JPS62224224A - Regeneration of plant body from protoplast of ligneous plant - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for regenerating a plant from protoplasts of a woody plant.

[Conventional technology]

A technology has been developed to obtain protoplasts, cells whose cell walls have been removed using enzymes that decompose plant cell walls (C0CKING, E, C, Nature+187
, 962-96, 1960), this method has recently been applied to many plants, and cell fusion technology has been attempted to artificially fuse protoplasts obtained from two types of plants obtained by this method. Became(
CARL8ON et er,t,, Proc.

NatL, Acad, Sci, US8 69.
2292-2294. 1972).

Cross breeding, which has been carried out as a method of improving plant varieties, is limited to plants of the same species, or very closely related plants such as Japanese black pine, which is obtained by crossing Japanese red pine and Japanese black pine. was. However, by using this cell fusion technology, it has become possible to hybridize not only plants of different species but also of different genera and families, increasing the possibility of obtaining hybrids. In other words, even if plants that are extremely distantly related in terms of plant classification have excellent properties, by using cell fusion technology, it is possible to add these properties to the target value, increasing the possibility of breeding. The range has become extremely large.

Furthermore, since woody plants such as poplars take many years from seed germination to flowering, it has taken a long time to carry out hybridization using traditional flower vases.

On the other hand, the method of improving plant varieties or traits using cell fusion technology is an extremely useful and important method because it can shorten this number of years.

However, for woody plants, there is an example in which a plant was regenerated not only from fused protoplasts but also from a single protoplast (5) in Tropita Orange (Shozo Kobayashi et al., Journal of Breeding, Vol. 34 (separate article). 2), 32-53.19
84), and Kozo (Narimi Oka, Katsuo Oyama, Journal of Breeding Science).

There are only a very small number of them, such as Volume 34 (Part 2, 26-27.1984).

Therefore, even if cell fusion technology were established, a method for culturing the fused cells would not be developed.
It is impossible to use the a-combination technology, and as a preliminary step, it is urgent to establish a technology for regenerating plants from protoplasts.

[Structure of the invention]

The present invention involves cell fusion treatment of woody plant protoplasts and herbaceous plant protoplasts: 2? This is a method for regenerating a differentiated plant body from woody protoplasts, which is characterized by treating with & and then differentiation.

The present inventors were conducting research on a method for regenerating plants from protoplasts of woody plants.
We have discovered that by treating the woody plant brotoplast with a chemical solution that causes cell fusion in the presence of herbaceous plant protoplasts and then allowing them to differentiate, it is possible to easily regenerate the differentiated plant body from the woody plant protoplast. .

Hereinafter, plants, cell fusion agents, protoplast culture media, and culture conditions that can be used in the present invention will be explained in detail.

Plants The plants to which the present invention can be applied are not particularly limited, but woody plants include, for example, evergreen broad-leaved trees such as podef, eucalyptus, acacia, puffa rubber, sumac, coffee, tangerine, lemon, cherry, apple, Include fruit trees such as pears, peaches, avocados, persimmons, walnuts, grapes, figs, almonds, and mangoes, flowering trees such as para, camellia, plum, and cherry trees, and coniferous trees such as pine, cedar, cypress, fir, and spruce. I can do it.

On the other hand, examples of herbaceous plants include kenaf, tobacco, and petunia.

Examples of the materials used include cultured cells derived from plant parts such as mesophyll tissue, stems, roots, and the like. Furthermore, it is also possible to use the shoot primordium obtained by culturing the shoot apex and the cutting ray obtained by cutting the stem into a nutrient medium after peeling the stem into a ring.

Cell fusion agent The cell fusion agent that can be used in the present invention includes polyvinyl alcohol (hereinafter abbreviated as PEC), but PEG is particularly preferred. Without using cell fusion agents'w. It is also possible to process using a gc method.

Protodefust culture medium The protoplast culture medium used in the present invention includes conventionally known plant tissue culture media, such as Gumbo μg's B5 medium, Murashige-Skoog's MS medium, etc. In particular, the B5 modified medium shown in Table 1 is suitable.

Examples of plant hormones in this medium include naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2
．． Examples include auxins such as 4-D), indo-p acetic acid (IAA), and indolebutyric acid (IBA), and cytokinins such as adenine and benzene μ adenine (BA), KT-30, kinetin, and zeatin.

Table 1 Composition of the medium used for protoplast culture Protoplast culture conditions In the present invention, in the early stage of culture of protoplasts obtained by treating protoplasts of both woody and herbaceous plants with a cell fusion treatment solution, Light irradiation is not necessary, and culturing in the dark is preferable for protoplast growth. Further, the temperature during the culture period is preferably 20 to 30°C, particularly preferably 25 to 28°C.

Protoplasts cultured in the dark are continued to be cultured by being irradiated with diffused light from midway through the culture while periodically replacing the culture medium.

The resulting cell mass is transplanted into a medium that promotes regeneration of the d-stripes. Subsequently, by transplanting the regenerated m-rows into a medium that promotes rooting and continuing culturing,
Complete regenerated plants can be obtained from protoplasts of woody plants.

The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples.

Example 1 1) Test body Populus charkowiensis XP
A branch of an adult poplar tree of the Caudina variety is cut, cut to a length of about 2 crn, sterilized by a conventional method, and the bark is aseptically stripped to the extent that the cambium layer is exposed. This is NAAo, 01rIII/l, B A
0.2■/L, sucrose 509/L and agar 8/i
The cuttings were placed in Ganpolleg's B5 solid medium containing a concentration of / / = to induce regeneration of foil streaks.

2) Test Kenaf Kenaf (Hlbiscus 5abdariffa)
The stems, leaves, and even roots of 2.4-D were sterilized in the usual way, and 2.4-D was 4μ/L and BA was 0.2η/L.

and gumbo μg B5 containing at a concentration of sucrose sag/l
'The cells were placed on a solid medium to induce pneumococcal growth.

5) Protoplast isolation method To isolate protoplasts from shoots regenerated from poplar cuttings and kenaf callus, cellulase (Onozuka R8) t=11 pectolyase Y-23 (
An enzyme solution containing 11% and sunnitol at a concentration of 131 was adjusted to pH 6. Both types of protoplasts were then isolated by enzymatic treatment for 6 hours in the light at a temperature of 30° C. with gentle shaking. After the treatment is complete, remove the enzyme solution and add 13 grams of mannide solution.
Washed twice, 5 x 105 pieces/each of both derotopfusts.
- 13% mandi to give a concentration of -/I/e,
A friend suspended in.

4) Treatment with protoplast fusion treatment solution Poplar and kenaf protoplast suspensions were mixed, and 200 μt of the mixture was placed on a 22 m diameter cover glass placed in a 6 G diameter petri dish. Next, around this, 40% by weight of PEG (molecular weight: 000) and 50 f of hydroxy V ethylbiverazine ethane μ fonic acid were added.
iM, and 200 μAm of PEG solution containing 100 mM chlorination power ciram were added and slowly mixed.

After leaving it at room temperature for about 30 minutes, the chloride power
@M1 A wash solution containing 13% mannitol was added 0.2 times, followed by another 1 time after 15 minutes. Additionally, the 1- and 4-wash solutions were added at 15 minute intervals. 1000
After centrifugation at rpm for 3 minutes, the supernatant was removed and fresh wash solution was added 4-i. After repeating the same centrifugation operation again, the cells were washed once with the protoplast culture medium shown in Table 1, and then culture was started.

5) Culture the poplar protoplasts that have been washed for 2 hours in the dark.
The cells were cultured at 8°C for about 1 month. The grown colonies were treated with a mannitol concentration of 6q6. They were successively transplanted to a medium containing gradually lowered S content to 0%, and about 4 months after the start of protoplast culture, they were transplanted to a medium for shoot regeneration shown in Table 2. Approximately two weeks after transplanting to a shoot regeneration medium, the shoots regenerated as shown in Figure 1, and when the shoots were further transplanted to a rooting medium shown in Table 3, roots elongated. The plant thus regenerated was morphologically identical to poplar, as shown in FIG.

Table 2 Composition of medium for shoot regeneration Table 3 Comparative example of composition of rooting medium 1 In Example 1, the protoplasts of poplar and kenaf were cultured alone without treatment with the drug that performs fusion treatment of protoplasts. As a result, the two-value item was not regenerated at all.

In this way, although each protoplast cannot differentiate and form a plant by itself,
The reason why poplar and kenaf protoplasts can be differentiated into υ plants by treating them with a fusion treatment solution is that some substance or stimulus that triggers poplar protoplasts to start dividing can cause kenaf protoplasts to differentiate into υ plants. This is thought to be caused by protoplasts.

〔Effect of the invention〕

As explained above, when protoplasts of woody plants and herbaceous plants are cultured alone without treatment with a fusion agent, no regeneration of the plants is observed. However, by culturing after treatment with a fusion agent, it has become possible to regenerate a complete woody plant differentiated from protoplasts.

[Brief explanation of drawings]

Figure 1 is a photograph showing the morphology of poplar shoots regenerated from protoplasts treated with poplar protoplasts and kenaf protoplasts with a chemical solution for cell fusion treatment, and Figure 2 is a photograph showing the morphology of poplar shoots obtained from the shoots shown in Figure 1. This is a photograph showing the morphology of a poplar. Calculation 2 t'EJ Procedural amendment 9/81? 6th Patent Office Commissioner Kuro 1) Mr. Akio 1, Indication of the case Patent Application No. 64800 of 1985 2
Title of the invention Method for regenerating a plant from protoplasts of a woody plant Relationship with the amended case Patent applicant address 4-7-5, Ginza, Chuo-ku, Tokyo Name Oji Paper Co., Ltd. Representative Kawa Keji Bunishi Shinbashi Chuo Bishi 502 Telephone (457) -5467 Name Patent Attorney (7850) Nakamoto
Hiroshi (and 2 others) & Subject of amendment (1) Detailed description of the invention in the specification 1 Explanation column 2 Contents of amendment (1) “Kena 7 (H1 biscuit)” on page 9, line 13 of the specification
a8abdariffa) J to [Kenaf (Hl
biscusCannabinus) J.

Claims (1)

[Claims] 1. A method for regenerating a differentiated plant from protoplasts of a woody plant, which comprises treating protoplasts of a woody plant and protoplasts of an herbaceous plant with a chemical solution for cell fusion treatment, and then causing them to differentiate.