Method for rapid propagation of phalaenopsis by tissue culture and seedling raising

Abstract

The application discloses a kind of flame phalaenopsis tissue culture rapid propagation and its seedling raising method.The method includes the steps of explant selection, explant disinfection, adventitious bud induction, adventitious bud proliferation, subculture, rooting culture and test-tube seedling transplanting.The flame phalaenopsis seedling cultivated by the application has stable genetic traits, maintains the characteristics of parent, has many advantages including invariability, less investment, high output, short cycle, etc., and can realize large-scale seedling production.

Description

Technical Field

[0001] This invention belongs to the field of rapid propagation technology of tissue culture, specifically relating to a method for rapid propagation of Phalaenopsis flamingo through tissue culture and its seedling cultivation. Background Technology

[0002] Renanthopsis is an intergeneric orchid formed through intergenous hybridization between Renanthopsis and Phalaenopsis. Currently, 163 intergeneric hybrid combinations of Renanthopsis and Phalaenopsis are registered with the Royal Horticultural Society (RHS). Renanthopsis combines the excellent characteristics of both, possessing the vibrant flower colors and strong plant resistance of Renanthopsis, and the rich flower colors and long flowering period of Phalaenopsis. Its overall plant and flowering traits lean towards Phalaenopsis, making it very popular with consumers. Currently, there is very little research on the rapid propagation of Renanthopsis through tissue culture, both domestically and internationally. The method of obtaining complete plants through aseptic sowing of seeds obtained from hybridization breeding to form callus and protocorms, followed by differentiation, seedling strengthening, and rooting culture, results in a high degree of segregation in the offspring, with significant individual differences, making it difficult to achieve a uniform population of plants, which is detrimental to the development and utilization of Renanthopsis in the flower market. Therefore, researching rapid propagation technology for clonal reproduction of Phalaenopsis flamingo and cultivating plants with stable and consistent traits is crucial for the development of Phalaenopsis flamingo, meeting the needs of the orchid market, and realizing large-scale artificial production. Summary of the Invention

[0003] In view of this, the technical problem solved by the present invention is to provide a method for rapid propagation and seedling cultivation of *Phalaenopsis flamingo* through tissue culture. By using the clustered bud approach for tissue culture of *Phalaenopsis flamingo*, a large number of genetically stable test-tube seedlings can be obtained, maintaining good parental characteristics, and enabling large-scale seedling production in a relatively short period. Therefore, this provides an effective method for the production of *Phalaenopsis flamingo* seedlings and lays a good foundation for the selection of superior varieties of *Phalaenopsis flamingo* and the cultivation of new intergeneric orchid varieties.

[0004] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0005] A method for rapid propagation of Phalaenopsis flamingo flower through tissue culture and its seedling cultivation includes the following steps:

[0006] (1) Selection of explants: Select flower stalks of the flame butterfly flower, preferably the flower stalks before the flower buds emerge, and use the flower stalk stem segments with axillary buds as explants;

[0007] (2) Disinfection of explants: After disinfecting the surface of the flower stalk and cleaning it with sterile water, cut the flower stalk into 2-3 cm long stem segments with axillary buds in the middle. Remove the outer bracts of the axillary buds on the flower stalk stem segments with a sterilized blade, immerse them in mercuric chloride solution for sterilization for 8-10 minutes, shaking them continuously during the process. Then, take out the flower stalk stem segments and rinse them with sterile water 3-5 times. After taking them out, use sterile filter paper to absorb the residual water on the flower stalk stem segments. Use a sterilized blade to cut off the tissue at both ends of the flower stalk stem segments that came into contact with the disinfectant solution to obtain the sterilized flower stalk stem segments.

[0008] (3) Bud induction: The sterilized flower stalk stem segments were inoculated into the induction medium and cultured for 30 to 50 days. When the buds turned green and enlarged, the next step of proliferation culture was carried out. The induction medium was 1 / 2 MS + 6-BA 0.5 to 1.0 mg / L + NAA 0.1 to 0.3 mg / L + coconut juice 10.00 to 20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5 to 5.8.

[0009] (4) Bud proliferation: Cut off the induced buds and transfer them to the proliferation medium for 60-90 days to obtain clustered buds. The proliferation medium is MS + 6-BA 1.0-1.5 mg / L + TDZ 1.0-1.5 mg / L + coconut juice 10.00-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8;

[0010] (5) Subculture: The clustered shoots were transferred to the subculture medium to obtain subculture seedlings. The subculture medium was MS + 6-BA 1.0-1.5 mg / L + TDZ 1.0-1.5 mg / L + coconut juice 10.00-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8;

[0011] (6) Rooting culture: Subcultured seedlings were cultured in rooting medium for 90-120 days to obtain rooted test-tube seedlings. The rooting medium was 1 / 2 MS + NAA 0.3-0.5 mg / L + activated carbon 0.5-1.0 g / L + coconut juice 10.0-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8.

[0012] (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 2-4 leaves, 3-5 cm long, 1-2 cm wide, 2-5 cm long roots, and 2-4 roots, they are placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 15-20 days of hardening, they are transplanted out of the bottle.

[0013] Preferably, in step (2), the surface disinfection of the flower stalk is specifically carried out by wiping the surface of the flower stalk with 75% alcohol by mass.

[0014] Preferably, in step (2), the concentration of the mercuric chloride solution is 0.1% by mass.

[0015] Preferably, in step (3), the conditions for induction culture are: culture temperature 25-28℃, dark culture for 20 days in the early stage, and culture for 30 days in the later stage under light intensity of 2000-2500 lx and light time of 12h / d.

[0016] Preferably, in step (4), the conditions for proliferation culture are: culture temperature 25-28℃, light intensity 2000-2500 lx, and light duration 12h / d.

[0017] Preferably, in step (5), the subculture is specifically: the subculture is carried out once every 60 to 90 days, and the number of subcultures is controlled within 15 times. The subculture conditions are: culture temperature 25 to 28°C, light intensity 2000 to 2500 lx, and light time 12h / d.

[0018] Preferably, in step (6), the conditions for rooting culture are: culture temperature 25-28℃, light intensity 2000-2500 lx, and light duration 12h / d.

[0019] Preferably, in step (7), the seedling transplantation specifically involves: using tweezers to remove the test-tube seedlings from the bottle and cleaning the culture medium on the roots, then soaking them in a 0.01% potassium permanganate solution for 2-3 minutes, taking them out and letting them dry, and then planting them directly in a small transparent plastic cup with sphagnum moss at a planting temperature of 20-28℃ and a humidity of 60-80%. Two days after transplanting the test-tube seedlings, the leaves are sprayed with water and a 0.1% carbendazim solution is sprayed, and then routine management is carried out.

[0020] The beneficial effects of this invention are:

[0021] The method of this invention is easy to operate, has low production costs, does not pollute the environment, and can realize large-scale seedling production. The flame orchid seedlings cultivated by this invention have stable genetic traits, maintain the characteristics of the parent plants, and have many advantages, including invariance, low input, high output, and short cycle. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1

[0024] (1) Selection of explants: Select flower stalks of Phalaenopsis flamingo flower, preferably the flower stalks before the flower buds emerge, and use the flower stalk stem segments with axillary buds as explants.

[0025] (2) Disinfection of explants: After wiping the surface of the flower stalk with 75% alcohol and cleaning it with sterile water, cut the flower stalk into 2-3 cm long stem segments with axillary buds in the middle. Remove the outer bracts of the axillary buds on the flower stalk stem segments with a sterilized blade, immerse them in a 0.1% mercuric chloride solution for 8 minutes for sterilization, shaking them continuously during the process. Then take out the flower stalk stem segments and rinse them 3 times with sterile water. After taking them out, use sterile filter paper to absorb the residual water on the flower stalk stem segments. Cut off 0.5 cm from each end of the flower stalk stem segments with a sterilized blade to obtain the sterilized flower stalk stem segments.

[0026] (3) Bud induction: The sterilized flower stalk segments were inoculated into the induction medium for culture. The induction medium was...

[0027] The culture medium consisted of 1 / 2 MS medium containing 0.5 mg / L 6-BA, 0.1 mg / L NAA, 10% v / v coconut juice, 30.0 g / L sucrose, and 7.0 g / L agar, with a pH of 5.5-5.8. The culture temperature was 25-28℃. Initially, the medium was cultured in the dark for 20 days, followed by 30 days of culture under a light intensity of 2000-2500 lx and a light duration of 12 h / d. During the culture process, the buds turned green and enlarged. After 50 days, the next stage of proliferation culture was initiated.

[0028] (4) Bud proliferation: The induced buds were cut off and transferred to a proliferation medium for culture. The proliferation medium consisted of MS + 6-BA 1.0 mg / L + TDZ 1.0 mg / L + coconut juice 10% v / v + sucrose 30.0 g / L + agar 7.0 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d. After 60-90 days of culture, clustered buds were obtained, with a proliferation ratio of up to 2.01.

[0029] (5) Subculture: The bud clusters were transferred to a subculture medium and cultured. Subculture was performed every 60-90 days, with the number of subcultures controlled within 15. The subculture medium was MS + 6-BA 1.0 mg / L + TDZ 1.0 mg / L + coconut juice 10% v / v + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0030] (6) Rooting Culture: Once a certain number of subcultured seedlings have been obtained, rooting culture is carried out using rooting medium. The rooting medium consists of 1 / 2 MS medium + 0.3 mg / L NAA + 0.5 g / L activated carbon + 10% v / v coconut juice + 30.0 g / L sucrose + 7.0 g / L agar, with a pH of 5.5-5.8. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d. After 90-120 days of culture, rooted test-tube seedlings are obtained.

[0031] (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 2-4 leaves, 3-5 cm long and 1-2 cm wide, and 2-5 cm long and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 15-20 days of hardening, they can be transplanted. Generally, transplanting is carried out in spring, from March to May. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them and let them dry. Then plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. If the temperature is higher than 30℃, fans and water curtains must be used to cool them down. The survival rate of transplanting can reach more than 92%.

[0032] Example 2

[0033] (1) Selection of explants: Select flower stalks of Phalaenopsis flamingo flower, preferably the flower stalks before the flower buds emerge, and use the flower stalk stem segments with axillary buds as explants.

[0034] (2) Disinfection of explants: After wiping the surface of the flower stalk with 75% alcohol and cleaning it with sterile water, cut the flower stalk into 2-3 cm long stem segments with axillary buds in the middle. Remove the outer bracts of the axillary buds on the flower stalk stem segments with a sterilized blade, immerse them in a 0.1% mercuric chloride solution for 9 minutes for sterilization, shaking them continuously during the process. Then take out the flower stalk stem segments and rinse them 4 times with sterile water. After taking them out, use sterile filter paper to absorb the residual water on the flower stalk stem segments. Cut off 0.5 cm from each end of the flower stalk stem segments with a sterilized blade to obtain the sterilized flower stalk stem segments.

[0035] (3) Bud induction: The sterilized flower stalk segments were inoculated into the induction medium for culture. The induction medium was...

[0036] The culture medium consisted of 1 / 2 MS medium containing 0.75 mg / L 6-BA, 0.2 mg / L NAA, 15% v / v coconut juice, 30.0 g / L sucrose, and 7.0 g / L agar, with a pH of 5.5-5.8. The culture temperature was 25-28℃. Initially, the medium was cultured in the dark for 20 days, followed by 30 days of culture under a light intensity of 2000-2500 lx and a light duration of 12 h / d. During the culture process, the buds turned green and enlarged. After 50 days, the next stage of proliferation culture was carried out.

[0037] (4) Bud proliferation: The induced buds were cut off and transferred to a proliferation medium for culture. The proliferation medium consisted of MS + 6-BA 1.25 mg / L + TDZ 1.25 mg / L + coconut juice 15% v / v + sucrose 30.0 g / L + agar 7.0 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d. After 60-90 days of culture, clustered buds were obtained, with a proliferation ratio of up to 2.52.

[0038] (5) Subculture: The clustered shoots were transferred to a subculture medium and cultured. Subculture was performed every 60-90 days, with the number of subcultures controlled within 15 times. The subculture medium was MS + 6-BA 1.25 mg / L + TDZ 1.25 mg / L + coconut juice 15% v / v + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0039] (6) Rooting Culture: Once a certain number of subcultured seedlings have been obtained, rooting culture is carried out using a rooting medium consisting of 1 / 2 MS medium + 0.4 mg / L NAA + 0.75 g / L activated carbon + 15% v / v coconut juice + 30.0 g / L sucrose + 7.0 g / L agar, with a pH of 5.5-5.8. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d. Rooted test-tube seedlings are obtained after 90-120 days of culture.

[0040] (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 2-4 leaves, 3-5 cm long and 1-2 cm wide, and 2-5 cm long and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 15-20 days of hardening, they can be transplanted. Generally, transplanting is carried out in spring, from March to May. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them and let them dry. Then plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. If the temperature is higher than 30℃, fans and water curtains must be used to cool them down. The survival rate of transplanting can reach more than 92%.

[0041] Example 3

[0042] (1) Selection of explants: Select flower stalks of Phalaenopsis flamingo flower, preferably the flower stalks before the flower buds emerge, and use the flower stalk stem segments with axillary buds as explants.

[0043] (2) Disinfection of explants: After wiping the surface of the flower stalk with 75% alcohol and cleaning it with sterile water, cut the flower stalk into 2-3 cm long stem segments with axillary buds in the middle. Remove the outer bracts of the axillary buds on the flower stalk stem segments with a sterilized blade, immerse them in a 0.1% mercuric chloride solution for 10 minutes for sterilization, shaking them continuously during the process. Then take out the flower stalk stem segments and rinse them 5 times with sterile water. After taking them out, use sterile filter paper to absorb the residual water on the flower stalk stem segments. Cut off 0.5 cm from each end of the flower stalk stem segments with a sterilized blade to obtain the sterilized flower stalk stem segments.

[0044] (3) Bud induction: The sterilized flower stalk segments were inoculated into the induction medium for culture. The induction medium was...

[0045] The formula consisted of 1 / 2 MS medium containing 1.0 mg / L 6-BA, 0.3 mg / L NAA, 20% v / v coconut juice, 30.0 g / L sucrose, and 7.0 g / L agar, with a pH of 5.5-5.8. The culture temperature was 25-28℃. The initial culture was in the dark for 20 days, followed by 30 days of culture under a light intensity of 2000-2500 lx and a light duration of 12 h / d. During the culture process, the buds turned green and enlarged. After 50 days, the next step of proliferation culture was carried out.

[0046] (4) Bud proliferation: The induced buds were cut off and transferred to a proliferation medium for culture. The proliferation medium consisted of MS + 6-BA 1.5 mg / L + TDZ 1.5 mg / L + coconut juice 20% v / v + sucrose 30.0 g / L + agar 7.0 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d. After 60-90 days of culture, clustered buds were obtained, with a proliferation ratio of up to 3.03.

[0047] (5) Subculture: The bud clusters were transferred to a subculture medium and cultured. Subculture was performed every 60-90 days, with the number of subcultures controlled within 15. The subculture medium was MS + 6-BA 1.5 mg / L + TDZ 1.5 mg / L + coconut juice 20% v / v + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0048] (6) Rooting Culture: Once a certain number of subcultured seedlings have been obtained, rooting culture is carried out using rooting medium. The rooting medium consists of 1 / 2 MS medium + NAA 0.5 mg / L + activated carbon 1.0 g / L + coconut juice 20% v / v + sucrose 30.0 g / L + agar 7.0 g / L, with a pH of 5.5-5.8. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d. After 90-120 days of culture, rooted test-tube seedlings are obtained.

[0049] (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 2-4 leaves, 3-5 cm long and 1-2 cm wide, and 2-5 cm long and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 15-20 days of hardening, they can be transplanted. Generally, transplanting is carried out in spring, from March to May. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them and let them dry. Then plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. If the temperature is higher than 30℃, fans and water curtains must be used to cool them down. The survival rate of transplanting can reach more than 92%.

[0050] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for rapid propagation of Phalaenopsis flamingo flower through tissue culture and its seedling cultivation, characterized in that, Includes the following steps: (1) Selection of explants: Select flower stalks of the flame butterfly flower, preferably the flower stalks before the flower buds emerge, and use the flower stalk stem segments with axillary buds as explants; (2) Disinfection of explants: After disinfecting the surface of the flower stalk and cleaning it with sterile water, cut the flower stalk into 2-3 cm long stem segments with axillary buds in the middle. Remove the outer bracts of the axillary buds on the flower stalk stem segments with a sterilized blade, immerse them in mercuric chloride solution for sterilization for 8-10 minutes, shaking them continuously during the process. Then, take out the flower stalk stem segments and rinse them with sterile water 3-5 times. After taking them out, use sterile filter paper to absorb the residual water on the flower stalk stem segments. Use a sterilized blade to cut off the tissue at both ends of the flower stalk stem segments that came into contact with the disinfectant solution to obtain the sterilized flower stalk stem segments. (3) Bud induction: The sterilized flower stalk stem segments were inoculated into the induction medium and cultured for 30 to 50 days. When the buds turned green and enlarged, the next step of proliferation culture was carried out. The induction medium was 1 / 2 MS + 6-BA 0.5 to 1.0 mg / L + NAA 0.1 to 0.3 mg / L + coconut juice 10.00 to 20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5 to 5.

8. (4) Bud proliferation: Cut off the induced buds and transfer them to the proliferation medium for 60-90 days to obtain clustered buds. The proliferation medium is MS + 6-BA 1.0-1.5 mg / L + TDZ 1.0-1.5 mg / L + coconut juice 10.00-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8; (5) Subculture: The clustered shoots were transferred to the subculture medium to obtain subculture seedlings. The subculture medium was MS + 6-BA 1.0-1.5 mg / L + TDZ 1.0-1.5 mg / L + coconut juice 10.00-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.8; (6) Rooting culture: Subcultured seedlings were cultured in rooting medium for 90-120 days to obtain rooted test-tube seedlings. The rooting medium was 1 / 2 MS + NAA 0.3-0.5 mg / L + activated carbon 0.5-1.0 g / L + coconut juice 10.0-20.0% + sucrose 30.0 g / L + agar 7.0 g / L, pH 5.5-5.

8. (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 2-4 leaves, 3-5 cm long, 1-2 cm wide, 2-5 cm long roots, and 2-4 roots, they are placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 15-20 days of hardening, they are transplanted out of the bottle.

2. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (2), the concentration of the mercuric chloride solution is 0.1% by mass.

3. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (3), the conditions for induction culture are: culture temperature 25-28℃, dark culture for 20 days in the early stage, and culture for 30 days in the later stage under light intensity of 2000-2500 lx and light time of 12h / d.

4. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (4), the conditions for proliferation culture are: culture temperature 25-28℃, light intensity 2000-2500 lx, and light duration 12h / d.

5. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (5), the subculture is specifically carried out by subculturing once every 60 to 90 days, and the number of subcultures is controlled within 15 times. The subculture conditions are: culture temperature 25 to 28℃, light intensity 2000 to 2500 lx, and light time 12h / d.

6. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (6), the conditions for rooting culture are: culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

7. The method for rapid propagation and seedling cultivation of *Phalaenopsis flamingoides* via tissue culture according to claim 1, characterized in that... In step (7), the seedling transplantation is specifically as follows: use tweezers to remove the test tube seedlings from the bottle and clean the culture medium on the roots, then soak them in a 0.01% potassium permanganate solution for 2-3 minutes, take them out and let them dry, and then plant them directly in a small transparent plastic cup with sphagnum moss. The planting temperature is 20-28℃ and the humidity is 60-80%. Two days after transplanting the test tube seedlings, spray water on the leaves and spray with a 0.1% carbendazim solution. After that, carry out routine management.