JPH0611210B2 - Mass multiplication method of woody plants by shoot primordia - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to woody plants which are long-term crops, such as poplar, eucalyptus, acacia, sumac, corana, kunugi, yellowfin,
The shoot primordia obtained by extracting and rotating culture of the shoot apices of industrially useful woody plants such as coffee, Hevea brasiliensis, grapes, apples, etc., can be rapidly statically cultivated in a large amount to rapidly and mass-produce these seedlings. The present invention 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 methods for propagating woody plants: sexual reproduction with seeds and asexual reproduction (cutting, tissue culture, etc.). In the former case, pollen is not constant in cross-fertilized plants, so it is not always the case. The nature of parents is not transmitted to children. Also, in excellent interspecific hybrids, F ₁ hybrids (first-generation hybrids) produced by hybrid vigor, and in polyploid plants, the parental genotype is not directly transmitted to children. On the other hand, the asexual reproduction method has been a cutting method for a long time and has been generalized as an excellent quality breeding method. However, in this case, due to the slow growth rate, low rooting ability, the need to install a heading garden to produce large amounts of cuttings, and the limited timing of cutting. However, it is not suitable as a method for growing any woody plant. In addition, tissue culture, which has been making remarkable progress recently, sterilizes plant stems, shoot tips, leaves, root tips, etc.
This is a technique to re-differentiate the plant body after forming callus (undifferentiated cell clumps) in an artificial medium containing plant growth hormone etc., but because of the frequent occurrence of chromosome mutations and gene mutations during the growth process, It can be difficult to grow large numbers of children of the same nature. Also, if you have been passing callus for a long time,
In general, the differentiation ability is often reduced and the proliferation rate is often reduced.

Incidentally, woody plants, especially in broad-leaved trees such as poplar and eucalyptus, shoot apex, lateral bud, cotyledon, hypocotyl,
There are examples of tissue culture of various organs such as stems. However, in the case of shoot apex, since the callus is induced and the shoots are redifferentiated, the variability of the shoots inevitably becomes a problem. On the other hand, there is also a method of directly inducing adventitious shoots from the stems (so-called micropropagation), but in this case, in order to continuously obtain shoots, it is necessary to constantly plant new stem sections at appropriate intervals. Since it is necessary, it has a big defect as a commercial mass-propagation method.

For nude plants such as conifers, by culturing young cotyledons in tissue, an embryoid body (a tissue that resembles the embryo of the seed and is bipolar, that is, an organ that has two primordia of shoots and roots) Can be created. For example, using Douglas fir (Pseudotsuga menziesii), Mostafa
M. Abo El-Nil et al. Produced the embryoid body of this by shaking culture (US Pat. No. 4,217,730, patent of August 19, 1980). However, even in this case, although there is an advantage that the regeneration period is shorter than that in the normal tissue culture (organ formation method), the rate of complete plant formation is 15 to
It is as low as 50%. A further disadvantage is that large numbers of seeds are required because young cotyledons are always used. 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 growing annual plants other than woody plants (Tanaka Ryoso et al., Jpn.J.Genet.Vol.
58, 65-70 (1983), JP-A-59-132823, JP-A-59-132823).

These seedling primordial laws refer to Takaso Tanaka et al. (Jpn.J.Genet.Vol.
58, 65-70 (1983) is a method of utilizing the "primitive" of the shoot found using Hapropappus which is an annual plant of the Asteraceae family. That is, the shoot primordium contained in the spinach-like cell clumps obtained by removing the shoot apex of an annual plant and culturing the mixture in an artificial medium having a constant composition under constant temperature, illuminance and rotation speed. It is a method of rapidly obtaining a large amount of genetically stable seedlings by culturing and seedlings.

We have watermelon, corn, rice, morning glory,
As a result of diligent research on the “shoot shoot primordia” method applied to annual plants such as assembly and poppy, it was previously proposed that this method can also be applied to perennial woody plants (Japanese Patent Application No. 60- 193881).

The shoot primordium is a Konpeito sugar-like proliferator, and by propagating this shoot primordia, a rapid and large amount of clone individuals can be produced from a plant.

Then, in the conventional method, a stationary medium is used to seedling the produced shoot primordia using a solid medium, for example, agar medium, but when this method is applied, the seedling rate is 5 to 30%.
There was a problem that it was nothing more than a degree.

[Object of the Invention]

The present invention, in the case of mass-propagating a woody plant by a shoot primordium method, solves the problems in the above-mentioned conventional techniques,
It is an object of the present invention to provide a method for improving the seedling conversion rate of shoot primordia of woody plants.

[Structure of Invention]

The present invention extracts the shoot apex of a woody plant, transplants it into an artificial medium containing an inorganic salt composition and a plant growth hormone, and obtains a shoot primordium obtained by spin culture under light irradiation in a liquid medium. In the temperature of 15 to 30 ° C. and 1,000 to 4,0
A genetically stable woody plant characterized in that it is cultivated statically under irradiation with light having an illumination intensity of 00 lux to produce a foliar body, and then transplanted to a rooting solid medium for rooting. It is a method to grow a large amount in a state.

The present inventors have conducted various studies on a method for growing a woody plant in a genetically stable state in a large amount, and shoots obtained by culturing the shoot apex of the woody plant under light irradiation. The inventors have found that the primordia are statically cultivated using a liquid medium containing an inorganic salt composition and a plant growth hormone similar to those used for cultivating shoot primordia, and have completed the present invention.

The woody plant which is the subject of the present invention is not particularly limited, but examples thereof include eucalyptus, acacia, Hevea brasiliensis, evergreen broad-leaved trees such as coffee, poplar, serrata, kunugi, deciduous broad-leaved trees such as sumac, mandarin orange, lemon. , Cherry, apple, pear, peach, avocado, kiwifruit,
Examples thereof include fruit trees such as oysters, walnuts, grapes, figs, almonds and mangos, and flowering trees such as roses, camellias, plums and sakura.

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

Shoot primordium production method Stem tips of woody plants are sterilized with a sterilizing solution and thoroughly washed with sterilized water, and then the stem tips are excised under a stereoscopic microscope in a clean bench, and these are used as an inorganic salt composition and plant growth. Transfer to an artificial liquid medium containing hormones. In this case, an organic substance such as coconut milk that promotes differentiation may be added.

The inorganic composition contained in the artificial liquid medium varies considerably depending on the plant, but basically it is B of Gamborg.
The composition contained in 5 medium (hereinafter referred to as B5 medium) and the like can be used with slight changes. Plant growth hormones include naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), indole-3.
-Butyric acid (IBA), phenylacetic acid (PAA), benzofuran-
Auxins such as 3-acetic acid (BFA) and phenylacetic acid (PBA) and 6-benzylaminopurine (BA), kinetin, 1- (2-chloro-4-pyridyl) 3-phenylurea (KT-30, Kyowa fermentation) Cytokinins such as Zeatin (Z) and Zeatin (Z) can be used.

The culture temperature for carrying out the rotary culture is preferably 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 becomes unstable. . Illuminance of light irradiation is 2,000 continuously
0 to 20,000 lux is suitable, and illuminance outside this range deteriorates the growth of shoot primordia. Further, the culture is characterized in that it is carried out while rotating, and if the stationary culture is carried out without rotating, the growth of shoot primordia is poor.

In the rotary culture, for example, a test tube containing a medium in which the stem apex is transplanted is placed on the rotary plate so that the rotary axis of a rotary culture device (Nihon Kaika Kikai Co., Ltd.) having a rotating disk with a diameter of about 100 cm is aligned. Install. In this case, even if the rotary plate rotates, the test tube is always directed in a fixed direction and is irradiated with light from above. The rotation speed of the rotating plate is 0.5-5r.
The pm rotation is good, and if the rotation speed is too high, the callus is large, whereas if it is small, the premature branching is large and good results cannot be obtained.

When applied to poplar, eucalyptus, and acacia, the breeding method of the present invention yields shoot primordia that actively grow.
The obtained shoot primordium is a hemispherical agglomerate, for example, in the case of poplar, it is a green lump, with callus near its base. In eucalyptus, it is a mass of black-purple, and it is the same in acacia with callus at its base as in poplar.
These shoot primordia have been actively growing for over 19 to 21 months.

The shoot primordia has a smooth surface in the initial stage and a diameter of 40 to 7
The constituent cells having a 0 μm ridge are uniformly small polygonal cells, and the division axis of the cells undergoes multiple divisions such as the stratum, parallel layer, and oblique layer. The shoot primordia (primary shoot primordia) gradually become larger, and when the diameter becomes 200 to 1,000 μm, they are differentiated into two layers of epidermis system and cortex system, and the outermost layer is 1 to 2 cells and their division axis is average. Only stratification is seen. The inner lining system is a collection of many rather large cells, and inside this cell are many well-developed chloroplasts, vacuoles, and storage granules. Furthermore, this shoot primordium (secondary shoot primordium) becomes a trapezoidal ridge with a diameter of about 400 to 2000 μm, and a large oil body was observed in the cells of the outermost epidermal system at this time, and the inner system of the endothelial system was observed. In the cells, the number of chloroplasts increases and vacuoles are greatly developed. At this time, several above-mentioned primary seedling primordia are newly formed around the trapezoidal ridge. The number of shoot primordia increases in the course of the above, and it increases about four times in one month. It should be noted that shoot primordia mutate only about once in a month with submutation at a low mutation rate (10 ^-6 order), which is the same as the natural mutation rate, and it is genetically extremely stable. Therefore, large-scale growth can be performed quickly.

Method for establishing seedling primordia The seedling primordia thus proliferated are transplanted to a liquid medium for seedling priming similar to that used for the production of shoot primordia.
When statically cultivated at a temperature of -30 ° C and an illuminance of 1,000 to 4,000 lux, a large number of minute stems and leaves are produced.
Next, when this is transplanted to a rooting medium and rooted, a complete plant is obtained. The period until the formation of a plant is about 3 months after the start of static culture, and the genotype of the obtained plant
The chromosome type and phenotype are exactly the same as the parent plant.

Example 1 Test Plant Poplar (Populus charkowiensis x P. cau-dina) Method for Producing Shoot Primitive Approximately 20 mm was cut off from the tip of the green shoots of poplar actively growing outdoors and 70% ethanol was used. After sterilizing for 15 minutes with 1-minute, 5-fold diluted antimine, it was washed with sterile water. Next, 0.5-1 mm of the shoot apex including the growth point is excised with a pincette and a scalpel under a stereoscopic microscope in a clean bench. The extracted shoot apex is transplanted to the Gamboorg B5 modified medium shown in Table 1. Prior to carrying out this experiment, the combination of hormones in the artificial medium in which the shoot primordium proliferated the fastest and was stable and its concentration were examined by the 25-squares method.
The results shown in and 3 were obtained.

Cultivation was carried out in a test tube with a diameter of 30 mm and a length of 200 mm containing 25 m of B5 modified medium, and the temperature was set at 28 ° C. at 2,0.
Rotation culture is performed at an illuminance of 00 to 20,000 lux and a rotation speed of 2 rpm.

Approximately 40 days after the start of culturing, a green shoot primordium agglomerate having a diameter of about 10 mm is obtained. After that, about every 1 month, this is about 5
Divide into 10 mm and subculture in the above-mentioned fresh medium.
Once the shoot primordium has been obtained, by substituting it, its growth rate will be about four times as much as in January. Therefore, when the number of months is n, the number of shoot primordia can be represented by 4 ⁿ . Next, the shoot primordium thus obtained is transplanted to a seedling medium.

Method for seedling primordia seedling Primitive shoot agglomerates obtained by spin-culture in B5 modified medium are used as they are at a temperature of 28 ° C. and an illuminance of 1,000 to 4,000 lux (16 hours light + 8 hours dark). The liquid static culture was carried out under the conditions. When culturing is continued for 3 weeks, 10 to 15 foliar bodies having a size of 3 to 5 mm are generated per shoot original mass (see FIG. 1).

When this foliage grows to 10 to 15 mm after further culturing for 4 weeks, 6 g / agar is added to B5 basal medium (medium obtained by removing only hormone from B5 modified medium) which is cut from the base and diluted 2-fold. When it was transplanted to roots and rooted, it was fully rooted in 3 weeks.

When the shoot primordium was transplanted directly to the agar medium for seedling formation without liquid static culture, the seedling formation ratio was only 30% of the shoot primordium. The conversion rate was 50%. Then, the grown seedlings were transplanted to a pot containing permikiurite, acclimated under low light for 2 weeks, transferred to a greenhouse, and grown into a healthy seedling by a normal seedling raising method.

Example 2 Test plant Eucalyptus saligna (E. grandis) Shoot primordium production method Approximately 1 from the tip of the actively growing shoot of a 3-year-old seedling.
A 0 mm piece was cut and sterilized with 70% ethanol for 30 seconds and 10-fold diluted antiformin for 20 minutes. After washing with sterilized water, the stem apex is extracted in the same manner as poplar and transplanted into an artificial medium.
The composition of the medium other than phytohormones is the same as that of poplar (Table 1). As for the plant hormone, as a result of examination by the 25th grid method, as shown in Table 4, NAA was 0 to 0.0.
2 mg /, BA was carried out in the range of 0.02-0.2 mg /. The optimum rotation speed was 1 rpm, which was different from that of poplar, but the culture temperature and illuminance were similar to those of poplar.

In the case of eucalyptus, the first shoot primordium agglomerate (diameter about 10 mm)
It took about 6 months, which is significantly longer than that of poplar, and the color was black purple. However, thereafter, similar to poplar, it showed a growth rate of about 4 times a month.

Method of seedling primordia seedlings The seed primordium agglomerates obtained by spin culturing were transferred to the same conditions as in the case of poplar while still in the liquid medium, and liquid stationary culture was performed.
The foliage was developed.

Root the obtained foliage in the same manner as for poplar,
Furthermore, I raised it to a seedling.

The seedling conversion rate was about 30% with respect to the shoot primordia. When the shoot primordium was transplanted directly to an agar medium without performing liquid static culture to carry out seedling formation, the seedling formation ratio was only 10%.

Example 3 Nude plant plant Acacia (Acacia auriculiformis) shoot primordium production method Approximately 10 mm was cut off from the tip of the actively growing branch of the 3rd-year-old shoot and sterilized in the same manner as in the case of eucalyptus. The composition of the artificial medium other than plant hormones is the same as that of poplar (Table 1). As a result of examining the plant hormone by the 25th grid method, a large number of shoot primordia appeared in the following combinations. 2,4-D: 0.02mg / + B
A: 0.02 mg /, 2,4-D: 0.02 mg / + B
A: 0.2 mg /. The conditions of temperature and illuminance were the same as those of poplar and eucalyptus, but the rotation speed was 2 rpm. The color of the obtained shoot primordium was blackish brown, and it took about 6 months to form a shoot primordia agglomerate with a diameter of about 10 mm. Propagated in.

Method of seedling primordia seedlings The shoot primordia agglomerates obtained by spin culturing were transferred to the same conditions as those for poplar and eucalyptus while still in the liquid medium, and liquid static culture was performed to generate shoots. .

The seedling conversion rate was about 20% based on the shoot primordia. When the shoot primordia were transplanted directly to an agar medium without performing liquid static culture to carry out seedling formation, the seedling formation ratio was only 5%.

Root the obtained foliage in the same manner as for poplar,
Furthermore, I raised it to a seedling.

(Effects of the Invention) According to the present invention, woody plants are maintained and propagated in a trophozoite genetically stable state for many years, and a large number of individual populations are produced efficiently as necessary. Technology was developed. The growth rate is extremely fast, and in the case of a broad-leaved tree, 4 ¹² pieces, that is, about 17 × 10 ⁶ times as many shoot primordia are obtained from one shoot apex, and the same number of plant seedlings are produced. It has become possible.

Although only the case of broad-leaved trees has been described above, it goes without saying that the method of the present invention can be applied to conifers.

[Brief description of drawings]

FIG. 1 is a photograph showing the morphology of the stems and leaves produced by liquid static culture of poplar shoot primordia.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Keigo Dohi 24-9 Nozono-cho, Kameyama City, Mie Prefecture Forest Tree Breeding Research Institute Kameyama Breeding Plant, Oji Paper Co., Ltd. (72) Masaki Ito Nome, Kameyama City, Mie Prefecture 24-9 Koinomachi Oji Paper Co., Ltd. Forest Tree Breeding Research Institute Kameyama Breeding Plant (56) References “Seed and Seedling Industry and Breeding Technology” CMC Co., Ltd. February 10, 1984, p. 171〜197 Supervised by Takaso Tanaka “Practical technology for mass production of cloned plants” CMC Co., Ltd. Showa December 25, 1960 38-47

Claims (3)

[Claims] 1. A shoot primordium derived from the shoot apex of a woody plant in a liquid medium at a temperature of 15 to 30 ° C. and 1,000 to
A woody plant based on a shoot primordia, which is characterized by performing static culture under irradiation with a light having an illumination intensity of 4,000 lux to generate a foliar, and then transplanting the foliage into a solid medium for rooting. Mass proliferation method. 2. The method according to claim 1, wherein the seedling formation by the liquid static culture is performed in a medium having the same composition as the artificial medium used for producing the shoot primordia. 3. The method according to claim 1, wherein the woody plant is a broad-leaved tree such as poplar, eucalyptus and acacia.