Rapid propagation method for orchid seedlings and application of rapid propagation method

Abstract

The invention discloses a rapid propagation method of orchid seedlings and application of the rapid propagation method. The rapid propagation method comprises the following steps: taking materials, disinfecting explants, inducing intermediate propagules, propagating the intermediate propagules, differentiating the intermediate propagules, rooting and strengthening seedlings and finally transplanting according to a traditional method. And the orchid seedlings with high survival rate are obtained under the condition of omitting higher sterilization cost and labor cost in cooperation with a disposable plastic packaging box with lower use cost and culture medium proportioning in different stages. The invention explores an industrialized orchid tissue culture technology, effectively solves the problems of high labor cost and tedious sterilization step in the conventional tissue culture technology, ensures the survival rate of orchid tissue culture, and improves the efficiency of industrialized production.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a rapid propagation method for orchid seedlings and an application thereof. Background Art

[0002] Orchids are monocotyledonous plants, epiphytic or terrestrial herbs, with racemes of several or many flowers. The colors include white, pure white, white-green, yellow-green, light yellow, light yellow-brown, yellow, red, blue and purple. They are very ornamental. Currently, tissue culture technology is commonly used to cultivate orchids on the market.

[0003] Plant tissue culture technology was first proposed by the famous German botanist G. Haberlandt in 1902, who proposed the theory that plant cells have "omnipotence", indicating that the organs and tissues of higher plants can be continuously divided until a single cell, and this single cell is potentially omnipotent, that is, plant cells have omnipotence. At the same time, it was boldly proposed that "each somatic cell, like an embryonic cell, can be cultured in vitro to become a complete plant, because each cell of the plant body contains all the genetic information of the individual and has a full set of genes to form a complete plant, so under suitable conditions, it can be induced to grow and differentiate to form a complete plant." After that, scientists did a lot of work. It was not until the 1930s that White in the United States successfully used tomato root tissue in vitro culture and established the first asexual reproduction system, also known as "clone", and then the tissue culture of tobacco stem cambium was also successful.

[0004] Nowadays, plant tissue culture technology has become quite mature, and a factory-based production method has been formed, so that demanders can obtain large quantities of plant seedlings in a relatively short period of time. CN106134801A discloses a method for cultivating multi-color magnolias, which mainly includes the steps of: parent plant cultivation, tissue grafting, post-grafting cultivation management, asexual reproduction stability traits, etc. The magnolias cultivated by this method can produce magnolias of various colors and flower shapes such as white, purple, lavender, yellow, and rose on the same magnolia tree. CN105340757A discloses a tissue culture method for Cymbidium orchid and its application. In this technology, LED lamps are mainly used as tissue culture light sources, saving 30%-50% of electricity resources, reducing costs, and improving the efficiency of factory production.

[0005] Although the above technology has been increasingly improved, there are still many problems in the tissue culture process, such as the culture medium usually needs to be sterilized at 121 degrees for 20 minutes, the cutting tools used need to be frequently sterilized, the plants need to be frequently transferred up to 18 times to obtain complete rooted seedlings, and the composition of the culture medium is complex and expensive. Considering that the key to plant tissue culture technology is that the tools used in the entire operation must be sterilized at high temperature, and the process of selecting tissues to suitable culture media must maintain a strict sterile environment, this has caused the technology itself to be a technology-intensive and labor-intensive industry. There is a lot of energy consumption, material consumption, time consumption and manpower consumption in the entire production process. Therefore, a fast and low-energy method for the rapid propagation of orchid seedlings is needed. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention aims to provide a method for rapid propagation of orchid seedlings and its application, which can solve the technical problems of high energy consumption, high labor cost and high cycle of existing orchid seedlings while ensuring a high propagation survival rate.

[0007] To achieve the above objectives, the present invention adopts the following solutions: A method for rapid propagation of orchid seedlings comprises the following steps: S1 Material collection: Select the middle lateral buds of healthy orchid plants without diseases and insect pests as the source of explants, rinse them with running water for 10 minutes, and gently brush off the dust on the surface with a brush while rinsing.

[0008] S2 Explant disinfection: Soak the explants in 0.1% mercuric chloride for 20 minutes, then rinse with sterile water 5 times, 15 minutes each time.

[0009] S3 Induce intermediate propagules: Spray alcohol for disinfection near the workbench, light the alcohol lamp, inoculate the explants in the culture medium within 30 cm around the alcohol lamp and culture for 30 days.

[0010] S4 intermediate propagule proliferation: spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the induced intermediate propagules into MS1-1 culture medium within 30 cm around the alcohol lamp and culture for 30 days. With 30 days as a transfer cycle, continue to transfer twice, and replace it with MS1-2 culture medium for the second transfer.

[0011] S5 Intermediate propagule differentiation: Spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the proliferated intermediate propagules into disposable plastic packaging box A1 containing 6-BA and NAA within 30 cm around the alcohol lamp for light culture for 30 days. With a transfer cycle of 45 days, continue to transfer twice, and replace it with disposable plastic packaging box A2 for the second transfer.

[0012] S6 rooting and strengthening seedlings: spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the buds differentiated from the intermediate propagules into disposable plastic packaging box A3 containing 6-BA and NAA within 30 cm around the alcohol lamp. Cultivate in natural sunlight for 30 days, then open the lid and continue rooting culture for 20 days.

[0013] S7 transplanting: Transplant the rooted and strong tissue culture seedlings of S6 into sphagnum moss and place them in a greenhouse for cultivation, maintaining the humidity at 70%-80% and the temperature at 20-24℃.

[0014] Preferably, the concentration of the culture medium in step S3 is 3 mg / L 6-BA, 0.5 mg / L NAA, 0.8 g / L potassium sorbate, 0.9 g / L ceftriaxone sodium, and 0.3 g / L benzoic acid; Preferably, the culture conditions in step S3 are continuous illumination for 16±1 hours per day, a light intensity of 1800 Lux, and a temperature of 26°C; Preferably, the concentration of the MS1-1 medium in step S4 is 2.5 mg / L 6-BA, 0.2 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid; the concentration of the MS1-2 medium is 2 mg / L 6-BA, 0.5 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid; Preferably, the culture conditions in step S4 are continuous illumination for 10-12 hours per day, a light intensity of 2000 Lux, and a temperature of 26° C.; Preferably, in step S5, the concentration of 6-BA in the disposable plastic packaging box A1 is 0.5 mg / L, the concentration of NAA is 1.0 mg / L, the concentration of potassium sorbate is 0.6 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.6 g / L; the concentration of 6-BA in the disposable plastic packaging box A2 is 0.3 mg / L, the concentration of NAA is 0.6 mg / L, the concentration of potassium sorbate is 0.6 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.6 g / L; Preferably, the culture conditions in step S5 are continuous illumination for 10-12 hours per day, the illumination intensity is 2000 Lux, and the temperature is 26° C.; Preferably, in the disposable plastic packaging box A3 in step S6, the concentration of 6-BA is 0.5 mg / L, the concentration of NAA is 1.5 mg / L, the concentration of arginine is 0.2 g / L, the concentration of potassium sorbate is 0.4 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.3 g / L; The application of the above orchid tissue culture method in large-scale orchid cultivation.

[0015] The present invention is a new tissue rapid culture technology based on the traditional orchid tissue culture technology and modern methods. It replaces the expensive tissue culture bottle with a lower-cost container and does not require sterility for the culture medium and the culture environment. While ensuring the survival rate of orchid tissue culture, it greatly shortens the culture time and reduces the cost. At the same time, it organically combines scientific research and teaching, agricultural science popularization and factory seedling cultivation, and uses the redundant intermediate propagule materials in scientific research or production, the antibacterial culture medium of the present invention, and the innovatively used simple container of the present invention to carry out science popularization teaching. While greatly reducing energy consumption and saving costs, it also takes into account economic benefits, social benefits and ecological benefits, making the selection and breeding of new orchid varieties more meaningful. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a diagram of the proliferation of intermediate propagules of hybrid orchids.

[0017] Figure 2 This is a picture of hybrid orchid rooting and growing strong seedlings. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] It should be noted that, although one of the practical problems solved by the technical solution adopted by the present invention is the high energy consumption caused by the frequent sterilization of equipment and materials in the production process, the necessary sterilization and disinfection are the key to realizing this technical solution. In the following embodiments, the sterilization time will be clearly stated. If it is not clearly stated, sterilization treatment is not required. Example

[0020] S1 Material collection: Select the middle lateral buds of healthy, pest-free hybrid orchid plants as the source of explants, rinse them with running water for 10 minutes, and gently brush off the dust on the surface with a brush while rinsing; S2 Explant disinfection: Soak the explants in 0.1% mercuric chloride for 20 minutes, then rinse with sterile water 5 times, 15 minutes each time.

[0021] S3 induces intermediate propagules: spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the explants in a disposable plastic packaging box containing 6-BA and NAA within 30 cm around the alcohol lamp for cultivation for 30 days. The concentrations are 6-BA 3 mg / L, NAA 0.5 mg / L, potassium sorbate 0.8 g / L, ceftriaxone sodium 0.9 g / L, and benzoic acid 0.3 g / L. Maintain 16 hours of continuous light per day, with a light intensity of 1800 Lux and a temperature of 26°C.

[0022] S4 intermediate propagule proliferation: spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the induced intermediate propagules into a disposable plastic packaging box containing 6-BA and NAA within 30 cm around the alcohol lamp for cultivation for 30 days. The concentration is 6-BA 2.5 mg / L, NAA 0.2 mg / L, potassium sorbate 0.8 g / L, ceftriaxone sodium 0.2 g / L, benzoic acid 0.4 g / L, with a transfer cycle of 30 days, and continue to transfer twice. During the second transfer, replace the 6-BA concentration in the disposable plastic packaging box with 2 mg / L, NAA 0.5 mg / L, potassium sorbate 0.8 g / L, ceftriaxone sodium 0.2 g / L, benzoic acid 0.4 g / L, maintain 10 hours of continuous light per day, the light intensity is 2000 Lux, and the temperature is 26°C.

[0023] S5 Intermediate propagule differentiation: Spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the proliferated intermediate propagules into a disposable plastic packaging box containing 6-BA and NAA within 30 cm around the alcohol lamp for light culture for 30 days. The concentrations are 6-BA 0.5 mg / L, NAA 1.0 mg / L, potassium sorbate 0.6 g / L, ceftriaxone sodium 0.2 g / L, and benzoic acid 0.6 g / L. The transfer cycle is 45 days, and the transfer is continued twice. During the second transfer, the concentration of 6-BA is replaced with 0.3 mg / L, NAA 0.6 mg / L, potassium sorbate 0.6 g / L, ceftriaxone sodium 0.2 g / L, and benzoic acid 0.6 g / L. Maintain 10 hours of continuous light per day, the light intensity is 2000 Lux, and the temperature is 26°C.

[0024] S6 rooting and strengthening seedlings: spray alcohol for disinfection near the workbench, light the alcohol lamp, and inoculate the buds differentiated from the intermediate propagules into a disposable plastic packaging box containing 6-BA and NAA within 30 cm around the alcohol lamp. After continuous cultivation in natural sunlight for 30 days, open the lid and continue to root and strengthen the seedlings for 20 days. The concentration of 6-BA is 0.5 mg / L, the concentration of NAA is 1.5 mg / L, the concentration of arginine is 0.2 g / L, the concentration of potassium sorbate is 0.4 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.3 g / L.

[0025] S7 transplanting: Transplant the rooted and strong tissue culture seedlings of S6 into sphagnum moss and place them in a greenhouse for cultivation. Maintain the humidity at 70%-80% and the temperature at 20-24℃.

[0026] Traditional orchid tissue culture technology requires strict aseptic operation in the process of cutting the propagule, proliferation and differentiation and rooting, which inevitably leads to the characteristics of this technology being cumbersome and cautious. Compared with the traditional orchid tissue culture technology, the above-mentioned orchid rapid tissue culture technology avoids the use of a clean bench in steps S2-S6, allowing workers to quickly handle the inoculation process in the same area of ​​space, greatly saving labor costs and enabling orchid tissue culture technology to achieve industrial development; Further statistics show that this technology can produce healthy hybrid orchid seedlings in about 290 days from the seven steps of material collection to transplantation, while the traditional orchid tissue culture technology requires at least 2-3 years to obtain seedlings of one variety. This technology greatly shortens the time of orchid tissue culture technology, and has far-reaching economic, social and ecological benefits, whether from the perspective of industrial production energy efficiency or classroom teaching. Precisely because the orchid tissue culture cycle is relatively long, no scientific research institutions, colleges or factories have been found to use disposable plastic packaging boxes as culture containers for production. This technology boldly uses disposable plastic packaging boxes as culture containers, replacing the expensive tissue culture bottles. According to statistics, the cost of using disposable plastic packaging boxes for a hybrid orchid tissue culture from start to finish is negligible, while the cost of using tissue culture bottles is more than 0.5 yuan per plant. Although tissue culture bottles can be reused, the cleaning and damage of tissue culture bottles must be considered during industrial production. The labor cost of workers and the time cost of the entire tissue culture cannot be ignored.

[0027] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for rapid propagation of orchid seedlings, characterized in that: The steps include: S1 Collection: Select the middle lateral buds of healthy orchid plants without diseases and insect pests as the source of explants, rinse them with running water for 10 minutes, and gently brush off the dust on the surface with a brush while rinsing; S2 Explant disinfection: Soak the explant in 0.1% mercuric chloride for 20 minutes, then rinse with sterile water 5 times, 15 minutes each time; S3 Induce intermediate propagules: Spray alcohol near the workbench for disinfection, light an alcohol lamp, and inoculate explants in culture medium within 30 cm around the alcohol lamp for 30 days; S4 Intermediate propagule proliferation: spray alcohol for disinfection near the workbench, light an alcohol lamp, and inoculate the induced intermediate propagules in MS1-1 medium within 30 cm around the alcohol lamp for 30 days. Take 30 days as a transfer cycle, and continue to transfer twice. Change to MS1-2 medium for the second transfer; S5 Intermediate propagule differentiation: Spray alcohol for disinfection near the workbench, light an alcohol lamp, and inoculate the proliferated intermediate propagules into disposable plastic packaging box A1 containing 6-BA and NAA within 30 cm around the alcohol lamp for light culture for 30 days. The transfer cycle is 45 days, and the transfer is continued twice. During the second transfer, the disposable plastic packaging box A2 is replaced; S6 rooting and strengthening seedlings: spray alcohol for disinfection near the workbench, light an alcohol lamp, and inoculate the buds differentiated from the intermediate propagules into disposable plastic packaging boxes A3 containing 6-BA and NAA within 30 cm around the alcohol lamp. Cultivate in natural sunlight for 30 days, then open the lid and continue rooting and cultivation for 20 days; S7 transplanting: Transplant the rooted and strong tissue culture seedlings of S6 into sphagnum moss and place them in a greenhouse for cultivation, maintaining the humidity at 70%-80% and the temperature at 20-24℃.

2. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: The culture medium concentration in step S3 is 3 mg / L 6-BA, 0.5 mg / L NAA, 0.8 g / L potassium sorbate, 0.9 g / L ceftriaxone sodium, and 0.3 g / L benzoic acid.

3. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: The culture conditions in step S3 are continuous illumination for 16±1 hours per day, a light intensity of 1800 Lux, and a temperature of 26°C.

4. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: The concentration of MS1-1 culture medium in step S4 is 2.5 mg / L 6-BA, 0.2 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid; the concentration of MS1-2 culture medium is 2 mg / L 6-BA, 0.5 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid.

5. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: The concentration of MS1-1 culture medium in step S4 is 2.5 mg / L 6-BA, 0.2 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid; the concentration of MS1-2 culture medium is 2 mg / L 6-BA, 0.5 mg / L NAA, 0.8 g / L potassium sorbate, 0.2 g / L ceftriaxone sodium, and 0.4 g / L benzoic acid.

6. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: Step S5: The concentration of 6-BA in the disposable plastic packaging box A1 is 0.5 mg / L, the concentration of NAA is 1.0 mg / L, the concentration of potassium sorbate is 0.6 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.6 g / L; the concentration of 6-BA in the disposable plastic packaging box A2 is 0.3 mg / L, the concentration of NAA is 0.6 mg / L, the concentration of potassium sorbate is 0.6 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.6 g / L.

7. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: The culture conditions in step S5 are continuous illumination for 10-12 hours per day, a light intensity of 2000 Lux, and a temperature of 26°C.

8. The method for rapid propagation of orchid seedlings according to claim 1, characterized in that: In step S6, the concentration of 6-BA in the disposable plastic packaging box A3 is 0.5 mg / L, the concentration of NAA is 1.5 mg / L, the concentration of arginine is 0.2 g / L, the concentration of potassium sorbate is 0.4 g / L, the concentration of ceftriaxone sodium is 0.2 g / L, and the concentration of benzoic acid is 0.3 g / L.

9. Use of the rapid propagation method for orchid seedlings as claimed in any one of claims 1 to 8 in large-scale production.

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