JP2631765B2 - Propagation method of shoot primordia of woody plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a perennial woody plant such as poplar,
The present invention relates to a method of producing shoot primordia of industrially useful woody plants such as eucalyptus, acacia, urushi and para rubber, and propagating them rapidly and in large quantities.

[Conventional technology]

There are two methods of growing woody plants: sexual reproduction using seeds and asexual reproduction (cutting, tissue culture).
In the former case, the pollen is not constant in the cross-fertilized plant, so that the nature of the parent is not necessarily transmitted to the child. The F ₁ hybrids were born by hybrid or hybrid vigor between the excellent species (F1 hybrid), and even the parent of the genotype in the polyploid plant, etc. is not transmitted as it is to the children. On the other hand, the asexual propagation method has a cutting method since ancient times, and has been popularized as a propagation method of excellent varieties. However,
In this case, it is necessary to set up a harvesting orchard to produce a large amount of cuttings, because the growth speed is slow, the rooting is low, and the cutting time is limited. It is not appropriate as a method for growing essential plants. Recently, a remarkable progress has been made in the tissue culture method in which the stems, shoot apices, leaves, root tips, etc. of plants are sterilized and then transformed into callus (undifferentiated tissue agglomeration) using an artificial medium supplemented with plant growth hormone. Although it is a technique for redifferentiating a plant, it is sometimes difficult to propagate a large number of children having the same properties as the parent due to frequent chromosomal mutations and gene mutations during the growth process. In addition, when the callus is passaged for a long period of time, the differentiation ability generally decreases, and the proliferation rate often decreases.

In the woody plants, especially in broadleaf trees such as poplar and eucalyptus, there are examples of tissue culture of various organs such as shoot tips, side buds, cotyledons, hypocotyls and stems for the purpose of mass propagation.
However, in the case of the shoot apex, once calli are induced and the shoots are regenerated, the variability of the obtained shoots is a problem. On the other hand, there is also a method of directly inducing an indefinite shoot from a stem or the like (so-called micropropagation).
However, in this case, it is necessary to constantly plant new stem sections or the like at appropriate intervals in order to continuously obtain shoots, which has a serious drawback as a commercial mass propagation method.

In addition, for gymnosperms such as conifers, embryo-like bodies (tissues similar to embryos of seeds, which are bipolar, ie, organs having two primordia, shoots and roots) are obtained by tissue culture of young cotyledons. It is possible to produce. For example, using Douglas fir (Pseudotsuga menziesii), Mostafa
M. et al. Have produced embryoid bodies by shaking culture (US Patent No. 4,217,730, August 19, 1980). However, even in this case, even though it has the advantage that the regeneration period is shorter than that of a normal tissue culture (organogenesis method), the rate of complete plant formation is low at 15 to 50%. On the downside, the use of young cotyledons always requires a large amount of seeds. Therefore, it is not a technique to propagate a certain excellent solid in large quantities without using seeds.

As described above, in the ordinary tissue culture method, at present, a technique for genetically stably and rapidly mass-producing a specific individual woody plant has not been established.

In recent years, the shoot primordium method has been proposed as a method for growing annual plants other than woody ones (Tanaka Ryuso et al. Jpn. J. Genet. Vol.
58, 65-70 (1983), JP-A-59-132823).

The shoot primordium refers to a hemispherical aggregate of cells having the shoot primordia, first discovered by Tanaka Ryuso using the annual plant Asteraceae, Haplopaps, and has the following characteristics. Generally, shoot primordia are produced by vertical rotation culture of shoot apex. This is the primary shoot primordia of 50-1,000 μm diameter cell clumps in which plastid-bearing cells are not stratified, and 2
It consists of stratified secondary shoot primordia with a diameter of 100 to 5,000 μm. This is a method that allows rapid and large-scale growth of hemispherical aggregates of cells, that is, "stalk primordia" by circulating this and growing vegetative bodies, and is a one-year freshman such as watermelon, corn, rice, morning glory, assembly, and poppy It has been applied to plants but has not yet been proposed for its application to permanent woody plants.

[Object of the invention]

An 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 the mass propagation method of the woody plant of the shoot basement method developed with annual plants. Another object is to provide a shoot primordia produced from the shoot apex that can grow in large quantities while maintaining the genotype and chromosome type of woody plants over generations. Another purpose is useful species (Species) and excellent varieties (variety) in woody plants.
It is an object of the present invention to provide a method for growing shoot primordia that can be applied to mass growth of corn. Other purposes are cross-fertilization plants,
Triploid not able to take the seed, aneuploid, male and female male individuals of a different strain plants, more F ₁ hybrids and interspecific hybrid born by heterosis, high woody of genetically hybridity as intergeneric hybrids It is an object of the present invention to provide a method for growing a main body (stalk primordium), which is the basis for maintaining and growing a plant genotype for many years. A further object is to provide a woody plant of wheelsfree.

[Configuration of the invention]

The present invention relates to a method comprising the steps of: `` Extracting the shoot apex of a woody plant, containing it at a concentration of up to 0.5 mg / day of a cytokinin-based plant hormone as an inorganic salt composition and a plant growth hormone, and at the same time, adding an auxin-based plant hormone of 0 to 0.2. transplanted into an artificial liquid medium containing a concentration of 1 mg / mg, at a temperature of 15 to 30 ° C., under a light intensity of 2,000 lux on the lower side and 10,000 to 20,000 lux on the upper side, and at a rotation speed of 0.5 to 5 rpm. A method of growing shoot primordia of a woody plant, comprising dividing the shoot primordia produced by spin-culture into a diameter of about 5 to 10 mm, and substituting and growing the fresh medium. .

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

1) Method of producing shoot basal substrate The shoot apex of a woody plant is sterilized with a bactericidal solution, washed with sterilized water, and the shoot apex is excised under a stereoscopic microscope in a clean bench, and this is used as an inorganic salt composition and plant growth hormone. And transplanted into an artificial liquid medium containing In this case, an organic substance that promotes differentiation such as coconut milk may be added. The conditions for the vertical (vertical) rotary culture are as follows:
The illuminance is 2,000 lux on the lower side and 10,000 to 20,000 lux on the upper side, and the rotation speed is 0.5 to 5 rpm.

Next, the produced shoot primordia are divided into a diameter of about 5 to 10 mm, and the resulting shoot primordia are propagated in the above fresh medium to propagate the shoot primordia.

Although the composition of the artificial liquid medium varies considerably from plant to plant, the basic inorganic salt composition is Gamborg (Gamborg).
The composition contained in the medium such as g5) B5 (hereinafter referred to as B5) can be used by slightly changing the composition. Examples of plant growth hormones include naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and indole-3-acetic acid (IA
A) auxins such as indole-3-propionic acid (IPA), indole-3-butyric acid (IBA), phenylacetic acid (PAA), benzofuran-3-acetic acid (BFA), phenylbutyric acid (PBA) and 1- ( 2-chloro-4-pyridyl) -3
-Phenylurea (KT-30, manufactured by Kyowa Hakko Co., Ltd.), 6
-Cytokinins such as benzylaminopurine (BA) and zeatin (Z) can be used. The culture temperature is especially 15 ~ 30 ℃
A constant temperature of 20 to 30 ° C. is suitable. If the temperature is lower than this, the progress of proliferation is delayed, and if the temperature is too high, the growth is poor and unstable. The cultivation of shoot primordia is characterized by vertical rotation culture, and the production of shoot primordia requires strong light, and the lower side is 2,000 lux continuously and the upper side is 10,000 to 20,000.
It is appropriate to irradiate light with an illumination of 0 lux. Illumination outside this range will degrade the growth of shoot primordia. In stationary culture, shoot primordia grow poorly. In the case of the vertical rotation culture, for example, the stem of a rotating wheel having a diameter of about 100 cm (manufactured by Nippon Medical Machine Co., Ltd.) and a plurality of stages from the circumferential surface in the direction of the rotation axis are placed in 204 test tubes in a stem top. The test tube is placed obliquely and the test tube is constantly oriented even when the rotating wheel rotates in the vertical direction, and the light is irradiated from above. The rotation speed is preferably 0.5 to 5 rpm. In this case, if the number of rotations is too high, the number of callus portions increases, and if the number of rotations decreases, the portion of premature branching increases, and in any case, good results cannot be obtained.

When the propagation method of the present invention is applied particularly to poplar, eucalyptus, and acacia, a shoot primordium that actively grows can be obtained.
Tables 1, 2, 3, and 4 show examples of the optimal medium in which these shoot primordia are produced by changing the composition and concentration of the medium. In Tables 1, 2, 3 and 4, the portions marked with a triangle are the combinations of hormone concentrations in which shoot primordia appeared, and in the combination of portions marked with x, early branching appeared and the white (no mark) portion Calli appeared in the combinations.

The artificial liquid medium in which the shoot primordia rapidly proliferated and was stable was 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 FIG. 2, four sets of optimal media were found by the combination of plant growth hormones. The rotation speed is 2r
pm was optimal.

(1) 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 optimal media were identified. Note that the rotation speed was optimally 1 rpm.

Here, two kinds of eucalyptus (E. saligna, E. grandis) were used, 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 optimal media were identified. In addition,
The optimal rotation speed was 2 rpm.

The resulting shoot primordia are hemispherical clumps, for example, poplar is a green mass with callus near its base. FIG. 1 is a photograph of shoot primordia of poplar about 40 days after culture. Eucalyptus is a black-purple mass with calli at its base as well. FIG. 2 is a photograph of eucalyptus shoot primordia about 6 months after culturing. Acacia is no different. Also,
These shoot primordia are now actively maintained and propagated for 10-12 months.

These shoot primordia are transplanted to a solid medium for seedling, and
Static culture at a temperature of 30 ° C and an illuminance of 1,000 to 4,000 lux produces a large number of fine foliates. Furthermore, when this is transplanted to a rooting medium, it will root and become a complete plant. The period until a plant is formed is about 20 days after static culture, and the genotype, chromosome type and phenotype of this plant are exactly the same as the parent plant.

The shoot primordia has a smooth surface at the initial stage and a diameter of 40-70.
μm (see FIG. 3), the constituent cells are uniformly small polygonal cells, and the cell division axis is vertical, parallel,
Diversifies the stratum and other parts. The shoot primordia (primary shoot primordia) gradually become larger and become 200-1,000 μm in diameter (see FIG. 4), differentiate into two layers, an epidermis system and a cortex system, and the outermost layer is 1-2 cells. In the cell division axis, only the parallel division is seen, and the inner lining of the cell is a collection of a number of rather large cells inside which well-developed chloroplasts, vacuoles, and storage substance granules Many are seen. Furthermore, this shoot primordium (secondary shoot primordium) becomes a trapezoidal ridge with a diameter of about 400 μm or more, and a large oil body is observed in the outermost epidermal cells at this time, and the inner endothelial system of the inner layer is observed. The number of chloroplasts increases in the cells, and the vacuoles are also largely developed. At this time, several primary shoot primordia are regenerated around the trapezoidal ridge. Through the above route, shoot primordia increases and increases about four times in one month.

On the other hand, secondary metabolites such as plastids are vigorously produced in shoot primordia, and thus can be said to be an effective method for producing useful substances that have not been produced by the conventional callus cell method.

In addition, shoot primordia can be rapidly mass-produced by subculturing about once a month in a genetically stable state (the mutation rate is the same as the spontaneous mutation rate, 10 ^-6 orders). It is.

Thus, according to the present invention, woody plants can be
Techniques have been developed for maintaining and growing vegetative bodies in a genetically stable state, and for mass-producing the population as needed. Its growth rate is extremely high, and in the case of broadleaf trees, about 4 ¹²年 間 17 × 10 ⁶ times the number of shoot primordia per year can be obtained from one shoot apex, and it is possible to mass-produce almost the same number of plants. It is now possible.

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

Example 1 (Populus Populus charkowiensis × P.caudi
na) i) Production of shoot primordium The artificial liquid medium used was the B5 modified medium shown in Table-5.
The B5 basal medium used here was 19
It means the composition of the ingredients announced in 1975, and the compound marked with ◯ is the substance modified this time. The appropriate concentration of plant growth hormone was examined by a 25-cross grid method.

First, a green part of a poplar growing actively in a greenhouse or a field is cut off by about 20 mm from the tip and washed with sterile water, and then, using a tweezer and a scalpel under a stereoscopic microscope in a clean bench, the stem apex (growing point) 0.5) to 1 mm. The removed stem apex is transplanted to a liquid medium shown in Table-5. Culture was performed by dispensing 25 ml of B5 modified medium into 30 mm
This is performed in a test tube of (φ) × 200 mm, and this is subjected to vertical rotation culture at a rotation speed of 2 rpm at a temperature of 20 to 30 ° C., a lower side of 2,000 lux, and an upper side of 20,000 lux. About 40 after the start of culture
A green shoot primordia agglomerate with a diameter of about 10 mm can be obtained in a day. Thereafter, the shoot primordia are divided into about 5 to 10 mm in diameter about every one month, and are subcultured and propagated in the above fresh medium. The growth rate increased about four times in January once the shoot primordia was formed. Accordingly, if the number of months is n, the number of shoot primordia growing can be represented by 4 ⁿ . That is, about 4 ¹²で per year
Since 17 million shoot primordia are produced, if this is transplanted to a seedling medium described below, the same plant can be rapidly produced in the plant industry while maintaining the chromosome type and genotype of the plant. It can proliferate in large quantities.

ii) Method for transforming shoot primordia In order to obtain complete plants from shoot primordia, naphthaleneacetic acid (NAA), 6-benzylaminopurine (BA) and KT-30 were obtained from the B5 modified medium in Table-5. Agar in basic medium
8 g / (0.8%) of naphthaleneacetic acid, 6-benzylaminopurine, or zeatin are added in the combinations shown in Tables 6.7.

For example, in Table 6, the combination of (a) NAA: 0 mg / + BA: 0.02 mg / (b) NAA: 0.001 mg / + BA: 0.02 mg / (c) NAA: 0.001 mg / + BA: 0.2 mg / Was appropriate. Table-
In No. 7, redifferentiation occurred in the group of NAA: 0 mg / + Z: 2 mg /. The pH (acidity) is adjusted to 5.5 to 5.8, and a solid medium is used. Add 40 ml of this medium to a 100 ml Erlenmeyer flask.
Dispense, and a shoot primordium agglomerate having a diameter of 5 to 10 mm is left on this. The temperature of the culture conditions at this time is 15-30 ° C, the illuminance is 1,
000-4,000 lux (16 hours light + 8 hours dark).
As a result, 8 to 15 shoots and shoots of 3 to 4 mm are produced per shoot original basal mass in a few weeks. FIG. 5 is a photograph showing the state of poplar shoot primordia three weeks after transplantation.

Next, when the shoots grow about 10 to 15 mm, the shoots are cut off from the base and transplanted to the above-mentioned basic medium (hormone-free state) containing 6 g / (0.6%) of agar and rooted. In this case, the reason why the concentration of the agar is low is to soften the medium to cause rooting, prevent root cutting at the time of transplantation, and promote growth after transplantation. Agar concentration is 0.4
~ 0.6% is preferred. After 2-3 weeks, when the roots were fully rooted, the plants were transplanted into a pot containing vermiculite and acclimated under low light for 1-2 weeks.
Transfer to greenhouse and grow to healthy seedlings according to normal seedling practices.

The seedling method of the present invention differs from the method of Tanaka Ryuso (1983) in which seedling primordia of annual plants are turned into seedlings in the following points. In other words, he uses a sample diluted in 1/5 of the B5 basal medium to produce seedlings, but in the present invention, he uses it without dilution and changes only the hormone concentration. Further, the present invention is characterized in that two types of culture media for shoot appearance and rooting appearance are prepared. As a result, the growth of shoots such as poplar, eucalyptus, and acacia was promoted. It is presumed that this is probably due to the abundance of Sakae light sources. In addition, by transplanting to a rooting medium, the rooting rate of the plant was increased by several tens of percent, and growth was promoted after rooting, which greatly contributed to the production of healthy seedlings.

Example 2 (Eucalyptus, Eucalyptus saligna, E.grand
is) i) Method of producing shoot primordium In the artificial liquid medium, the composition shown in Table 5 (for Oprah) was examined for the plant growth hormone by the 25-cross grid method. changed. That is, NAA
Was performed in the range of 0 to 0.02 mg /, and BA was performed in the range of 0.02 to 0.2 mg /. The rotation speed was 1 rpm, which was different from that of poplar, but the other conditions were the same as those of poplar.

In the case of eucalyptus, the first shoot primordia agglomerate (about 10m in diameter)
m), but it was significantly longer than poplar and took about 6 months. And the color was black purple. However, thereafter, as with poplar, the growth rate was about four times a month, indicating that large-scale growth was possible.

ii) Seedling primordia cultivation method As in the case of poplar, plant growth hormone is added to the basic medium of B5 in a combination as shown in Table-8. For example, when (a) NAA: 0 mg / + BA: 0.02 mg / (b) NAA: 0.001 mg / + BA: 0.02 mg /, the appearance of shoots was highest.

The other conditions such as temperature, illumination, and rooting medium are the same as those of poplar.

In eucalyptus, it takes time to make the first shoot primordium agglomerate, but once it is made, it can grow best at about four times a month in January, like poplar. Therefore,
Since one of the shoot apex portion is increased to approximately 4 ¹² ≒ 1,700 thousand times a year, it is possible to sufficiently factory production.

Example 3 (Acacia auriculiformis) i) Method of producing shoot primordium As a result of examining only the components of plant growth hormone in the B5 modified medium shown in Table 5 by a 25-cross grid method, a large number of shoot primordia were obtained in the following combinations. Appeared.

(A) 2,4-D: 0.02mg / + BA: 0.02mg / (b) 2,4-D: 0.02mg / + BA: 0.2mg / The conditions of temperature and illuminance are the same as for poplar and eucalyptus. However, the rotation speed was 2 rpm. The color of shoot primordia was black-purple, similar to eucalyptus. It took about 6 months for the shoot primordia agglomerates with a diameter of about 10 mm to form, but thereafter grew at a rate of about 4 times a month, similar to poplar.
Therefore, it has been found that can grow about 4 ¹² ≒ 1,700 thousand times a year.

ii) Method of regenerating shoot primordia Shoots appear by adding eucalyptus regeneration medium to the base medium of B5 in Table-8. Subsequent rooting operations
It is exactly the same as poplar and eucalyptus.

The hardwood tree has been described above, but it goes without saying that the present invention can also be applied to coniferous trees.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photograph showing shoot primordia of poplar about 40 days after culture, FIG. 2 is a photograph showing shoot primordia of eucalyptus about 6 months after culture, FIG. Fig. 4 shows a photograph of a cross section of the primary shoot primordium of poplar, Fig. 4 shows a photograph of a cross section of the secondary shoot primordium of poplar, and Fig. 5 shows the establishment of a single shoot primordium of poplar three weeks after transplantation. It is a photograph which shows the state which performed.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Keigo Doi 24-9 Nojonocho, Kameyama-shi, Mie Oji Paper Co., Ltd. Kameyama Breeding Laboratory, Forestry and Tree Breeding Research Institute Co., Ltd. New Technology ”CMC Corporation (Showa 59-2-10) 171-197

Claims (1)

(57) [Claims] The present invention relates to a method for preparing a stem apex of a woody plant, comprising a cytokinin-based plant hormone as an inorganic salt composition and a plant growth hormone at a concentration of 0.5 mg / max. transplanted into an artificial liquid medium containing a concentration of mg / mg, at a temperature of 15-30 ° C,
At 2,000 lux, the upper side is under illumination of 10,000 to 20,000 lux, and the shoot primordia created by vertical rotation culture at a rotation speed of 0.5 to 5 rpm is divided into a diameter of about 5 to 10 mm and the above. A method of growing shoot primordia of woody plants, wherein the method is subcultured and propagated in a fresh medium.