of protoplasts. In addition, when culturing in a liquid medium, it is not only necessary to avoid the harmful effects of polyphenols, but also to prevent protoplasts floating in the liquid medium from aggregating and inhibiting their division and survival. It is necessary to avoid this. "Dierite" used in the present invention
("GELRITE", manufactured by Kelco, USA),
The main component is polysaccharides released outside the bacterial body by Pseudomonas elodea, a type of bacteria. By adding this to a liquid medium at a low concentration, a layer of gelatinized dierite is formed at the bottom of the culture container. The protoplasts are attached and supported to this. For this reason, even when exchanging the medium, the protoplasts are adhered to the dierite layer, and the exchange can be performed without flowing out together with the medium. In this way, a callus of a woody plant is formed for the purpose of regenerating the plant body. Plant regeneration method Callus obtained by co-cultivating protoplasts,
Plant hormones such as naphthalene acetic acid (NAA),
2,4-dichlorophenoxyacetic acid (2,4-D),
Easily regenerate shoots by using auxins such as indole acetic acid (IAA) and cytokinins such as benzyladenine (BA), KT-30, kinetin, and zeatin.
Further rooting is possible. In this way, complete plants are regenerated from protoplasts. EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited to these Examples. [Example] Test Plant Poplar (Populus charkowiensis×P.
caudina, OP-20), and kenaf (Hibiscus
sabdariffa) was used. Method for producing shoots of poplar After sterilizing the stems of adult poplar trees with 70% ethanol for 5 minutes, they were aseptically cut into approximately 2 cm long pieces, the epidermis was peeled until the cambium layer was reached, and the stems of the plants shown in Table 1 were cut. 0.01 mg of naphthalene acetic acid as a plant hormone in Gamborg B5 medium, which is a tissue culture medium.
Cuttings were placed in agar medium (0.6% agar added) to which KT-30 was added at a rate of 0.02 mg/day and the pH was adjusted to 5.6. This was cultured at a temperature of 28°C and an illuminance of 3000 lux.

[Table] A large number of adventitious buds were observed to form about 40 days after the start of culture, and shoots of about 1 to 2 cm were obtained by further culturing. In addition, by sub-planting these cuttings into fresh multilayer soil, it is possible to repeatedly regenerate shoots. Induction of kenaf callus After sterilizing kenaf seeds with 70% ethanol and 10-fold diluted antiformin solution, 3% sucrose was added aseptically to Gamborg B5 medium, which is a plant tissue culture medium. Germinated on agar medium, cut the stems including the stem nodes into lengths of about 1 cm, and added them to B5 medium as plant hormones, such as 2 mg/2,4-dichlorophenoxyacetic acid and 0.01 mg/benzyladenine.
The mixture was placed on top of agar agar added at a ratio of . This was cultured at a temperature of 28°C and an illuminance of 3000 lux. A yellow-white callus was obtained about 40 days after the start of culture.
This was used as a material for isolation and preparation of protoplasts. Isolation and preparation of protoplasts For each 1 g of poplar shoots and kenaf callus obtained by the above method, 20 ml of enzyme solution (1% cellulase Onozuka RS, 0.1% pectolyase Y-23, 13% mannitol, PH5.6) at a temperature of 20-30℃ and shaking at 20-30 rpm for 6-24 hours.
Protoplasts of both plants were isolated separately by enzymatic treatment for several hours. After treatment, the protoplast suspension was passed through a nylon mesh to remove undigested cell mass, and further centrifuged to separate the enzyme solution and protoplasts, and washed with a washing solution (13% mannitol, 50mM CaCl ₂ ). After washing twice with water, the cells were subjected to culture. Cultivation of protoplasts and regeneration of plants The culture medium for protoplasts was prepared by adjusting the concentration of substances other than mannitol to twice the concentration of Gamborg's B5 medium shown in Table 1, and on the other hand,
Prepare a dierite solution by adding dierite at a ratio of 0.05% to a 9% mannitol solution (PH5.6), and autoclave both. After the liquid has cooled down sufficiently,
A mixture of poplar and kenaf protoplasts in a ratio of 3:1 (3 parts poplar to 1 part kenaf) was suspended in the dierite solution at a concentration of 10 ⁵ cells/ml. This mixed protoplast suspension and the culture medium were mixed at a ratio of 1:1 and plated on a 6 cm diameter petal. The amount to be plated was 2 ml per share. The plated protoplasts were initially cultured in the dark, and the concentration of mannitol was increased to 6% at 15 days and 3% at 40 days after the start of culture, depending on the division of the protoplasts.
% and 0% in 50 days, while sucrose was increased to 3% and cultured at a temperature of 28°C. After culturing, from about 30 days onwards, NAA was added to Gamborg's B5 medium shown in Table 1, under light conditions, and from about 50 days onwards.
Poplar colonies, the target plants, were taken out and transplanted onto a shoot regeneration medium made by adding 0.02 mg/0.02 mg/BA, 0.2 mg/3% sucrose, and 0.6% agar to harden it. This was cultured at 28°C under a 16 hour photoperiod condition. Approximately 30 days after transplanting to a shoot regeneration medium, shoots regenerated, and when these were further transplanted to a rooting medium shown in Table 2 and cultured under the same conditions, roots were formed and the plants were regenerated. Ta. The state of this rooted plant is shown in FIG.

[Table] [Effects of the invention] As explained above, until now it has been extremely difficult to regenerate plants from protoplasts isolated and prepared from tissues of plants, especially woody plants. By mixing and culturing the protoplasts of a plant with herbaceous plant protoplasts that have high division activity or herbaceous plant protoplasts that have the ability to regenerate a plant body, it becomes possible to easily regenerate the plant body. , provides an extremely powerful method for creating new plant varieties.

[Brief explanation of drawings]

FIG. 1 is a photograph showing a plant body from poplar protoplasts that was regenerated by co-cultivating poplar protoplasts and kenaf protoplasts.

Claims (1)

[Scope of Claims] 1. A method for regenerating a plant from protoplasts of a woody plant, which comprises culturing the protoplasts of a woody plant in the coexistence of protoplasts of an herbaceous plant, and then differentiating the protoplasts. 2 Calli or shoots of woody plants and herbaceous plants are individually treated with enzymes to isolate protoplasts, and both types of protoplasts obtained are mixed at a certain ratio to form an inorganic salt composition and a support system for protoplasts. In an artificial liquid medium containing plant hormones, culture is performed at a temperature of 20 to 30°C to form a callus (cell mass) in which protoplasts grow and divide, and only the callus of woody plants is transferred to a regeneration medium. 2. The method according to claim 1, wherein the plant is regenerated by regenerating shoots and rooting. 3. The method according to claim 1 or 2, wherein the mixing ratio of woody plant protoplasts and herbaceous plant protoplasts is 1:3 to 3:1.