JPH0611209B2 - Mass growth method for woody plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention provides industrially useful woody plants such as poplar, eucalyptus, acacia, sumac, and paragum which are long-term crops. primordia) is a method for rapid and large-scale propagation using a primordia), and relates to a method for mass-propagating genetically excellent varieties in the fields of forestry, agriculture, landscaping and greening industries.

[Prior Art] There are two types of methods for growing woody plants, a sexual reproduction method using seeds and an asexual reproduction method (cutting and tissue culture).
In the former case, the pollen is not constant in the cross-fertilized plant, and the nature of the parent is not always transmitted to the child. In addition, excellent interspecific hybrids and F ₁ hybrids (first-generation hybrids) created by hybrid vigor,
Furthermore, in a polyploid plant, the genotype of the parent is not directly transmitted to the child. On the other hand, the asexual breeding method has long had a cutting method, and has been generalized as an excellent quality breeding method. However, in this case, the growth speed is slow, the rooting is low,
It is not suitable as a method for propagating all woody plants due to the necessity of setting up a heading garden to produce large amounts of cuttings, and because of limited cutting time. In addition, tissue culture, which has made remarkable progress in recent years, is to sterilize plant stems, shoot tips, leaves, root tips, etc., and then callus (undifferentiated tissue agglomeration) with artificial medium containing plant growth hormone etc.
This is a technique for redifferentiating a plant body after it has turned into a plant, but it is sometimes difficult to grow a large number of children of the same nature as the parent because many chromosomal and gene mutations occur during the process of multiplication. Further, when the callus is subcultured for a long time, the differentiation ability generally decreases and the proliferation rate often decreases.

Regarding woody plants, particularly in broad-leaved trees such as poplar and eucalyptus, there are tissue culture examples of various organs such as shoot tips, lateral buds, cotyledons, hypocotyls, and stems for the purpose of mass growth.
However, in the case of shoot apex, since callus is once induced and shoots are redifferentiated, the variability of the shoots inevitably becomes a problem. On the other hand, there is also a method to directly induce adventitious shoots from stems (so-called micropropagation).
However, in this case, in order to continuously obtain the shoots, it is necessary to continuously plant new stem slices and the like at appropriate intervals, which is a big drawback as a commercial mass-propagation method.

For coniferous plants such as conifers, by culturing young cotyledons in tissue, embryoid bodies (tissues similar to the embryos of seeds, which are bipolar, that is, organs having two primordia of shoots and roots) are obtained. It is possible to create. For example, using Douglas fir (Pseudotsuga menziesii), Mosta-fa
M. et al. Produced an embryoid body of this by shaking culture (US Pat. No. 4,217,730, 1980, 8).
19th). However, even in this case, although the advantage that the regeneration period is shorter than that in the normal tissue culture (organ formation method), the rate of becoming a complete plant is as low as 15 to 50%. A further disadvantage is that a large amount of seeds are always needed due to the use of young cotyledons. Therefore, it does not become a technique for growing a specific excellent individual in large quantities without using seeds.

As described above, in the conventional tissue culture method, a technology for genetically stable and rapid mass multiplication of a specific individual of woody plant has not been established at present.

In recent years, the shoot primordium method has been proposed as a method for propagating annual plants other than woody plants (Tanaka Ryoso et al. Jpn. J. Genet. Vol. 5).
8, 65-70 (1983), JP-A-59-132823).

The shoot primordium is a hemispherical aggregate of cells having the "primordium" of the shoot, which was first discovered by Ryoso Tanaka using the annual plant Hapropappus of the family Asteraceae, and has the following characteristics. Generally, shoot primordia are produced by rigid (vertical) rotary culture of shoot tips. It consists of primary shoot primordia of cell aggregates of 50 to 1000 μm in diameter in which plastid-bearing cells are not stratified and secondary shoot primordia of 100 to 5,000 μm in diameter that are two-layered. And a hemispherical agglomerate of cells by circulating this and vegetative growth, that is, a method of rapidly growing a large amount of "shooting primordia", such as watermelon, corn, rice, morning glory, assembly, poppy, etc. It has been applied to annual plants, but has not yet been proposed for application to perennial woody plants.

[Object of the Invention]

The object of the present invention is to solve the problems of the conventionally known sexual reproduction method and asexual reproduction method, and to apply the method to a mass-propagation method of woody plants of the shoot primordium method developed for annual plants. Another object is to provide a method for mass-growing woody plants using shoot primordia created from shoot apex capable of mass-growing while maintaining the genotype and chromosome type of woody plants for generations. To aim. Another object is to provide a shoot primordium method that can be applied to mass multiplication of useful species and excellent variety among woody plants. Other purposes include cross-fertilized plants, seedless triploids, aneuploids, male individuals of dioecious plants, as well as F ₁ hybrids, interspecific hybrids and intergeneric hybrids produced by hybrid vigor. Another object of the present invention is to provide a method for maintaining and growing the genotype of a woody plant highly genetically hybridized for many years and a method for mass-producing a woody plant applicable to the production of secondary metabolites. Yet another object is to provide a wheelsfree woody plant.

[Structure of Invention]

The present invention extracts the shoot apex of a woody plant, transplants it into an artificial liquid medium containing an inorganic salt composition and a plant growth hormone, and at a temperature of 15 to 30 ° C., 2,000 to 20,0.
Under the illumination of 00 lux and at a rotation speed of 0.5 to 10 rpm, a solid rotation culture is performed to produce and propagate a shoot primordia, and the obtained shoot primordia is statically cultivated on a solid medium. Is a method for mass-propagating woody plants, which comprises forming shoots by transplanting them to a solid medium for rooting and then rooting.

Next, the proliferation method of the present invention will be described in detail.

1) Shoot primordium production method After sterilizing the shoot tips of a woody plant with a sterilizing solution and washing with sterilized water, the shoot apex is extracted under a stereoscopic microscope in a clean bench, which is used as an inorganic salt composition and a plant growth hormone. To an artificial liquid medium containing
Incubate at a temperature of -30 ° C under illumination of 2,000-20,000 lux and at 0.5-10 rpm.

In this case, an organic substance that promotes differentiation such as coconut milk may be added to the culture.

The composition of the artificial liquid medium varies considerably depending on the plant, but the basic inorganic salt composition is Gambor (Gambor).
The composition contained in the medium such as B5 of g) (hereinafter referred to as B5) can be used after slightly changing the composition. As plant growth hormone, naphthalene acetic acid (NAA), 2,4 dichlorophenoxyacetic acid (2,4-D), indole-3
-Acetic acid (IAA), indole-3-propionic acid (I
PA), indole-3-butyric acid (IBA), phenylacetic acid (PAA), benzofuran-3-acetic acid (BFA), phenylacetic acid (PBA) and other auxins and 1- (2
-Chloro-4-pyridyl) 3-phenylurea (KT-
30, cytokinins such as 6-benzylaminopurine (BA) and zeatin (Z) may be used. The culture temperature is preferably a constant temperature of 15 to 30 ° C., particularly 20 to 30 ° C., and if the temperature is lower than this, the progress of growth is delayed, and if the temperature is too high, the growth is poor and the culture becomes unstable. Intense light is required for producing shoot primordia and growing culture, and 2,000 lux is continuously on the lower side and 10,0 on the upper side.
It is suitable to irradiate with light with an illumination degree of 00 to 20,000 lux. In static culture, shoot primordia grow poorly. In the case of rigid type rotary culture, for example, the stem apex placed in 204 test tubes over several stages in the direction of the rotation axis from the cylindrical surface and the circumferential surface of a rotating wheel (made by Nihon Kaika Kikai Co., Ltd.) with a diameter of about 100 cm. The test tube should be placed so that the medium in which the cells were transplanted is directed in a fixed direction even if the rotating wheel rotates in a rigid direction, and the light is emitted from above. The rotation speed is preferably 0.5 to 5 rpm and gentle rotation. in this case,
If the number of rotations is too high, the number of callus will increase, and conversely, if the number of rotations will be low, the number of premature branches will increase, and good results will not be obtained.

When applied to poplar, eucalyptus, and acacia, the breeding method of the present invention yields shoot primordia that grow actively. Tables 1, 2, 3, and 4 show examples of the optimum medium in which these shoot primordia were produced by changing the composition and concentration of the medium. In Tables 1, 2, 3 and 4, the circles show the combination of hormone concentrations in which the shoot primordia appeared, and in the combination of the crosses, precocious branches appeared and white (unmarked) Callus appeared in the combination.

The artificial liquid media in which the shoot primordia grew fastest and were stable were as follows.

(1) Poplar NAA: 0.02 mg / + BA: 0.2 mg / NAA: 0.02 mg / + BA: 0.4 mg / NAA: 0.05 mg / + BA: 0.4 mg / NAA: 0.2 mg / + KT-30: 0.2 mg / As shown in Tables 1 and 2, four sets of optimum medium were found by the combination of plant growth hormones. The rotation speed is 2rp
m was optimal.

(2) Eucalyptus NAA: 0 mg / + BA: 0.2 mg / NAA: 0.02 mg / + BA: 0.02 mg / NAA: 0.02 mg / + BA: 0.2 mg / As shown in Table 3, three sets of optimum The medium was revealed. The optimum rotation speed was 1 rpm.

Two kinds of eucalyptus (E.saligna, E.grandis) were used here, but almost the same results were shown.

(3) Acacia 2,4-D: 0.02 mg / + BA: 0.02 mg / 2,4-D: 0.02 mg / + BA: 0.2 mg / As shown in Table 4, two sets of optimum media were found. In addition,
The optimum rotation speed was 2 rpm.

The shoot primordium obtained is a hemispherical agglomerate, for example, poplar is a green mass with callus near its base. First
The figure is a photograph of shoot primordia of poplar about 40 days after culture. In eucalyptus, it is a black-purple mass with callus at its base as well. Figure 2 is a photograph of shoot primordia of eucalyptus about 6 months after culturing. Acacia is the same. Also,
These shoot primordia are now actively maintained and proliferated for already 10-12 months.

Then, transplant these shoot primordia to a solid medium for seedling,
When statically cultivated at a temperature of 15 to 30 ° C. and an illumination degree of 1,000 to 4,000 lux, a large number of minute stems and leaves are produced. Furthermore, when this is transplanted to a rooting medium, it roots and becomes a complete plant. In addition, the period until the plant is formed is
About 20 days after stationary culture, the genotype, chromosome type and phenotype of this plant are exactly the same as those of the parent plant.

The shoot primordia has a smooth surface in the initial stage and a diameter of 40 to 7
It has ridges of 0 μm (see FIG. 3), and its constituent cells are uniformly small polygonal cells, and the division axis of the cells is vertical, parallel layer, oblique layer, and other polygonal divisions. This shoot primordia (primary shoot primordia) gradually becomes larger and has a diameter of 200 to 1,00.
When it becomes 0 μm (see FIG. 4), 2 of the epidermal system and the cortical system
The outermost layer is 1 to 2 cells, the division axis of the cells is only parietal division, and the inner layer system inside is a group of many rather large cells. There are many developed chloroplasts, vacuoles, and granules of stored substances. Furthermore, this shoot primordia (secondary shoot primordia) has a diameter of approximately 400 μm.
The above trapezoidal ridges were formed, and large oil bodies were observed in the epidermal cells of the outermost layer at this time, the number of chloroplasts increased in the cells of the endothelium of the inner layer, and vacuoles also developed greatly. is doing. At this time, several above-mentioned primary shoot primordia are newly formed around this trapezoidal ridge. Through the above route, shoot primordia increase and about four times per month.

On the other hand, since shoot metabolites actively produce secondary metabolites such as plastids, it can be said that this method is also effective as a method for producing useful substances that were not produced by the conventional callus cell method.

In addition, shoot primordium is obtained by subculturing about once a month,
It is genetically stable (mutation rate is on the order of 10 ^-6, which is the same as the natural mutation rate), and it is capable of rapid mass growth.

Therefore, the present invention has developed a technique for maintaining and growing woody plants in a vegetative, genetically stable state for many years, and for producing a large amount of the population as needed. The growth rate is extremely high, and in the case of a broad-leaved tree, approximately 4 ¹² ≈17 × 10 ⁶ times as many shoot primordia are obtained from one shoot apex,
Furthermore, it has become possible to mass-produce almost the same number of plants.

Hereinafter, examples of the present invention will be described in detail.

Example 1 (Populus Populus charkowiensis x P. caudin
a) As the artificial liquid medium, B5 modified medium shown in Table 5 was used.
The basal medium of B5 used here means the component composition announced by Gambok in 1975, and the compound marked with ◯ is the substance modified this time. The appropriate concentration of the plant growth hormone was examined by the 25-grid method.

First, cut about 20 mm from the tip of the green part of poplar that is actively growing in a greenhouse or field, wash it with sterilized water, and then use tweezers and a scalpel under a stereoscopic microscope in a clean bench to shoot the stem apex (growing point). Neighborhood) 0.5-1
mm excise. The extracted shoot apex is transplanted to an artificial liquid medium shown in Table-5. Cultivation is carried out in a 30 mm (φ) × 200 mm test tube into which 25 m of B5 modified medium is dispensed, and this is performed at a temperature of 20 to 30 ° C. at a lower side of 2,000 lux and an upper side of 10,000 to 20,000 lux. Incubate at 2 rpm for solid rotation under the irradiation of light having a light intensity of. About 40 after the start of culture
A green shoot primordia agglomerate with a diameter of about 10 mm is obtained per day. Thereafter, the shoot primordia are divided into about 5 to 10 mm in diameter about every one month, and subcultured and grown in the fresh medium. Once the shoot primordia were produced, the growth rate increased by about four times in January. Therefore, if the number of months is n, the growth of shoot primordia can be represented by 4 ⁿ . That is, about 4 ¹² per year
Approximately 17 million shoot primordia are produced, so if this is transplanted to a seedling medium described later, it is possible to quickly produce homogenous plants in a plant factory while maintaining the chromosome type and genotype of the plant. Can grow in large quantities.

In order to obtain a complete plant from shoot primordium, agar 8 g / (was added to the B5 modified medium in Table 5 except for naphthalene acetic acid (NAA), 6-benzylaminopurine (BA) and KT-30. 0.8%), naphthalene acetic acid, 6-
Benzylaminopurine or zeatin is added in the combination shown in Tables -6 and 7.

For example, in Table 6, the combination of NAA: 0 mg / + BA: 0.02 mg / NAA: 0.001 mg / + BA: 0.02 mg / NAA: 0.001 mg / + BA: 0.2 mg / was suitable as the regeneration medium. Also, the table −
In No. 7, redifferentiation occurred in the NAA: 0 mg / + Z: 2 mg / section. The pH (acidity) is 5.5 to 5.8.
And use a solid medium. In addition, 100m of this medium
40m in a Erlenmeyer flask with a diameter of 5
Let the 10 mm shoot primordium agglomerate stand still. At this time, the culture condition temperature is 15 to 30 ° C., and the illuminance is 1,000 to 4,000.
Lux (16 hours light + 8 hours dark). As a result, 8 to 15 foliar bodies of 3 to 4 mm are produced per shoot primordium agglomerate in 2 to 3 weeks. FIG. 5 is a photograph showing the seedling primordia of poplar seedlings three weeks after transplantation.

Next, when this shoot grows to about 10 to 15 mm, this shoot is cut from the base and transplanted to the above-mentioned basic medium (hormone-free state) containing 6 g / (0.6%) of agar to start rooting. Let In this case, the concentration of agar is low because the medium is softened to facilitate rooting, root cutting is prevented during transplantation, and growth after transplantation is promoted. The agar concentration is preferably 0.4 to 0.6%. After 2-3 weeks, when the roots are fully rooted, this plant is transplanted to a pot containing permiculite, acclimated under low light for 1-2 weeks, and then transferred to a greenhouse. Raise a sapling.

The method of seedling production of the present invention differs from the method of producing seedling primordia of annual plants by Tanaka Ryoso (1983) in the following points. That is,
He uses B5 basal medium diluted to 1/5 for seedling formation, but in the present invention, it is used undiluted and only the hormone concentration is changed. In addition, it is characterized in that two types of medium for shoot appearance and medium for root emergence are prepared. This promoted the growth of shoots such as poplar, eucalyptus and acacia. It is speculated that this is probably due to the abundant nutritional sources. Moreover, the rooting rate of the plants was increased by several tens of percent by transplanting the rooting medium, and the growth promotion after rooting was also recognized, which greatly contributed to the production of healthy seedlings.

Example 2 (Eucalyptus, Eucalyptus saligna, E. grandi
s) The artificial liquid medium has the composition shown in Table 5 (for poplar),
As a result of examining the plant growth hormone by the 25th base method, the composition was changed to the one indicated by ○ in Table-3. That is, NAA
Is 0 to 0.02 mg /, BA is 0.02 to 0.2 mg /
It went within the range of. Also, the number of rotations is 1 unlike Poplar.
The rpm was optimal, but other temperature and lighting conditions were the same as those of poplar.

In the case of eucalyptus, the first shoot primordium agglomerate (diameter about 10 m
It took about 6 months, which is significantly longer than that of poplar. And the color was blackish purple. However, thereafter, similar to poplar, it showed a growth rate of about 4 times per month, indicating that mass growth was possible.

As with poplar, plant growth hormones are added to the basal medium of B5 in the combinations shown in Table-8. For example, when NAA: 0 mg / + BA: 0.02 mg / NAA: 0.001 mg / + BA: 0.02 mg /, the appearance of shoots was highest.

Other temperatures, illuminance, rooting medium conditions, etc. are the same as those of poplar.

In eucalyptus, it takes time to make the first shoot primordium agglomerate, but once it is done, the rest is the same as poplar.
It can grow in large quantities at a rate of about 4 times a month. Therefore, one shoot apex increases by about 4 ¹² ≈17 million times in one year, which enables sufficient factory production.

Example 3 (Acacia auriculifor-mis) As a result of examining only the components of plant growth hormone in the B5 modified medium shown in Table 5 by the 25th disc method, a large number of shoot primordia appeared in the following combinations.

2,4-D: 0.02 mg / + BA: 0.02 mg / 2,4-D: 0.02 mg / + BA: 0.2 mg / The conditions of temperature and illuminance were the same as those of poplar and eucalyptus, The rotation speed was 2 rpm. The color of shoot primordium was dark brown, similar to eucalyptus. It took about 6 months for the shoot primordium agglomerates with a diameter of about 10 mm to form, but thereafter, like Poplar, it grew at a rate of about 4 times a month.
Therefore, it was revealed that the cells could grow about 4 ¹² ≈17 million times in one year.

By adding the Eucalyptus regeneration medium shown in Table 8 to the basal medium of B5, shoots appear. Subsequent rooting operations are exactly the same as for poplar and eucalyptus.

Although a broad-leaved tree has been described above, it goes without saying that the present invention can be applied to a conifer.

[Brief description of drawings]

Figure 1 is a photograph showing the shoot primordia of poplar about 40 days after culturing, Figure 2 is a photograph showing the shoot primordia of eucalyptus about 6 months after culturing, and Figure 3 is the primary shoot primordia of poplar. Photograph of the cross section of the base, Fig. 4 is a photograph of the cross section of the secondary shoot primordia of poplar,
FIG. 5 is a photograph showing the seedling primordia of poplar seedlings three weeks after transplantation.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keigo Dohi 24-9 Nonoshino-cho, Kameyama City, Mie Prefecture Forest Tree Breeding Research Institute, Kameyama Breeding Plant, Oji Paper Co., Ltd. (56) Reference JP-A-59-132823 ( JP, A) Masayuki Takeuchi, External Edition, "New Plant Tissue Culture," Asakura Shoten, published on September 20, 1979, p. 229-243 Kato Yukio "Plant Tissue Culture Method" Seibundo Shinkosha, published on September 25, 1968, p. 17 ~ 18

Claims (4)

[Claims] 1. A stem apex of a woody plant is extracted and transplanted into an artificial liquid medium containing an inorganic salt composition and a plant growth hormone, and the temperature is set at 15 to 30 ° C. to 2,000 to 20,
Vertically cultivated at a rotation rate of 0.5 to 10 rpm under an illumination of 000 lux to produce and multiply shoot primordia,
A method for mass-proliferating woody plants, which comprises statically culturing the obtained shoot primordia on a solid medium to generate shoots, and then transplanting the shoots onto a solid medium for rooting and rooting. 2. The propagation method according to claim 1, wherein the woody plant is a broad-leaved tree such as poplar, eucalyptus and acacia. 3. The growth method according to claim 1 or 2, wherein a Gambouk B ₅ medium is used as the inorganic salt composition, and cytokinin and auxin compounds are used as the plant growth hormone. 4. Stationary culture is carried out using Gambouk B ₅ medium as a basic medium and cytokinin and auxin compounds as plant growth hormones at a temperature of 15 to 30 ° C. and an illumination of 1,000 to 4,000 lux. The proliferation method according to any one of claims 1 to 3, which is performed once.