JP3463756B2 - Agglomerate of shoot primordia of Eucalyptus plant, method of producing the same, and method of growing Eucalyptus plant - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of producing a shoot-primordium agglomerate of a woody plant and a method for producing the same, and a method of growing a woody plant using the shoot-primoral agglomerate. More specifically, from a piece of tissue extracted from a vegetative organ other than the shoot apex of a woody plant, a shoot primordium agglomerate, which is an aggregate of shoot primordia, is efficiently produced to produce a genetically excellent woody plant. Is a method for stably and massively multiplying. 2. Description of the Related Art A shoot primordium agglomerate is a cell agglomerate capable of mass-producing plants having excellent and useful traits without mutating the traits. etc(
Jpn. Genet., 58: 65-70, 1983), is an annual plant of the Asteraceae family, cultivating a stalk apex containing its growing point and rotating vertically under a certain temperature, illuminance and rotation speed. It is named for the cell clumps obtained by rotating culture on a machine. When a plant is regenerated from a shoot original base mass, a part thereof is cut out, transferred to a seedling medium, and cultured. [0003] Tanaka et al. Use this shoot primordium agglomerate to produce an annual plant, haplopups (Japanese Patent Laid-Open No. 59-1328).
No. 22), useful annual plants (JP-A-59-1328).
No. 23) and Stevia (JP-A-61-96994). Further, the present inventors have found that a method using a shoot primordium agglomerate is also effective for mass propagation of woody plants (Japanese Patent Application Laid-Open No. 62-55020). This method
The shoot apex of a woody plant is excised, transplanted into an artificial medium containing an inorganic salt composition and a plant growth hormone, and
A method of producing a shoot original base mass by rotating culture with a rotary culture machine that rotates in a vertical direction under constant conditions for 50 days, further transplanting this to a seedling culture medium, culturing it still, and regenerating a plant body It is. The present inventors have further improved this method,
The shoot apex of a woody plant grown aseptically is excised, and 1- (2-chloro-4-pyridyl) -3 is used as a plant hormone.
By using phenylurea (hereinafter abbreviated as 4PU), a method for more stably producing a shoot primordium agglomerate has been found (JP-A-2-265419). [0006] However, the conventional propagation method using shoot primordium agglomerates is characterized in that a shoot apex including a growing point of a plant is all removed, and this is used as a starting material to produce a shoot primordium agglomerate. Given that the number of shoot tips is limited, plants with industrially useful genetic traits cannot always be efficiently propagated, and the shoot tips of plants collected outdoors are used. In this case, this is very sensitive to sterilization treatment, and there are many operational difficulties such as the shoot tip turning brown immediately after planting and the need to remove the shoot tip under a stereoscopic microscope. Efficiency is limited, so improvements remain. [0007] Therefore, the present inventors have intensively studied a method for stably and efficiently producing a shoot primordium agglomerate. As a result, using aseptically grown eucalyptus plants or explants extracted from the leaves of eucalyptus plants in the field as a material, shoot primordia agglomerates can be more efficiently produced, and plants can be planted therefrom. We have invented this method of regeneration. Namely, the present inventors have an even, shoot apex portion case of producing seedlings JoHara Motoshu mass in a conventional manner using organs other than the shoot apical portion (1) Eucalyptus plant <br/> object It is possible to easily produce a shoot primordia agglomerate in the same manner as when forming a shoot primordium agglomerate. (2) From a shoot primordia agglomerate produced from an organ other than the stem apex of a eucalyptus plant, It is possible to regenerate plants by inducing shoots by rooting method, further rooting and regenerating plants, and by combining the above methods (1) and (2), it is much more stable and efficient than conventional methods. It was found that excellent Eucalyptus plants could be multiplied in large quantities. SUMMARY OF THE INVENTION An object of the present invention is to reduce the efficiency of producing shoot primary base clumps from woody plants because the starting material is limited to the shoot apex when mass-proliferating. In solving the problem. [0010] SUMMARY OF THE INVENTION The present invention, seedlings JoHara Motoshu masses from leaves of Eucalyptus plant <br/> thereof, and leaves of Eucalyptus plants
Cut out parts, inorganic salts composition and a plant hormone
And transplanted into an artificial liquid medium supplemented with 1- (2-chloro-pyridyl) -phenylurea, and at a temperature of 15 to 30 ° C., a lower side of 1,000 to 2,000 lux and an upper side of 10,000.
Under illumination of 0-20,000 lux and 0.5-10 rp
m, a method of producing shoot primary base agglomerates characterized by performing vertical rotation culture at a rotational speed of m, and producing the shoot primary base agglomerates by static culture to produce seedlings, and genetically transforming woody plants. The present invention relates to a method for growing woody plants, which is characterized in that it grows in large amounts in a stable state. [0011] The method of producing a shoot primordium agglomerate and the method of regenerating a plant according to the present invention will be described in detail below. A method for producing a shoot primordium agglomerate A method for producing a shoot primordium agglomerate of a woody plant has already been disclosed in
No. 5,550,020 and Japanese Patent Application Laid-Open No. 2-265419, which have been almost established, the present inventors have made the following points in order to more stably and efficiently produce shoot original base agglomerates. Was improved. Use of vegetative organs other than the shoot apex of the plant . That is, without cutting out the shoot apex portion including a growing point, outright sterilized leaves of field plants in the usual pasteurization
Or out, aseptically excised leaves of training plants, which tissue culture medium of the plant, for example, gumbo - naphthalene grayed of B5 medium (see Table 1), the 4PU as cytokinins in plant hormones, as auxins Acetic acid (NA
A), 2,4-dichlorophenoxyacetic acid (2,4-D)
Alternatively, indole acetic acid (IAA) or the like is added, and the cells are inoculated on a liquid medium further containing sucrose. This is 15 ~
Vertical rotation culture is performed at a temperature of 30 ° C., an illuminance of 1,000 to 20,000 lux on the lower side and 10,000 to 20,000 lux on the upper side, and a rotation speed of 1 to 10 rpm in a vertical direction for 10 to 50 days. [ Table 1] A method of seedling primordium agglomeration is used.
Shoots are obtained by transplanting them into a fixed medium for raising seedlings. That is, NAA and the like as auxins and BA and the like as cytokinins were added to a B5 medium of gambog or an MS medium of Murashige-Skoog, and BA was added.
The shoot original base mass is transplanted to a seedling culture medium supplemented with -3% sucrose and 0.4-0.6% agar and the like, at a temperature of 15-30 ° C and 1,000-4,000 lux. Incubation under light for 12 to 16 hours results in a large number of minute shoots. [0016] It was then transplanted into a fixed medium for rooting and to rooting, the whole plant. The period until the plant is regenerated is about three months after the start of stationary culture, and the genotype, chromosome type and phenotype of the obtained plant are exactly the same as the parent plant. The present invention will be described more specifically below with reference to examples. [0018] Example 1 Test Plants Monoyu - Cali (Eucalyptus saligna) seedlings JoHara Motoshu mass Yu obtained by production methods Hei method of creating a sterile seedlings No. disclosed in Japanese 2-265419 of - potassium (Eucalyptus saligna) A leaf portion having a size of about 5 mm was cut out, and 3% of sucrose was added to B5 medium of gambog, which is a tissue culture medium of a plant, to give 0.02 mg / L NA as a plant hormone.
A and 0.5 mg / l of 4PU were added, and the cells were inoculated in a liquid medium adjusted to pH 5.6. Furthermore, from <br/> tip portion of the branch of fourth grade seedlings grown outdoors 70% concentration cut leaves of about 10mm alcohol - in antiformin diluted 30 seconds and 10 times Le Sterilized for 20 minutes. After washing this three times with sterile water,
It was planted in the organization culture medium in accordance with the above-mentioned method. [0020] at a temperature of these 28 ℃, the lower side is 2,00
Lux of 20,000 lux at 0 lux and 2 rp
Vertical rotation culture was performed in a vertical direction at an inclination angle of 76 degrees for 30 days at a rotation speed of m. Each 100 specimens were used to examine the formation rate of shoot primordia agglomerates. Table 2 shows the results. [ Table 2] [0022] As shown by the experimental results, with a useful genetic traits, it is impossible to remove the 100 of the stem top simultaneously from one Eucalyptus plant, if the 1 /
Even if there are ten, it requires a great deal of effort. However, according to this method using a vegetative organ composed of leaves , a large amount of shoot primordium agglomerates can be easily obtained from the same Eucalyptus plant. It is possible. A method for producing seedling primordium agglomerates The shoot primordium agglomerates obtained by vertical rotation culture in the vertical direction were added to a B5 medium of Gamborg as a plant hormone at 0.02 mg / kg.
1 NAA, 0.2 mg / l BA, and 1 sucrose
% And agar was added to the seedling medium adjusted to pH 5.6. Then, as a result of culturing at 27 ° C. and illuminance of 4,000 lux under light conditions for 16 hours, seedling formation was recognized on the 60th day. Next gumbo To rooting this - the addition of NAA and 1% sucrose further 0.4% agar 0.01 mg / l to B5 medium grayed was adjusted to pH5.6 originating The cells were transplanted to a root medium and cultured under the same conditions. As a result, on the 30th day, the roots became complete plants. [0025] EXAMPLE 2 Test Plants Monoyu - Cali (Eucalyptus grandis) seedlings JoHara Motoshu mass Yu obtained by production method of producing method aseptic seedlings - Cali (Eucalyptus
grandis) was cut out in the same manner as in Example 1, planted in a liquid medium, and cultivated vertically in a vertical direction. As a result, as in Example 1, shoot primordium agglomerates could be produced with high frequency (60%). [0026] The Naeka method resulting seedlings JoHara Motoshu mass seedlings JoHara Motoshu mass was plated on Naeka medium in the same manner as in Example 1. The culture was continued at a temperature of 27 ° C. and an illuminance of 4,000 lux under light conditions for 16 hours. As a result, seedlings were observed on the 60th day. Next, Example 1 to make them rooting
The cells were transplanted to the same rooting medium and cultured under the same conditions. As a result, on the 30th day, roots were established and complete plants were regenerated. The genotype, chromosome type and phenotype of the obtained plant were exactly the same as the parent plant. According to the present invention, it has become possible to efficiently and stably produce plants having useful and excellent genes without mutating their traits. That is, compared with the case of using the shoot apex, it is possible to supply an unlimited number of seedlings having the same genetic trait in a short time and at the same time, and it is possible to sufficiently cope with industrial afforestation projects without using seedlings It became so.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Masaru Shibata 24-9 Nojonocho, Kameyama-shi, Mie Oji Paper Co., Ltd. Kameyama Laboratory, Forest Tree Breeding Laboratory Co., Ltd. (56) References JP-A-4-4828 (JP, A) JP-A-2-265419 (JP, A) Agriculture and horticulture Vol. 63 No. 1 (1988) pp. 133-137 Chemistry and Biology Vol. 16, No. 12 (1978) pp. 776-782 Research Report of Forestry Research Institute No. 360 (1991) pp. 57-64 (58) Fields investigated (Int. Cl. ⁷ , DB name) A01H 4/00

Claims (1)

(57) [Claims 1] A leaf is cut out from a plant of the genus Eucalyptus, and 1- (2-chloro-4-pyridyl) -3-phenylurea is added as an inorganic salt composition and a plant hormone. Transplanted into an artificial liquid medium, at a temperature of 15 to 30 ° C.,
000-2,000 lux and the upper side is 10,000-2
Under illumination of 000 lux and 0.5 to 10 rpm
Vertical rotation culture at a rotation speed of を 得to obtain a shoot primordium agglomerate of Eucalyptus plants , and then the shoot primordium agglomerates are seeded by stationary culture.
Eucalyptus plants in large quantities in a genetically stable state
A method for growing eucalyptus plants, characterized by multiplying
Law .