WO2015101009A1

WO2015101009A1 - Inducing medium for butterfly orchid and method for improving survival rate of butterfly orchid pedicel

Info

Publication number: WO2015101009A1
Application number: PCT/CN2014/082047
Authority: WO
Inventors: 陈永得
Original assignee: 佛山市顺德区今日景艺生物科技有限公司
Priority date: 2013-12-30
Filing date: 2014-07-11
Publication date: 2015-07-09
Also published as: CN103688865B; CN103688865A

Abstract

The present invention provides an inducing medium for butterfly orchid and a method for improving a survival rate of a butterfly orchid pedicel. The inducing medium is prepared by adding 7-9 g/L of agar, 15-30 g/L of white granulated sugar, 1.5-3 mg/L of 6-benzylaminopurine, 0.2-0.5 mg/L of naphthylacetic acid, 0-50 mg/L of carbenicillin, 0-100 g/L of coconut milk and 1-3 g/L of activated carbon to a 1/2 MS culture medium. The inducing medium can be used for inducing the butterfly orchid pedicel. The method for improving the survival rate of the butterfly orchid pedicel has low sterilizing contamination rate, and can effectively improve the survival rate of the sterilized explant.

Description

Phalaenopsis induction medium and method for improving the survival rate of peduncle

The invention belongs to the technical field of plant tissue culture and seedling culture, and particularly relates to a method for effectively improving the survival rate and the induction effect of the butterfly orchid stem when the butterfly orchid stem is used as the explant for primary culture.

BACKGROUND OF THE INVENTION Phalaenopsis Bl. spp. is a tropical aerial orchid with large flowers, long flowering period, beautiful flowers, rich color, beautiful flower shape, such as butterfly dancing, hence the name. Among the tropical orchids, it is known as the "Queen of Orchids" and is one of the most popular orchids in recent years.

Phalaenopsis is a monosexual aerial orchid. Plants rarely develop lateral buds, and seeds are extremely difficult to germinate. They are routinely propagated and the proliferation rate is very slow. Therefore, tissue culture is used to rapidly propagate them. Explant disinfection is the first step in tissue rapid propagation. The thorough disinfection and the degree of damage to the explants determine whether the in vitro culture system can be successfully established. This is also a factor limiting the further application of tissue culture and rapid propagation techniques. one. At present, in the process of inducing the cultivation of phalaenopsis sinensis by using peduncle bud as an explant, it is often incompletely sterilized during the initial induction culture, and the damage to the explants during sterilization is large, resulting in low survival rate of stalks. Carbenicillin is an antibiotic. It is currently used in medicine. It is mainly used for inflammation caused by Pseudomonas aeruginosa and some Proteus and Escherichia coli. It is rarely found in tissue culture applications.

Summary of the invention

The object of the present invention is to provide an induction medium which can be used as a butterfly orchid stem inducing medium; the present invention also provides a method for improving the survival rate of a butterfly orchid stem, which has low cost The sterilization contamination rate is low, which can effectively improve the survival rate of explants after sterilization.

In order to achieve the object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides an induction medium for adding agar, white sugar, 6-benzylamino gland to 1/2 MS medium. The concentration of the following components in the induction medium is as follows: agar 7~9g/L, white sugar 15~30g/L, carbenicillin 0. ~50 mg/L, 6-benzylaminoadenine 1.5~3 mg/L, naphthaleneacetic acid 0.2~0.5mg/L, coconut juice 0~100 g/L, activated carbon l~3g/L.

The concentrations of the following components in the induction medium were: carbenicillin 40~50 mg/L, 6-benzylaminoadenine

2.5~3mg/L, naphthaleneacetic acid 0.4~0.5mg/L, coconut juice 80~100g/L.

The concentrations of the following components in the induction medium were: carbenicillin 50 mg/L, 6-benzylaminoadenine 3 mg/L, naphthaleneacetic acid 0.5 mg/L, and coconut juice 100 g/L. In a second aspect, the present invention provides a method for improving the survival rate of a butterfly orchid stem, comprising the following steps:

1) Cut the mature peduncle on the robust mother plant of Phalaenopsis into a segment with a bud as an explant, soak it in

200mg / L carbenicillin solution, soaked for 30min, then taken out to dry; 2) The external implants were routinely sterilized on a sterile and clean bench, and then inoculated into the induction medium for about 14 days, and the axillary buds sprouted and expanded.

The induction medium is prepared by adding agar, white granulated sugar, 6-benzylaminoadenine, naphthaleneacetic acid, carbenicillin, coconut juice and activated carbon to 1/2 MS medium. The concentrations of the following components in the induction medium were: agar 7~9g/L, white sugar 15~30g/L, carbenicillin 0~50 mg/L, 6-benzylaminoadenine 1.5~3 mg/ L, naphthaleneacetic acid 0.2~0.5mg/L, coconut juice 0~100g/L, activated carbon l~3g/L.

Preferably, the concentrations of the following components in the induction medium are: carbenicillin 40-50 mg/L, 6-benzylaminoadenine 2.5-3 mg/L, naphthaleneacetic acid 0.4-0.5 mg/L, coconut juice 80~100 g/L. The pedicel was induced and cultured by the preferred medium, and the contamination rate was as low as 5.11%, and the germination rate was as high as 94.13%, and the germination time was short, only 14 to 16 days, and the comprehensive score was high.

Further preferably, the concentrations of the following components in the induction medium are: carbenicillin 50 mg/L, 6-benzylaminoadenine 3 mg/L, naphthaleneacetic acid 0.5 mg/L, coconut juice 100 g/L . With this further preferred induction medium, the induction of the culture is optimal.

In step 2, before inoculation, the incision that has been contacted with the disinfectant at both ends of the peduncle is cut off with a sterilized scalpel, and then inoculated into the medium, and each stem is inoculated with 5 stem segments.

Preferably, in the step 2, the culture conditions are as follows: temperature 25 ± 1 ° C, light intensity 1500 ~ 18001 x, light and dark alternate 12 h / 12 h, that is, 12 hours of light and 12 hours of no light.

The conventional disinfection means soaking for 30 s with 75% alcohol and then soaking for 15 min with 0.1% liters of mercury.

The technical solution of the present invention has the following beneficial effects:

The invention firstly uses the 200mg/L carbenicillin solution to soak the peduncle in the sterilization process of the butterfly orchid stem, thereby killing the bacteria on the surface of the peduncle, reducing the pollution rate, and less damage to the explant, which helps to improve The survival rate after stalk sterilization.

The invention further adds carbenicillin to the induction medium, and can further significantly inhibit the endophytic bacteria of the explants during the cultivation process, thereby reducing the pollution in the tissue culture process, and at the same time, the growth of the peduncle is not obvious. The inhibitory effect, but also promotes the germination of axillary buds and shortens the germination time.

The invention further adds coconut juice to the induction medium, which can effectively improve the robustness of the bud of the Phalaenopsis, and the coconut juice is rich in active substances, and has obvious promoting effects on the germination and bud development of the axillary bud.

Adding 0.3% activated carbon to the induction medium of the present invention can effectively inhibit the browning phenomenon and increase the survival rate of the pedicel. The use of white granulated sugar as the carbon source in the induction medium can effectively reduce the cost without affecting the survival rate and germination rate of the pedicel.

The method of the invention sterilizes and cultures the explants, and has the characteristics of low cost, low sterilization pollution rate, high survival rate of explants, and rapid sprouting of axillary buds, and the pollution rate can be reduced to 4.25%. The method of using the carbenicillin to soak the peduncle and adding the medium can effectively improve the survival rate of the phalaenopsis stalk after sterilization, reduce the damage of the bactericide to the explant, and promote the germination of the axillary bud and shorten the germination time.

detailed description

The technical solution of the present invention will be further described below in conjunction with the embodiments.

Example 1~7

1) Preparation of the induction medium used in Examples 1 to 7

The induction medium of Examples 1 to 7 was prepared by adding agar, white granulated sugar, activated carbon, coconut milk, carbenicillin, 6-benzylaminoadenine, naphthalene acetic acid to 1/2 MS medium, agar, white granulated sugar. The concentrations of activated carbon, coconut milk, carbenicillin, 6-benzylaminoadenine, and naphthaleneacetic acid in the induction medium of Examples 1-7 are shown in Table 1. The induction medium Nos. 1 to 7 in Table 1 correspond to the induction medium of Examples 1 to 7, respectively. After the induction medium of Examples 1 to 7 was placed, they were separately placed in bottles 1 to 7. The 1/2 MS medium used, the formulation table of which is shown in Table 2 below, the amount described in Table 2 refers to the amount used in the preparation of 1 liter of 1/2 MS medium, and the amount of a large amount of elements in 1/2 MS medium. It is half the amount of a large amount of elements in standard MS medium.

Table 1 concentration of each component of the induction medium

Table 2 1/2 MS medium composition and its dosage (the amount refers to the amount in 1 liter 1/2 MS medium) Type of ingredients and dosage type Component potassium nitrate KN0 ₃ 950mg Manganese sulfate tetrahydrate MnS0 ₄ 4H ₂ 0 22.3 Mg large amount of trace

Ammonium nitrate NH ₄ N0 ₃ 825mg Zinc sulphate heptahydrate ZnS0 ₄ -7H ₂ 0 8.6mg Elemental element

Calcium Chloride Dihydrate CaCl 2H ₂ 0 220mg Boric Acid H ₂ B0 ₃ 6.2mg Magnesium sulfate heptahydrate

185mg potassium iodide KI 0.83mg

MgS0 ₄ -7H ₂ 0

Sodium molybdate dihydrate

Potassium dihydrogen phosphate KH ₂ P0 ₄ 85mg 0.25mg

NaMo0 ₄ -2H ₂ 0

Inositol lOOmg Copper sulfate pentahydrate CuS0 ₄ .5H ₂ 0 0.025mg Glycine 2mg Cobalt chloride CoCl 6H ₂ 0 0.025mg Organic

Niacin 0.5mg

Ingredient Na ₂ · EDTA 37.3mg Vitamin B ₆ 0.5mg Iron salt Vitamin O.lmg Iron sulfate heptahydrate FeS0 ₄ -7H ₂ 0 27.8mg When preparing the induction medium, a) Prepare 1/2MS medium according to Table 2 in advance. As a base mother liquor, then add 6-benzylaminoadenine, naphthaleneacetic acid, coconut juice, activated carbon to the base mother liquor, mix and reserve; b) pour the agar into boiling tap water, then pour it into white After the sugar is dissolved, add the agar-white sugar solution to the base mother liquor and dilute to 1L, and sterilize it with spare; c) After disinfecting the antibiotic carbenicillin vial with 75% alcohol, use a disposable medical syringe to the vial. Injecting sterile water into caropenicillin mother liquor, filtering and sterilizing with filter sterilizer for use; d) After sterilizing the medium, the temperature of the medium is cooled to 40~50 °C, in sterile super On the net workbench, the caropenicillin mother liquor after filtration and sterilization in c was added to the culture medium, and mixed to prepare an induction medium. The amounts of agar, white granulated sugar, activated carbon, coconut milk, carbenicillin, 6-benzylaminoadenine, and naphthaleneacetic acid in each of the induction media of Examples 1 to 7 are shown in Table 1. In the preparation of the induction medium, in addition to the preparation of various nutrient mother liquors using distilled water, other components can be prepared by replacing tap water with tap water, which indirectly simplifies the tissue culture procedure and cost.

2) stalk disinfection

Cut the mature peduncle of Phalaenopsis into segments with one bud, leave the length of 1.5~2cm on the upper and lower sides of the bud, soak it in 200mg/L carbenicillin solution, take it out after drying for 30min; then put it in sterile and clean work. On the stage, soak for 30s with 75% alcohol, wash 3 times with sterile water, then soak for 15 min with 0.1% liters of mercury, and wash 5 times with sterile water.

3) Inoculation and culture of peduncle

Inoculation was carried out in a sterile ultra-clean workbench, and the incision in contact with the disinfectant at both ends of the peduncle was cut out with a sterilized scalpel, and inoculated into each of the induction mediums of Examples 1 to 7, each of which was inoculated with 5 bud segments. After inoculation, seal with a sterilized stopper. After inoculation, the culture flasks were placed in an artificial culture chamber. The parameters of the culture environment in each flask were as follows: temperature 25 ± 1 °C, light intensity 1500~18001x, light and dark alternate 12h/12h.

Comparative example 1~7

Comparative Examples 1 to 7 were basically the same as Examples 1 to 7, respectively, except that Comparative Examples 1 to 7 were not in Step 2). There is a step of soaking the peduncle in a 200 mg/L carbenicillin solution. The peduncle cultured in the induction medium in Examples 1 to 7 was cultured for 14 to 18 days, and it was found that the axillary bud sprouted and expanded. After inoculation of Examples 1-7 and Comparative Examples 1-7 in the induction medium for 30 days, the contamination rate and germination rate of the orchid orchid stems in each culture flask were counted. The experimental data are shown in Table 3.

Table 3 Experimental data after 30 days of culture

In Table 3, the germination rate after deducting the contaminated bottle, the number of germination / the number of sterile buds X 100%

In Table 3, comprehensive evaluation y = [(1 - pollution rate) + germination rate] X 100 / germination time

It can be seen from Table 3 that under the same induced culture conditions, the contamination rate of the carbenicillin of the present invention is significantly decreased after soaking seeds, especially in the induction medium, adding a certain amount of carbenicillin (No. 3, No. 4, 5). When the No. 6 and No. 6 induction medium were used, the overall score was not reduced, but increased. Compared with Example 7 and Examples 3-6, the contamination rate was significantly increased due to the absence of carbenicillin, the germination rate decreased, and the axillary bud sprouting time was slightly delayed. It can be seen that the addition of carbenicillin can effectively reduce the peduncle disinfection. The pollution rate is beneficial to increase the germination rate and shorten the germination time. It can be seen from Table 3 that 6-ammonium adenine is the main cause of germination germination, which can promote the germination and growth of peduncle in a certain concentration range. The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Therefore, any of the above embodiments can be made according to the technical essence of the present invention without departing from the technical solution of the present invention. Simple modifications, equivalent changes and modifications are still within the scope of the technical solutions of the present invention.

Claims

Wo tl ¾

1. An induction medium, characterized in that the induction medium is a mixture of agar, white sugar, 6-benzylaminoadenine, naphthylacetic acid, carbenicillin, coconut juice, and activated carbon added to 1/2MS medium. Prepared, the concentrations of the following components in the induction medium are: agar 7~9g/L, sugar 15~30g/L, carbenicillin 0~50 mg/L, 6-benzylaminoadenine 1.5 ~3 mg/L, naphthalene acetic acid 0.2~0.5mg/L, coconut juice 0~100 g/L, activated carbon 1~3g/L.

2. The induction medium according to claim 1, characterized in that the concentrations of the following components in the induction medium are: carbenicillin 40~50 mg/L, 6-benzylaminoadenine 2.5~3 mg /L, naphthalene acetic acid 0.4~0.5 mg/L, coconut juice 80-100 g/L.

3. The induction medium according to claim 2, wherein the concentrations of the following components in the induction medium are: carbenicillin 50 mg/L, 6-benzylaminoadenine 3 mg/L, Naphthalene acetic acid 0.5 mg/L, coconut juice 100 g/L.

4. A method for improving the survival rate of Phalaenopsis peduncles, which is characterized by including the following steps:

1) Cut the mature flower stalks on the strong mother plant of Phalaenopsis into segments with one bud as explants, soak them in 200mg/L carbenicillin solution, soak them for 30 minutes and then take them out to dry;

2) Routinely disinfect the explants on a sterile ultra-clean workbench, and then inoculate them into the induction medium for induction culture;

The induction medium is prepared by adding agar, white sugar, 6-benzylaminoadenine, naphthylacetic acid, carbenicillin, coconut juice, and activated carbon to 1/2MS medium. The following components are in the induction medium The concentrations are: agar 7~9g/L, white sugar 15~30g/L, carbenicillin 0~50 mg/L, 6-benzylaminoadenine 1.5~3 mg/L, naphthylacetic acid 0.2~0.5mg /L coconut juice 0~100 g/L, activated carbon 1~3g/L.

5. The method according to claim 4, characterized in that the concentrations of the following components in the induction medium are: carbenicillin 40~50 mg/L, 6-benzylaminoadenine 2.5~3 mg/L , naphthalene acetic acid 0.4~0.5 mg/L, coconut juice 80~100 g/L.

6. The method according to claim 5, characterized in that the concentrations of the following components in the induction medium are respectively: carbenicillin 50 mg/L, 6-benzylaminoadenine 3 mg/L, naphthylacetic acid 0.5 mg/L, coconut juice 100 g/L.

7. The method according to claim 4, characterized in that, in step 2, before inoculation, first use a sterilized scalpel to remove the incisions at both ends of the pedicel that have been in contact with the disinfectant, and then inoculate it into the culture medium. Each bottle was inoculated with 5 stem segments.

8. The method according to claim 4, characterized in that in step 2, the conditions for induction culture are: temperature 25±1°C, light intensity 1500~18001x, light and dark alternating 12h/12h.

9. The method according to claim 4, characterized in that the conventional disinfection refers to soaking in 75% alcohol for 30 seconds, and then soaking in 0.1% mercury chloride for 15 minutes.