A method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings

Abstract

The present invention provides a method for inducing the rhizomes of spring orchid test tube seedlings to bloom. It belongs to the field of plant tissue culture technology, and the specific steps are: taking out the seeds and inoculating them in a germination medium and culturing them for 2-10 days to obtain rhizomes; then placing the rhizomes in a proliferation medium and culturing them into robust rhizomes, and then inoculating the robust rhizomes into a flower bud induction medium to induce the formation of flower buds, and finally inoculating the flower buds into a flowering medium, culturing for 20 days until the flower buds are obviously enlarged and form a complete flower. The method adopted by the present invention directly promotes the rhizomes of spring orchid test tube seedlings to transition from the vegetative growth stage to the reproductive growth stage, induces flowering, determines the shape of hybrid offspring in advance, greatly shortens the traditional spring orchid breeding cycle, and improves the induction rate and flowering rate of flower buds by regulating the concentration of each component in the flower bud induction medium and the flowering medium.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings. Background Art

[0002] Spring orchid (Cymbidium goeringii) has a fragrant flower and elegant leaves. For Chinese people, orchids have extremely special meaning and importance and are deeply loved by the people. Traditional spring orchid hybrid breeding requires sterile sowing and cultivation in the laboratory, and outdoor planting to achieve flowering to determine whether the hybrid offspring has good shape. It takes at least 10 years from pollination, germination to flowering and large-scale planting. It has defects such as long R&D cycle, high investment and low output, which makes it difficult to meet the rapidly changing market demand. In addition, the existing induced rhizome flowering has the problem of too low flower bud induction rate and too low normal flower rate. Summary of the invention

[0003] 1. Technical issues to be solved

[0004] In view of the shortcomings of the prior art, the present invention provides a method for inducing flowering of rhizomes of spring orchid test tube seedlings, which directly promotes the transition of the rhizomes of spring orchid test tube seedlings from the vegetative growth stage to the reproductive growth stage, induces flowering, and determines the shape of hybrid offspring in advance, greatly shortening the traditional spring orchid breeding cycle, improving the induction rate and flowering rate of flower buds, and solving the problems raised in the above-mentioned background technology.

[0005] (II) Technical solution

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] According to a first aspect of the present invention, there is provided a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings, comprising the following steps:

[0008] (1) Aseptic germination to obtain rhizomes: Prepare sterile spring orchid capsule seeds and evenly disperse them in a germination medium and seal it. Move the inoculated germination medium into a culture room for culture to obtain 0.5 cm rhizomes. The germination culture medium is: 1 / 2MS + 0.5 mg / L 6-BA + 1.5 mg / L NAA + 1 g / L activated carbon + 30 g / L sucrose, pH 5.5;

[0009] (2) Rhizome proliferation: The rhizome obtained in step (1) is inoculated into a proliferation medium to obtain a robust rhizome of 2 to 8 cm, wherein the proliferation medium is: MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA + peptone + 10% coconut water, with a pH of 5.8;

[0010] (3) Rhizome flower bud induction: The robust rhizome obtained in step (2) is inoculated into a flower bud induction medium and cultured for 45 days to allow obvious flower buds to grow on the robust rhizome. The flower bud induction medium is: MS + 8 mg / L 6-BA + 0.02 mg / L NAA + 0.05 mg / L TDZ + 50 g / L sucrose + 7.5 g / L agar + 0.02 g / L activated carbon, pH 5.8;

[0011] (4) Rhizome flowering culture: The rhizome with flower buds obtained in step (3) is inoculated into a flowering medium and cultured for 15 to 25 days to form a fully formed flower. The flowering medium is 1 / 2MS + (1.5 to 3 mg / L) 6-BA + (0.1 to 0.5 mg / L) NAA + 30 g / L sugar + 5% coconut milk, with a pH of 5.5.

[0012] Preferably, the culture conditions in step (1), step (2), step (3) and step (4) are: temperature of 24-26°C, light intensity of 1500-2000lx, relative humidity of 65-80%, and light exposure of 16h per day.

[0013] Preferably, in step (1), the preparation of the sterile spring orchid capsule seeds comprises the following steps: washing the spring orchid capsule with sterile water, first placing it in 75% ethanol and letting it stand for 10 seconds, then washing it three times with sterile water, then soaking it in 0.1% sodium hypochlorite for 15 to 20 minutes, then washing it three times with sterile water, and finally soaking it in 0.1% sodium hypochlorite for 3 to 4 minutes, then washing it three times with sterile water.

[0014] Preferably, the spring orchid capsules are selected from spring orchid capsules harvested before maturity and cracking.

[0015] (III) Beneficial effects

[0016] The present invention provides a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings. The method has the following beneficial effects:

[0017] (1) This scheme provides a method for inducing flowering of rhizomes of spring orchid test tube seedlings. By preparing the base liquid in the culture medium, the rhizomes of spring orchids can be directly transitioned from the vegetative growth stage to the reproductive growth stage. From the germination of spring orchid seeds to flowering, it only takes about one year to induce flowering of spring orchids. The shape of orchids can be evaluated more quickly, which greatly shortens the breeding cycle of traditional spring orchids and improves breeding efficiency.

[0018] (2) The scheme provides a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings, which increases the flower bud induction rate of rhizomes of Cymbidium orchid test tube seedlings to more than 18% by adjusting the concentrations of NAA and TDZ in the flower bud induction medium.

[0019] (3) This scheme provides a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings, which increases the flowering rate of rhizomes of Cymbidium orchid test tube seedlings to more than 69% by adjusting the concentrations of 6-BA and NAA in the flowering culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of flower buds, leaf buds and flower buds generated from the rhizomes of Cymbidium orchid test tube seedlings after rhizomes are induced to flower in the present invention;

[0021] Figure 2 It is a visual diagram of the induction of rhizomes of Cymbidium orchid test tube seedlings into flowers during the process of rhizomes flower bud induction of the present invention, wherein (a) (b) (c) (d) are state diagrams of 350 to 360 days of induction culture;

[0022] Figure 3 The abnormal flowers are produced in the process of rhizome induction of Cymbidium orchid test tube seedlings in the process of rhizome flower cultivation of the present invention;

[0023] Figure 4 It is a schematic diagram of inducing rhizomes of Cymbidium orchid test tube seedlings into flowers of the present invention. DETAILED DESCRIPTION

[0024] In order to better illustrate the content of the present invention, a detailed description is given below in conjunction with specific embodiments.

[0025] Example 1

[0026] This embodiment provides a method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings, comprising the following steps:

[0027] Sterilization of explants: Place the spring orchid capsules with the seed clip turning from green to yellow in 75% ethanol for 10 seconds, wash with sterile water 3 times, then place in 0.1% sodium hypochlorite for 15 minutes, wash with sterile water 3 times, and finally place in 0.1% sodium hypochlorite for 3 minutes, and wash with sterile water 3 times;

[0028] Aseptic germination to obtain rhizomes: After wiping the water on the surface of spring orchid capsules with sterile filter paper, cut the spring orchid capsules, take out the seeds and inoculate them in the germination medium. The germination medium is: 1 / 2MS+0.5mg / L6-BA+1.5mg / L NAA+1g / L activated carbon+30g / L sucrose, pH 5.5, shake after inoculation to evenly spread the seeds in the medium, seal, move the inoculated culture bottle into the culture room, set the culture temperature to 25°C, the light intensity to 1500lx, the relative humidity to 65, and light for 16h per day. After 90 days, the seeds begin to germinate, and after 150 days, the protocorms grow to 3-4mm, and after 210d, 0.5cm rhizomes are obtained;

[0029] Rhizome proliferation: The obtained rhizomes were transferred to a proliferation medium for inducing rhizome proliferation, the proliferation medium was MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA + peptone + 10% coconut water, pH 5.8, the culture temperature was set at 25°C, the light intensity was set at 1500 lx, the relative humidity was set at 65, the light was illuminated for 16 hours per day, and the culture was carried out for 60 days to obtain 8 cm strong rhizomes;

[0030] Rhizome flower bud induction: Inoculate the robust rhizome into the flower bud induction medium, set the culture temperature to 25℃, the light intensity to 1500lx, the relative humidity to 65, and light for 16h per day for 45 days, so that obvious flower buds grow on the robust rhizome, and the flower bud induction rate is 25.83%. Figure 1 As shown, the flower bud induction medium is MS + 8 mg / L 6-BA + 0.02 mg / L NAA + 0.05 mg / L TDZ + 50 g / L sucrose + 7.5 g / L agar + 0.02 g / L activated carbon, pH 5.8;

[0031] The rhizome of Chunlan test tube seedlings is induced into a flower state during the process of rhizome flower bud induction. Figure 2 As shown in Figure 2, during the induction process, the induced abnormal flowers are as follows Figure 3 As shown, they were removed during the subsequent culture process;

[0032] Rhizome flowering culture: The rhizome with flower buds was inoculated into a flowering medium, which was: 1 / 2MS + 3mg / L 6-BA + 0.5mg / L NAA + 30g / L sugar + 5% coconut juice, pH 5.5, culture temperature was set at 25°C, light intensity was set at 1500lx, relative humidity was set at 65, and light was on for 16h per day. After 20 days of culture, the flower buds were significantly enlarged, the normal flowering rate was 87.31%, and the flowers with complete morphology were formed. Figure 4 shown.

[0033] Example 2

[0034] The method adopted in this embodiment is the same as that in Example 1, except that during the process of rhizome flower bud induction, the flower bud induction medium is MS + 8 mg / L 6-BA + 0.02 mg / L NAA + 0.1 mg / L TDZ + 50 g / L sucrose + 7.5 g / L agar + 0.02 g / L activated carbon, the pH is 5.8, and the flower bud induction rate is 18.25%.

[0035] Example 3

[0036] The method used in this embodiment is the same as that in Example 1, except that the flowering medium in the rhizome flowering culture process is 1 / 2MS + 3mg / L 6-BA + 0.1mg / L NAA + 30g / L sugar + 5% coconut juice, the pH is 5.5, and the normal flowering rate is 82.64%.

[0037] Example 4

[0038] The method used in this embodiment is the same as that in Example 1, except that the flowering medium in the rhizome flowering culture process is 1 / 2MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sugar + 5% coconut juice, the pH is 5.5, and the normal flowering rate is 73.43%.

[0039] Example 5

[0040] The method used in this embodiment is the same as that in Example 1, except that the flowering medium in the rhizome flowering culture process is 1 / 2MS+1.5mg / L 6-BA+0.1mg / L NAA+30g / L sugar+5% coconut juice, the pH is 5.5, and the normal flowering rate is 69.16%.

[0041] Based on the process of rhizome flower bud induction, the effects of flower bud induction medium NAA and TDZ on the flower bud induction rate of rhizome of spring orchid test tube seedlings are shown in Table 1;

[0042] Table 1

[0043] NAA(mg / L) TDZ(mg / L) Flower bud induction rate (%) Example 1 0.02 0.05 25.83 Example 2 0.02 0.1 18.25 Comparative Example 1 0.02 0.2 6.91 Comparative Example 2 0.2 0.05 17.71 Comparative Example 3 0.2 0.1 5.28 Comparative Example 4 0.2 0.2 6.39

[0044] Based on the process of rhizome flowering culture, the effect of the ratio of 6-BA and NAA in the flowering culture medium on the normal flowering rate is shown in Table 2;

[0045] Table 2

[0046]

[0047]

[0048] According to the data in Tables 1 and 2, by adjusting the concentrations of substances in the flower bud induction medium and the flowering medium, the induction rate and flowering rate of rhizomes of Cymbidium orchid test tube seedlings can be increased, thereby achieving large-scale breeding.

[0049] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings, characterized in that: The following steps are involved: (1) Aseptic germination to obtain rhizomes: Prepare sterile spring orchid capsule seeds and evenly disperse them in a germination medium and seal it. Move the inoculated germination medium into a culture room for culture to obtain 0.5 cm rhizomes. The germination culture medium is: 1 / 2MS + 0.5 mg / L 6-BA + 1.5 mg / L NAA + 1 g / L activated carbon + 30 g / L sucrose, pH 5.5; (2) Rhizome proliferation: The rhizome obtained in step (1) is inoculated into a proliferation medium to obtain a robust rhizome of 2 to 8 cm, wherein the proliferation medium is: MS + 1.5 mg / L 6-BA + 0.2 mg / L NAA + peptone + 10% coconut water, with a pH of 5.8; (3) Rhizome flower bud induction: The robust rhizome obtained in step (2) is inoculated into a flower bud induction medium and cultured for 45 days to allow obvious flower buds to grow on the robust rhizome. The flower bud induction medium is: MS + 8 mg / L 6-BA + 0.02 mg / L NAA + 0.05-0.1 mg / L TDZ + 50 g / L sucrose + 7.5 g / L agar + 0.02 g / L activated carbon, pH 5.8; (4) Rhizome flowering culture: The rhizome with flower buds obtained in step (3) is inoculated into a flowering medium and cultured for 15 to 25 days to form a fully formed flower. The flowering medium is 1 / 2MS + 1.5 to 3 mg / L 6-BA + 0.1 to 0.5 mg / L NAA + 30 g / L sugar + 5% coconut juice, with a pH of 5.

5.

2. A method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings according to claim 1, characterized in that: The culture conditions in step (1), step (2), step (3) and step (4) are: temperature of 24-26° C., light intensity of 1500-2000 lx, relative humidity of 65-80%, and light exposure of 16 hours per day.

3. A method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings according to claim 1, characterized in that: In step (1), the capsule of spring orchid is washed with sterile water, first placed in 75% ethanol and allowed to stand for 10 seconds, then washed three times with sterile water, then soaked in 0.1% sodium hypochlorite for 15 to 20 minutes, then washed three times with sterile water, and finally soaked in 0.1% sodium hypochlorite for 3 to 4 minutes, then washed three times with sterile water.

4. A method for inducing flowering of rhizomes of Cymbidium orchid test tube seedlings according to claim 3, characterized in that: The spring orchid capsules are selected from spring orchid capsules harvested before maturity and cracking.

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