JP3611484B2 - How to save and replicate moth orchids - Google Patents

Description

表１に示すように、第二処理区のように除葉処理を行った結果、２９％の割合で娘シュートが側生した。一方、粗調整のみ行った第一処理区では娘シュートの側生が認められなかった。
【００４３】
この結果から、除葉／除根処理のみにより、娘シュートの側生を促進させることができることが示された。
【００４４】
［実施例２］ 誘導培地中に添加された植物ホルモンの濃度の影響
次に、実施例１の除葉処理したシュートを用い、植物ホルモンの含有濃度の異なる誘導培地で娘シュートの側生を試みた。
【００４５】
ここで用いたシュート誘導培地は、次の通り調整した。誘導培地は、Ｂ５培地に蔗糖１％、ココナツウオーター１０％、ベンジルアデニン（ＢＡ）ｌｐｐｍを添加して、寒天０．７％で固形化した。この培地を「培地ａ」とし、培地ａに植物ホルモンであるベンジルアデニン（ＢＡ）を５ｐｐｍ添加したものを「培地ｂ」とし、さらに、上記培地ａのベンジルアデニン（ＢＡ）を１０ｐｐｍ添加したものを「培地ｃ」として調整した。
【００４６】
各培地ａ〜ｃにそれぞれ除葉処理をした親シュートを置床し、２５℃で２０００ｌｕｘの１６時間照明下で培養し、２ヶ月後に調査を行った。その結果を表２に示した。
【００４７】
【表２】

表２に示したように、培地にＢＡの５ｐｐｍ添加区では最も高い６５％の割合で娘シュートの側生が観察された。
【００４８】
［実施例３］ 培養時の温度条件の影響
実施例１の除葉処理したシュートを用い、異なる温度条件で培養した際の娘シュートの側生率への影響を調べた。
【００４９】
シュート誘導培地は上記実施例２の結果に従って、Ｂ５培地に蔗糖１％、ココナツウオーター１０％、ベンジルアデニン（ＢＡ）５ｐｐｍを添加して寒天０．７％で固形化したものを調整した。
【００５０】
当該培地に除葉処理をしたシュートを置床し、２５℃、２８℃および３０℃で２０００ｌｕｘの１６時間照明下で培養し、２ヶ月後に調査を行った。その結果を表３に示した。
【００５１】
【表３】

表３に示したように、２８℃の高温培養では、最も高い７２％の割合で娘シュートの側生が観察された。一方、この温度より高い３０℃の条件では、葉が枯れるなどの高温障害が現れ、結果として娘シュートの側生率が低下した。
【００５２】
【発明の効果】
以上の通り、本発明によれば、親シュートが潜在的に有する腋芽を側生する能力を効果的に引き出させ、高効率で娘シュートを側生させることが可能となる。従って、本発明の方法は、遺伝的に保存された娘シュートを側生させることができるとともに、この方法により順次娘シュートを側生させることにより、親株の品種を長期に保存することが可能となる。
【図面の簡単な説明】
【図１】実施例１における未処理の親シュート（Ｉ）、粗処理の親シュート（ＩＩ）、除葉、除根処理された親シュート（ＩＩＩ）を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for preserving and replicating a moth orchid parent strain by shoot culture.
[0002]
[Prior art]
The moth orchid is one of the most popular horticultural plants among the orchids. Conventionally, as a method for long-term preservation and stable replication so as not to extinguish excellent strains obtained by breeding, firstly, plant hormonal agents such as BA (b-benzylaminopurine) are used for parental flower stem buds. And shoot (so-called high buds) to obtain shoots (so-called high shoots) (AT Tse, RJ Smith and WP Hackett. 1971. Adventitious short formation of Phalaenopsis nodes. Amer. Orchid Soc. Bull. 40: 807-810.).
[0003]
Further, as a second method, there is a method in which shoots are obtained by bactericidal treatment of flower stem buds and aseptic culture (DM Reisinger, EA Ball and J. Arditi. 1976. Clonal propagation of Phalaenopsis by means). of flow-stalk node cultures. The Orchid Review. 84: 45-52).
[0004]
Further, as a third method, there is a method of cultivating a sterile shoot node to obtain a shoot (JX Duan, H. Chen and S. Yamazawa. 1996. In vitro propagation of Pharaenopsis via culture of cytokinin-induced nodes. J. Plant Growth Reg. 15: 133-137.).
[0005]
[Problems to be solved by the invention]
However, in the first and second conventional methods, flower stems can be obtained only at the flowering time once a year, and moreover, only one shoot can usually be obtained from one bud obtained from the flower stem. Was a problem.
[0006]
In addition, in the third conventional method, even if a plurality of shoots are obtained, the survival rate of the nodes is low in each shoot, and a multi-shoot with low genetic conservation is caused by a high concentration of plant hormones. There is a problem that it is likely to occur.
[0007]
From these results, first, in the former method (the first and second conventional methods), the shoots obtained from the flower stem buds were originally used with a plurality of buds that could potentially be lateral shoots. It was thought that it was not. In the latter method, the obtained axillary buds were effectively used, but it was considered that the method of use was not appropriate for storage / replication of the parent strain.
[0008]
Therefore, an object of the present invention is to provide a method for replicating moth orchid that can store and replicate a parent strain more stably and aseptically in a long term in order to solve these problems.
[0009]
[Means for Solving the Problems]
In order to achieve the above object, the present invention provides a method for conserving and replicating the moth orchid by laterally generating a daughter shoot from a parent shoot obtained based on a flower stem of a parent strain moth orchid. A leaf removal / root removal step for removing leaves and roots from the base while leaving one or two leaves, and a culture step for cultivating the parent shoots after the leaf removal / root removal step in an induction medium to cause daughter shoots to grow laterally; , Is included.
[0010]
According to the above invention, in the leaf removal / root removal step, leaves and roots are removed from the base of the parent shoot obtained based on the flower stem of the parent strain moth orchid to expose the axillary bud. Growth can be promoted to shoots.
[0011]
That is, according to the present invention, it is possible to efficiently grow the potential buds of the parent shoots that have been difficult to grow. In addition, unlike the conventional one in which a high concentration of plant hormones is added and a plurality of shoots are unnaturally produced from the node fragments, in the present invention, one daughter shoot is basically grown from the buds of the parent shoot. Therefore, the daughter shoot can be replicated while preserving the genetic characteristics of the parent strain. Moreover, in this method, in the parent shoot, at least the upper one or two leaves are left and the leaves are removed to prevent the formation of multi-shoots with low genetic conservation.
[0012]
In the present specification, the term “parent shoot” means a shoot having the potential to produce a daughter shoot. Therefore, the `` parent shoot obtained based on the flower stalk of the parent strain moth orchid '' is a shoot that has been directly sprouting and grown from the flower stalk of the parent strain moth orchid, as long as it has the potential to side-produce daughter shoots, and This daughter shoot is also included because the daughter shoot obtained from the parent shoot can be a parent shoot . The “daughter shoot” means a shoot obtained by growing from the parent shoot.
[0013]
The present invention is characterized in that in the above-mentioned invention, 1 to 4 leaves are removed from a base part of a parent shoot in the leaf removal / root removal step. In this way, by removing 1 to 4 leaves from the base of the parent shoot, it is possible to appropriately expose the potential buds present in the leaf buds of each leaf and to help the growth.
[0014]
The present invention is characterized in that, in the above-mentioned invention, the induction medium contains a plant hormone at a concentration capable of promoting the lateral growth of daughter shoots.
[0015]
According to the above invention, the addition of plant hormones can help the growth of the buds exposed in the leaf removal / root removal step, and increase the growth efficiency on the daughter shoots. Thus, since the plant hormone added here is at a concentration that causes the axillary buds potentially possessed by the parent shoot to grow laterally, it is possible to maintain genetic preservability.
[0016]
More specifically, the concentration of plant hormone that can promote the lateralization of this daughter shoot is 1-15 ppm benzyladenine.
[0017]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, preferred embodiments of the present invention will be described in detail.
[0018]
There are no particular limitations on the varieties of moth orchids to which this method of preservation and replication of moth orchids can be applied. For example, Dtps. White Castle, Dtps. Quevedo, Dtps. Dortenopsis genus such as Zuma White Buttefly, Phal. Zuma Rascal, Phal. Zuma's Pixie, Phal. Bald Mountain, Phal. A Phalaenopsis genus such as Alfenso Morenon is included, and many other moth orchid varieties and original species can also be included.
[0019]
In this method, first, it is necessary to prepare a first parent shoot obtained based on the flower stem of the parent strain moth orchid.
[0020]
The first parent chute is preferably aseptic in order to prevent the invasion of diseases from the outside and to be able to replicate stably for a long time. Moreover, as long as it is prepared aseptically, the parent shoot obtained from the flowering stalk flower stem may be directly surface sterilized, but preferably, the flower stalk of the flowering moth orchid itself is cultured aseptically. It is a sterile parent chute obtained by Since the parent chute prepared by the latter can be used as an aseptic condition directly without performing surface sterilization as in the former, it is possible to prevent damage to the obtained parent chute. Can do.
[0021]
The parent shoot is subjected to leaf removal / root removal treatment in order to form buds that become daughter shoots from the first parent shoot. In this leaf removal treatment, if the leaves are removed so that the potential buds existing at the roots of the leaves can be exposed, the number of leaves to be removed can be appropriately adjusted. For example, when about 4 leaves are growing on the parent chute, about 1 to 3 leaves located sequentially below the base can be removed. However, when all the leaves of the parent shoot are removed, there is a tendency to form multishoots and PLBs with low genetic preservation, so it is preferable to leave at least one or two young leaves at the top.
[0022]
In addition, when removing the leaves, it is desirable to completely remove the leaves from the root of the leaves so that the buds can be exposed rather than removing only the leaf tips. Moreover, with this leaf removal treatment, the roots are removed from the parent shoot. These leaf removal / root removal processing operations are not particularly limited as long as the leaves and roots to be removed can be removed from the roots. For example, the following operations can be performed.
[0023]
First, the prepared parent chute is roughly adjusted, the root is excised with a sterilized scalpel, and a large leaf of about 3 cm or more, that is, about one-third of the leaf tip side 2 to 3 from the shoot base to be removed. Excised. The leaves to be removed are then completely excised from the root.
[0024]
The parent shoots after the above-mentioned leaf removal / root removal step are cultivated using an induction medium because of daughter shoot side effects.
[0025]
The induction medium that can be used in this culture step is not particularly limited as long as the daughter shoot can be laterally generated from the parent shoot. For example, a tissue culture leaf medium such as a 1 / 2MS medium, a B5 medium, or a WV medium is used. Can be used. A support is added to the induction medium, and a gelling agent such as gellan gum or agar can be used as the support. When agar is used as the support, it can be added so as to be 0.6 to 1.0%, preferably 0.7%.
[0026]
In addition, it is preferable to add sugars, natural organic substances, plant hormones and the like to the induction medium. Examples of the saccharide added to the induction medium include polysaccharides, monosaccharides, and sugar alcohols (for example, sucrose and sorbitol). For example, when sucrose is added to the induction medium, it can be added to a concentration of 0.5 to 3%, more preferably 1%. In addition, for other saccharides, the addition concentration can be set in consideration of the sucrose concentration.
[0027]
Examples of the natural organic material include coconut water, fresh banana, and fresh potato. When coconut water is used as this natural organic substance, it can be added so as to be 5 to 25%, preferably 10%. Even when other natural products are added, the addition amount can be appropriately determined in consideration of the concentration of the coconut water.
[0028]
Furthermore, examples of plant hormones added to the induction medium include benzyladenine. When the plant hormone is added to the induction medium, it is preferably added at a concentration that can promote the lateral growth of daughter shoots. For example, in the case of the benzyladenine, preferably 1 to 15 ppm. It can add so that it may become 5 ppm.
[0029]
The parent shoot is placed on the induction medium prepared as described above and cultured. When the parent shoot is placed on the induction medium, the base of the parent shoot that has undergone leaf removal treatment is brought into contact with the induction medium.
[0030]
In addition, the culture of the parent shoot after placement is performed in the range of 18 to 30 ° C, preferably 28 ° C. In this culture, the parent shoot is placed under illumination continuously for all day or about 16 hours per day. The illumination intensity of this illumination can be 500-5000 lux, preferably 2000-3000 lux. Under such conditions, a daughter shoot is normally produced by culturing the parent shoot for 30 to 90 days. However, the ability to form a laterally produced daughter shoot varies among varieties, so the above range. An appropriate culture period can be determined depending on the variety. If necessary, a culture period outside this range can be determined.
[0031]
The daughter shoots produced by the culture are cut out from the bonded portion of the parent shoot with a sterile scalpel, and the plant body is regenerated from the cut out daughter shoots. To regenerate the daughter shoots into plant bodies, transplant the daughter shoots into a normal method, for example, a normal seedling culture medium or aseptic seeding medium, and keep the brightness and breathability constant. It can be grown on the body.
[0032]
As described above, in addition to the parent shoot, one or more genetically preserved daughter shoots can be replicated in addition to the parent shoot, and these can be grown on plants. The daughter shoots obtained here also have the ability to become parent shoots and potentially cause side buds to grow laterally.
[0033]
In order to bring out the ability of this daughter shoot to laterally grow the axillary buds, after culturing and growing for 2 to 3 months as described above, this is used as a parent shoot for the above-described series of steps (leaf removal / root removal). Steps and culture steps using induction medium) are repeated.
[0034]
In detail, basic culture media, such as MS culture medium, B5 culture medium, and V & W culture medium, can be used for the seedling culture medium which transplants the cut daughter shoot, for example. In addition, the medium contains, for example, fresh banana 50-200 g / l, preferably 100 g / l, sucrose 0.5-3%, preferably 1%, activated carbon 0.55-2%, preferably 0.1%. It is good to add. Moreover, it is preferable to add agar as a support for this medium to 0.5 to 0.8%, preferably 0.6%, and to solidify the medium.
[0035]
Moreover, as seedling raising conditions, the temperature is maintained at 20 to 28 ° C., preferably 23 to 26 ° C., and illumination is continued for 8 to 24 hours, preferably 16 hours. The illumination intensity of this illumination can be 200 to 5000 lux, preferably 2000 to 3000 lux.
[0036]
The daughter shoot grown under such conditions grows up to a parent shoot that causes the next daughter shoot to grow laterally. By growing daughter shoots in this way and using them as parent shoots in order, daughter shoots are sequentially generated, thereby making it possible to obtain a large number of seedlings that inherit the characteristics of the parent strain moth orchid. At the same time, the parent strain as a variety can be passaged one after another, and the variety can be preserved for a long time.
[0037]
【Example】
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
[0038]
[Example 1] Influence on side production rate of daughter shoot in leaf removal treatment In this example, a kind of moth orchid, Dtps. A parent shoot having aseptic 4 to 5 leaves derived from seedlings obtained by culturing the flower stem buds of Quevedo was used. The method for preparing and cultivating flower buds is Tanaka method (M. TANAKA, 1987. STUDIES ON THE CLOONAL PROPAGATION OF PHALAENOPSSIS THROUGH IN VITRO CULTURE. MEMORS OF FACULT. It was.
[0039]
As shown in FIG. 1 as a test plot, a rough adjustment treatment plot (II) in which the untreated parent shoot (I) is roughly adjusted, and three to four leaves are sequentially cut from the base after the coarse adjustment. In addition, a defoliation treatment section (III) in which one or two young leaves at the top were left was provided.
[0040]
As a medium for shoot culture, 1% sucrose, 10% coconut water, and 1 ppm benzyladenine (BA) were respectively added to B5 medium and solidified with agar 0.7%.
[0041]
The parent shoot of each treatment group was placed on the above medium, cultured at 25 ° C. under a certification of 2000 lux for 16 hours, and the lateral viability was examined after 2 months. The results are shown in Table 1.
[0042]
[Table 1]

As shown in Table 1, as a result of the leaf removal treatment as in the second treatment section, daughter shoots were laterally produced at a rate of 29%. On the other hand, side effects of daughter shoots were not observed in the first treatment area where only rough adjustment was performed.
[0043]
From this result, it was shown that the side growth of daughter shoots can be promoted only by the leaf removal / root removal treatment.
[0044]
[Example 2] Effect of the concentration of plant hormones added to the induction medium Next, the shoots subjected to the defoliation treatment of Example 1 were used, and the daughter shoots were laterally grown on the induction medium having different concentrations of plant hormones. It was.
[0045]
The shoot induction medium used here was prepared as follows. As the induction medium, 1% sucrose, 10% coconut water and 1 ppm benzyladenine (BA) were added to B5 medium, and solidified at 0.7% agar. This medium is referred to as “medium a”, the medium a added with 5 ppm of plant hormone benzyladenine (BA) is referred to as “medium b”, and the medium a is further added with 10 ppm of benzyladenine (BA). Prepared as “medium c”.
[0046]
The parent shoots that had undergone defoliation treatment were placed on each of the media a to c, cultured at 25 ° C. under 2000 lux for 16 hours, and examined after 2 months. The results are shown in Table 2.
[0047]
[Table 2]

As shown in Table 2, the side growth of daughter shoots was observed at the highest rate of 65% in the group where 5 ppm of BA was added to the medium.
[0048]
[Example 3] Effect of temperature conditions during culture Using the shoots subjected to leaf removal in Example 1, the influence on the lateral growth rate of daughter shoots when cultured under different temperature conditions was examined.
[0049]
The shoot induction medium was prepared by adding 1% sucrose, 10% coconut water, 5 ppm benzyladenine (BA) to B5 medium and solidifying with 0.7% agar according to the results of Example 2 above.
[0050]
The shoots subjected to leaf removal treatment were placed on the medium, cultured at 25 ° C., 28 ° C. and 30 ° C. under 2000 lux for 16 hours, and examined after 2 months. The results are shown in Table 3.
[0051]
[Table 3]

As shown in Table 3, in the high-temperature culture at 28 ° C., side shoots of daughter shoots were observed at the highest rate of 72%. On the other hand, under the condition of 30 ° C., which is higher than this temperature, a high temperature disorder such as leaf dying appeared, and as a result, the lateral rate of daughter shoots decreased.
[0052]
【The invention's effect】
As described above, according to the present invention, it is possible to effectively draw out the ability of the parent shoot to side-produce the axillary bud, and to efficiently grow the daughter shoot side by side. Therefore, the method of the present invention can laterally grow genetically preserved daughter shoots, and can sequentially preserve the cultivar of the parent strain for a long time by laterally growing daughter shoots by this method. Become.
[Brief description of the drawings]
FIG. 1 is a diagram showing an untreated parent shoot (I), a rough treated parent shoot (II), and a parent shoot (III) subjected to leaf removal and root removal treatment in Example 1. FIG.

Claims (5)

A method for preserving and replicating the moth orchid by laterally generating a daughter shoot from a parent shoot obtained based on the flower stem of the parent strain moth orchid,
Leave-off / root-removing step for removing leaves and roots from the base, leaving at least one or two top leaves in the parent shoot;
A method for preserving and replicating moth orchids, comprising: culturing a parent shoot after the defoliation / root removal treatment step in an induction medium, and causing a daughter shoot to grow laterally. The method according to claim 1, wherein 1 to 4 leaves are removed from the base of the parent shoot in the leaf removal / root removal step. 3. The method for preserving and replicating moth orchids according to claim 1 or 2, wherein the induction medium contains a plant hormone at a concentration capable of promoting side production of daughter shoots. 4. The method for conserving and replicating moth orchids according to claim 3, wherein the plant hormone at a concentration capable of promoting the side production of the daughter shoots is 1 to 15 ppm of benzyladenine. The method for preserving and replicating moth orchids according to any one of claims 1 to 4, wherein in the culturing step, the parent shoot is cultured at 20 to 30 ° C.

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