JPH06189643A - Large-scale multiplication of rhododendron - Google Patents

Abstract

PURPOSE:To multiply a Rhododendron on a large scale according to the tissue culture method. CONSTITUTION:A piece of shoot apex tissue of a Rhodedendron is subjected to a first culture in a culture medium containing a plant hormone belonging to auxin and a plant hormone belonging to cytokinin so as to aseptically grow the shoot apex tissue. Thereby, a cultured seedling substantially not having a root is obtained and the resultant seedling is then transferred into a culture medium containing a plant hormone belonging to auxin and a plant hormone belonging to cytokinin and subjected to a second culture for multiplication. Further, the resultant multiplied seedlings not having a root are transferred into a culture medium containing a plant hormone belonging to auxin so as to allow the seedling initially to take roots and subsequently to grow into a complete plant body.

Description

Detailed Description of the Invention

[0001]

The present invention relates to the genus Rhododen.
dron) A method for mass-propagating a plant tissue culture.

[0002]

BACKGROUND OF THE INVENTION Rhododendron plants are distributed in the boreal and temperate zones of the northern hemisphere, and are found in mountains such as South Asia, Malaysia, New Guinea, and Australia, and there are over 600 species. Rhododendron plants are used for potted plants, cut flowers, etc. in addition to garden trees. Clonal multiplication of these rhododendron plants has been conventionally performed by cutting or grafting. However, clonal growth by cuttings and grafts has a low growth rate, and it is almost impossible to grow in large quantities. Therefore, it is necessary to establish a mass proliferation method by tissue culture.

As an example of tissue culture of a rhododendron plant, Fordham & Stimart (HortScience, Vol.17, No.
5, 1982) using Exbury Azalea and Anderson's Rhodod
We are trying tissue culture with 5 kinds of cytokinin plant hormones on endron medium. As a result, it is reported that the proliferation rate is increased by using zeatin.
The medium and hormones used in this report and the present invention are different, and in this report, the shoots were successfully grown but not rooted.

Martin (HortScience, Vol.17, N
Rhododendron using the same medium as the former.
We are performing tissue culture on flower buds of 5 varieties of catawbiense . This report also differs from the present invention in the tissues and medium used, and the growth rate is still at a low level. In addition, Anderson (Journal of the American Society for
Horiticultural Science, Vol.109, No.3, 1984) proposes a medium having a new composition by modifying the medium used in the above two reports to improve the growth rate. The composition of the reported medium is different from that of the present invention, and the growth rate is not yet at a satisfactory level. All of the various tissue culture methods of the rhododendron plants illustrated here are beyond the scope of basic experiments, and have not been able to be mass-produced on a commercial scale as in the present invention.

[005]

DISCLOSURE OF THE INVENTION The present invention provides a method for mass-growing a rhododendron plant clone, which has hitherto relied on grafting, cutting, etc., by tissue culture. Furthermore, the present invention replaces the conventional tissue culture technology of rhododendron plants, which has been still limited to basic research, with tissue culture of apical shoot apices and axillary shoot apices to mass-proliferate plants at a high growth rate. It is intended to provide a method.

[0005]

[Means for Solving the Problems] The present invention uses a tissue piece at the apex of a rhododendron plant as a first culture of auxin-type plant hormones 0.01 to 5.0 mg / l, cytokinin-type plant hormones 0.01 to 5.0 mg / l. Aseptically elongate the shoot apex using a medium containing l, and cultivate the shoots with substantially no roots, and then as the second culture, the shoots are auxin plant hormones 0.01 to 5.0 mg / l, Transplant into a medium containing 0.01 to 5.0 mg / l of cytokinin plant hormones for growth, and then transplant the grown rootless shoots into a medium containing auxin plant hormones for rooting to complete plant body. It is a method of mass-proliferating Rhododendron plants, which is characterized in that

The present invention will be described in more detail below. Tissues of Rhododendron Plants Applicable to the present invention are Rhododendron plants, such as Azuma Rhododendron, Giant Rhododendron, Tsukushi Rhododendron, Hakusan Rhododendron, Rhododendronia japonica, Rhododendronia japonica, Rhododendronia japonicus, Rhododendronia japonicus , Amaranthaceae, Genji Azalea, Vladivostoki Azalea, White-spotted Azalea, Ezomurasaki Azalea, Sakai Azalea, Renge Azalea, Kojira-tsuji-koji, Tsukihaji-tsuji, Yamatsuji, Oshimatsuji-tsujimitsu, Kirishimatsuji, Kirishimajitsu, Fuji azalea, Ooyama azalea, Ashitaka azalea, Unzen azalea, Yakushima azalea, Saku Azalea, Yodogawa azalea, Korean azalea, Japanese azalea, Cypress azalea, Kerama azalea, Mochi azalea, purple azalea, azalea, azalea, azalea, azalea, azalea, azalea, azalea, azalea, azalea, azalea In addition to tree species such as Kunitsutsutsutsutsutsutsu, Kiyosumitsutsutsutsutsuji, Ontsutsuji, Azalea, Jingoutsutsu, Goyoutsutsuji, Kurofunetsutsuji, purple azalea, Akebonotsutsu, Akashinoto, Obaku azalea, these cultivated varieties and interspecific hybrids. As the tissue pieces (explants) used for tissue culture, shoot apices and apical shoots of axillary buds can be used.

Medium As the medium used in the present invention, a medium generally used for tissue culture of plants can be widely used. For example, Gamboug B5 medium, Murashige-Skoog medium, Litvay medium (LM
Medium), Woody pland medium (WPM medium), Whit
Examples of the medium (e medium) (White medium) or a medium in which the composition of these mediums are modified can be exemplified, but in the present invention, it is particularly preferable to use the WPM medium or the medium in which the WPM medium is modified.

[0008] Plant hormones As the phytohormone to be added to the medium naphthalene acetic acid (NAA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4 -D), indole-3-propionic acid (IPA), benzofuran-3-acetic acid (BFA),
Auxins such as phenylbutyric acid (PBA) and their derivatives, and benzyladenine (BA), kinetin, zeatin, 2-isopentenyladenine (2i
P), (2-chloro-4-pyridyl) -3-phenylurea (4PU), and other cytokinins.

Regeneration of shoots without roots (first cultivation
(Nourishment) A tissue piece of a rhododendron plant surface-sterilized is planted on a solid medium in which plant hormones and sucrose obtained by combining cytokinins and auxins are added to the above medium. The culture conditions are as follows: room temperature is 10 to 30 ° C., illuminance is 1000 to 20000 lux, light period is 10 to 16 hours, and dark period is 14 to 8 hours. After planting the tissue piece, the regeneration of the shoots starts from the tissue piece in about 4 weeks, and after about 8 weeks, the height of the seedling grows to about 1 to 2 cm.

Propagation of shoots without roots (second cultivation
Feeding ) The rootless shoots regenerated as described above are transplanted to a solid medium in which the above-mentioned medium is supplemented with plant hormones and sucrose by the combination of cytokinins and auxins. The culture conditions are the same as the conditions for regeneration of shoots without roots, but preferable conditions are room temperature 15 to 25 ° C., 5000 to 15000 lux, light period for 12 to 16 hours, and dark period for 12 to 8 hours. At this time, the growth rate may be improved by using an aseptically aerated container other than the Erlenmeyer flask as the container used for the culture.

After transplanting the rootless shoots, the shoots start to grow in about 1 to 3 weeks. The manner of this proliferation varies depending on the individual, but roughly divided into the following two types. One growth morphology is one in which a large number of adventitious buds are formed and become multiblasts to grow. Another form of growth is to form callus at the base of transplanted rootless shoots and regenerate and grow adventitious shoots from this callus. Further, by cutting off the callus formed at the base of the shoot and transplanting it into a fresh medium, the shoot without roots can be redifferentiated and proliferated.

Rooting from rootless shoots Rootless shoots grown as described above were diluted to about 1/2 to 1/4 of the above medium, and auxin plant hormone and sucrose were added thereto. Transfer to solid medium. At this time, it is desirable that the sucrose added to the medium is kept as low as possible. In some cases, sucrose may not be added. Agar, gellan gum and the like are used as a solidifying agent for the medium, but in particular gellan gum may improve the rooting rate. The culture conditions at the time of rooting are the same as the regeneration of shoots without root and the growth of shoots without root.

Rooting is recognized as early as 2 weeks after transplanting to a rooting medium. After 2-4 weeks, the root length is 1
Extends to about 2 cm. There is no problem if the culture is continued as it is and is cultivated to such an extent that it can be acclimated, but at this point, plant roots should not be added and the rooted shoots should be transplanted to a fresh medium similar to the other ingredients. It is possible to promote shoot growth. Shoots 2-3 cm (6 leaves)
Approximately about 7 sheets), the process shifts to acclimation. A large number of healthy seedlings can be obtained by using a conventionally known method as the acclimation method.

[0014]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Example 1 Individual explants selected from the Ghent hybrid of the Astragalus azalea and its surface sterilization method A branch of about 2 cm in length including the shoot apex was cut from a 20-year-old individual selected from the Ghent hybrid of the Astragalus azalea, and the following Surface sterilization was performed by the method of. Perform ultrasonic cleaning while soaking in 70% ethanol for 20 seconds, followed by 0.3% Tween20.
Immerse in 20-fold diluted antiformin containing 3 minutes (1 minute of which is ultrasonic cleaning). After this, rinse anti-formin 3 times with sterile water.

Regeneration of shoots without roots (medium and plant hormones) A partially modified WPM medium (Table 1) was diluted to 1/2 and sucrose 3% was added thereto. 2 in this medium as cytokinin type plant hormone
iP2 mg / l and IAA0.
The pH was adjusted to 5.4 by adding 1 mg / l. 40 ml of this medium is put into a 100 ml glass Erlenmeyer flask (bottom area: about 20 cm ² ).
Each aliquot was added, 0.7% of agar was added, and the mixture was sterilized by an autoclave (121 ° C, 1.2 kg / cm ² , 15 minutes).

[0016]

[Table 1] Table 1 Woody plant medium (WPM) Components of modified medium ─────────────────────────── Components (mg / l ) ─────────────────────────── NH ₄ NO ₃ 400 K ₂ SO ₄ 990 MgSO ₄・ 7H ₂ O 370 CaCl ₂・ 2H ₂ O 96 Ca (NO ₃ ) ₂・ 4H ₂ O 556 KH ₂ PO ₄ 170 H ₃ BO ₄ 6.2 MnSO ₄・ 4H ₂ O 22.3 ZnSO ₄・ 7H ₂ O 8.6 CuSO ₄・ 5H ₂ O 0.025 Na ₂ MoO ₂・ 2H ₂ O 0.25 CoCl ₂・ 6H ₂ O 0.025 Na ₂・ EDTA 37.3 FeSO ₄・ 7H ₂ O 27.8 myo-Inositole 100.0 Thiamin HCl 0.1 Pyridoxine HCl 0.5 Nicotinic acid 0.5 Glycine 2.0 ────────────── ──────────────

(Planting and culture conditions for explants) From the branch containing the above-mentioned surface-sterilized shoot apex, the shoot apical part having a size of 1 to 2 mm including the growth point is cut into the above-mentioned medium, and 2 stems per flask are cut. Planted top by top, at 23 ° C for 16 hours light period (2000lux
), And cultured for 8 hours in the dark. Elongation of shoots was observed about 4 weeks after planting the shoot apices, and seedling height was 1 cm after about 8 weeks.
Extend to a degree.

Growth of rootless shoots (medium and plant hormones) A medium containing sucrose and plant hormones similar to the medium used for regeneration of rootless shoots was added to pH.
Adjusted to 5.4, aseptically ventilated polycarbonate culture pot with Millipore filter (bottom area: approx. 44 cm
60 ml each was added to ² ), 0.7% agar was further added, and the mixture was sterilized by an autoclave (121 ° C, 1.2 kg / cm ² , 15 minutes).

(Planting and Culture Conditions of Shoots) About 5 to 7 seedlings having no root described above were planted in this medium, and the explants were cultured under the conditions. From shoots without one root about 4 to 8 weeks after transplantation of growth medium
10 to 20 shoots without root grow. By dividing these shoots without grown roots and transplanting them into a fresh growth medium, the growth can be repeated semipermanently.

Rooting of rootless shoots (medium and plant hormones) The partially modified WPM medium used for the regeneration of rootless shoots was diluted 1/6,
Add 1.5% sucrose. Furthermore, 0.1 mg / l of IBA is added. Dispense 40 ml of this medium into a 100 ml glass Erlenmeyer flask and add 0.4% gellan gum.
Sterilize by autoclave (121 ℃, 1.2kg / cm
² , 15 minutes). Two rootless shoots are transplanted into each flask and the cells are cultured under the conditions used for regeneration of rootless shoots and growth of rootless shoots. Approximately 4 weeks after transplanting the rooting medium, the root length grows to about 1 cm.

At this point, in order to promote the elongation of shoots and roots, they are transplanted to the following medium. Partially modified WPM
Dilute the medium to 1/4, add 1.5% sucrose and no plant hormones. Dispense 60 ml of this medium into a culture pot with a sterile aseptic polycarbonate polycarbonate Millipore filter, add 0.4% gellan gum, and sterilize by autoclaving (121 ° C, 1.2 kg / c).
m ² , 15 minutes). Two rooted shoots are transplanted per pot. 4 to 6 weeks after transplantation, the shoots grow to about 2.5 to 4.0 cm. The rooting rate in this example is 95% or more.

As described above, when the seedling height reached about 2.5 to 4.0 cm, each individual was transplanted to volcanic ash (particle diameter of 3 mm or less) by the method already known, the humidity was gradually lowered, and the seeds were transferred to high illuminance. Acclimation is completed in about 4 weeks and normal shoots are obtained. When culturing is performed using this Example, a normal seedling can be obtained from one shoot apex in about 16 to 22 weeks. Further, theoretically, it is possible to obtain 1 × 10 ⁶ shoots from one shoot apex in half a year by repeating the culture.

Example 2 Rengue Azalea cultivar Exbury Aza
Individual explants selected from lea and its surface sterilization method A 20 cm long plant selected from Exbury Azalea, a varieties of long-aged azaleas, was cut into branches of about 2 cm in length, including the shoot apex, and surface sterilized by the following method. It was Perform ultrasonic cleaning while soaking in 70% ethanol for 20 seconds, followed by 0.3% Tween2
Immerse in 20-fold diluted antiformin containing 0 for 3 minutes (1 minute of which is ultrasonic cleaning). After this, rinse anti-formin 3 times with sterile water.

Regeneration of shoots without roots (medium and plant hormones) 1 partly modified WPM medium
Diluted to / 2, and sucrose 3% was added thereto. 2 iP 2 mg / l as a cytokinin-type plant hormone and IAA 0.1 mg / l as an auxin-type plant hormone were added to this medium to adjust the pH to 5.4. 40 ml of this medium was dispensed into a 100 ml glass Erlenmeyer flask, 0.7% of agar was added and sterilized by an autoclave (121 ° C, 1.2 kg /
cm ² , 15 minutes).

(Planting and culture conditions for explants) In addition to the above-mentioned surface-sterilized shoot-containing apices, a branch containing growth points 1 to
2 mm-sized shoot tips were cut out, 2 shoot tips were planted per flask, and cultured at 23 ° C. for 16 hours light period (2000 lux) and 8 hours dark period. Elongation of the shoots is observed about 4 weeks after the shoot apical planting, and the seedling height grows to about 1 cm after about 8 weeks.

Growth of rootless shoots Sucrose similar to the medium used for regeneration of rootless shoots,
The medium containing phytohormones was adjusted to pH 5.4, and 60 ml each was aseptically dispensed into a polycarbonate culture pot with a Millipore filter that could be aseptically ventilated, 0.7% of agar was added, and sterilized by an autoclave (121 ° C, 1.2 kg / cm
² , 15 minutes).

1 shoot of the above-mentioned rootless shoot was added to this medium
About 5 to 7 plants were planted per pot, and the explants were cultured under the same conditions. Callus is formed at the root of a shoot without one root about 4 to 8 weeks after the growth medium is transplanted, and 2-3 shoots are redifferentiated from this callus.

In addition, about 2 to 3 adventitious buds grow from the axils of the planted rootless shoots. By removing the rootless shoots from the callus formed at the root of the shoots and transplanting them into a fresh medium having the same composition, the rootless shoots can be propagated through the same process. Further, by transplanting the callus formed at the root of the shoot into a fresh medium containing the same components and culturing, the shoot is redifferentiated from the callus, and the shoot can be proliferated. By repeating these operations, the shoots can be proliferated semipermanently. The proliferation rate in this example is 4 to 6 times.

Rooting from rootless shoots One WPM medium was prepared by partially modifying the above rootless shoots.
/ 6 dilution and add 1.5% sucrose. Furthermore, IBA
0.05 mg / l is added. Dispense 60 ml each of this medium into culture pots with sterile aseptic polycarbonate Millipore filters, add 0.4% gellan gum, and sterilize by autoclaving (121 ° C, 1.2 kg / cm ² ,
15 minutes). Seven rootless shoots are transplanted into each pot, and the seedlings are cultured under the conditions used for regeneration of rootless shoots and growth of rootless shoots. Approximately 4 weeks after transplanting the rooting medium, the root length grows to about 1 cm.

At this point, in order to promote the elongation of shoots and roots, the medium is transplanted to the following medium. Partially modified WPM
Dilute the medium to 1/4, add 1.5% sucrose and no plant hormones. Dispense 60 ml of this medium into a culture pot equipped with a polycarbonate Millipore filter, add 0.4% gellan gum, and sterilize by autoclaving (121 ° C, 1.2 kg / cm ² , 15 minutes). Two rooted shoots are transplanted per pot. 4 after transplant
The shoots grow to about 2.5-4.0 cm in 6 weeks. The rooting rate in this example is 30% or more.

As described above, when the seedling height reached about 2.5 to 4.0 cm, each individual was transplanted to volcanic ash (particle diameter of 3 mm or less) by a method that has already been clarified, the humidity was gradually lowered, and it was transferred to high illuminance. Acclimation is completed in about 4 weeks and normal shoots are obtained. When culturing is performed using this Example, a normal seedling can be obtained from one shoot apex in about 16 to 22 weeks. Further, by repeating the culture, 5.3 × 10 ⁵ shoots can be obtained from one shoot apex in one year.

[0032]

INDUSTRIAL APPLICABILITY According to the present invention, clonal growth of rhododendron plants, which have so far relied on grafting and cutting, can be grown in large quantities by tissue culture using apical shoots and axillary shoot shoot tips. By this method, it became possible to efficiently propagate a large amount of rhododendron plants developed as a new variety.

Claims (1)

[Claims] 1. A auxin-type plant hormone 0.01 to 5. as a first culture of a tissue piece at the apex of a rhododendron plant.
0 mg / l, cytokinin plant hormone 0.01-5.0 mg / l
Aseptically elongate the shoot apex using a medium containing, and cultivate the shoots with substantially no roots, and then as a second culture, the shoots are auxin plant hormone 0.01-5.0 mg /
l, Cytokinin plant hormones were transplanted to a medium containing 0.01 to 5.0 mg / l for growth, and the grown rootless shoots were transferred to a medium containing auxin plant hormones for rooting. A method for mass-producing a rhododendron plant, which comprises growing the plant.