JPH05236835A - First generation culturing method for auxiliary bud of phalaenopsid - Google Patents

Abstract

PURPOSE:To improve the efficiency in the formation of a protocorm-like body(PLB) by checking and reducing the dormancy and death of an auxiliary bud in a stage of the most important initial culture in a culture process in forming the PLB for inducing a clonal seedling derived from a scape auxiliary bud of a phalaenopsid. CONSTITUTION:A basal solid culture medium is prepared by using 3g/l hyponex, 20g/l sucrose, 8g/l agar and 2g/l peptone as a composition and keeping the pH at 5.0-5.1 before sterilization. A combination of naphthaleneacetic acid (NAA) with zeatin as an additive is then added to the basal culture medium to prepare a culture medium. An auxiliary bud collected from a scape of a phalaenopsid is bedded in the prepared culture medium and cultured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a primary culture method for Phalaenopsis axillary buds, and more specifically, PLB for inducing cloned seedlings (mericlone seedlings) derived from flower stalk axillary buds of Phalaenopsis.
In the formation of (protocorm-like body), dormancy and death of axillary buds can be reduced as much as possible at the stage of initial culture, which is the most important stage in the culture process, and thus the formation of PLB can efficiently promote the formation of PLB. It relates to the primary culture method.

[0002]

2. Description of the Related Art The development of the Western orchid industry as it is today is due to the practical application of clone seedlings (Mericron seedlings) by biotechnology.
Among the western orchids, Phalaenopsis has been growing in demand and demand in recent years, but the production of cloned seedlings, which has long been desired, has not yet been put to practical use. The reason for this is that because Phalaenopsis is a monostem plant, when the shoot apex is removed, the mother strain disappears, and thus it is not possible to divide and reproduce. Also, the shoot apex of Phalaenopsis turns black during culture and easily dies. Is mentioned.

Therefore, in order to produce cloned seedlings of Phalaenopsis, tissues other than the shoot apex are used as explants. The main method is to cultivate flower stems with axillary buds and A method of collecting leaf pieces from shoots obtained by development and forming PLB by culturing the same; collecting only axillary buds of flower stems
Although a method of forming PLB, a method of forming PLB directly from a flower stem slice, and the like have been proposed, the present situation is that they have not yet been put to practical use.

[0004]

SUMMARY OF THE INVENTION In view of the above situation, the present invention collects only flower axillary buds as an explant in forming PLB for inducing clone seedlings derived from flower stalk axillary buds of Phalaenopsis. (EN) A primary culture method for Phalaenopsis axillary buds, which employs a method of culturing but can effectively promote the formation of PLB by reducing dormancy and death of axillary buds at the stage of initial culture. This is the purpose of the task.

[0005]

In order to solve the above problems, the first technical means taken by the present invention is to compose 3 g / l of Hyponex, 20 g / l of sucrose, 8 g / l of agar and 2 g / l of peptone. Then, a basic solid medium is prepared by maintaining pH 5.0-5.1 before sterilization, and (a) 6-benzylaminopurine (BAP) 1-5 mg as an additive to this basic medium.
/ L, (b) N6- (Δ ² isopentenyl) adenamine hemihydrate (2-ip) 1-5 mg / l, (c) a combination of naphthalene acetic acid (NAA) and zeatin, Is added to prepare a medium, and axillary buds collected from flower stalks of Phalaenopsis are placed on the prepared medium and cultured. The second technical means taken by the present invention is Kano containing peptone.
Medium (Hyponex 3,000 mg / l, sucrose 2
50,000mg / l, agar 8,000 ~ 10,000m
(g / l composition medium), a methanol extract collected from a Phalaenopsis flower stem piece is added to prepare a medium,
An axillary bud collected from the flower stalk of Phalaenopsis is placed on the prepared medium and cultured.
Furthermore, the third technical means taken by the present invention is to add the protein hydrolyzate obtained from legume grain to Kano medium to prepare the above medium, and to the prepared medium, axillary buds collected from flower stalks of Phalaenopsis Is placed and cultured.

[0006]

Therefore, according to the present invention, since only the axillary buds of the Phalaenopsis flower stalk are cultivated, contamination (propagation of various bacteria in the incubator) is suppressed as much as possible, and the axillary buds at the stage of initial culture are acted by the action of the prepared medium. Dormancy and mortality are reduced, and the formation of PLB is efficiently promoted.

[0007]

EXAMPLES Examples of the present invention will be described in detail below. Axillary buds wrapped in bracts are present in several nodes at the base of the flower stalk of Phalaenopsis, and in order to efficiently induce the formation of PLB by culturing the axillary buds, dormancy and death of axillary buds at the stage of initial culture. It is an indispensable condition to reduce as much as possible. The present invention was devised to satisfy these conditions, and is characterized by culturing by the following method.

[First Invention] First, a basic medium for axillary bud culture is prepared. That is, the basic medium is 3 g of Hyponex
/ L, sucrose 20g / l, agar 8g / l, peptone 2g
The basic solid medium is prepared by keeping the pH of 5.0 to 5.1 before sterilization with the composition of 1 / l. Next, 1 to 5 mg of (a) 6-benzylaminopurine (BAP) as an additive to this basal medium
/ L, (b) N6- (Δ ² isopentenyl) adenamine hemihydrate (2-ip) 1-5 mg / l, (c) a combination of naphthalene acetic acid (NAA) and zeatin, Was added to prepare a medium, axillary buds collected from flower stalks of Phalaenopsis were placed on the prepared medium, and the culture conditions were 25 ° C. constant temperature, 12 hours continuous day length,
After culturing at 2,000 lux, the following experimental results were obtained.

[Table 1]

[Table 2]

[Table 3]

[Table 4]

The above experimental results show the survival rate after 16 days of culturing. As is clear from this, when culturing in a medium prepared by adding additives to the basal medium, A high survival rate was obtained as compared with the case of culturing only in the medium (without addition), and particularly when a combination of NAA and zeatin was added, a high survival rate was obtained in an extremely stable state. From this, according to the culture method of the first invention, it was confirmed that dormancy and death of axillary buds were prevented at the stage of initial culture, and then 2 to
After culturing for 3 months, PLB was formed from axillary buds.

[Second Invention] Kano with peptone added
Medium (Hyponex 3,000 mg / l, sucrose 2
50,000mg / l, agar 8,000 ~ 10,000m
(g / l composition medium), a methanol extract collected from a Phalaenopsis flower stem piece is added to prepare a medium,
Axillary buds collected from flower stalks of Phalaenopsis are placed in this conditioned medium, and the culture conditions are 2,000 lux at a constant temperature of 25 ° C. for 12 hours. In this case, Ka
The flower stem piece methanol extract to be added to the no medium is collected by the following procedure. Cut the flower stalk of Phalaenopsis into several pieces with a length of 5 to 10 mm. 10 g of the cut flower stem pieces are weighed and placed in a mortar. The above flower stem pieces are stored in a freezer for one day. Prepare 150 ml of 80% methanol. Place frozen flower stem pieces and 75 ml of methanol in a mortar. After crushing the flower stem pieces, store in a cool dark place for 5 to 6 hours. The liquid in the mortar was filtered with gauze to collect the filtrate, and the residue was put in the mortar again, and the remaining amount of methanol prepared in
Add ml. Grind the flower stem pieces. The liquid in the mortar is filtered through gauze, the filtrate is added to the filtrate collected in, 150 ml of the above filtrate is filtered, and then the filtrate is concentrated. Next, to culture axillary buds, 2 g of peptone was added to Kano medium.
The culture medium is prepared by adding 5 to 10% of the concentrated liquid collected by the above-mentioned procedure to the culture medium containing 1 / l, and axillary buds are placed on the prepared culture medium. As a result of the experiment, at the stage of initial culture, dormancy and death of axillary buds were reduced.
Viability of ~ 100% could be obtained. And, it was presumed that such good results were obtained, in combination with culturing only axillary buds as explants, and that the medium had the effect of suppressing the occurrence of contamination as much as possible. ..

[Third invention] A protein hydrolyzate obtained from legume seeds is added to Kano medium to prepare the above medium, and axillary buds collected from flower stalks of Phalaenopsis are placed on the prepared medium, and culture conditions are set. Incubation is carried out at 2,000 lux at a constant temperature of 25 ° C. for 12 hours. In this case, the protein hydrolyzate in pea grains to be added to Kano medium is collected by the following procedure. Bean seeds 10 g / phosphoric acid 2 in phosphate buffer
Grind the legume under conditions of 0-300 ml. Filter and collect the filtrate. The filtrate is centrifuged under the conditions of 12,000 rpm and 15 min, and the supernatant is collected. An appropriate amount of (NH ₄ ) SO ₂ is added to the supernatant so that the supernatant becomes a 80% solution, and salting out is performed. The salted-out solution is centrifuged under the conditions of 15,000 rpm and 15 min. The precipitate is collected and dissolved in pure water to give an aqueous solution. 4% H ₂ SO ₄ is added to the aqueous solution in a volume ratio of 1: 1. The mixed solution is heated at 80 ° C. for 30 minutes and then cooled. Next, in order to culture axillary buds, the mixed solution collected by the above procedure is added to Kano medium to prepare a medium, and the axillary buds are placed on the prepared medium and cultured. results of the experiment,
At the stage of initial culture, dormancy and death of axillary buds were reduced, and a survival rate of 70% to 100% could be obtained. The reason why such favorable results were obtained is presumed to be that the presence of amino acids in the culture medium promoted the growth of plants (axillary buds). In addition, in the above-mentioned Examples, legumes containing a large amount of protein were used as the amino acid source, but it seems that the amino acid source does not need to be particularly limited.

As described above, when axillary buds of Phalaenopsis are cultivated by the culturing method according to the first to third inventions, a high survival rate is secured at the stage of initial culture, but the culturing is continued for 2 to 3 months. Then PLB from axillary bud
Is formed. However, a PLB that induces a cloned seedling (young plant) can usually be obtained from only one individual young plant from one PLB. Therefore, in order to obtain a large amount of seedlings, it is necessary to grow them at the PLB stage. Therefore,
When the formed PLBs were divided and placed in a sterile seeding medium and cultured, the PLBs were separated from the individual PLBs after 60 days had elapsed.
When the agglomerates of B are formed, and the agglomerates of PLB are further divided and the culture is continued, a clone seedling (young plant) is obtained from each PLB after 2 months.

In this case, when the clone seedlings are obtained,
It was confirmed that the growth of clone seedlings was promoted when CO ₂ was fertilized in the clone seedlings in the dark period under the condition of the light-dark period photoperiod of 12 hours each. Figure 1, there is shown an example of an instrument that is used to fertilization of CO ₂ in the clones seedlings were plated on sterile seed medium, the instrument CO ₂ and air and a higher mixing concentration CO ₂ Mixer 1 for producing gas,
A communicating pipe 3 provided with a filter 2 for filtering the above-mentioned gas to remove microorganisms, a flask 4 in which sterile water is stored so as to give steam to the filtered gas to make it wet, and a wet gas from the flask 4 Is connected to the sterile seeding medium 6 in the culture chamber 5.

By the way, the cloned seedlings cultivated as described above are acclimatized and gradually switched to the conditions outside the incubator, that is, the conditions such as humidity, temperature and light intensity. Water moss was used as a medium for acclimation. However, when water moss is used as the medium, the water retention and drainage properties are likely to differ because the density of the moss varies depending on the pot, and this has the drawback that irrigation management cannot be automated. Therefore, in the present invention, a clonal seedling is spread on the laid non-woven fabric, irrigated with a mist, and an acclimation method of controlling temperature and humidity with an air conditioner, a boiler, etc. is adopted, so that irrigation can be automated and irrigation management can be facilitated. became.

[0015]

As is apparent from the above description, according to the present invention, in forming PLB that induces clone seedlings derived from flower stalk axillary buds of Phalaenopsis, the axillary buds of axillary buds at the stage of initial culture, which is the most important in the culture process, are formed. Dormancy,
PLB formation can be efficiently promoted by suppressing or reducing mortality as much as possible, and PLB by subsequent subculture
, The mass production of cloned seedlings, and further contribute to the establishment of nutritional varieties of Phalaenopsis, which was previously impossible.

[Brief description of drawings]

[Fig. 1] Schematic configuration of CO ₂ fertilizer application to clone seedlings

[Explanation of Codes] 1 Mixer 2 Filter 3 Communication tube 4 Flask 5 Culture room 6 Sterile seeding medium 7 Communication tube

Claims (3)

[Claims] 1. Hyponex 3 g / l, sucrose 20 g
/ L, agar 8 g / l, peptone 2 g / l, pH 5.0 to 5.1 before sterilization is maintained to prepare a basic solid medium, and (a) 6-benzylamino is added to this basic medium as an additive. Pudding (BAP) 1-5 mg
/ L, (ii) N6-(delta ² isopentenyl) Adenominhemi hydrate (2-ip) 1~5mg / l , ( c) a combination of a zeatin naphthalene acetic acid (NAA), 1 kind from among Is added to prepare a medium, and axillary buds collected from flower stalks of Phalaenopsis are placed on the prepared medium and cultured. 2. Kano medium supplemented with peptone (hyponex 3,000 mg / l, sucrose 20,000 m)
g / l, agar having a composition of 8,000 to 10,000 mg / l), a methanol extract collected from a piece of Phalaenopsis flower stem was added to prepare a medium, and the prepared medium was collected from the flower stem of Phalaenopsis. 1. A primary culture method for Phalaenopsis axillary buds, which comprises culturing the axillary buds placed on the bed. 3. A protein hydrolyzate obtained from legume seeds is added to Kano medium to prepare the above medium, and axillary buds collected from flower stalks of Phalaenopsis are placed on the prepared medium and cultured. Characteristic Phalaenopsis axillary bud primary culture method.