JPH09224513A - Growing point cultivating method for citrus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently raise seedlings of citruses by performing growing point cultivation by new culture medium composition different from a conventional method. SOLUTION: When a seedling body is formed by cultivating growing points of citruses in a culture medium containing cytokinin, etc., cytokinin having an N,N'-substituted urea structure such as thidiazuron and CPPU is used and/or the culture medium contains no starch, etc. The use of the culture medium 1a by this growing point cultivating method for citrus accelerates speedy and normal growth as to the growing points of the citruses. Therefore, plant bodies 3a1 and 3a2 of the citruses can be reproduced with high quality through the medium and virus-free, viroid-free plants of citruses can be produced.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a very practical method for growing citrus cultivars that can be used for large-scale growth using tissue culture and for producing healthy plants from virus-affected strains.

[0002]

2. Description of the Related Art Heretofore, a tissue piece containing a growth point has been cut out from a plant, and this tissue piece has been cultured in a test tube or the like to give a healthy individual not infected with a viral disease, that is, a virus-free seedling. There is known a technique for reproducing the. The growth point here is present at the tips of shoots, side buds, etc. of shoots and consists of meristems of many cells, and is also called shoot apex. In the growing point culture method, the meristem of this growing point is cut off and cultured in a test tube.When cutting out the growing point for the purpose of obtaining virus-free seedlings, the growing point should be taken into consideration in consideration of virus distribution. It is necessary to carefully cut out only the very small part that contains it.

On the other hand, from the horticultural standpoint, citrus is said to refer to a group of plants included in three genera of the genus Citrus, the genus Kalachi and the genus Kumquat ("Fruit gardening", Asakura Shoten, p.
59). In addition, there is a document that "the technology for growing point culture of citrus is established (" fruit tree seedling production and biotechnology ", Hakutosha, p129)", which is the basis for this. (Sonogaku Zakka., Vol. 58, Supplement 2, p62-p63, 1989) clearly states that "problems remain in the growth in primary culture".

That is, the growth point culture of citrus has a problem in its growth, and it is the current situation that it has not reached a technical level at which the main use thereof, such as the production of virus / viroid-free (detoxified) plants, can be put to practical use. . Therefore, in order to produce virus and viroid-free citrus fruits, it is the actual situation that complicated techniques such as shoot apex grafting had to be used (“Fruit and Seedling Production and Biotechnology”, Hirotosha, p143-p146). .

[0005]

DISCLOSURE OF THE INVENTION The present inventors have recognized that the conventional method for cultivating the growth point of citrus fruits has the above-mentioned problems, and the growth point is changed by a novel medium composition different from the conventional method. A method for cultivating and efficiently growing citrus seedlings was examined.

[0006]

As a result, the present inventors have succeeded in solving the above-mentioned problems of the prior art by adding a substance, which has not been used until now, to a culture medium during the growth point culture of citrus. The present invention has been completed by discovering that the present invention can be solved and further conducting research based on this new finding. That is, according to the present invention, in culturing the growth points of citrus fruits in a medium containing cytokinins to form seedlings, cytokinins having an N, N′-substituted urea structure were used as the cytokinins. A method for cultivating a citrus growing point is provided.

[0007] A method for cultivating a citrus growing point is provided, in which the citrus growing point is cultivated in a medium containing cytokinins to form seedlings, and the medium contains starch. To be done. Furthermore,
As the cytokinins in the above structure, N, N'-
A cytokinin having a substituted urea structure may be used.

[0008]

Embodiments of the present invention will be described below. Citrus applied to the present invention includes citrus genus (Citrus) and citrus genus (Ponciru).
s) and a plant belonging to three genera of the genus Fortunella (Fortunella) are not particularly limited. The citrus plant is not particularly limited, but specifically, citrus fruits,
Grapefruit, sweet orange, lemon, tangor, etc. can be exemplified.

In the present invention, the growth points of citrus plants are tissue-cultured in a specific medium to form seedlings. The growth points used for tissue culture in this case include apical or lateral buds of cultivated plants and cultured plants, callus in culture,
Growth points such as embryos and shoot primordia and tissues in the vicinity thereof can be used.

The cytokinins used in the present invention include cytokinins having an N, N'-substituted urea structure. Examples of the cytokinin having this N, N′-substituted urea structure include thidiazuron [Thi
diazon: N-phenyl-N '-(1,
2,3-thiadiazol-5-yl) urea:
Hereinafter, abbreviated as TDZ], N- (2-chloro-4-pyridyl) -N'-phenylurea [N- (2-chlor
o-4-pyridyl) -N'-phenyurere
a: hereinafter abbreviated as CPPU], or N-phenyl-N '-(4-pyridyl) urea [N-phenyl]
-N '-(4-pyridyl) urea], N-phenyl-N'-(2,6-dichloro-4-pyridyl) urea [N-phenyl-N '-(2,6-dichloro)
o-4-pyridyl) urea], or N-phenyl-N ′-(2,6-dibromo-4-pyridyl) urea [N-phenyl-N ′-(2,6-dibromo)
o-4-pyridyl) urea] and the like can be exemplified, but it is not particularly limited as long as it is a urea type cytokinin having a heterocycle in which one benzene ring of the diphenylurea structure is replaced with another ring. The cytokinin having this N, N'-substituted urea structure is prepared to have a concentration of 10 ^{-6 to} 10 ^-12 M in the medium.

The starches used in the present invention include:
Examples thereof include those derived from corn, wheat, potato and the like. In the case of the present method used for citrus, corn starch, wheat starch and the like are particularly preferable. This starch is preferably prepared to have a concentration of 0.1% to 10% in the medium.

The medium used in the growth point culture of citrus fruits of the present invention contains cytokinins having an N, N'-substituted urea structure of 10 ^-6 to 10 ^-12 M, preferably 10 ^-7.
It is preferable to use a medium containing 10 ^-10 M. Alternatively, starch is 0.1% to 10%, preferably 0.5% to 1%
It is also possible to use a medium added so that the concentration becomes.
Further, a medium containing both cytokinin and N-N'-substituted urea structure-containing starch can be used, and in that case, the growth of the growing point is further promoted.

The medium used in the present invention is preferably a medium containing an inorganic component and a carbon source as essential components, to which plant hormones and vitamins are added, and further amino acids if necessary. . The inorganic components used in the medium include nitrogen, phosphorus, potassium, sodium,
Inorganic salts containing elements such as calcium, magnesium, sulfur, iron, manganese, zinc, boron, molybdenum, chlorine, iodine and cobalt can be mentioned, and specifically,
Potassium nitrate, sodium nitrate, ammonium nitrate, potassium chloride, calcium chloride, potassium monohydrogen phosphate,
Sodium dihydrogen phosphate, magnesium sulfate, magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate,
Examples thereof include compounds such as manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid and cobalt chloride.

As the carbon source used in the medium, in addition to the starches described above, for example, sucrose (Sucros) is used.
Examples thereof include carbohydrates such as e) and their derivatives, organic acids such as fatty acids, and primary alcohols such as ethanol. As the plant hormone used in the medium, other than urea-type cytokinins such as TDZ or CPPU, for example, benzyladenine (hereinafter abbreviated as BA), kinetin, adenine-type cytokinins such as zeatin, and naphthalene acetic acid (hereinafter, NAA), indoleacetic acid (IAA), p-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-
D), indole butyric acid (IBA), and auxins of these derivatives can be exemplified.

As vitamins used in the medium
Is biotin, thiamine (vitamin B ₁), Pyridoxy
(Biamine B₆), Pyridoxal, pyridoxami
, Calcium pantothenate, ascorbic acid (vitamin
C), inositol, nicotinic acid, nicotinic acid
And riboflavin (vitamin B₆) Etc.
Wear. As the amino acids used in the medium, for example,
For example, glycine, alanine, glutamine, cystine, fe
Nylalanine, proline, lysine, etc. can be exemplified.
You. Usually, the medium contains about 0.1 μm of the above-mentioned inorganic components.
M to about 100 mM, 10 plant hormones^-12M
Chair 10^-FiveM and amino acids from 0 to about 1000
It is desirable to use it by including mg / l.

Specific examples of the medium used for the tissue culture of the present invention include conventionally known medium for plant tissue culture, for example, Murashige Scoog ('62) [Mur.
Ashige &Skoog's medium, Murashige Tucker's ('69) [Murashige & Tucker] medium, Rinsmeier Skoog (RM-1965)
[Linsmaier & Skoog] medium, White ('63) [White] medium, Gamborg [Gam
Borg's B-5 medium, Mitsui's M-9 medium, niche.
A medium prepared by adding the above-mentioned carbon source and plant hormones to a niche [Nitch & Nitch] medium, a Lloyd & McCown WPM medium, etc., and further adding the above-mentioned vitamins and amino acids as necessary. Can be illustrated.

In the present invention, those prepared based on the niche niche, Rinsmeier Skoog, Murashige Scoog, and Murashige Tucker medium are particularly preferable. Regarding the composition of the conventionally known medium described above, for example, "New plant tissue culture" by Takeuchi, Nakajima, and Furuya
(P386-p391, Asakura Shoten, 1979). In addition, the medium that can be used in the present invention is preferably a solid medium or a liquid medium in which a gelling agent such as agar or gellite is usually contained in an amount of 0.1 to 2%.

Therefore, according to the present invention constructed as described above, the growth points of citrus can be efficiently grown. In addition, the plant obtained by the present invention can be grown into a healthy seedling having a constant property when it is normally cultivated, and it can also be applied to the production of virus / viroid-free plants.

[0019]

EXAMPLES Next, the method of the present invention will be specifically illustrated by Examples. Embodiment 1 FIG. The growth point is 0.2-under a stereoscopic microscope from the cultured plants of Sanhokan and Ponkan that have been maintained and passaged in a sterile condition.
It was cut into a size of 0.3 mm and placed on a medium of a 25 mm diameter test tube. The medium is Murashige-Skoog's medium (MS
Medium) as a basic medium, sucrose 20 g / l, biotin 0.05 mg / l, folic acid 0.5 mg / l, gibberellin (GA ₃ ) 3 × 10 ⁻⁵ M, NAA10 ⁻⁷ M, gellan gum 2.5 g / l Sterile solid MS medium containing
5.6) was added with TDZ prepared at 10 ⁻⁸ and 10 ⁻⁹ M concentration, respectively. Five citrus vegetative growth points were placed on each plate and cultured at 27 ° C., 500 lux, and 16-hour photoperiod for 40 days.

Comparative Example 1 Compared with Example 1, except that BA10 ^-6 M was added instead of TDZ, the growth point of citrus was cultured in the same manner as in Example 1. The results of Comparative Example 1 are shown in Table 2.

The culture results according to the above Example 1 are shown in Table 1 by using the length (shoot length) of the growing point as a criterion. According to this, in the medium to which 10 ⁻⁹ M TDZ was added, the growth was promoted in both cultivars as compared with Comparative Example 1 shown in Table 2.

Here, in FIG. 1, Sanhokan (FIGS. (A) and (b)) cultured in a medium supplemented with 10 ⁻⁹ M TDZ,
Sanhokan (cultivated in the medium of Comparative Example 1 (FIG. 2 (c),
(D)) shows the culture state. That is, FIG.
As shown in (b), the seedlings 3a ₁ , 3a _{2 of} Sanhokan were cultured in the medium 1a in the test tube 2 according to this Example 1.
Is free of viruses and viroids and has a height of 20-
It grows well to about 50 mm and has many nodes. Therefore, the Sanhokan seedlings can be subjected to tissue culture in large numbers when cut into nodes, leaving a possibility of large-scale growth.

On the other hand, according to the comparative example, the seedlings 3b ₁ and 3b of Sanhokan were cultured in the medium 1b in the test tube 2.
_As shown in FIGS. 1 (c) and 1 (d), ₂ can be cultured only up to about 3 to 5 mm, and no node formation is observed. Such seedlings have a very poor survival rate when grafted and are not practical.

Embodiment 2 FIG. In Example 1, except that TDZ was replaced by 10 ⁻⁶ M of conventional general-purpose BA, and 1% of corn starch (hereinafter abbreviated as CS) or wheat starch (hereinafter abbreviated as WS) was added. In the same manner as in Example 1, citrus vegetative points were cultured. Table 1 shows the results. Here, starch is 1
The ones that had been pregelatinized by heating at 21 ° C. for 20 minutes were added. In addition, the added amount of CS and WS is% by volume with respect to the entire medium. According to the results of Example 2, both varieties were slightly inferior to Example 1, but compared to Comparative Example 1, growth could be promoted.

Embodiment 3 FIG. In Example 1, TDZ is 1
0 ^-9 M, and 0% CS, except that 0.5%, was added in 1%, respectively, were cultured growing point of the to citrus same manner as in Example 1. Table 2 shows the obtained results.
That is, at this time, both varieties were able to further promote growth as compared with Comparative Example 1.

[0026]

[Table 1]

[0027]

[Table 2]

Embodiment 4 FIG. Only Ponkan was used as a variety, and a growing point cut out to a size of 0.5 to 0.6 mm was used, CS was 0.5%, and TDZ
CPPU instead of 10 ^-7 M, 10 ^-8 M, 10 ^-9 M,
Example 1 except that 10 ^-10 M each was added.
The culture was performed in the same manner as described above. The results obtained are shown in Table 3 below.

Comparative Example 2 The same size of poncan growing point as used in Example 4 was used and BA was 10 ^-7 M.
The growing point was cultured in the same manner as in Comparative Example 1 except that it was added. The results of this Comparative Example 2 are shown in Table 3.

[0030]

[Table 3]

That is, the CPPU 10 as in the fourth embodiment.
^It can be seen that, when cultured in a medium containing ^-7 M, the growth is promoted as compared with that in Comparative Example 2. In addition, in Example 4 and Comparative Example 2, the shoot growth was generally good because the cut out growth points were Examples 1 to 3.
This is because the one used is larger than that used in Comparative Example 1.

[0032]

As described above, ^10-6 to ^{10-12 of} cytokinin having an N, N'-substituted urea structure such as TDZ or CPPU is added to the medium according to the present invention.
M is added or / and starch is 0.1% to 10%.
%, The growth point of citrus can be tissue-cultured, and the growth point of citrus can be rapidly promoted to promote normal growth. Therefore, by passing through this, it is possible to regenerate a healthy citrus plant having a constant property. As a result, according to the method of the present invention, it is possible to efficiently reproduce a high-quality plant from the growth point of citrus fruits as compared with the conventional method, and to produce a large amount of virus-viroid-free plants of citrus fruits. That is, the tissue culture method based on the novel knowledge according to the present invention could be realized.

The method of culturing the growth point of citrus in tissue by adding starch to the above medium, for example, 0.1% to 10%, also promotes the normal growth of the growth point of citrus. It

Further, when the growth points of citrus fruits were cultured in a medium to which both cytokinins having N, N'-substituted urea structure and starches were added, compared to the case where each of them was added alone. Can further promote the growth.

[Brief description of drawings]

FIG. 1 shows three treasure citrus cultivated by the growing point culturing method, (a) and (b) are external views showing the three treasure citrus plantlets cultivated by the method of the present invention, (c) and (d). [Fig. 4] is an external view showing a Sanbokan seedling that has been cultured by a conventional method.

[Explanation of symbols]

1a medium 1b medium 2 test tube 3a ₁ young plant 3a ₂ young plant 3b ₁ young plant 3b ₂ young plant

Claims (3)

[Claims] 1. A cytokinin having an N, N′-substituted urea structure is used as the cytokinins for culturing the growth points of citrus fruits in a medium containing the cytokinins to form seedlings. A method for cultivating a citrus growing point. 2. A method for cultivating a citrus growing point, characterized in that, when a citrus growing point is cultivated in a medium containing cytokinins to form a seedling, the medium contains starch. 3. The method for cultivating citrus growing point according to claim 2, wherein cytokinins having an N, N′-substituted urea structure are used as the cytokinins.