Rapid propagation method for butterfly orchid tissue culture

Abstract

The invention discloses a rapid propagation method for tissue culture of phalaenopsis amabilis, which comprises the following steps: selection and treatment of explants: selecting healthy and disease-free explants, and properly treating the explants to improve the success rate of subsequent culture; primary induction culture: putting the treated explant into an induction culture medium to promote primary growth and differentiation; cluster bud induction and subculture multiplication culture: inducing the explant to form a plurality of cluster buds by optimizing culture conditions, and carrying out subculture multiplication culture so as to improve the propagation coefficient; rooting culture: carrying out rooting culture on the formed buds to promote the development of root systems and improve the transplanting survival rate; transplanting and seedling hardening: transplanting the plants with good rooting into a proper matrix, and carrying out seedling hardening to enhance the adaptive capacity of the plants; through the steps, rapid propagation of phalaenopsis amabilis can be achieved, the output period is remarkably shortened, and the production cost is reduced.

Description

Technical Field

[0001] The present application relates to the field of flower tissue culture, and in particular to a rapid propagation method for Phalaenopsis tissue culture. Background Art

[0002] Phalaenopsis, belonging to the Orchidaceae family, is a popular ornamental plant that is loved by consumers for its bright colors and long flowering period. As an important fresh cut flower and potted plant, Phalaenopsis occupies an important position in the flower market and is widely used in flower gardening, interior decoration, and gift giving, which has promoted the development of related industries. At present, the propagation of Phalaenopsis mainly adopts traditional methods such as tissue culture and division. Although these methods have achieved planting to a certain extent, due to the long propagation cycle and high technical requirements, the propagation efficiency is not high and cannot meet the growing market demand. In addition, traditional propagation methods are often affected by diseases, environmental factors, etc., which affect the survival rate and quality of seedlings and cause waste of resources. For this reason, it is particularly important to explore and establish a rapid propagation system for Phalaenopsis. Summary of the invention

[0003] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for rapid propagation of Phalaenopsis tissue culture to solve the problems raised in the above-mentioned background technology.

[0004] According to one aspect of the present application, a method for rapid propagation of Phalaenopsis tissue culture comprises the following steps:

[0005] Step S1: Selection and processing of explants:

[0006] S11. Select the top young stem segments and healthy bulbs of robust and disease-free Phalaenopsis plants as explants;

[0007] S12, pre-treating the selected explants at low temperature for two weeks, taking them out and cleaning them, soaking them in thiophanate-methyl, then rinsing them with running water, and then rinsing them with sterile water;

[0008] S13, sterilizing the cleaned explants, then rinsing with sterile water, then treating with mercuric chloride, and then wiping the surface moisture with sterile filter paper;

[0009] S14, using a sterilized blade to cut the treated explants longitudinally into bulbs with a width of 3-4 mm, and cut the stem segments into explants with a thickness of about 2-3 mm;

[0010] Step S2, primary induction culture:

[0011] S21, inoculating the treated explants into primary induction medium;

[0012] S22, 10 days of dark culture at the beginning of the culture, followed by 45 days of culture under the condition of light intensity of 3000 lux;

[0013] S23, screening out adventitious buds and bulblets that meet the standards, the standards being that the adventitious buds are 3-4 cm high and the bulblets are 1.5-2 cm in diameter;

[0014] Step S3, cluster bud induction and subculture proliferation culture:

[0015] S31, inoculating the adventitious buds that grow well in the primary culture into a cluster bud induction medium and culturing them under light for 30 days;

[0016] S32, inoculating the clustered buds into the subculture proliferation medium and culturing them under light for 30 days;

[0017] Step S4, rooting culture: cut the small cluster of seedlings into individual plants and inoculate them into rooting medium, culture them under light for 15 days, observe the growth of the root system, and ensure that the root system is well developed;

[0018] Step S5: Transplanting and hardening:

[0019] S51. Move the Phalaenopsis tissue culture seedlings with a root length of 3-5 cm to a place with scattered light for seedling hardening. After one week, gradually move them to a place with better light.

[0020] S52. Wash the root culture medium of the prepared tissue culture seedlings and plant them in a hole tray filled with peat. Water them for the first time until the soil can be squeezed into a ball without dripping. Build a small arch shed and cover it with film and culture them under natural light.

[0021] Preferably, in step S12, the low-temperature pretreatment temperature is 5°C, and the explants are cleaned by soaking them in 500-fold thiophanate-methyl for 20 minutes, then rinsing them with running water for 30 minutes, and then rinsing them with sterile water for 4 times;

[0022] In step S13, the cleaned explants are immersed in 75% ethanol for 30 seconds for disinfection, then rinsed with sterile water for 4 times, then treated with 0.1% mercuric chloride for 5 minutes, and finally wiped dry with sterile filter paper.

[0023] Preferably, in step S13, the method for disinfecting the explant further comprises placing the explant on a clean bench and irradiating it with ultraviolet light for 30 minutes and then ventilating it for 15 minutes for disinfection.

[0024] Preferably, in step S2, the components of the primary induction medium are: MS medium, sucrose 25 g / L, agar 6 g / L, 6-benzylaminopurine 1 mg / L and α-naphthylacetic acid 0.2 mg / L.

[0025] Preferably, in step S2, the culture conditions of the primary induction culture medium are: LED lighting intensity 2000-3000 lux, 10 hours of illumination per day, and the culture temperature is maintained at 23±2°C.

[0026] Preferably, in step S31, the components of the clustered shoot induction medium are: MS medium, 6-BA 1.5 mg / L and NAA 0.1 mg / L;

[0027] In step S32, the components of the subculture proliferation medium are: MS medium, 6-BA 0.5 mg / L and NAA 0.05 mg / L.

[0028] Preferably, in step S3, during the cluster bud induction and subculture proliferation culture stage, LED lighting is used and the culture temperature is 23±2°C.

[0029] Preferably, in step S4, the ingredients of the rooting medium are: MS medium, sucrose 20 g / L, agar 4 g / L, NAA 0.3 mg / L.

[0030] Preferably, in step S4, the rooting culture is performed with illumination for 8 hours per day, the culture temperature is maintained at 25±1° C., and the relative humidity is maintained at 60%-75%.

[0031] Preferably, in step S5, the bottle body is rotated periodically during the seedling hardening treatment to ensure that the tissue culture seedlings are evenly exposed to light, and a nitrogen-containing nutrient solution is added after transplanting to promote the growth of the seedlings.

[0032] The present application discloses a method for rapid propagation of Phalaenopsis tissue culture. Through the above-mentioned design steps, the present application can realize a rapid propagation system for Phalaenopsis, shorten the output cycle of Phalaenopsis, and reduce production costs; by treating with 75% ethanol for 30 seconds and 0.1% mercuric chloride for 5 minutes, the contamination rate of tissue culture inoculation can be reduced to less than 10%, thereby reducing the contamination rate and saving costs; scale cutting method: longitudinal cutting method increases the induction rate of adventitious buds and bulblets, while reducing the cutting surface and thus reducing the contamination rate; dark culture for 10 days is conducive to the formation of bulblets, providing possibility and technical support for exploring rapid propagation of Phalaenopsis tissue culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is an overall flow chart according to an embodiment of the present invention. DETAILED DESCRIPTION

[0034] In order to make the contents of the present application easier to understand, the technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application.

[0035] like Figure 1As shown, a method for rapid propagation of Phalaenopsis tissue culture, characterized in that the method comprises the following steps:

[0036] Step S1: Selection and processing of explants:

[0037] S11. Select the top young stem segments and healthy bulbs of robust and disease-free Phalaenopsis plants as explants;

[0038] S12, pre-treating the selected explants at 5°C for two weeks, taking them out and cleaning them, soaking them in 500-fold thiophanate-methyl for 20 minutes, then rinsing them in running water for 30 minutes, and then rinsing them in sterile water for 4 times;

[0039] S13, the cleaned explants were immersed in 75% ethanol for 30 seconds for disinfection or placed on a clean bench and irradiated with ultraviolet light for 30 minutes and then ventilated for 15 minutes for disinfection, then rinsed with sterile water for 4 times, then treated with 0.1% mercuric chloride for 5 minutes, and finally wiped the surface moisture with sterile filter paper;

[0040] S14. Use a sterilized blade to cut the treated explants longitudinally into bulbs with a width of 3-4 mm, and cut the stem segments into explants with a thickness of about 2-3 mm.

[0041] Step S2, primary induction culture:

[0042] S21, inoculating the treated explants into a primary induction medium, the components of the medium are: MS medium, sucrose 25g / L, agar 6g / L, 6-benzylaminopurine 1mg / L and α-naphthaleneacetic acid 0.2mg / L, the culture conditions of the primary induction medium are: LED lighting intensity 2000-3000lux, 10 hours of light per day, and the culture temperature is maintained at 23±2℃;

[0043] S22, 10 days of dark culture at the beginning of the culture, followed by 45 days of culture under the condition of light intensity of 3000 lux;

[0044] S23. Screen out adventitious buds and bulblets that meet the standards. The standards are that the adventitious buds are 3-4 cm high and the bulblets are 1.5-2 cm in diameter. Screening of adventitious buds and bulblets after the induction culture is completed can improve the success rate of subsequent reproduction.

[0045] Step S3, cluster bud induction and subculture proliferation culture:

[0046] S31, inoculating the adventitious buds that grow well in the primary culture into a cluster bud induction medium, and culturing them under light for 30 days. The components of the cluster bud induction medium are: MS medium, 6-BA 1.5 mg / L and NAA 0.1 mg / L;

[0047] S32, inoculating the clustered buds into a subculture proliferation medium, and culturing under light for 30 days, wherein the components of the subculture proliferation medium are: MS medium, 6-BA 0.5 mg / L and NAA 0.05 mg / L;

[0048] During the cluster bud induction and subculture proliferation culture stage, LED lighting was used, the culture temperature was 23±2℃, and the observation indicators during the proliferation culture stage included the bud cluster proliferation rate and growth rate to ensure that they met the subsequent transplanting standards.

[0049] Step S4, rooting culture: cut the small cluster of seedlings into individual plants and inoculate them into rooting medium. The ingredients of the rooting medium are: MS medium, sucrose 20g / L, agar 4g / L, NAA0.3mg / L. Culture under light for 15 days, observe the growth of the root system to ensure that the root system is well developed. The rooting culture is illuminated for 8 hours / day, the culture temperature is maintained at 25±1°C, and the relative humidity is maintained at 60%-75%.

[0050] Step S5: Transplanting and hardening:

[0051] S51. Move the Phalaenopsis tissue culture seedlings with a root length of 3-5 cm to a place with scattered light for seedling hardening. After one week, gradually move them to a place with better light.

[0052] S52, washing the root culture medium of the refined tissue culture seedlings, planting them in a plug tray filled with peat, watering them for the first time until the soil can be squeezed into a ball without dripping, and then building a small arch shed with a film and culturing them under natural light;

[0053] During the seedling hardening treatment, the bottle body is rotated regularly to ensure that the tissue culture seedlings receive even light, and nitrogen-containing nutrient solution is added after transplanting to promote seedling growth.

[0054] In summary, the present application can realize a rapid propagation system for Phalaenopsis, shorten the output cycle of Phalaenopsis, and reduce production costs; by treating with 75% ethanol for 30 seconds and 0.1% mercuric chloride for 5 minutes, the contamination rate of tissue culture inoculation can be reduced to less than 10%, thereby reducing the contamination rate and saving costs; scale cutting method: longitudinal cutting method increases the induction rate of adventitious buds and bulblets, while reducing the cutting surface and thus reducing the contamination rate; dark culture for 10 days is conducive to the formation of bulblets, providing possibility and technical support for exploring the rapid propagation of Phalaenopsis tissue culture.

[0055] The above embodiments are only used to illustrate the technical solutions of the embodiments of the present application, rather than to limit them. Although the embodiments of the present application are described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents, without departing from the spirit and scope defined by the claims of the present application.

Claims

1. A rapid propagation method for Phalaenopsis tissue culture, characterized in that: The method comprises the following steps: Step S1: Selection and processing of explants: S11. Select the top young stem segments and healthy bulbs of robust and disease-free Phalaenopsis plants as explants; S12, pre-treating the selected explants at low temperature for two weeks, taking them out and cleaning them, soaking them in thiophanate-methyl, then rinsing them with running water, and then rinsing them with sterile water; S13, sterilizing the cleaned explants, then rinsing with sterile water, then treating with mercuric chloride, and then wiping the surface moisture with sterile filter paper; S14, using a sterilized blade to cut the treated explants longitudinally into bulbs with a width of 3-4 mm, and cut the stem segments into explants with a thickness of about 2-3 mm; Step S2, primary induction culture: S21, inoculating the treated explants into primary induction medium; S22, 10 days of dark culture at the beginning of the culture, followed by 45 days of culture under the condition of light intensity of 3000 lux; S23, screening out adventitious buds and bulblets that meet the standards, the standards being that the adventitious buds are 3-4 cm high and the bulblets are 1.5-2 cm in diameter; Step S3, cluster bud induction and subculture proliferation culture: S31, inoculating the adventitious buds that grow well in the primary culture into a cluster bud induction medium and culturing them under light for 30 days; S32, inoculating the clustered buds into the subculture proliferation medium and culturing them under light for 30 days; Step S4, rooting culture: cut the small cluster of seedlings into individual plants and inoculate them into rooting medium, culture them under light for 15 days, observe the growth of the root system, and ensure that the root system is well developed; Step S5: Transplanting and hardening: S51. Move the Phalaenopsis tissue culture seedlings with a root length of 3-5 cm to a place with scattered light for seedling hardening. After one week, gradually move them to a place with better light. S52. Wash the root culture medium of the prepared tissue culture seedlings and plant them in a hole tray filled with peat. Water them for the first time until the soil can be squeezed into a ball without dripping. Build a small arch shed and cover it with film and culture them under natural light.

2. The rapid propagation method of Phalaenopsis tissue culture according to claim 1, characterized in that: In step S12, the low-temperature pretreatment temperature is 5°C, and the explants are cleaned by soaking them in 500-fold thiophanate-methyl for 20 minutes, then rinsing them with running water for 30 minutes, and then rinsing them with sterile water for 4 times; In step S13, the cleaned explants are immersed in 75% ethanol for 30 seconds for disinfection, then rinsed with sterile water for 4 times, then treated with 0.1% mercuric chloride for 5 minutes, and finally wiped dry with sterile filter paper.

3. A rapid propagation method for Phalaenopsis tissue culture according to claim 2, characterized in that: In step S13, the method for disinfecting the explant further includes placing the explant on a clean bench and irradiating it with ultraviolet light for 30 minutes and then ventilating it for 15 minutes for disinfection.

4. The rapid propagation method of Phalaenopsis tissue culture according to claim 1, characterized in that: In step S2, the components of the primary induction medium are: MS medium, 25 g / L sucrose, 6 g / L agar, 1 mg / L 6-benzylaminopurine and 0.2 mg / L α-naphthylacetic acid.

5. The rapid propagation method of Phalaenopsis tissue culture according to claim 4, characterized in that: In step S2, the culture conditions of the primary induction culture medium are: LED lighting intensity 2000-3000 lux, 10 hours of illumination per day, and the culture temperature is maintained at 23±2°C.

6. The method for rapid propagation of Phalaenopsis tissue culture according to claim 1, characterized in that: In step S31, the components of the clustered shoot induction medium are: MS medium, 6-BA 1.5 mg / L and NAA 0.1 mg / L; In step S32, the components of the subculture proliferation medium are: MS medium, 6-BA 0.5 mg / L and NAA 0.05 mg / L.

7. The rapid propagation method of Phalaenopsis tissue culture according to claim 6, characterized in that: In step S3, during the cluster bud induction and subculture proliferation culture stage, LED lighting is used and the culture temperature is 23±2°C.

8. The rapid propagation method of Phalaenopsis tissue culture according to claim 1, characterized in that: In step S4, the ingredients of the rooting medium are: MS medium, sucrose 20 g / L, agar 4 g / L, and NAA 0.3 mg / L.

9. The method for rapid propagation of Phalaenopsis tissue culture according to claim 8, characterized in that: In the step S4, the rooting culture is illuminated for 8 hours per day, the culture temperature is maintained at 25±1° C., and the relative humidity is maintained at 60%-75%.

10. The method for rapid propagation of Phalaenopsis tissue culture according to claim 1, characterized in that: In step S5, the bottle body is rotated regularly during the seedling hardening treatment to ensure that the tissue culture seedlings receive light evenly, and nitrogen-containing nutrient solution is added after transplanting to promote the growth of the seedlings.

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