JP2004121133A - Induction method of adventitious root and induction method of adventitious bud of fig tree - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain an adventitious root and adventitious bud of a fig tree. <P>SOLUTION: The induction method of the adventitious root involves culturing tissue fragments of the adventitious root or the adventitious bud in the presence of phloroglucinol to induce the adventitious root or the adventitious bud. <P>COPYRIGHT: (C)2004,JPO

Description

【００２４】
表１から、２，４−Ｄを単独で含む培地を用いても葉片から不定根を誘導できることが明らかとなった（図１参照）。さらに表１から、２，４−Ｄに加えてＰＧを添加した培地を用いた場合には、不定根発生率を大幅に高められることも明らかとなった。特に、２，４−Ｄ　１．０　ｐｐｍとＰＧ　０．５　ｍＭで最も効率よく不定根を誘導できた。
以上の結果から、ＰＧは、イチジクの不定根をより効果的に誘導する作用を有することが判明した。
【００２５】
〔実施例２〕不定芽の誘導
実施例１と同様に採取した葉片を、様々な濃度の２，４−Ｄとチジアズロン（ＴＤＺ、シグマ社製）を添加したＭＳ培地（３％ショ糖、０．８％寒天、ｐＨ　５．８、０．５　ｍＭ　ＰＧを含む）に植え付けた。なお、本例においては、２，４−Ｄの濃度を０．０（無添加）、０．５、１．０、２．０或いは５．０　ｐｐｍとし、ＴＤＺの濃度を０．０（無添加）、０．５、１．０、２．０或いは５．０　ｐｐｍとした。なお、各試験区においては、１０〜１２個の葉片を用いて３回以上の反復試験で行った。
葉片を植え付けた後、２５℃暗黒化で４９日間培養した。各試験区で観察された発芽率及び発芽数を表２に示す。
【００２６】
【表２】

【００２７】
表２から、ＰＧの存在下において、ＴＤＺを単独で含む培地を用いた場合、如何なる器官形成も認められず、不定芽を誘導できないことが明らかとなった。また、表２から、ＰＧの存在下において、２，４−ＤとＴＤＺとを含む培地を用いた場合、不定芽を誘導できることが明らかとなった（図２参照）。特に、ＰＧの存在下においてＴＤＺ　１．０　ｐｐｍ及び２，４−Ｄ　１．０　ｐｐｍを含む培地を用いた場合には、不定芽を最も効果的に誘導できることが明らかとなった。
一方、不定芽の誘導に対するＰＧの影響を検討するために、ＰＧの濃度を０．０（無添加）、０．５或いは１．０　ｍＭとした培地に葉片を植え付け、同様に培養した。なお、使用した培地に含まれるＴＤＺの濃度を１．０　ｐｐｍとし、２，４−Ｄの濃度を１．０　ｐｐｍとした。また、培養は２５℃暗黒化で６０日間とした。結果を表３に示す。
【００２８】
【表３】

【００２９】
表３から、ＰＧ無添加区では、不定芽が誘導されなかったのに対し、ＰＧを添加した試験区では、不定芽が誘導されることが明らかとなった。また、表３から、ＰＧの濃度が０．５　ｍＭの試験区では、ＰＧの濃度が１．０　ｍＭの試験区よりも優れた発芽率を示すことが明らかとなった。
以上、表２及び表３の結果から、イチジクから不定芽を誘導するには、ＴＤＺ及びＰＧを含む培地が有効であることが判明した。
【００３０】
〔実施例３〕不定芽からの植物体再生
実施例２で得られた不定芽を用いて以下のように植物体を再生した。具体的には、先ず、得られた不定芽を６−ベンジルアミノプリン（ＢＡ）とα−ナフタレン酢酸（ＮＡＡ）を含むＭＳ培地に移植して成長させた。このとき、培養条件としては、温度２５±２℃、白色蛍光管下（５５．６μｍｏｌ／ｓ／ｍ^２）の１６時間日長とした。
【００３１】
その後、茎長が１〜２ｃｍになったときに、発根培地としてインドール酪酸（ＩＢＡ）１．０　ｐｐｍを含むＭＳ培地または植物ホルモンを含まないＭＳ培地に移植した。このとき、培養条件としては、温度２５±２℃、白色蛍光管下（５５．６μｍｏｌ／ｓ／ｍ^２）の１６時間日長とした。移植１０〜３０日後に発根を確認することができた（図３参照）。
【００３２】
そして、発根後、馴化・鉢上げすることでイチジク植物体へ再生させることができた（図４参照）。具体的に馴化は、以下のように行った。根に付いた寒天を取り除いた後、イチジク植物体をバーミキュライトの入ったプラスチック製ポットに移植した。そのポットを密閉可能な透明プラスチック容器に入れ、約２週間の間、高湿度条件を保つために密閉状態で育成した。その後、イチジク植物体の生育状況を見ながら上部のふたを順次ずらして外部の湿度（相対湿度６０〜７５％）に馴化させた。このときの培養条件としては、温度２５±２℃、白色蛍光管（２９．４μｍｏｌ／ｓ／ｍ^２）の１６時間日長にした。
また、具体的に鉢上げは以下のように行った。外部の湿度に十分馴化したイチジク植物体を培養土の入った素焼き鉢に移植し、自然光下の温室内で育成した。
【００３３】
【発明の効果】
以上、詳細に説明したように、本発明により、イチジクにおける不定根及び不定芽を誘導できる全く新規な方法及び誘導剤を提供することができる。すなわち、本発明に係るイチジクの不定根誘導方法及び不定根誘導剤によれば、従来において得ることができなかったイチジク不定根を作出することができる。また、本発明に係るイチジクの不定芽誘導方法及び不定芽誘導剤によれば、従来において得ることができなかったイチジク不定芽を作出することができる。
【図面の簡単な説明】
【図１】実施例１で作出した不定根の写真である。
【図２】実施例２で作出した不定芽の写真である。
【図３】実施例３で作出した発根培地における不定芽由来植物の写真である。
【図４】実施例３で作出した鉢上げ後の不定芽由来植物の写真である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for inducing adventitious roots and adventitious buds of figs (Ficus carica L.).
[0002]
[Prior art]
In recent years, adventitious buds, adventitious roots and adventitious embryos have been derived from plant fragments in some fruit trees, and the production of transgenic plants by the Agrobacterium method has been reported. However, in the case of FIG., There have been no reports of adventitious bud induction, adventitious embryo induction or plant regeneration from protoplasts. Therefore, with respect to the fig, a technique for producing a recombinant has not been established.
[0003]
Fig is one of the remarkably growing tree species in recent years. However, in order to overcome disease resistance and cold resistance and elucidate physiological functions, it is indispensable to establish a technique for producing a recombinant. In tissue culture of figs (Ficus carica L.), growth point culture (see Non-Patent Documents 1, 2 and 3), axillary bud culture (see Non-Patent Document 4), and shoot formation from flower buds (see Non-Patent Documents 5 and 6) ) Is reported. However, neither method is accompanied by organogenesis. In addition, there are no reports on plant regeneration from protoplasts or induction of somatic embryos.
The establishment of the adventitious shoot and adventitious root induction technology for figs will solve the important constraints of the technology for producing transgenics, and may become one of the core technologies of fig transgenic technology in the future.
[0004]
[Non-patent document 1]
Murithi LM, Rangan TS, Waite BH (1982) In vitro propagation of fig through shot tipure. HortScience 17: 86-87.
[Non-patent document 2]
Pontikis CA, Melas P (1986) Micropropagation of Ficus carica L. HortScience 21: 153.
[Non-Patent Document 3]
Furukawa, S. (1996) Growth point culture in figs. Abstracts of the 1996 Horticultural Society Kinki Chapter Research Presentation: 4.
[Non-patent document 4]
Kumar V, Radha A, Kumar Chitta S (1998) In vitro plant regeneration offig (Ficus carica L. cv. Gumul) using optical bubbles frommatures. Plan Cell Reports 17: 717-720.
[Non-Patent Document 5]
Utsunomiya, N., Takano, H., Nakahara, K. (1996) Shoot formation from fig flower buds in vitro. Abstracts of the 1996 Horticultural Society Kinki Chapter Research Presentation: 2.
[Non-Patent Document 6]
Sengou Ai, Shinzaki Kanzaki, Yoshihiro Takamatsu, Naoki Utsunomiya (2000) Study on shoot formation from fig fruits under in vitro. Horticultural Society Magazine (separate volume 2) 69: 130.
[0005]
[Problems to be solved by the invention]
Accordingly, the present invention has been made in view of the above-described circumstances, and has been shown a novel novel adventitious root induction method and an adventitious root inducer capable of obtaining adventitious roots and adventitious shoots of figs, and an adventitious shoot induction method and an adventitious shoot inducer of figs. The purpose is to provide.
[0006]
[Means for Solving the Problems]
In order to achieve the above-mentioned object, as a result of intensive studies by the present inventors, adventitious roots can be induced by culturing fig-derived tissue pieces in the presence of phloroglucinol, and in the presence of phloroglucinol and cytokinin. It has been found that adventitious buds can be induced by culturing the tissue piece, and the present invention has been completed.
That is, the present invention includes the following.
[0007]
(1) A method for inducing adventitious roots of figs, comprising culturing a piece of tissue derived from a fig (Ficus carica L.) in the presence of phloroglucinol.
(2) The method for inducing adventitious roots of fig according to (1), wherein the concentration of phloroglucinol is in the range of 0.1 to 1.0 mM.
(3) An adventitious root inducer for figs, comprising phloroglucinol as an active ingredient.
[0008]
(4) A method for inducing adventitious buds of figs, comprising culturing a tissue piece derived from a fig (Ficus carica L.) in the presence of phloroglucinol and cytokinin.
(5) The adventitious bud of the fig according to (4), wherein the concentration of phloroglucinol is in the range of 0.1 to 1.0 mM and the concentration of cytokinin is in the range of 0.01 to 10.0 ppm. Guidance method.
[0009]
(6) The method for inducing adventitious buds of a fig according to (4) or (5), wherein the cytokinin is thidiazuron.
(7) An agent for inducing adventitious shoots of figs, comprising phloroglucinol and cytokinin as active ingredients.
(8) The agent for inducing adventitious buds of fig according to (7), wherein the cytokinin is thidiazuron.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
In order to induce the adventitious root of FIG., First, a tissue piece collected from FIG. Is cultured in the presence of phloroglucinol. On the other hand, in order to induce adventitious buds of the fig, first, a tissue piece collected from the fig is cultured in the presence of phloroglucinol and thidiazuron. That is, adventitious roots can be induced by culturing a tissue piece collected from FIG. In the presence of phloroglucinol, and adventitious buds can be induced by culturing the tissue piece in the presence of phloroglucinol and thidiazuron. it can.
The fig here has a scientific name of Ficus carica L. It may belong to any sub-class as long as it belongs to Ficus carica.
[0011]
As the tissue piece, for example, a leaf piece of a plant from the growing point culture of the fig to the subculture can be used. In addition, the tissue piece may be collected from any organ of the fig plant. For example, a tissue piece such as a leaf, petiole, seed embryo, fruit stalk, flower arrangement, flower, floral pattern, or root can be used. In addition, sterilized collected tissue pieces, aseptic seedlings, buds, roots, rhizomes, petiole or leaf sections of the fig plant that has grown the aseptic seedlings are preferably used. Among these, leaf tissue fragments are particularly preferred.
[0012]
On the other hand, as a medium for culturing tissue pieces, any medium can be used as long as it is a medium generally used for plant tissue culture. Specifically, as the medium, for example, an MS (Murashige & Skoog) medium, an LS (Linsmeier & Skoog) medium, a B5 medium, a Nitsch & Nitsch medium, a Gamborg medium, a White medium, a Tuleke medium, and a modified medium thereof can be used. .
[0013]
In particular, when inducing adventitious roots, a medium containing phloroglucinol is used. As phloroglucinol, commercially available products and synthetic products can be used. As commercial products, phloroglucinol dihydrate, phloroglucinol, and phloroglucinol (anhydrous) can be used. These commercial products can be purchased from Kanto Chemical Co., Ltd. and Wako Pure Chemical Industries, Ltd.
[0014]
In the case of inducing adventitious roots, the concentration of phloroglucinol is preferably in the range of 0.1 to 1.0 mM. When the concentration of phloroglucinol is less than 0.1 mM, there is a possibility that adventitious roots cannot be induced. Further, when the concentration of phloroglucinol exceeds 1.0 mM, there are disadvantages such as growth inhibition and death of tissue pieces.
[0015]
On the other hand, when inducing adventitious buds, a medium containing phloroglucinol and cytokinin is used. The cytokinin may be any of natural and synthetic cytokinins. Specific examples of cytokinins include thidiazuron (TDZ), furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), 2-chloro-4-pyridyl-phenylurea (CPPU), isopentenyladenine (2iP) and Zeatin can be mentioned, and it can be used alone or in combination of two or more. In particular, it is preferable to use thiadiazuron (TDZ) as cytokinin. TDZ has a formal name of 1-phenyl-3-1,2,3-thiadiazol-5-ylurea and can be purchased from, for example, Sigma and Wako Pure Chemical Industries.
[0016]
When inducing adventitious buds, the concentration of phloroglucinol is preferably in the range of 0.1 to 1.0 mM, and the concentration of thidiazuron is preferably in the range of 0.1 to 5.0 ppm. When the concentration of phloroglucinol is less than 0.1 mM, adventitious shoots may not be induced. Further, when the concentration of phloroglucinol exceeds 1.0 mM, there are disadvantages such as growth inhibition and death of tissue pieces. When the concentration of thidiazuron is less than 0.01 ppm, there is a possibility that adventitious buds cannot be induced. In addition, when the concentration of thidiazuron exceeds 10.0 ppm, there are disadvantages such as inhibition of growth and death of tissue pieces.
[0017]
In addition, the medium may contain auxins and cytokinins. Auxins include 2,4-dichlorophenoxyacetic acid (2,4-D), α-naphthylacetic acid (NAA), 4-chlorophenoxyacetic acid (4-CPA), indoleacetic acid (IAA), and indolebutyric acid (IBA) , 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), picloram and the like, which can be used alone or in combination of two or more. Examples of cytokinins include thidiazuron (TDZ), furfurylaminopurine (kinetin), 6-benzylaminopurine (BA), 2-chloro-4-pyridyl-phenylurea (CPPU), isopentenyladenine (2iP) and Zeatin and the like can be mentioned, and they can be used alone or in combination of two or more. A preferred range of the concentration of auxins is 0.1 to 50 ppm, and a preferred range of the concentration of cytokinins is 0.01 to 50 ppm.
[0018]
Furthermore, in addition to auxins and cytokinins, saccharides commonly used in tissue culture, such as sucrose, glucose, and fructose, can be used as a carbon source in the medium. A preferred concentration of the saccharide is 10 to 50 g / l. It is also effective to change the content of the constituent components of the medium, add or delete vitamins and amino acids, etc., as necessary. The medium may further contain gibberellins, abscisic acid, brassinolides, ethylene, and the like.
[0019]
In the case of inducing adventitious roots, the pH of the medium is preferably in the range of 5.0 to 6.5, the culture temperature is preferably in the range of 15 to 35 ° C, and the culture time is in the range of 20 to 60 days. Preferably, culture in the dark is preferred. In the case of inducing adventitious shoots, the pH of the medium is preferably in the range of 5.0 to 6.5, the culture temperature is preferably in the range of 15 to 35 ° C, and the culture time is 20 to 90 days. And culture in the dark is preferred.
[0020]
As described above, by using a medium containing phloroglucinol, it is possible to induce adventitious roots and adventitious buds using a piece of fig tissue. In addition, the adventitious shoots can be used to regenerate fig plants by applying a usual method. Specifically, for example, the obtained adventitious buds are transplanted and grown in a mixed medium of cytokinin and auxin. Thereafter, when the stem length becomes 1-2 cm, the cells are transplanted to an MS medium or an MS medium containing auxin. And after rooting, it can be regenerated into a fig plant by acclimatization and potting.
[0021]
【Example】
Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these Examples.
[Example 1] Induction of adventitious roots First, leaf pieces of a plant during subculture were collected from a growing point culture of a fig. The growth point culture of the fig was performed as follows. The shoot tip of the fig variety "Masui Dauphin" cultivated in the field was used. The leaf tip of “Masui Dauphin” with its leaves removed is immersed in an aqueous solution of sodium hypochlorite (diluted to an effective chlorine content of about 0.5%) from which a few drops of Tween 20 have been removed for about 15 minutes, and then sterilized by distillation. Washed three times with water. Under a stereoscopic microscope, a growth point portion of about 0.5 mm was cut out and inoculated in a growth point growth medium. As a growth point growth medium, an MS medium adjusted to pH 5.8 by adding 3% sucrose, 0.7% agar, and 1.0 ppm of 6-benzylaminopurine (BA) was used.
The subculture was performed under the following conditions. The plant expanded with the growing point growing medium was adjusted to pH 5.8 by adding 3% sucrose, 0.9% agar, 0.04 ppm indolebutyric acid (IBA) and 1.0 ppm BA. The cells were transplanted to MS medium and subcultured.
[0022]
Next, the collected leaf pieces were subjected to MS to which various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mM phloroglucinol (PG, manufactured by Wako Pure Chemical Industries, Ltd.) were added. Inoculated in medium (3% sucrose, 0.8% agar, pH 5.8). In this example, the concentration of 2,4-D was 0.0 (no addition), 0.1, 0.5, 1.0, or 5.0 ppm. In addition, in each test plot, 12 leaf pieces were used in a four-time repetition test.
After planting the leaf pieces, the cells were cultured in the dark at 25 ° C. for 30 days. Table 1 shows the rooting rate and rooting number observed in each test plot.
[0023]
[Table 1]

[0024]
From Table 1, it has been clarified that adventitious roots can be induced from leaf pieces even when a medium containing 2,4-D alone is used (see FIG. 1). Further, Table 1 also revealed that the use of a medium containing PG in addition to 2,4-D significantly increased the adventitious root generation rate. In particular, adventitious roots could be induced most efficiently with 1.0 ppm of 2,4-D and 0.5 mM of PG.
From the above results, it was found that PG has an effect of more effectively inducing adventitious roots of figs.
[0025]
[Example 2] Induction of adventitious buds Leaf pieces collected in the same manner as in Example 1 were subjected to MS medium (3%) supplemented with various concentrations of 2,4-D and thidiazuron (TDZ, manufactured by Sigma). Sucrose, 0.8% agar, pH 5.8, containing 0.5 mM PG). In this example, the concentration of 2,4-D was 0.0 (no addition), 0.5, 1.0, 2.0 or 5.0 ppm, and the concentration of TDZ was 0.0 (no addition). Addition), 0.5, 1.0, 2.0 or 5.0 ppm. In each test plot, three or more repetitive tests were performed using 10 to 12 leaf pieces.
After inoculating the leaf pieces, the cells were cultured in the dark at 25 ° C. for 49 days. Table 2 shows the germination rate and the number of germination observed in each test plot.
[0026]
[Table 2]

[0027]
From Table 2, it was clarified that when a medium containing TDZ alone was used in the presence of PG, no organogenesis was observed and adventitious buds could not be induced. In addition, Table 2 revealed that adventitious shoots can be induced when a medium containing 2,4-D and TDZ is used in the presence of PG (see FIG. 2). In particular, when a medium containing 1.0 ppm of TDZ and 1.0 ppm of 2,4-D in the presence of PG was used, it was revealed that adventitious shoots could be most effectively induced.
On the other hand, in order to examine the effect of PG on the induction of adventitious buds, leaf pieces were inoculated in a medium having a PG concentration of 0.0 (no addition), 0.5 or 1.0 mM, and cultured similarly. The concentration of TDZ contained in the medium used was set to 1.0 ppm, and the concentration of 2,4-D was set to 1.0 ppm. The culture was performed at 25 ° C. for 60 days in the dark. Table 3 shows the results.
[0028]
[Table 3]

[0029]
From Table 3, it was revealed that adventitious shoots were not induced in the PG-free group, whereas adventitious shoots were induced in the test group to which PG was added. Also, from Table 3, it was clarified that the test group in which the PG concentration was 0.5 mM exhibited a higher germination rate than the test group in which the PG concentration was 1.0 mM.
As described above, the results in Tables 2 and 3 show that a medium containing TDZ and PG is effective for inducing adventitious buds from figs.
[0030]
[Example 3] Plant regeneration from adventitious buds Using the adventitious buds obtained in Example 2, plants were regenerated as follows. Specifically, first, the obtained adventitious buds were transplanted and grown on an MS medium containing 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). At this time, the culture conditions were a temperature of 25 ± 2 ° C. and a photoperiod of 16 hours under a white fluorescent tube (55.6 μmol / s / m ² ).
[0031]
Thereafter, when the stem length became 1-2 cm, the cells were transplanted to an MS medium containing 1.0 ppm of indolebutyric acid (IBA) or an MS medium containing no plant hormone as a rooting medium. At this time, the culture conditions were a temperature of 25 ± 2 ° C. and a photoperiod of 16 hours under a white fluorescent tube (55.6 μmol / s / m ² ). Rooting could be confirmed 10 to 30 days after transplantation (see FIG. 3).
[0032]
Then, after rooting, it was able to be regenerated into a fig plant by acclimatization and potting (see FIG. 4). Concrete adaptation was performed as follows. After removing the agar from the roots, the fig plant was transplanted to a plastic pot containing vermiculite. The pot was placed in a sealable transparent plastic container and grown in a sealed state for about two weeks to maintain high humidity conditions. Thereafter, the upper lid was sequentially displaced while observing the growth condition of the fig plant to acclimate to the external humidity (relative humidity: 60 to 75%). The culture conditions at this time were a temperature of 25 ± 2 ° C. and a 16-day photoperiod of a white fluorescent tube (29.4 μmol / s / m ² ).
Moreover, the potting was specifically performed as follows. The fig plant, which had been sufficiently adjusted to the external humidity, was transplanted into a clay pot containing culture soil and grown in a greenhouse under natural light.
[0033]
【The invention's effect】
As described in detail above, the present invention can provide a completely novel method and an inducer capable of inducing adventitious roots and adventitious buds in figs. That is, according to the method for inducing adventitious roots of figs and the adventitious root inducer according to the present invention, it is possible to produce adventitious roots of figs which could not be obtained conventionally. Further, according to the method for inducing adventitious buds of a fig and the adventitious bud inducer according to the present invention, it is possible to produce an adventitious bud of figs which could not be obtained conventionally.
[Brief description of the drawings]
FIG. 1 is a photograph of an adventitious root created in Example 1.
FIG. 2 is a photograph of an adventitious bud produced in Example 2.
FIG. 3 is a photograph of an adventitious bud-derived plant in a rooting medium created in Example 3.
FIG. 4 is a photograph of an adventitious bud-derived plant after potting created in Example 3.

Claims (8)

A method for inducing adventitious roots of figs, comprising culturing a piece of tissue derived from a fig (Ficus carica L.) in the presence of phloroglucinol. 2. The method for inducing adventitious roots of fig according to claim 1, wherein the concentration of phloroglucinol is in the range of 0.1 to 1.0 mM. An adventitious root inducer for figs, comprising phloroglucinol as an active ingredient. A method for inducing adventitious buds of figs, comprising culturing a tissue piece derived from Fig (Ficus carica L.) in the presence of phloroglucinol and cytokinin. 5. The method for inducing adventitious shoots of fig according to claim 4, wherein the concentration of phloroglucinol is in the range of 0.1 to 1.0 mM and the concentration of cytokinin is in the range of 0.01 to 10.0 ppm. The method for inducing adventitious buds of a fig according to claim 4 or 5, wherein the cytokinin is thidiazuron. An agent for inducing adventitious buds of figs comprising phloroglucinol and cytokinin as active ingredients. The agent for inducing adventitious buds of fig according to claim 7, wherein the cytokinin is thidiazuron.

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