Method for promoting seed germination and seedling formation of paphiopedilum by wide-energy-spectrum white light neutron irradiation

Abstract

The application discloses a method for promoting seed germination and seedling formation of Paphiopedilum by wide-energy-spectrum white light neutron irradiation. The method comprises the following steps: after sterilization, Paphiopedilum seeds are subjected to wide-energy-spectrum white light neutron irradiation treatment, are sowed into sowing culture medium for seed germination culture, are transferred to a strong seedling culture medium for strong seedling culture, and are transplanted. The wide-energy-spectrum white light spectrum is 1eV-200MeV. The energy spectrum of the Chinese spallation neutron source anti-angle white light neutron is 1eV-200MeV, and the most probable energy is 1MeV, which has a spectrum property very close to that of atmospheric neutrons. Low-dose irradiation significantly promotes the germination rate and seedling formation rate of Paphiopedilum. The germination rate is 80.1% under an irradiation dose of 15 minutes, the germination rate is 86.9% under an irradiation dose of 30 minutes, and the germination rate is 93.5% under an irradiation dose of 1 hour.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of Paphiopedilum breeding, and particularly relates to a method for promoting seed germination and seedling growth of Paphiopedilum by wide-energy-spectrum white light neutron irradiation. BACKGROUND

[0002] Paphiopedilum is one of the most endangered plant groups in the world. The seed of Paphiopedilum lacks endosperm and needs to rely on fungal symbiosis to germinate, and the germination rate under natural conditions is extremely low. The artificial breeding of Paphiopedilum adopts the method of sterile sowing, and the germination rate and seedling growth rate are not high. Paphiopedilum is a very rare high-grade flower with great development potential. The difficulty in breeding greatly hinders the conservation and commercial production of Paphiopedilum. In recent years, different seed maturity and pretreatment, different culture medium and pH, different hormones and organic additives have been used to improve the germination rate of Paphiopedilum, but the effect is not high. SUMMARY

[0003] The present application provides a method for promoting seed germination and seedling growth of Paphiopedilum by wide-energy-spectrum white light neutron irradiation. The Paphiopedilum seeds treated by wide-energy-spectrum white light neutron irradiation are sowed by sterile sowing according to the technical scheme of the present application.

[0004] In order to achieve the purpose of solving the above problems, the present application adopts the following technical scheme:

[0005] A method for promoting seed germination and seedling growth of Paphiopedilum by wide-energy-spectrum white light neutron irradiation, comprising the following steps: sterilizing Paphiopedilum seeds and then treating them by wide-energy-spectrum white light neutron irradiation, sowing them in sowing medium for seed germination culture, transferring them to seedling culture medium for seedling culture, and transplanting them; the wide-energy-spectrum white light has a spectrum of 1eV-200MeV.

[0006] Preferably, the wide-energy-spectrum white light has a spectrum peak near 1MeV, and when the beam power is 125kW, the beam spot is 50x50mm, and the fluence rate is 2x10 7 n / cm 2 / s.

[0007] Preferably, the irradiation time is 15-60 minutes.

[0008] Preferably, the Paphiopedilum seeds are taken from the capsule of Paphiopedilum 350 days after pollination.

[0009] Preferably, the Paphiopedilum is Paphiopedilum spicerianum.

[0010] Preferably, the seeding medium is: Florabak 1.5 g / L + sucrose 15 g / L + agar 4.8 g / L + activated carbon 0.5 g / L + peptone 2 g / L + inositol 0.1 g / L + coconut juice 50 mL / L + B5 vitamins 1 mL (1000 times) / L + iron salt 10 mL (100 times iron salt) / L + NAA 1 mg / L, pH 6.0; the seedling medium is: Florabak 3.0 g / L + NAA 1.0 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + agar 5 g / L + 10% banana homogenate by mass, pH 5.4.

[0011] Preferably, the seed germination culture condition is: dark culture for 40 days after sowing, culture temperature is 26-28℃, 40 days later, light intensity is 2000-2500 Lx, daily light is 12 h / d, temperature is 26-28℃; the seedling culture condition is: light intensity is 2000-2500 Lx, daily light is 12 h / d, temperature is 26-28℃.

[0012] Preferably, the transplanting is when the test-tube seedling grows to 8-12 cm, the out-bottle transplanting, the transplanting matrix is a mixed matrix of planting stone and pine bark at a volume ratio of 1:9.

[0013] Preferably, the method comprises the following steps:

[0014] (1) disinfect and aseptically distribute Paphiopedilum seed;

[0015] (2) treat the distributed seed with wide-spectrum white light neutron irradiation, irradiation time is 0, 15, 30 or 60 minutes;

[0016] (3) aseptically sow the seed treated with irradiation on a germination medium;

[0017] (4) transfer the seedling formed by germination to a seedling culture medium for seedling culture;

[0018] (5) when the seedling grows to about 10 cm, out-bottle transplanting, and then normal management.

[0019] Preferably, the distribution method of Paphiopedilum seed is carried out in a super-clean workbench, the tube opening is sealed with PM-996 culture dish sealing film and then placed in an enzyme-free and sterile sealing bag for carrying

[0020] Preferably, the specific steps are as follows:

[0021] The capsule of Paphiopedilum insigne is disinfected 350 days after pollination in a clean bench: first, the surface of the capsule is wiped with cotton dipped in 75% (V / V) alcohol, then the capsule is picked up with sterilized tweezers and put into a sterilized glass bottle, 75% (V / V) alcohol is poured into the glass bottle to immerse the capsule for 30 seconds, during which the glass bottle is shaken constantly, the alcohol is poured out, and the capsule is washed with sterile water once; then 0.1% mercury is poured into the glass bottle and two drops of Tween 20 are added to immerse the capsule for 20 minutes, during which the glass bottle is shaken constantly, the mercury is poured out, and the capsule is washed with sterile water three times, and after the capsule is dried, the capsule is cut open with a sterilized scalpel, and the seeds in the capsule are put into a 1.5 mL enzyme-free sterile centrifuge tube;

[0022] The centrifuge tube containing the seeds is placed in the center of the beam spot for irradiation, the irradiation time is 15-60 minutes, the irradiation beam line is the anti-angle white light neutron beam line, the beam line energy spectrum is 1 eV-200 MeV, the energy spectrum peak is near 1 MeV, when the beam power is 125 kW, the beam spot is 50*50 mm, and the fluence rate is 2*10 7 n / cm 2 / s;

[0023] An agar solution with an m / v of 0.01% is prepared, sterilized at 121°C for 20 minutes, and placed to cool but not solidify, the seeds are dispersed into the agar solution, and a uniform suspension of the seeds is prepared, the agar suspension of the seeds is sucked with a sterilized gun head and dropped onto the culture medium, and the seed suspension is evenly distributed on the surface of the culture medium by gently shaking, the seed culture medium is: flower treasure No. 1 1.5 g / L + sucrose 15 g / L + agar 4.8 g / L + activated carbon 0.5 g / L + peptone 2 g / L + inositol 0.1 g / L + coconut water 50 mL / L + B5 vitamin 1 mL (1000 times) / L + iron salt 10 mL (100 times iron salt) / L + NAA 1 mg / L, pH 6.0, after sowing, dark culture for 40 days, temperature 26-28°C, after 40 days, light for 12 h / d, light intensity 2000-2500 Lx, temperature 26-28°C;

[0024] After sowing for 4 months, the seedlings are transferred to a strong seedling culture medium for strong seedling culture, the strong seedling culture medium is: flower treasure No. 1 3.0 g / L + NAA 1.0 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + agar 5 g / L + mass ratio 10% banana homogenate, pH 5.4, light intensity 2000-2500 Lx, daily light for 12 h / d, temperature 26-28;

[0025] When the seedlings grow to 8-12 cm, they are taken out of the bottle and transplanted, the test tube seedling transplanting medium is a mixture of plant gold stone and pine bark at a volume ratio of 1:9, the pine bark is rinsed and soaked before use, and the plant gold stone is rinsed before use.

[0026] The application also provides the application of the above method in promoting seed germination and seedling formation of Phalaenopsis.

[0027] Compared with the prior art, the application has the advantages and positive effects that:

[0028] The energy spectrum of the anti-angle white light neutron of the Chinese Spallation Neutron Source is 1eV-200MeV, and the peak value of the energy spectrum is near 1MeV. The energy spectrum property is very close to that of atmospheric neutrons. Neutrons are neutral particles generated by nuclear reactions, and have high biological effects. Low-dose white light neutron irradiation significantly promotes the germination rate and seedling formation rate of Phalaenopsis. In the method for promoting seed germination and seedling formation of Phalaenopsis by wide-energy-spectrum white light neutron irradiation provided by the application, the germination rate of Phalaenopsis is 80.1% under an irradiation dose of 15 minutes, the germination rate is 86.9% under an irradiation dose of 30 minutes, and the germination rate is 93.5% under an irradiation dose of 1 hour. The germination rate of the control group is 77.8%. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The results of 60-day sowing observation of Phalaenopsis seed treated by wide-energy-spectrum white light neutron irradiation are provided in the application.

[0030] Figure 2 The test tube seedlings of Phalaenopsis seed treated by wide-energy-spectrum white light neutron irradiation and sowed for 120 days are provided in the application.

[0031] Figure 3 The test tube seedlings of Phalaenopsis seed treated by wide-energy-spectrum white light neutron irradiation and sowed for 240 days and the seedlings transplanted for 30 days are provided in the application. DETAILED DESCRIPTION

[0032] The following examples are further illustrations of the application, but are not limiting of the application.

[0033] Example 1:

[0034] A method for promoting seed germination and seedling formation of Phalaenopsis by wide-energy-spectrum white light neutron irradiation comprises the following steps:

[0035] 1. Seed disinfection and sub-packaging

[0036] The seed of Paphiopedilum exapparitum is the capsule 350 days after pollination. The seed disinfection method is to disinfect the surface of the capsule in a clean bench: first wipe the surface of the capsule with cotton dipped in 75% (V / V) alcohol, then use a sterilized forceps to clamp the capsule into a sterilized glass bottle, pour 75% (V / V) alcohol to immerse the capsule for 30 seconds, shake the glass bottle constantly during the process, pour out the alcohol, and wash it with sterile water once; then pour 0.1% mercury and add two drops of Tween 20 to immerse the capsule for 20 minutes, shake the glass bottle constantly during the process, pour out the mercury, and wash it with sterile water three times. After the capsule is dried, use a sterilized scalpel to cut open the capsule, and evenly distribute the seeds in 3 capsules into 3 groups of 1.5 mL non-enzyme sterile centrifuge tubes, with each capsule corresponding to one group, a total of 3 groups of 9 tubes. Seal the tube opening with PM-996 Petri dish sealing film, then put it into a non-enzyme sterile sealing bag, and carry it to the China Spallation Neutron Source for irradiation.

[0037] 2. Irradiation treatment

[0038] The irradiation experiment was carried out at the China Spallation Neutron Source. One tube was randomly taken from each group corresponding to each capsule, and 3 tubes of seeds were fixed horizontally on the central irradiation table, and irradiated at the same time, with an irradiation time of 15 minutes. The irradiation beam line was an anti-angle white light neutron beam line, with a beam line energy spectrum of 1eV-200MeV and a peak energy spectrum near 1MeV. When the beam power was 125kW, the beam spot was 50x50mm, the fluence rate was 2x10 7 n / cm 2 / s. The control group was not irradiated.

[0039] 3. Seed sowing

[0040] After irradiation, the seeds were immediately taken to the tissue culture room of South China Botanical Garden for sowing. 0.01% (m / v) agar solution was prepared, sterilized at 121°C for 20 minutes, and placed until it was cool but not solidified. Each tube of seeds was separately dispersed into the agar solution, and a uniform suspension of seeds was prepared, and was marked as a group after sowing. The agar suspension of seeds was taken with a sterilized syringe, and was dropped onto the culture medium, and was gently shaken to evenly distribute the seed suspension on the surface of the culture medium. The seed sowing medium was H26 medium (Huaobao No. 1 1.5g / L + sucrose 15g / L + agar 4.8g / L + activated carbon 0.5g / L + peptone 2g / L + inositol 0.1g / L + coconut water 50mL / L + B5 vitamin 1mL (1000 times) / L + iron salt 10mL (100 times iron salt) / L + NAA 1mg / L), pH 6.0. Each tube of seeds was sowed in not less than 10 bottles, and was numbered. After sowing, dark culture was carried out for 40 days with black cloth, and the temperature was 26-28°C. After 40 days, the black cloth was removed, the light intensity was 2000-2500Lx, the daily light was 12h / d, and the temperature remained unchanged.

[0041] 4. Germination rate statistics

[0042] Zettler (2002) proposed a grading system for orchid seed germination: grade 0 indicates the embryo has not yet germinated; grade 1 indicates the embryo has expanded but not broken through the seed coat; grade 2 indicates expansion to the point of breaking through the seed coat; grade 3 indicates the appearance of a bulge at the stem apex; grade 4 indicates the differentiation and development of the first leaf; and grade 5 indicates the elongation of the first leaf. Germination at stage 4 or above is considered successful. Sixty days after sowing, three bottles were randomly selected from each seed group. Approximately 10 mm x 10 mm of culture medium was removed using a sterilized spoon. Germination rates were counted under a stereomicroscope. This was repeated three times. The germination rate was 84.1%.

[0043] 5. Cultivation of strong seedlings

[0044] Four months after sowing, the seedlings were transferred to a seedling growth medium for seedling growth. The medium was Huabao No. 1 medium (3.0 g / L Huabao No. 1 + 1.0 mg / L NAA + 0.5 g / L activated carbon + 30 g / L sucrose + 5 g / L agar + 10% banana homogenate by weight, pH 5.4). The light intensity was 2000-2500 Lx, the daily light intensity was 12 hours / day, and the temperature was 26-28 degrees.

[0045] 6. Transplanting of test tube seedlings

[0046] When the seedlings reach approximately 10 cm, they are removed from the jars and transplanted. The medium for transplanting the test tube seedlings is a mixture of phytosanitary stone and pine bark (volume ratio 1:9). The pine bark is thoroughly soaked and the phytosanitary stone and pine bark are thoroughly rinsed. Plant them in 1.7-inch pots. Maintain appropriate humidity and ventilation. After two weeks, move them into a greenhouse for cultivation and receive normal watering, fertilizer, and pesticide management.

[0047] Example 2:

[0048] The irradiation time in the irradiation treatment step is 30 minutes, and the remaining steps are the same as in Example 1.

[0049] In this embodiment, the seed germination rate was 86.9%.

[0050] Example 3:

[0051] The irradiation time in the irradiation treatment step is 1 hour, and the remaining steps are the same as in Example 1.

[0052] In this embodiment, the seed germination rate was 93.5%.

[0053] The results of astronomical observation of the white flag Paphiopedilum seeds of Example 1-3 after being irradiated with broad energy spectrum white light neutrons for 60 days are as follows: Figure 1 The germination status is shown in Table 1. Figure 2 As shown in the figure, the test tube seedlings 240 days after sowing and the plants 30 days after transplanting are as follows: Figure 3 shown.

[0054] Table 1 Germination of Paphiopedilum seed treated with different irradiation times

[0055]

[0056] The above only is the preferred embodiment of the present application, it should be pointed out, for the ordinary skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered the scope of protection of the present application.

Claims

1. A method for promoting seed germination and seedling formation of Paphiopedilum by irradiation with broad-spectrum white light neutrons, characterized in that: The invention comprises the following steps: sterilizing Paphiopedilum seeds and then irradiating them with wide-spectrum white light neutrons, sowing them into a sowing medium for seed germination culture, transferring them into a seedling culture medium for seedling culture, and transplanting them; the wide-spectrum white light energy spectrum is 1 eV to 200 MeV, the peak of the wide-spectrum white light energy spectrum is near 1 MeV, and when the beam is supplied at 125 kW, the beam spot is 50×50 mm, and the fluence rate is 2×10 7 n / cm 2 / s, and the irradiation time is 15-60 minutes.

2. The method according to claim 1, characterized in that The Paphiopedilum seeds are taken from the capsules of Paphiopedilum 350 days after pollination.

3. The method according to claim 1, characterized in that The Paphiopedilum is Paphiopedilum whitskinensis ( Paphiopedilum spicerianum ).

4. The method according to claim 1, wherein The sowing culture medium is: Huabao No. 1 1.5 g / L + sucrose 15 g / L + agar 4.8 g / L + activated carbon 0.5 g / L + peptone 2 g / L + inositol 0.1 g / L + coconut milk 50 mL / L + 1000 times B5 vitamin mother solution 1 mL / L + 100 times iron salt mother solution 10 mL / L + NAA 1 mg / L, pH 6.0; the seedling culture medium is: Huabao No. 1 3.0 g / L + NAA 1.0 mg / L + activated carbon 0.5 g / L + sucrose 30 g / L + agar 5 g / L + 10% banana homogenate by mass, pH 5.

4.

5. The method according to claim 1, wherein The seed germination culture conditions are: dark culture for 40 days after sowing, the culture temperature is 26-28°C, after 40 days, the light intensity is 2000-2500 Lx, the daily light is 12 h / d, and the temperature is 26-28°C; the seedling culture conditions are: light intensity is 2000-2500 Lx, the daily light is 12 h / d, and the temperature is 26-28°C.

6. The method according to claim 1, characterized in that The transplanting is carried out when the test tube seedlings grow to 8-12 centimeters, and the transplanting medium is a mixed medium of plant stone and pine bark in a volume ratio of 1:

9.

7. The method according to claim 1, characterized in that The specific steps are as follows: Capsules of Paphiopedilum leucoderma 350 days after pollination were sterilized in a clean bench: first, the capsule surface was wiped with cotton dipped in 75% alcohol, then the capsule was clamped into a sterilized glass bottle with sterilized tweezers, and 75% alcohol was poured into the capsule for immersion and disinfection for 30 seconds. During this period, the glass bottle was shaken continuously, the alcohol was poured out, and the capsule was rinsed with sterile water. Then, 0.1% mercuric chloride and two drops of Tween 20 were poured into the capsule for immersion and disinfection for 20 minutes. During this period, the glass bottle was shaken continuously, the mercuric chloride was poured out, and the capsule was rinsed three times with sterile water. After the pods were dried, they were cut open with a sterilized scalpel, and the seeds in the pods were placed in a 1.5 mL enzyme-free sterile centrifuge tube. The centrifuge tube containing the seeds was placed in the center of the beam spot for irradiation. The irradiation time was 15-60 minutes. The irradiation beam line was an off-angle white light neutron beam line with an energy spectrum of 1 eV to 200 MeV and an energy spectrum peak near 1 MeV. When the beam was supplied with 125 kW, the beam spot was 50×50 mm and the injection rate was 2×10 7 n / cm 2 / s; Prepare an agar solution with an m / v of 0.01%, sterilize it at 121°C for 20 minutes, place it until it is cool but not solidified, disperse the seeds in the agar solution, and prepare a uniform suspension of seeds. Use a sterilized pipette tip to absorb the agar suspension of seeds, hit it on the culture medium, and shake it gently to make the seed suspension evenly distributed on the surface of the culture medium. The seed sowing medium is: Huabao No. 1 1.5 g / L + sucrose 15 g / L + agar 4.8 g / L + activated carbon 0.5 g / L + peptone 2 g / L + inositol 0.1 g / L + coconut milk 50 mL / L + 1000 times B5 vitamin mother solution 1 mL / L + 100 times iron salt mother solution 10 mL / L + NAA 1 mg / L, pH 6.0, dark culture for 40 days after sowing, the temperature is 26-28°C, and after 40 days, the light is 12 h / d and the light intensity is 2000-2500 Lx, temperature 26-28°C; Four months after sowing, the seedlings were transferred to a seedling growth medium for seedling growth culture. The seedling growth medium consisted of 3.0 g / L Huabao No. 1, 1.0 mg / L NAA, 0.5 g / L activated carbon, 30 g / L sucrose, 5 g / L agar, and 10% banana homogenate (pH 5.4). The medium was maintained at a light intensity of 2000–2500 Lx, a daily light intensity of 12 h / d, and a temperature of 26–28°C. When the seedlings grow to 8-12 cm, they are taken out of the bottle and transplanted. The transplanting medium for the test tube seedlings is a mixture of planting stone and pine bark in a volume ratio of 1:

9. The pine bark should be rinsed and soaked before use, and the planting stone should be rinsed before use.

8. Use of the method according to any one of claims 1 to 7 in promoting seed germination and seedling formation of Paphiopedilum.

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