JPH044828A - Shoot primodium of arboreous 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 is directed to the rapid and large-scale propagation of industrially useful woody plants such as perennial crops such as poplar, eucalyptus, acacia, sumac, and paragum. It relates to the shoot base of woody plants.

[Conventional technology]

There are two ways to propagate woody plants: sexual reproduction using seeds and asexual propagation (cuttings, tissue culture), but in the former case, pollen is not constant in cross-fertilized plants, so it is not always possible to reproduce from the parent. The nature of the child is not conveyed to the child. In addition, excellent interspecific hybrids and F1 hybrids (-hybrid hybrids) produced by hybrid vigor.Furthermore, in polyploid plants, the genotypes of the parents are not directly transmitted to their offspring. On the other hand, asexual propagation methods include the seed method, which has been popularized as a method for propagating superior varieties. just,
In this case, due to the slow propagation speed, low rooting ability, the need to set up a scion garden to produce a large amount of scions, and the limited time for cuttings, etc.
It cannot be said to be an appropriate propagation method for all woody plants.

In addition, the tissue culture method, which has recently made remarkable progress, sterilizes plant stems, shoot tips, leaves, root tips, etc., and then converts them into callus (undifferentiated tissue aggregates) in an artificial medium containing plant growth hormones. , is a technology for redifferentiating plants, but because chromosomal and genetic mutations frequently occur during the propagation process, it may be difficult to reproduce a large number of offspring with the same characteristics as their parents. Furthermore, when calli are subcultured for a long period of time, the differentiation ability generally decreases and the proliferation rate often decreases.

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

In addition, for gymnosperms such as conifers, embryoid bodies (tissues similar to embryos in seeds and bipolar, that is, organs with two primordia, shoots and roots) can be produced by tissue culturing young cotyledons. It is possible to create one. For example, Douglas fir (Pseudotsuga menziesii)
), MostafaMo et al. produced embryoid bodies of this by shaking culture (US patent, Nα4.
217.730.August 19, 1980) However,
Although this method also has the advantage of a shorter regeneration period than normal tissue culture (organ formation method), the rate of complete plant formation is low at 15-50%. An additional drawback is that always
Since young cotyledons are used, large amounts of seeds are required. Therefore, it cannot be used as a technology to propagate a large number of specific, excellent solids without using seeds.

As described above, with conventional tissue culture methods, the current situation is that no technology has been established for rapidly and genetically stable mass propagation of specific individuals of woody plants.

In recent years, the shoot primordium method has been proposed as a propagation method for annual plants other than woody plants (Takashi Tanaka et al. Jpn, J. Gen.
et, Shil, 58, 65-70
(1983), Japanese Unexamined Patent Publication No. 132823/1983).

A shoot primordium refers to a hemispherical agglomeration of cells containing a shoot primordium, which was first discovered by Takashi Tanaka using Habropapus, an annual plant of the Asteraceae family, and has the following characteristics. . Generally, shoot primordia are produced by vertical rotational culture of shoot apices. This is a diameter of 50 to 1.5 mm without stratified cells with plastids. It consists of a primary planar streak primordium of a cell aggregate of OO0μm and a two-layered two-layer primordium of a diameter of 100 to 5000μm, and by circulating this and multiplying trophozoites, a hemispherical aggregate of cells is formed. body, or “shoot primordium”
It is a method that allows for rapid and large-scale propagation of plants, and has been applied to annual plants such as watermelon, corn, rice, morning glory, Japanese commonweed, and poppy, but no application to permanent woody plants has yet been proposed.

[Purpose of the invention]

The purpose of the present invention is to solve the problems of conventionally known sexual and asexual reproduction methods, and to apply the shoot primordia method developed for living plants in 2011 to a method for mass propagation of woody plants. shall be. Another purpose is to provide shoot primordia produced from the top of a vine stem by mass propagation while maintaining the genotype and chromosome type of a woody plant over generations. Another purpose is to find useful species (5pecies) and excellent varieties (5pecies) among woody plants.
The purpose of the present invention is to provide shoot primordia that can be applied to large-scale propagation of various species. Other purposes include cross-fertilized plants, male individuals of triploid, aneuploid, and dioecious plants that cannot produce seeds, as well as F1 hybrids produced by hybrid vigor, interspecific hybrids, and interlayer hybrids. The purpose is to provide the main body (shoot primordium) that is the basis for maintaining and propagating the genotypes of woody plants with high hybridity over many years. Still another object is to provide a virus-free woody plant.

[Structure of the Invention] The present invention is directed to shoot primordia derived from the shoot apices of woody plants, and by statically culturing the shoot primordia, woody plants can be propagated in large quantities in a genetically stable state. .

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

1) Shoot primordium production method After sterilizing the shoot apex of a woody plant with a sterilizing solution and washing it with sterile water, the shoot apex is extracted under a stereomicroscope in a clean bench and treated with an inorganic salt composition and a plant growth hormone. Transplant into artificial liquid medium containing. In this case, an organic substance that promotes differentiation, such as coconut milk, may be added.

The conditions for vertical (vertical) rotational culture are a temperature of 15 to 30°C;
Illumination intensity is 2,000 lux at the bottom and 10,000 lux at the top
20.000 lux and rotation speed 0.5-5 rp
It is m.

The composition of the artificial liquid medium varies considerably depending on the plant, but the basic inorganic salt composition is Gambog.
The composition contained in a medium such as B5 (hereinafter referred to as B5) of Rg) can be used with a slightly different composition. Naphthalene vinegar is a plant growth hormone! ! 112 (NAA
) 2.4 dichlorophenoxyacetic acid (2,4-D)
Indole-3 acetic acid (IAA) Indole-3
-Propionic acid (IPA) Indole-3-acetic acid (
IBA) Phenylacetic acid (FAA), benzofuran-3-acetic acid (BFA), auxins such as phenylamine (PBA), and 1-(2-chloro-4=pyridyl)
-3-Phenylurea (KT-30, manufactured by Kyowa Hakko Co., Ltd.) Cytokinins such as 6-benzylaminopurine (BA) and zeatin (Z) are used. A constant culture temperature of 15 to 30°C, especially 20 to 30°C, is appropriate.
As a result, growth is delayed at low temperatures, and growth is poor and unstable at temperatures that are too high. The culture of shoot primordia is characterized by vertical rotational culture, and the production of shoot primordium requires strong light, and the lower side is continuous at 2.00 m
At 0 lux, the top side is 10. It is appropriate to irradiate the light with an illumination intensity of D00~2 [1,000 lux. Illumination levels outside this range impair the growth of shoot primordia. In static culture, the growth of shoot primordia is poor. For vertical rotary culture, for example, a rotary wheel with a diameter of about 100 cm (manufactured by Nihon Ika Kikai Seisakusho) is used.
The circumferential surface and several steps from the circumferential surface in the direction of the rotation axis,
The medium containing the transplanted stem tips placed in 204 test tubes was placed diagonally, and the test tubes were placed so that they always faced the same direction even if the rotary wheel rotated vertically, and the light beam was irradiated from above. Make it. Further, the rotation speed is preferably a gentle rotation of 0.5 to 5 rpm. In this case, if the rotation speed is too high, there will be a lot of callus, and if it is too low, there will be a lot of early branches, and in any case, good results will not be obtained.

The propagation method of the present invention, when applied particularly to poplar, eucalyptus, and acacia, can produce actively proliferating shoot primordia.
Examples 1, 2, 3, and 4 show examples of optimal media in which these shoot primordia are produced by changing the composition and concentration of the media. In Tables 1.2.3 and 4, the parts marked with ○ are the hormone concentration combinations where shoot primordia appear, the combinations marked with X show the appearance of early branches, and the white (unmarked) parts are the combinations of hormone concentrations where shoot primordia appear. In the combination, callus appeared.

The artificial liquid medium in which shoot primordia grew fastest and most stably was as follows.

(1) Poplar ■NAA: 0.02mg/A+BA: 0.2m
g/f■NAA: 0.02mg/ji! +BA:0.4
mg/A■NAA: 0.05mg/jl'+BA
: 0.4mg/A■NAA: 0.2mg/j! +
KT-30: 0.2 mg/β As shown in Tables 1 and 2, four sets of optimal media were found depending on the combination of plant growth hormones. In addition, the rotation speed is 2r
pm was optimal.

Table 1 Combinations of optimal hormone concentrations for forming poplar shoot primordia Unit: mg/I1 Table Combinations for optimal hormone concentrations for forming poplar shoot primordia However, NAA: naphthaleneacetic acid, BA: 6-benzylamino However, T-30 (cytokinin type) (2) Eucalyptus ■NAA: Omg/j! +BA: 0.2mg/
It■NAA: 0.02mg/R+BA:
0.02mg/R■NAA: 0.02mg/j! +
BA: 0.2mg/ji! As shown in Table-3, 3
A set of optimal media was found. Note that the optimum rotation speed was 1 rpm.

Table 3: Combinations of optimal hormone concentrations for formation of shoot primordia of Eucalyptus. Here, two types (B, saligna, B.

Eucalyptus (G. grandis) was used, but the same results were obtained.

(3) Acacia ■2.4-D: 0.02mg/R+B A:
0.02mg/I! ■2.4-D: 0.02m
g/Il+BA: 0.2 mg/11 As shown in Table-4, two sets of optimal media were found. Note that the optimum rotation speed was 2 rpm.

Table 4: Optimal hormone concentration combinations for forming acacia shoot primordia, where BA: 6-benzylaminopurine, NAA: naphthalene acetic acid, BA: 6-benzylaminopurine, 2.4-D: 2 acids 4 Dichlorophenoxy vinegar The shoot primordium obtained is a hemispherical agglomerate, for example, in poplar, it is a green clump with a callus near its base. 1st
The figure is a photograph of a poplar primordium approximately 40 days after culture. In eucalyptus, it is a black-purple mass with a callus at its base. Figure 2 is a photograph of a eucalyptus shoot primordium approximately 6 months after culture. The same goes for acacia.

Furthermore, these shoot primordia have been actively maintained and proliferated for 10 to 12 months.

These shoot primordia were transplanted to a solid medium for seedling formation, and
When cultured statically at a temperature of 30° C. and an illumination intensity of 1,000 to 4,000 lux, a large number of minute shoots are produced. moreover,
When transplanted into a rooting medium, it will root and become a complete plant. The period of time for the plant to emerge is about 20 days after static culture, and the genotype, chromosome type, and phenotype of this plant are completely the same as the parent plant.

Initially, the shoot primordium has a smooth surface and a diameter of 40 to 7 mm.
(See Figure 3), whose constituent cells are uniformly small polygonal cells, and the cell division axes undergo polygonal division such as stratified, parallel, and oblique layers. This shoot primordium (1 whistle primordium) gradually becomes larger and has a diameter of 200 to 1000.
μm (see Figure 4), it differentiates into two layers: the epidermal system and the skin layer, the outermost layer has 1 to 2 cells, and the cell division axis shows only parallel division, and the inner layer is the inner layer. The system is a collection of many rather large cells, and inside these cells there are many well-developed chloroplasts, vacuoles, and storage substance granules. Furthermore, this shoot primordium (two-blowout primordium) has a diameter of approximately 400 μm.
At this stage, large oil bodies are observed in the cells of the epidermal system in the outermost layer, and the number of chloroplasts increases in the cells of the endothelial system in the inner layer, and vacuoles also develop greatly. are doing. At this stage, several new primordia of the above-mentioned 1-blow-flute ray are generated around this trapezoidal bulge. Through the above-mentioned route, the shoot primordium increases 6 and approximately quadruples in one month.

On the other hand, since secondary metabolites such as plastids are actively produced in shoot primordia, this method can be said to be effective as a method for producing useful substances that cannot be produced by the conventional callus cell method.

In addition, shoot primordia are subcultured approximately once a month.
It is genetically stable (the mutation rate is on the order of 10-6, the same rate as the natural mutation rate), and can be rapidly grown in large quantities.

Therefore, according to the present invention, a technique has been developed to maintain and propagate woody plants in a vegetative state in a genetically stable state for many years, and to produce large numbers of their populations as needed. Its multiplication rate is extremely high; in the case of broad-leaved trees, 412-17 x 106 times as many shoot primordia can be obtained from one shoot apex per year;
Furthermore, it has become possible to mass-produce approximately the same number of plants.

Examples of the present invention will be described in detail below.

■Example 1 (Populus charkow)
i) Creation of shoot primordium The B5 modified medium shown in Table 5 was used as the artificial liquid medium.

The B5 basic medium used here means the component composition announced by Gamborg in 1975, and the compounds marked with ○ are the substances modified this time. The appropriate concentration of plant growth hormone was examined using the 25-base method.

Table 5 Gamborg B5 modified medium (for poplar) Unit: (mg/A) First, cut off about 20 mm from the tip of a poplar actively growing in a greenhouse or field, wash it with sterile water, and place it on a clean bench. Under a stereomicroscope, remove the shoot apex (near the growing point) using a vinset and a scalpel.
Extract 1 mm. The excised stem tip is transplanted into the liquid medium shown in Table 5. The culture was carried out in a 30-hole (φ) x 200-diameter test tube into which B5 modified medium 25-cm was dispensed, at a temperature of 20 to 30°C, a lower side of 2000 lux, an upper side of 2Ω,
Vertical rotary culture at a rotation speed of 2 rpm under an illumination intensity of 000 lux. Approximately 40 days after the start of culture, green shoot primordium aggregates with a diameter of 100% are obtained. Thereafter, this shoot primordium is divided into 5 to 10 mm diameter pieces approximately every month and subplanted into the fresh medium described above for multiplication. The proliferation rate increased approximately four times in January once the shoot primordium was formed. Therefore,
If the number of months is n, the number of shoot primordia multiplied can be expressed as 4'', that is, approximately 414''-, 170 in one year.
Since 10,000 shoot primordia are produced, if these are transplanted into the seedling medium described later, it is possible to quickly produce large quantities of plants of the same quality in a plant factory while maintaining the plant's chromosome type and genotype. Can multiply.

ii) Seedling method for shoot primordia To obtain complete plants from shoot primordia, naphthalene acetic acid (NAA), 6-benzylaminopurine (BA) and KT-30 were removed from the B5 modified medium in Table 5. Basic medium: agar 8 g/l (0.8%) naphthalene acetic acid, 6
-Benzylaminopurine or zeatin Table-6,7
Add in the combinations shown.

Table 6 Redifferentiation medium for poplar shoot primordia (basal medium: B5) Unit: mg/R BA: 6-benzylaminopurine Table 7 Regeneration medium for poplar shoot primordia (basic medium 2B5) However, NAA: naphthaleneacetic acid, Z: zeatin For example, in Table 6, (a) NAA: Omg/j! +BA: 0.02
IIg/l(b)NAA: 0.001 mg/f
10BA: 0.02n+g/ii However, NAA:
Naphthalene acetic acid, (C)NAA: 0.001 mg/Il +BA
: A combination of 0.2 mg/1 was suitable as a regeneration medium.

Also, in Table 7, NAA: Omg/f+Z: 2
Regeneration occurred in the mg/β group. Note that the pH (acidity) is adjusted to 5.5 to 5.8, and a solid medium is used. Also, add 40ml of this medium to a 100-sized Erlenmeyer flask! Dispense and place a shoot primordia aggregate with a diameter of 5 to 10 (0111) on top of this.The temperature of the culture conditions at this time is 15 to 30℃,
The illumination intensity is 1,000 to 4,000 lux (16 hours of light and 18 hours of darkness). As a result, 3 to 4
8 to 15 shoot primordia are produced per shoot primordium mass. FIG. 5 is a photograph showing the state of a poplar shoot primordium that has turned into a seedling three weeks after transplantation.

Next, when the shoots have grown to about 10-15 mm, cut them from the base and add 6 g/f (0.6%) agar.
Transplant to the above-mentioned basal medium (hormone-free) containing . In this case, the reason why the concentration of agar is low is to soften the medium to facilitate rooting, prevent root breakage during transplantation, and promote growth after transplantation. The agar concentration is preferably 0.4 to 0.6%. After 2 to 3 weeks, when the plants are fully rooted, transplant them into a pot containing vermiculite, allow them to acclimatize for 1 to 2 weeks, and then transfer them to a greenhouse and grow them into strong seedlings using normal seedling cultivation methods. Grow into a sapling.

The seedling production method of the present invention differs from the method of Takashi Tanaka (1983) for producing seedlings from shoot primordia of student plants in the following points. That is, although he uses a B5 basic medium diluted to 175 to grow seedlings, in the present invention he uses it without dilution and only changes the hormone concentration.

It is also unique in that it has two types of media: one for sprouting and one for rooting. This promoted the growth of shoots of poplar, eucalyptus, acacia, etc. This is probably due to the abundance of nutritional sources. Also,
By transplanting the plants to a rooting medium, the rooting rate of the plants increased by several tens of percent, and post-rooting growth was also promoted, greatly contributing to the production of healthy seedlings.

■Example 2 (Eucalyptus, Bucalyptus sa
llgna.

B. grandis) i) Method for producing shoot primordia The artificial liquid medium has the composition shown in Table 5 (for poplar),
As a result of examining plant growth hormones using the 25-substrate method, the composition was changed to the one shown in Table 3 with an O mark. That is, N.A.A.
is θ~0.02mg/i! , BA is 0.02-0.2
It was carried out within the range of mg/1. Also, the rotation speed was different from that of poplar, and the optimal rotation speed was 1 rpm, but the other conditions of temperature and illumination were the same as for poplar.

In the case of eucalyptus, the first shoot primordium conglomerate (diameter 10+
nm), but it is significantly longer than poplar, about 6 nm).
It took months. The color was blackish-purple. However, similar to poplars, the growth rate was approximately four times that per month, indicating that large-scale growth is possible.

ii) Seedling formation method of shoot primordium As with poplar, plant growth hormones are added to the B5 basic medium in the combinations shown in Table 8. For example, (a) NAA: Omg/f+BΔ: 0.02mg/
A et al.) NAA: 0.001 mg/fl +BA
: At 0.02 mg/R, the appearance of shoots was the highest.

Note that other conditions such as temperature, illumination level, rooting medium, etc. are the same as for poplar.

In eucalyptus, it takes time to form the first shoot primordium, but once the first shoot primordium is formed, the rest is 1 minute, just like in poplar.
They can multiply in large numbers at a rate of about four times a month. Therefore, the size of one shoot apex increases by approximately 4"!=1,700 months in one year, which is sufficient for factory production.

■Example 3 (Acacia, Acacia auricu)
liformis) l) Method for producing shoot primordia As a result of examining only the plant growth hormone components of the B5 modified medium shown in Table 5 using the 25-base method, a large number of shoot primordia appeared in the following combinations.

(a) 2.4-D: 0.02mg/R+ B A
: 0.02mg/It(b)2.4-D : 0
．． 02mg/R+BA: 0.2mg/R
The conditions of temperature and illuminance were the same as those for poplar and eucalyptus, but the rotation speed was 2 rpm. The color of the shoot primordium was blackish-purple, similar to eucalyptus. It took about 6 months to form a shoot primordium cluster with a diameter of about 10 mm, but after that, like poplar, it multiplied at a rate of about 4 times per month.

Therefore, it has been found that the number of cells can be increased approximately 412″, or 17 million times, in one year.

ii) Shoot primordium regeneration method By adding the eucalyptus regeneration medium shown in Table 8 to the basic medium of B5, shoots appear. The subsequent rooting procedure is exactly the same as for poplar and eucalyptus.

Table 8 Regeneration medium for eucalyptus shoot primordia (Basic medium 285) However, NAA: naphthalene acetic acid Although the above description has been made regarding broad-leaved trees, it goes without saying that the present invention can also be applied to coniferous trees.

[Brief explanation of drawings]

Figure 1 is a photograph showing a poplar shoot primordium approximately 40 days after culture, Figure 2 is a photograph showing a eucalyptus shoot primordium approximately 6 months after cultivation, and Figure 3 is a photograph showing a poplar primary shoot primordium. Figure 4 is a photo of a cross section of a poplar secondary shoot primordium.
FIG. 5 is a photograph showing the state of a poplar shoot primordium that has turned into a seedling three weeks after transplantation. ′蓼、? Seki

Claims (1)

[Claims] 1. Shoot primordium derived from the shoot apex of a woody plant. 2. Extract the stem top of a woody plant and use it as an inorganic salt composition and a plant growth hormone containing cytokinin plant hormones at a maximum concentration of 0.5 mg/l, and at the same time 0 to 0.2 mg of auxin plant hormones. transplanted into an artificial liquid medium containing a concentration of
The lower side is 2,000 lux and the upper side is 10,000~20,
Under the illumination intensity of 000 lux and 0.5~10rp
A shoot primordium of a woody plant according to claim 1, which is produced by vertical rotational culture at a rotation speed of m.