WO2022247259A1 - Isolation-based breeding method for solid dendrobium - Google Patents

Isolation-based breeding method for solid dendrobium Download PDF

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WO2022247259A1
WO2022247259A1 PCT/CN2021/141023 CN2021141023W WO2022247259A1 WO 2022247259 A1 WO2022247259 A1 WO 2022247259A1 CN 2021141023 W CN2021141023 W CN 2021141023W WO 2022247259 A1 WO2022247259 A1 WO 2022247259A1
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culture
solid
protocorm
dendrobium
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PCT/CN2021/141023
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French (fr)
Chinese (zh)
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谢光明
李秀梅
刘进平
陈银华
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海南大学
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008Methods for regeneration to complete plants
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001Culture apparatus for tissue culture

Definitions

  • the invention relates to the technical field of separation and breeding, in particular to a method for separation and breeding of solid-colored dendrobium orchid flowers.
  • Dendrobium orchid, Dendrobium orchidaceae is a perennial herbaceous epiphytic plant, with huge racemes, various flower colors, and gorgeous and colorful.
  • Dendrobium has many flower color chimeras, such as red and white flower color chimeras. The flower color and pattern of such flower color chimeras are unstable during reproduction, and it is difficult to keep consistent with the mother plant. And pure color flower or single color flower varieties can maintain the stability of flower color in the breeding process, and have higher commerciality.
  • Dendrobium orchid conventional propagation methods are branching, branching and cuttings, but these methods have larger propagules and low reproductive rates.
  • Dendrobium orchid can be rapidly propagated by plant tissue culture, usually through protocorm induction, protocorm proliferation, protocorm differentiation into seedlings, strong seedlings and rooting culture. During this process, protocorms (similar to somatic embryos originating from a few cells) proliferate indefinitely, and the local solid-colored areas of flower-color chimeras will regenerate stable solid-colored or monochromatic individuals. The adventitious proliferation of protocorms with a few cell origins can effectively separate stable pure-colored or monochromatic flower individuals from flower-color chimeras.
  • the application number CN103314861A discloses a method of in vitro hybrid breeding of Dendrobium orchids, which adopts sterile inoculation, in vitro flowering induction and in vitro hybrid breeding, selects hybrid combinations with excellent flower shape and color for tissue culture, shortens the hybrid breeding cycle, and breeds new varieties of Dendrobium orchids.
  • Neither of the two patents involves the method for the separation and breeding of pure-colored flowers of Dendrobium orchids, but only involves the technical issues of improving the cultivation efficiency of Dendrobium orchids and cultivating new hybrid varieties.
  • the purpose of the present invention is to provide a method for separation and breeding of Dendrobium orchid solid-color flowers, which can effectively separate stable solid-color flowers or single-color flowers from Dendrobium orchid color chimeras.
  • a method for separating and breeding dendrobium pure color flowers comprising the following steps:
  • Selection of S1 explants Use the stems of the lateral buds of Dendrobium orchids as explants, rinse them with tap water, soak in diluted detergent for 5-15 minutes, rinse them with tap water, soak them in 70% alcohol by volume for 50-70 seconds, and then soak them in sterile water. Rinse 4-6 times, remove leaves and membranous leaf sheaths on a clean bench, transfer to 0.1% HgCl 2 solution for disinfection for 4-12 minutes, rinse 4-6 times with sterile water, absorb with sterile gauze or sterile filter paper dry water.
  • S2 protocorm induction inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 1 ⁇ 4mg/L+NAA 0.1 ⁇ 0.2mg/L for the first solid culture; when the new axillary buds grow to 0.8 ⁇ 1.2cm, after 2 ⁇ 3 pieces of leaves, cut off the new shoots, peel off the leaves, remove the terminal buds, take the axillary buds, re-insert the same formula as 1/2MS+BA 1 ⁇ 4mg/L+NAA 0.1 ⁇ 0.2mg/
  • the new induction medium of L carries out second solid-state culture, obtains protocorm;
  • the first solid-state culture cultured for 45-55 days, wherein, cultured in the light for 10-14 hours a day, cultured in the dark for 10-14 hours; light culture conditions are light intensity 1300-1700Lux, temperature 24-28 ° C, dark culture temperature 24 ⁇ 28°C.
  • each second solid-state culture is repeated 2-3 times, and each second solid-state culture is 25-35 days; each second solid-state culture includes 9-11 days of total darkness culture, followed by 10-14 hours of light culture every day, and dark Cultivate for 10-14 hours; light culture conditions are light intensity 1300-1700Lux, temperature 24-28°C, dark culture temperature is 24-28°C;
  • S3 protocorm proliferation cut off the top buds of the protocorm, select the protocorm containing 5-15 protocorm particles and inoculate to the formula of 1/2MS+BA 1-4mg/L+NAA 0.1-0.2mg/L+banana 20-40g /L+Potatoes 10-30g/L proliferation medium, 24-28°C, 100-120rpm liquid suspension culture, every 3-7d, cut off the newly grown buds of the protocorm, insert the same formula as 1/2MS+ BA 1 ⁇ 4mg/L+NAA 0.1 ⁇ 0.2mg/L+banana 20 ⁇ 40g/L+potato 10 ⁇ 30g/L in the new proliferation medium culture, co-culture 4 ⁇ 6 cycles, each cycle is 25 ⁇ 35d, get protocorm clusters;
  • S4 protocorm differentiation Inoculate protocorms in a group into a differentiation medium with a formula of NAA0-0.2mg/L+banana 20-40g/L+potato 10-30g/L, solid-state culture, culture for 35-45 days, including total darkness After culturing for 6 to 8 days, cultivate in light for 10 to 14 hours and in dark for 10 to 14 hours every day to obtain seedlings; the conditions for light culture are light intensity of 1300 to 1700 Lux, temperature of 24 to 28°C, and dark culture temperature of 24 to 28°C;
  • Rooting culture of S5 strong seedlings The seedlings are cultivated for 45-55 days in the strong seedling medium formulated as 1/2MS+banana 20-40g/L+potatoes 10-30g/L+activated carbon 10-20g/L.
  • the beneficial effects of the present invention are: (1) the present invention utilizes the protocorm adventitious proliferation of a few cell origin bodies in the tissue culture and rapid propagation process of Dendrobium orchid, which can be effectively separated from flower-color chimeras to produce stable The individual of pure color flower or monochromatic flower, this is Dendrobium orchid conventional propagation method (such as division, division bud and cutting) can't be done effectively.
  • the present invention adopts the protocorm induction medium, cultures repeatedly, successfully induces the protocorm, and uses the protocorm inoculation to separate a certain number of pure-color flower plants;
  • the present invention adopts the processing method of liquid suspension culture and cutting off new buds, further suppresses the differentiation of protocorm proliferation stage buds, effectively improves the proliferation rate of protocorms, and makes the number of flowering plants and the number of pure-color flower plants obtained more, and the number of pure-color flowers High flower separation rate;
  • the protocorm differentiation medium of the present invention removes the cytokinin BA on the basis of the protocorm proliferation medium, so that the protocorm becomes larger and greener, the number of protocorms differentiated into seedlings increases, and the differentiated seedlings are larger and stronger ;
  • Adding banana 30g/L and potato 20g/L in the rooting medium of the present invention can promote strong seedlings while promoting rooting, not only easier to go out, and the plant leaves are thicker and wider, and the roots produced are thicker and thicker. strong.
  • S1 explant selection use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
  • S2 protocorm induction Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 1mg/L+NAA 0.1mg/L for the first solid-state culture, culture for 45 days, culture in the light for 14 hours every day, and culture in the dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the formula of 1/2MS+BA 1mg/L+NAA 0.1mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are light intensity
  • S3 Protocorm Proliferation Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the multiplication of the formula 1/2MS+BA 1mg/L+NAA 0.1mg/L+banana 20g/L+potato 10g/L
  • the culture medium 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, and the insertion formula is 1/2MS+BA 1mg/L+NAA 0.1mg/L+banana 20g/L+potato 10g/
  • the new proliferation medium of L was cultured, co-cultured for 4 cycles, each cycle was 25 days, and the protocorms were obtained into agglomerates;
  • S4 protocorm differentiation Inoculate the protocorm in a group in the differentiation medium with the formula of banana 20g/L + potato 10g/L, solid-state culture for 35 days, and get seedlings, including 6 days of total dark culture, 14 hours of light culture every day, and dark culture 10h; light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature 26°C;
  • Rooting culture of S5 strong seedlings the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 20g/L+potatoes 10g/L+activated carbon 10g/L for 45 days, cultivated in light for 14 hours and dark for 10 hours every day to obtain seedlings and transfer them into the formula
  • the rooting medium is 1/2MS+banana 20g/L+potato 10g/L+activated carbon 10g/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting seedlings are obtained.
  • Cultivate for 10 hours; light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature is 26°C;
  • S1 explant selection use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
  • S2 protocorm induction Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 45 days, culture in light for 14 hours every day, and culture in dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are light intensity 1500L
  • S3 Protocorm Proliferation Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L+banana 40g/L+potato 30g/L
  • the culture medium 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, and the insertion formula is 1/2MS+BA 4mg/L+NAA 0.2mg/L+banana 40g/L+potato 30g/
  • the new proliferation medium of L was cultured, co-cultured for 4 cycles, each cycle was 25 days, and the protocorms were obtained into agglomerates;
  • S4 protocorm differentiation Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.2mg/L+banana 40g/L+potato 30g/L, culture in solid state for 35 days, and get seedlings, including culturing in total darkness for 6 days, and light every day Cultivate for 14 hours, and cultivate in the dark for 10 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
  • Rooting culture of S5 strong seedlings the seedlings are cultivated in the strong seedling medium with the formula of 1/2MS+banana 40g/L+potato 30g/L+activated carbon 10g/L for 45 days, 14 hours in light and 10 hours in darkness every day to obtain seedlings and transfer into the formula
  • the rooting medium is 1/2MS+banana 20g/L+potato 10g/L+activated carbon 10g/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting seedlings are obtained.
  • Cultivate for 10 hours; light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature is 26°C;
  • S1 explant selection use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
  • S2 protocorm induction inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 45 days, culture in the light for 14 hours every day, and culture in the dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are
  • S3 protocorm proliferation Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L In the culture medium, 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g /L of new proliferation medium culture, co-cultivate 4 cycles, each cycle is 25d, to get protocorms into agglomerates;
  • S4 protocorm differentiation Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, solid-state culture for 35 days, and get seedlings, including culturing in total darkness for 6 days, and light every day Cultivate for 14 hours, and cultivate in the dark for 10 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
  • Rooting culture of S5 strong seedlings the seedlings are cultivated in the strong seedling medium with the formula of 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 45 days, with light for 14 hours and dark for 10 hours every day to obtain seedlings and transfer them into the formula
  • the rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting shoots are obtained.
  • the difference between this comparative example and Example 3 is that in the S3 step, the proliferation medium formula is 1/2MS+BA 3mg/L+NAA 0.2mg/L, and the culture medium is prepared by adding 9g/L carrageenan Culture medium, using solid culture instead of liquid suspension culture.
  • Example 3 The difference between this comparative example and Example 3 is that in the S3 step, the medium is prepared by adding 9 g/L carrageenan to the medium to prepare a solid medium, and solid culture is used instead of liquid suspension culture.
  • Example 3 The difference between this comparative example and Example 3 is that in the S2 step, when the new axillary buds grow to 0.8cm, after 2 leaves, the new axillary buds are inoculated to a formula of 1/2MS+BA 3mg/L+NAA 0.2mg In the proliferation medium of /L+banana 30g/L+potato 20g/L; All the other steps are with embodiment 3.
  • the separation rate of dendrobium pure-color flowers in Examples 1 to 3 is more than 60%, and the solid-color flowers are effectively separated from the dendrobium orchid color chimera, and the solid-color flowers of dendrobium are realized. Fast, stable and effective separation.
  • the role of sex can inhibit the differentiation of buds in the proliferation culture stage to the greatest extent, thereby increasing the proliferation rate of protocorms and further improving the separation rate of pure-colored flowers; in comparative example 3, it is axillary buds instead of protocorms that are inoculated into the proliferation medium, and the protocorms are finally produced. Almost all of the plants are variegated flower plants. Only by inoculating with protocorms can a certain number of pure-colored flower plants be isolated. At the same time, with the increase of protocorm proliferation rate, the effectiveness of this pure-colored flower separation is also improving.
  • S1 explant selection use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 10 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
  • S2 protocorm induction Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 55 days, culture in light for 12 hours every day, and culture in dark 12h, the light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 1.2cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time for the second time Solid-state culture for 35 days, each second solid-state culture includes 11 days of total dark culture, 12 hours of light culture every day, and 12 hours of dark culture; the light culture conditions are light intensity 1500Lux
  • Proliferation of S3 protocorms cut off the top buds of protocorms, select protocorms containing 15 protocorm particles and inoculate them to multiply with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L
  • the culture medium 26°C, 120rpm liquid suspension culture for 30 days, every 7 days, cut off the newly grown shoots of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potatoes 20g/L of new proliferation medium culture, co-cultivation for 5 cycles, each cycle is 35d, to get protocorms into agglomerates;
  • S4 protocorm differentiation Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, culture in solid state for 45 days, and get seedlings, including culturing in total darkness for 8 days, and light every day Cultivate for 12 hours, and cultivate in the dark for 12 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
  • Rooting culture of S5 strong seedlings the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 55 days, cultivated in light for 12 hours and dark for 12 hours every day to obtain seedlings and transfer them into the formula
  • the rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 95 days, and the rooting shoots are obtained.
  • the test results show that the proliferation rate of the protocorm is 3.26, the number of flowering plants is 4592, the number of pure-color flower plants is 3297, and the separation rate of pure-color flowers is 71.80%.
  • S1 explant selection use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 10 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
  • S2 protocorm induction inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 50 days, and culture in light for 12 hours every day, and culture in dark 12h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 1cm, after 3 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re-inoculate Enter the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture is repeated for 3 times, each time the second solid-state Cultivate for 30 days, and each second solid-state culture includes 10 days of full-dark culture, followed by 12 hours of light culture and 12 hours of dark culture every day;
  • S3 Protocorm Proliferation Cut off the top buds of the protocorm, select the protocorm containing 10 protocorm particles and inoculate to the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L for proliferation and cultivation Medium, 26°C, 110rpm liquid suspension culture for 30 days, every 5 days, cut off the newly grown shoots of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g /L of new proliferation medium culture, co-cultivation for 5 cycles, each cycle is 30d, to get protocorms into agglomerates;
  • S4 protocorm differentiation Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, culture in solid state for 40 days, and get seedlings, including culturing in total darkness for 7 days, and light every day Cultivate for 12 hours, and cultivate in the dark for 12 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
  • Rooting culture of S5 strong seedlings the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 50 days, cultivated in the light for 12 hours a day, and cultivated in the dark for 12 hours to obtain seedlings and transfer them into the formula
  • the rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 90 days, and the rooting shoots are obtained.
  • the test results show that the proliferation rate of the protocorm is 3.48, the number of flowering plants is 5237, the number of pure-color flower plants is 3985, and the separation rate of pure-color flowers is 76.09%.
  • Example 5 The difference between this comparative example and Example 5 is that in the S3 step, the medium is prepared by adding 9 g/L of carrageenan to the medium to prepare a solid medium, and solid culture is used instead of liquid suspension culture; every 5 days, the protocorm is directly inserted into Cultivate with the new proliferation medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L, and the newly grown buds of the protocorm are not cut off before inoculation.
  • the test results show that the proliferation rate of the protocorm is 1.62, the number of flowering plants is 1219, the number of pure-color flower plants is 482, and the separation rate of pure-color flowers is 39.54%.
  • Protocorm proliferation The protocorm proliferation medium uses the same hormone combination as the protocorm induction medium (BA 3mg/L+NAA 0.2mg/L), and the effects of organic additives banana and potato on protocorm proliferation are tested , the results are shown in Table 4.
  • the separation and breeding method of Dendrobium orchid solid-color flowers uses the stem section of Dendrobium orchid lateral buds as explants, scientifically prepares each medium component, utilizes the protocorm proliferation stage to adopt liquid suspension culture and cuts off
  • the processing method of newly grown buds from the protocorm, the control of the cultivation light and the cultivation time have realized better, stable and rapid separation of the solid-colored flowers in the dendrobium orchid color chimera.

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Abstract

The present invention provides an isolation-based breeding method for solid dendrobium, comprising: taking a lateral bud stem segment of dendrobium as an explant; using a protocorm induction culture medium: 1/2MS+BA 3 mg/L+NAA 0.2 mg/L; using a protocorm proliferation culture medium: 1/2MS+BA 3 mg/L+NAA 0.2 mg/L+banana 30 g/L+potato 20 g/L; and performing isolation-based breeding of solid dendrobium by combining liquid suspension culture and a treatment method of cutting off a new bud. According to the present invention, the proliferation rate of protocorms can be increased, and stable solid flowers can be effectively isolated from a dendrobium chimera, with an isolation rate of 76.09%.

Description

一种石斛兰纯色花的分离育种方法A kind of separation breeding method of dendrobium orchid pure color flower 技术领域technical field
本发明涉及分离育种技术领域,特别涉及一种石斛兰纯色花的分离育种方法。The invention relates to the technical field of separation and breeding, in particular to a method for separation and breeding of solid-colored dendrobium orchid flowers.
背景技术Background technique
石斛兰,兰科石斛属多年生草本附生类植物,总状花序硕大,花色繁多,艳丽多彩。石斛兰有很多花色嵌合体品种,如红白花色嵌合体,这类花色嵌合体品种在繁殖过程中花色和图案不稳定,难以与母株保持一致。而纯色花或单色花品种在繁殖过程中能保持花色的稳定性,具有较高的商品性。Dendrobium orchid, Dendrobium orchidaceae is a perennial herbaceous epiphytic plant, with huge racemes, various flower colors, and gorgeous and colorful. Dendrobium has many flower color chimeras, such as red and white flower color chimeras. The flower color and pattern of such flower color chimeras are unstable during reproduction, and it is difficult to keep consistent with the mother plant. And pure color flower or single color flower varieties can maintain the stability of flower color in the breeding process, and have higher commerciality.
石斛兰常规繁殖方法为分株、分芽和扦插,但是这些方法繁殖体较大,繁殖率低。采用植物组织培养可对石斛兰进行快速繁殖,通常通过原球茎诱导、原球茎增殖、原球茎分化成苗、壮苗和生根培养等环节进行培育和繁殖。在此过程中原球茎(类似于由少数细胞起源的体细胞胚胎)会不定增殖,花色嵌合体局部的纯色区域会再生出稳定的纯色或单色个体。利用少数细胞起源体的原球茎进行不定增殖,可以有效地从花色嵌合体中分离出稳定的纯色花或单色花个体。只有成功诱导出原球茎,并采用原球茎增殖的方式,才能分离到一定数量纯色花植株。而现有技术缺少将石斛兰的花色嵌合体中分离出纯色花的分离育种方法。如,申请号CN107711508A公开一种石斛兰的组培快繁方法,对石斛兰外植体的伤害小,提供的培养基配方可显著提高石斛兰的诱导分化率、外植体增殖系数和生根率,缩短培育周期,提高石斛兰组培苗的质量。申请号CN103314861A公开一种石斛兰试管内杂交育种方法,采用无菌接种、试管开花诱导和试管杂交育种,选择杂交组合优良花形和花色进行组织培养,缩短杂交育种周期,培育出石斛兰新品种。两篇专利都未涉及到石斛兰纯色花分离育种的方法,仅是涉及提高石斛兰培育成效和培育杂交新品种的技术问题。Dendrobium orchid conventional propagation methods are branching, branching and cuttings, but these methods have larger propagules and low reproductive rates. Dendrobium orchid can be rapidly propagated by plant tissue culture, usually through protocorm induction, protocorm proliferation, protocorm differentiation into seedlings, strong seedlings and rooting culture. During this process, protocorms (similar to somatic embryos originating from a few cells) proliferate indefinitely, and the local solid-colored areas of flower-color chimeras will regenerate stable solid-colored or monochromatic individuals. The adventitious proliferation of protocorms with a few cell origins can effectively separate stable pure-colored or monochromatic flower individuals from flower-color chimeras. Only by successfully inducing protocorms and adopting the protocorm multiplication method can a certain number of pure color flower plants be isolated. And prior art lacks the separation breeding method that isolates pure color flower in the color chimera of Dendrobium orchid. For example, application number CN107711508A discloses a tissue culture and rapid propagation method of Dendrobium orchid, which has little damage to Dendrobium orchid explants, and the medium formula provided can significantly improve the induced differentiation rate, explant proliferation coefficient and rooting rate of Dendrobium orchid , shorten the cultivation period, and improve the quality of Dendrobium orchid tissue culture seedlings. The application number CN103314861A discloses a method of in vitro hybrid breeding of Dendrobium orchids, which adopts sterile inoculation, in vitro flowering induction and in vitro hybrid breeding, selects hybrid combinations with excellent flower shape and color for tissue culture, shortens the hybrid breeding cycle, and breeds new varieties of Dendrobium orchids. Neither of the two patents involves the method for the separation and breeding of pure-colored flowers of Dendrobium orchids, but only involves the technical issues of improving the cultivation efficiency of Dendrobium orchids and cultivating new hybrid varieties.
发明内容Contents of the invention
鉴于此,本发明的目的在于提供一种石斛兰纯色花的分离育种方法,能够有效地从石斛兰花色嵌合体中分离出稳定的纯色花或单色花。In view of this, the purpose of the present invention is to provide a method for separation and breeding of Dendrobium orchid solid-color flowers, which can effectively separate stable solid-color flowers or single-color flowers from Dendrobium orchid color chimeras.
本发明的技术方案是这样实现的:Technical scheme of the present invention is realized like this:
一种石斛兰纯色花的分离育种方法,包括以下步骤:A method for separating and breeding dendrobium pure color flowers, comprising the following steps:
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡5~15min,自来水冲洗干净,用体积浓度70%酒精浸泡50~70s,然后无菌水冲洗4~6次,在超净台上除去叶片及膜质叶鞘,转入质量浓度0.1%HgCl 2溶液消毒4~12min,无菌水冲洗4~6次,用无菌纱布或无菌滤纸吸干水。 Selection of S1 explants: Use the stems of the lateral buds of Dendrobium orchids as explants, rinse them with tap water, soak in diluted detergent for 5-15 minutes, rinse them with tap water, soak them in 70% alcohol by volume for 50-70 seconds, and then soak them in sterile water. Rinse 4-6 times, remove leaves and membranous leaf sheaths on a clean bench, transfer to 0.1% HgCl 2 solution for disinfection for 4-12 minutes, rinse 4-6 times with sterile water, absorb with sterile gauze or sterile filter paper dry water.
进一步说明,质量浓度0.1%HgCl 2溶液加入1-2滴吐温-80。 To further illustrate, add 1-2 drops of Tween-80 to the 0.1% HgCl 2 solution.
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 1~4mg/L+NAA0.1~0.2mg/L的诱导培养基上进行第一次固态培养;当新生腋芽长至0.8~1.2cm,叶片2~3片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入同配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L的新诱导培养基,进行第二次固态培养,得原球茎;S2 protocorm induction: inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L for the first solid culture; when the new axillary buds grow to 0.8~1.2cm, after 2~3 pieces of leaves, cut off the new shoots, peel off the leaves, remove the terminal buds, take the axillary buds, re-insert the same formula as 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/ The new induction medium of L, carries out second solid-state culture, obtains protocorm;
进一步说明,第一次固态培养,培养45~55d,其中,每天光照培养10~14h,黑暗培养10~14h;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度为24~28℃。Further explanation, the first solid-state culture, cultured for 45-55 days, wherein, cultured in the light for 10-14 hours a day, cultured in the dark for 10-14 hours; light culture conditions are light intensity 1300-1700Lux, temperature 24-28 ° C, dark culture temperature 24 ~28°C.
进一步说明,第二次固态培养反复培养2-3次,每次第二次固态培养25~35d;每次第二次固态培养包括全黑暗培养9~11d后,每天光照培养10~14h,黑暗培养10~14h;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度均为24~28℃;To further illustrate, the second solid-state culture is repeated 2-3 times, and each second solid-state culture is 25-35 days; each second solid-state culture includes 9-11 days of total darkness culture, followed by 10-14 hours of light culture every day, and dark Cultivate for 10-14 hours; light culture conditions are light intensity 1300-1700Lux, temperature 24-28°C, dark culture temperature is 24-28°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有5~15个原球茎颗粒接种至配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+香蕉20~40g/L+土豆10~30g/L的增殖培养基中,24~28℃、100~120rpm液体悬浮培养,每3~7d,切掉原球茎新长出的芽,接入同配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+香蕉20~40g/L+土豆10~30g/L的新增殖培养基培养,共培养4~6个周期,每个周期为25~35d,得原球茎成团;S3 protocorm proliferation: cut off the top buds of the protocorm, select the protocorm containing 5-15 protocorm particles and inoculate to the formula of 1/2MS+BA 1-4mg/L+NAA 0.1-0.2mg/L+banana 20-40g /L+Potatoes 10-30g/L proliferation medium, 24-28℃, 100-120rpm liquid suspension culture, every 3-7d, cut off the newly grown buds of the protocorm, insert the same formula as 1/2MS+ BA 1~4mg/L+NAA 0.1~0.2mg/L+banana 20~40g/L+potato 10~30g/L in the new proliferation medium culture, co-culture 4~6 cycles, each cycle is 25~35d, get protocorm clusters;
S4原球茎分化:将原球茎成团接种在配方为NAA0~0.2mg/L+香蕉20~40g/L+土豆10~30g/L的分化培养基,固态培养,培养35~45d,其中,包括全黑暗培养6~8d后,每天光照培养10~14h,黑暗培养10~14h,得小苗;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度为24~28℃;S4 protocorm differentiation: Inoculate protocorms in a group into a differentiation medium with a formula of NAA0-0.2mg/L+banana 20-40g/L+potato 10-30g/L, solid-state culture, culture for 35-45 days, including total darkness After culturing for 6 to 8 days, cultivate in light for 10 to 14 hours and in dark for 10 to 14 hours every day to obtain seedlings; the conditions for light culture are light intensity of 1300 to 1700 Lux, temperature of 24 to 28°C, and dark culture temperature of 24 to 28°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉20~40g/L+土豆10~30g/L+活性炭10~20g/L的壮苗培养基,培养45~55d,其中,每天光照培养10~14h,黑暗培养10~14h,得苗株,移入配方为1/2MS+香蕉20~40g/L+土豆10~30g/L+活性炭10~20g/L+IBA 0~0.4mg/L+NAA 0~0.4mg/L的生根培养基,生根培养85~95d,其中,包括全黑暗培养6~8d后,每天光照培养10~14h,黑暗培养10~14h,得生根苗;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度均为24~28℃;Rooting culture of S5 strong seedlings: The seedlings are cultivated for 45-55 days in the strong seedling medium formulated as 1/2MS+banana 20-40g/L+potatoes 10-30g/L+activated carbon 10-20g/L. 14h, cultivated in the dark for 10-14h, to get seedlings, transplanted into the formula 1/2MS+banana 20-40g/L+potato 10-30g/L+activated carbon 10-20g/L+IBA 0-0.4mg/L+NAA 0-0.4mg /L of rooting medium, rooting culture for 85-95 days, including 6-8 days of total darkness culture, 10-14 hours of light culture every day, 10-14 hours of dark culture, to get rooting seedlings; light culture conditions are light intensity 1300-1700Lux , the temperature is 24-28°C, and the dark culture temperature is 24-28°C;
S6优良后代单株选择:将长至5~8cm,根支4条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of excellent offspring of S6: Transplant rooted seedlings with a length of 5-8cm, more than 4 root branches, and a root length of more than 2cm, eliminate chimera plants with variegated flowers, and retain the offspring with pure-colored or monochromatic flowers.
与现有技术相比,本发明的有益效果为:(1)本发明利用石斛兰组织培养与快速繁殖过程中少数细胞起源体的原球茎不定增殖,可以有效地从花色嵌合体中分离产生稳定的纯色花或单色花个体,这是石斛兰常规繁殖方法(如分株、分芽和扦插)无法有效做到的。本发明采用原球茎诱导培养基,反复培养,成功诱导出原球茎,采用原球茎接种,才能分离到一定数量的纯色花植株;Compared with the prior art, the beneficial effects of the present invention are: (1) the present invention utilizes the protocorm adventitious proliferation of a few cell origin bodies in the tissue culture and rapid propagation process of Dendrobium orchid, which can be effectively separated from flower-color chimeras to produce stable The individual of pure color flower or monochromatic flower, this is Dendrobium orchid conventional propagation method (such as division, division bud and cutting) can't be done effectively. The present invention adopts the protocorm induction medium, cultures repeatedly, successfully induces the protocorm, and uses the protocorm inoculation to separate a certain number of pure-color flower plants;
(2)本发明增殖培养基加入香蕉利于原球茎的增殖,土豆利于原球茎的生长,产生的原球茎健壮饱满、颗粒较大,活力强,在原球茎增殖过程中不易分化产生小芽,减少芽出芽的不利现象;(2) Adding bananas to the proliferation medium of the present invention is beneficial to the proliferation of protocorms, and potatoes are beneficial to the growth of protocorms, and the protocorms produced are strong and full, with larger particles and strong vitality. Unfavorable phenomenon of germination;
(3)本发明采用液体悬浮培养和切掉新芽的处理方式,进一步抑制原球茎增殖阶段芽的分化,有效地提高原球茎的增殖率,使获得的开花植株数和纯色花植株数多,纯色花分离率高;(3) The present invention adopts the processing method of liquid suspension culture and cutting off new buds, further suppresses the differentiation of protocorm proliferation stage buds, effectively improves the proliferation rate of protocorms, and makes the number of flowering plants and the number of pure-color flower plants obtained more, and the number of pure-color flowers High flower separation rate;
(4)本发明原球茎分化培养基在原球茎增殖培养基基础上,去除细胞分裂素BA,可使原球茎变大变绿,分化成苗的原球茎增多,分化的苗株较大、较壮;(4) The protocorm differentiation medium of the present invention removes the cytokinin BA on the basis of the protocorm proliferation medium, so that the protocorm becomes larger and greener, the number of protocorms differentiated into seedlings increases, and the differentiated seedlings are larger and stronger ;
(5)本发明生根培养基中附加香蕉30g/L和土豆20g/L可在促进生根的同时促进壮苗,不仅更易出根,且植株叶片更厚、更宽,产生的根更粗、更壮。(5) Adding banana 30g/L and potato 20g/L in the rooting medium of the present invention can promote strong seedlings while promoting rooting, not only easier to go out, and the plant leaves are thicker and wider, and the roots produced are thicker and thicker. strong.
具体实施方式Detailed ways
为了更好理解本发明技术内容,下面提供具体实施例,对本发明做进一步的说明。In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.
本发明实施例所用的实验方法如无特殊说明,均为常规方法。The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.
本发明实施例所用的材料、试剂等,如无特殊说明,均可从商业途径得到。The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.
实施例1Example 1
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡10min,自来水冲洗干净,用体积浓度70%酒精浸泡60s,然后无菌水冲洗5次,在超净台上除去叶片及膜质叶鞘,转入含有1滴吐温-80的质量浓度0.1%HgCl 2溶液消毒8min,无菌水冲洗5次,用无菌纱布或无菌滤纸吸干水。 S1 explant selection: use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 1mg/L+NAA 0.1mg/L的诱导培养基上进行第一次固态培养,培养45d,每天光照培养14h,黑暗培养10h,光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;当新生腋芽长至0.8cm,叶片2片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入配方为1/2MS+BA 1mg/L+NAA 0.1mg/L的新诱导培养基进行第二次固态培养,得原球茎;第二次固态培养反复培养2次,每次第二次固态培养25d,每次第二次固态培养包括全黑暗培养9d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;S2 protocorm induction: Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 1mg/L+NAA 0.1mg/L for the first solid-state culture, culture for 45 days, culture in the light for 14 hours every day, and culture in the dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the formula of 1/2MS+BA 1mg/L+NAA 0.1mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有5个原球茎颗粒接种至配方为1/2MS+BA 1mg/L+NAA 0.1mg/L+香蕉20g/L+土豆10g/L的增殖培养基中,26℃、100rpm液体悬浮培养,每3d,切掉原球茎新长出的芽,接入配方为1/2MS+BA 1mg/L+NAA 0.1mg/L+香蕉20g/L+土豆10g/L的新增殖培养基培养,共培养4个周期,每个周期为25d,得原球茎成团;S3 Protocorm Proliferation: Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the multiplication of the formula 1/2MS+BA 1mg/L+NAA 0.1mg/L+banana 20g/L+potato 10g/L In the culture medium, 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, and the insertion formula is 1/2MS+BA 1mg/L+NAA 0.1mg/L+banana 20g/L+potato 10g/ The new proliferation medium of L was cultured, co-cultured for 4 cycles, each cycle was 25 days, and the protocorms were obtained into agglomerates;
S4原球茎分化:将原球茎成团接种在配方为香蕉20g/L+土豆10g/L的分化培养基,固态培养35d,得小苗,其中,包括全黑暗培养6d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;S4 protocorm differentiation: Inoculate the protocorm in a group in the differentiation medium with the formula of banana 20g/L + potato 10g/L, solid-state culture for 35 days, and get seedlings, including 6 days of total dark culture, 14 hours of light culture every day, and dark culture 10h; light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature 26°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉20g/L+土豆10g/L+活性炭10g/L的壮苗培养基,培养45d,每天光照培养14h,黑暗培养10h,得苗株,移入配方为1/2MS+香蕉20g/L+土豆10g/L+活性炭10g/L+NAA 0.2mg/L的生根培养基,生根培养85d,得生根苗,其中,包括全黑暗培养6d后,每天光照培养14h,黑暗培养10h;光照培养条件均为光照强度1500Lux,温度26℃,,黑暗培养温度均为26℃;Rooting culture of S5 strong seedlings: the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 20g/L+potatoes 10g/L+activated carbon 10g/L for 45 days, cultivated in light for 14 hours and dark for 10 hours every day to obtain seedlings and transfer them into the formula The rooting medium is 1/2MS+banana 20g/L+potato 10g/L+activated carbon 10g/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting seedlings are obtained. Cultivate for 10 hours; light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature is 26°C;
S6优良后代单株选择:将长至4cm,根支3条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of S6 excellent progeny: transplant the rooted seedlings with a length of 4 cm, more than 3 root branches, and a root length of more than 2 cm, eliminate chimera plants with variegated flowers, and keep the offspring with pure or monochromatic flowers.
实施例2Example 2
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡10min,自来水冲洗干净,用体积浓度70%酒精浸泡60s,然后无菌水冲洗5次,在超净台上除去叶片及膜质叶鞘,转入含有1滴吐温-80的质量浓度0.1%HgCl 2溶液消毒8min,无菌水冲洗5次,用无菌纱布或无菌滤纸吸干水。 S1 explant selection: use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 4mg/L+NAA 0.2mg/L的诱导培养基上进行第一次固态培养,培养45d,每天光照培养14h,黑暗培养10h,光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;当新生腋芽长至0.8cm,叶片2片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入配方为1/2MS+BA 4mg/L+NAA 0.2mg/L的新诱导培养基进行第二次固态培养,得原球茎;第二次固态培养反复培养2次,每次第二次固态培养25d,每次第二次固态培养包括全黑暗培养9d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;S2 protocorm induction: Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 45 days, culture in light for 14 hours every day, and culture in dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有5个原球茎颗粒接种至配方为1/2MS+BA 4mg/L+NAA 0.2mg/L+香蕉40g/L+土豆30g/L的增殖培养基中,26℃、100rpm液体悬浮培养,每3d,切掉原球茎新长出的芽,接入配方为1/2MS+BA 4mg/L+NAA 0.2mg/L+香蕉40g/L+土豆30g/L的新增殖培养基培养,共培养4个周期,每个周期为25d,得原球茎成团;S3 Protocorm Proliferation: Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the formula of 1/2MS+BA 4mg/L+NAA 0.2mg/L+banana 40g/L+potato 30g/L In the culture medium, 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, and the insertion formula is 1/2MS+BA 4mg/L+NAA 0.2mg/L+banana 40g/L+potato 30g/ The new proliferation medium of L was cultured, co-cultured for 4 cycles, each cycle was 25 days, and the protocorms were obtained into agglomerates;
S4原球茎分化:将原球茎成团接种在配方为NAA0.2mg/L+香蕉40g/L+土豆30g/L的分化培养基,固态培养35d,得小苗,其中,包括全黑暗培养6d后, 每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;S4 protocorm differentiation: Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.2mg/L+banana 40g/L+potato 30g/L, culture in solid state for 35 days, and get seedlings, including culturing in total darkness for 6 days, and light every day Cultivate for 14 hours, and cultivate in the dark for 10 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉40g/L+土豆30g/L+活性炭10g/L的壮苗培养基,培养45d,每天光照培养14h,黑暗培养10h,得苗株,移入配方为1/2MS+香蕉20g/L+土豆10g/L+活性炭10g/L+NAA 0.2mg/L的生根培养基,生根培养85d,得生根苗,其中,包括全黑暗培养6d后,每天光照培养14h,黑暗培养10h;光照培养条件均为光照强度1500Lux,温度26℃,,黑暗培养温度均为26℃;Rooting culture of S5 strong seedlings: the seedlings are cultivated in the strong seedling medium with the formula of 1/2MS+banana 40g/L+potato 30g/L+activated carbon 10g/L for 45 days, 14 hours in light and 10 hours in darkness every day to obtain seedlings and transfer into the formula The rooting medium is 1/2MS+banana 20g/L+potato 10g/L+activated carbon 10g/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting seedlings are obtained. Cultivate for 10 hours; light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature is 26°C;
S6优良后代单株选择:将长至4cm,根支3条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of S6 excellent progeny: transplant the rooted seedlings with a length of 4 cm, more than 3 root branches, and a root length of more than 2 cm, eliminate chimera plants with variegated flowers, and keep the offspring with pure or monochromatic flowers.
实施例3Example 3
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡10min,自来水冲洗干净,用体积浓度70%酒精浸泡60s,然后无菌水冲洗5次,在超净台上除去叶片及膜质叶鞘,转入含有1滴吐温-80的质量浓度0.1%HgCl 2溶液消毒8min,无菌水冲洗5次,用无菌纱布或无菌滤纸吸干水。 S1 explant selection: use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 8 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的诱导培养基上进行第一次固态培养,培养45d,每天光照培养14h,黑暗培养10h,光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;当新生腋芽长至0.8cm,叶片2片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的新诱导培养基进行第二次固态培养得原球茎;第二次固态培养反复培养2次,每次第二次固态培养25d,每次第二次固态培养包括全黑暗培养9d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;S2 protocorm induction: inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 45 days, culture in the light for 14 hours every day, and culture in the dark 10h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 0.8cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time the second solid-state Cultivate for 25 days, and each second solid-state culture includes 9 days of total dark culture, followed by 14 hours of light culture and 10 hours of dark culture every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有5个原球茎颗粒接种至配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的增殖培养基中,26℃、100rpm液体悬浮培养,每3d,切掉原球茎新长出的芽,接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的新增殖培养基培养,共培养4个周期,每个周期为25d,得原球茎成团;S3 protocorm proliferation: Cut off the top buds of the protocorm, select the protocorm containing 5 protocorm particles and inoculate to the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L In the culture medium, 26°C, 100rpm liquid suspension culture, every 3 days, cut off the newly grown buds of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g /L of new proliferation medium culture, co-cultivate 4 cycles, each cycle is 25d, to get protocorms into agglomerates;
S4原球茎分化:将原球茎成团接种在配方为NAA0.1mg/L+香蕉30g/L+土豆20g/L的分化培养基,固态培养35d,得小苗,其中,包括全黑暗培养6d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;S4 protocorm differentiation: Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, solid-state culture for 35 days, and get seedlings, including culturing in total darkness for 6 days, and light every day Cultivate for 14 hours, and cultivate in the dark for 10 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L的壮苗培养基,培养45d,每天光照培养14h,黑暗培养10h,得苗株,移入配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L+IBA 0.2mg/L+NAA 0.2mg/L的生根培养基,生根培养85d,得生根苗,其中,包括全黑暗培养6d后,每天光照培养14h,黑暗培养10h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;Rooting culture of S5 strong seedlings: the seedlings are cultivated in the strong seedling medium with the formula of 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 45 days, with light for 14 hours and dark for 10 hours every day to obtain seedlings and transfer them into the formula The rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 85 days, and the rooting shoots are obtained. Cultivate in light for 14 hours and in dark for 10 hours every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S6优良后代单株选择:将长至4cm,根支3条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of S6 excellent progeny: transplant the rooted seedlings with a length of 4 cm, more than 3 root branches, and a root length of more than 2 cm, eliminate chimera plants with variegated flowers, and keep the offspring with pure or monochromatic flowers.
对比例1Comparative example 1
本对比例与实施例3的区别在于,所述的S3步骤中,增殖培养基配方为1/2MS+BA 3mg/L+NAA 0.2mg/L,培养基添加9g/L的卡拉胶制备成固体培养基,采用固体培养替代液体悬浮培养。The difference between this comparative example and Example 3 is that in the S3 step, the proliferation medium formula is 1/2MS+BA 3mg/L+NAA 0.2mg/L, and the culture medium is prepared by adding 9g/L carrageenan Culture medium, using solid culture instead of liquid suspension culture.
对比例2Comparative example 2
本对比例与实施例3的区别在于,所述的S3步骤中,培养基添加9g/L的卡拉胶制备成固体培养基,采用固体培养替代液体悬浮培养。The difference between this comparative example and Example 3 is that in the S3 step, the medium is prepared by adding 9 g/L carrageenan to the medium to prepare a solid medium, and solid culture is used instead of liquid suspension culture.
对比例3Comparative example 3
本对比例与实施例3的区别在于,所述的S2步骤中,当新生腋芽长至0.8cm,叶片2片后,将新生腋芽接种至配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的增殖培养基中;其余步骤同实施例3。The difference between this comparative example and Example 3 is that in the S2 step, when the new axillary buds grow to 0.8cm, after 2 leaves, the new axillary buds are inoculated to a formula of 1/2MS+BA 3mg/L+NAA 0.2mg In the proliferation medium of /L+banana 30g/L+potato 20g/L; All the other steps are with embodiment 3.
1、石斛花纯色花株分离测定1. Determination of separation of dendrobium flower pure color flower strains
测定实施例1~3和对比例1~3不同培养条件对石斛兰纯色花株分离的影响,其中,增殖率=增殖产生的原球茎数÷接种的原球茎数,纯色花分离率(%)=纯色花植株数÷开花植株数×100%,测定结果如表1。Measure the impact of different culture conditions of Examples 1 to 3 and Comparative Examples 1 to 3 on the separation of dendrobium orchid solid-colored flower strains, wherein, the protocorm number of multiplication rate=proliferation ÷ the protocorm number of inoculation, the pure-color flower separation rate (%) =Number of pure-color flowering plants÷number of flowering plants×100%, the measurement results are shown in Table 1.
表1Table 1
Figure PCTCN2021141023-appb-000001
Figure PCTCN2021141023-appb-000001
经本发明提供的石斛兰纯色花的分离育种方法,实施例1~3的石斛兰纯色花分离率在60%以上,有效地从石斛兰花色嵌合体中分离得到纯色花,实现石斛兰纯色花快速、稳定、有效的分离。Through the separation and breeding method of dendrobium pure-color flowers provided by the invention, the separation rate of dendrobium pure-color flowers in Examples 1 to 3 is more than 60%, and the solid-color flowers are effectively separated from the dendrobium orchid color chimera, and the solid-color flowers of dendrobium are realized. Fast, stable and effective separation.
对比例1在原球茎增殖阶段,采用不含香蕉和土豆的培养基,进行固态培养,纯色花的分离率仅有10.92%;对比例2在原球茎增殖阶段,采用固态培养替代液体悬浮培养,原球茎增殖率为2.30,纯色花分离率为51.69%,植物组织培养过程中,组织培养物的极性作用会促进芽的分化,降低组织培养物的增殖率,液体悬浮培养具有破坏组织培养物发育极性的作用,可以最大程度地抑制增殖培养阶段芽的分化,进而提高原球茎增殖率,进一步提高纯色花的分离率;对比例3接种至增殖培养基的是腋芽而不是原球茎,则最后产生的植株几乎全部都是杂色花植株,只有采用原球茎接种,才能分离到一定数量纯色花植株,同时,随着原球茎增殖率的提高,这种纯色花分离的有效性也在提高。In comparative example 1, in the protocorm multiplication stage, solid-state culture was carried out using a medium not containing bananas and potatoes, and the separation rate of pure-colored flowers was only 10.92%; in comparative example 2, in the protocorm multiplication stage, solid-state culture was used instead of liquid suspension culture, and the The proliferation rate is 2.30, and the separation rate of pure color flowers is 51.69%. In the process of plant tissue culture, the polarity of tissue culture will promote the differentiation of buds and reduce the proliferation rate of tissue culture. Liquid suspension culture has the ability to destroy the growth pole of tissue culture. The role of sex can inhibit the differentiation of buds in the proliferation culture stage to the greatest extent, thereby increasing the proliferation rate of protocorms and further improving the separation rate of pure-colored flowers; in comparative example 3, it is axillary buds instead of protocorms that are inoculated into the proliferation medium, and the protocorms are finally produced. Almost all of the plants are variegated flower plants. Only by inoculating with protocorms can a certain number of pure-colored flower plants be isolated. At the same time, with the increase of protocorm proliferation rate, the effectiveness of this pure-colored flower separation is also improving.
实施例4Example 4
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡10min,自来水冲洗干净,用体积浓度70%酒精浸泡60s,然后无菌水冲洗5次,在超净台上除去叶片及膜质叶鞘,转入含有1滴吐温-80的质量浓度0.1%HgCl 2溶液消毒10min,无菌水冲洗5次,用无菌纱布或无菌滤纸吸干水。 S1 explant selection: use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 10 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的诱导培养基上进行第一次固态培养,培养55d,每天光照培养12h,黑暗培养12h,光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;当新生腋芽长至1.2cm,叶片2片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的新诱导培养基进行第二次固态培养,得原球茎;第二次固态培养反复培养2次,每次第二次固态培养35d,每次第二次固态培养包括全黑暗培养11d后,每天光照培养12h,黑暗培养12h;光照培养条件均为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;S2 protocorm induction: Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 55 days, culture in light for 12 hours every day, and culture in dark 12h, the light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 1.2cm, after 2 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re- Insert the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture was repeated twice, each time for the second time Solid-state culture for 35 days, each second solid-state culture includes 11 days of total dark culture, 12 hours of light culture every day, and 12 hours of dark culture; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are 26°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有15个原球茎颗粒接种至配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的增殖培养基中,26℃、120rpm液体悬浮培养30d,每7d,切掉原球茎新长出的芽,接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的新增殖培养基培养,共培养5个周期,每个周期为35d,得原球茎成团;Proliferation of S3 protocorms: cut off the top buds of protocorms, select protocorms containing 15 protocorm particles and inoculate them to multiply with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L In the culture medium, 26°C, 120rpm liquid suspension culture for 30 days, every 7 days, cut off the newly grown shoots of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potatoes 20g/L of new proliferation medium culture, co-cultivation for 5 cycles, each cycle is 35d, to get protocorms into agglomerates;
S4原球茎分化:将原球茎成团接种在配方为NAA0.1mg/L+香蕉30g/L+土豆20g/L的分化培养基,固态培养45d,得小苗,其中,包括全黑暗培养8d后,每天光照培养12h,黑暗培养12h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;S4 protocorm differentiation: Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, culture in solid state for 45 days, and get seedlings, including culturing in total darkness for 8 days, and light every day Cultivate for 12 hours, and cultivate in the dark for 12 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L的壮苗培养基,培养55d,每天光照培养12h,黑暗培养12h,得苗株,移入配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L+IBA 0.2mg/L+NAA 0.2mg/L的生根培养基,生根培养95d,得生根苗,其中,包括全黑暗培养8d后,每天光照培养12h,黑暗培养12h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;Rooting culture of S5 strong seedlings: the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 55 days, cultivated in light for 12 hours and dark for 12 hours every day to obtain seedlings and transfer them into the formula The rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 95 days, and the rooting shoots are obtained. Cultivate in light for 12 hours and in dark for 12 hours every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S6优良后代单株选择:将长至8cm,根支4条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of excellent offspring of S6: Transplant rooted seedlings with a length of 8 cm, more than 4 root branches, and a root length of more than 2 cm, eliminate chimera plants with variegated flowers, and retain offspring with pure-colored or single-colored flowers.
经试验结果表明,原球茎的增殖率为3.26,开花植株数为4592株,纯色花植株数为3297株,纯色花分离率为71.80%。The test results show that the proliferation rate of the protocorm is 3.26, the number of flowering plants is 4592, the number of pure-color flower plants is 3297, and the separation rate of pure-color flowers is 71.80%.
实施例5Example 5
S1外植体选择:以石斛兰侧芽茎段为外植体,自来水冲洗干净,稀释的洗洁精浸泡10min,自来水冲洗干净,用体积浓度70%酒精浸泡60s,然后无菌水冲洗5次,在超净台上除去叶片及膜质叶鞘,转入含有1滴吐温-80的质量浓度0.1%HgCl 2溶液消毒10min,无菌水冲洗5次,用无菌纱布或无菌滤纸吸干水。 S1 explant selection: use Dendrobium orchid lateral bud stems as explants, rinse with tap water, soak in diluted detergent for 10 minutes, rinse with tap water, soak in 70% alcohol by volume for 60 seconds, then rinse with sterile water for 5 times, Remove the leaves and membranous leaf sheaths on an ultra-clean bench, transfer to a 0.1% HgCl solution containing 1 drop of Tween- 80 for disinfection for 10 minutes, rinse with sterile water for 5 times, and dry the water with sterile gauze or sterile filter paper .
S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的诱导培养基上进行第一次固态培养,培养50d,每天光照培养12h,黑暗培养12h,光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;当新生腋芽长至1cm,叶片3片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L的新诱导培养基进行第二次固态培养,得原球茎;第二次固态培养反复培养3次,每次第二次固态培养30d,每次第二次固态培养包括全黑暗培养10d后,每天光照培养12h,黑暗培养12h;光照培养条件均为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;S2 protocorm induction: inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L for the first solid-state culture, culture for 50 days, and culture in light for 12 hours every day, and culture in dark 12h, light culture conditions are light intensity 1500Lux, temperature 26°C, dark culture temperature is 26°C; when the new axillary bud grows to 1cm, after 3 leaves, cut off the new bud, peel off the leaf, remove the terminal bud, take the axillary bud, and re-inoculate Enter the new induction medium with the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L for the second solid-state culture to obtain protocorms; the second solid-state culture is repeated for 3 times, each time the second solid-state Cultivate for 30 days, and each second solid-state culture includes 10 days of full-dark culture, followed by 12 hours of light culture and 12 hours of dark culture every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
S3原球茎增殖:切掉原球茎的顶部芽,挑选原球茎含有10个原球茎颗粒接种至配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L增殖培养基中,26℃、110rpm液体悬浮培养30d,每5d,切掉原球茎新长出的芽,接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的新增殖培养基培养,共培养5个周期,每个周期为30d,,得原球茎成团;S3 Protocorm Proliferation: Cut off the top buds of the protocorm, select the protocorm containing 10 protocorm particles and inoculate to the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L for proliferation and cultivation Medium, 26°C, 110rpm liquid suspension culture for 30 days, every 5 days, cut off the newly grown shoots of the protocorm, insert the same formula as 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g /L of new proliferation medium culture, co-cultivation for 5 cycles, each cycle is 30d, to get protocorms into agglomerates;
S4原球茎分化:将原球茎成团接种在配方为NAA0.1mg/L+香蕉30g/L+土豆20g/L的分化培养基,固态培养40d,得小苗,其中,包括全黑暗培养7d后,每天光照培养12h,黑暗培养12h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度为26℃;S4 protocorm differentiation: Inoculate the protocorm in a group into the differentiation medium with the formula of NAA0.1mg/L+banana 30g/L+potato 20g/L, culture in solid state for 40 days, and get seedlings, including culturing in total darkness for 7 days, and light every day Cultivate for 12 hours, and cultivate in the dark for 12 hours; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;
S5壮苗生根培养:将小苗经配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L的壮苗培养基,培养50d,每天光照培养12h,黑暗培养12h,得苗株,移入配方为1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L+IBA 0.2mg/L+NAA 0.2mg/L的生根培养基,生根培养90d,得生根苗,其中,包括全黑暗培 养7d后,每天光照培养12h,黑暗培养12h;光照培养条件为光照强度1500Lux,温度26℃,黑暗培养温度均为26℃;Rooting culture of S5 strong seedlings: the seedlings are cultivated in the strong seedling medium formulated as 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L for 50 days, cultivated in the light for 12 hours a day, and cultivated in the dark for 12 hours to obtain seedlings and transfer them into the formula The rooting medium is 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L, and the rooting culture is 90 days, and the rooting shoots are obtained. Cultivate in light for 12 hours and in dark for 12 hours every day; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;
S6优良后代单株选择:将长至7cm,根支4条以上,根长2cm以上的生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of S6 excellent progeny: transplant the rooted seedlings with a length of 7 cm, more than 4 root branches, and a root length of more than 2 cm, eliminate chimera plants with variegated flowers, and keep the offspring with pure or monochromatic flowers.
经试验结果表明,原球茎的增殖率为增殖率3.48,开花植株数为5237株,纯色花植株数为3985株,纯色花分离率为76.09%。The test results show that the proliferation rate of the protocorm is 3.48, the number of flowering plants is 5237, the number of pure-color flower plants is 3985, and the separation rate of pure-color flowers is 76.09%.
对比例4Comparative example 4
本对比例与实施例5的区别在于,所述的S3步骤中,培养基添加9g/L的卡拉胶制备成固体培养基,采用固体培养替代液体悬浮培养;每5d,直接将原球茎接入同配方为1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L的新增殖培养基培养,接入前未切掉原球茎新长出的芽。The difference between this comparative example and Example 5 is that in the S3 step, the medium is prepared by adding 9 g/L of carrageenan to the medium to prepare a solid medium, and solid culture is used instead of liquid suspension culture; every 5 days, the protocorm is directly inserted into Cultivate with the new proliferation medium with the formula of 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L, and the newly grown buds of the protocorm are not cut off before inoculation.
经试验结果表明,原球茎的增殖率为增殖率1.62,开花植株数为1219株,纯色花植株数为482株,纯色花分离率为39.54%。The test results show that the proliferation rate of the protocorm is 1.62, the number of flowering plants is 1219, the number of pure-color flower plants is 482, and the separation rate of pure-color flowers is 39.54%.
2、本发明的单一实验2. Single experiment of the present invention
(1)侧芽消毒:对石斛兰侧芽用0.1%HgCl 2采用不同时间进行消毒,消毒效果如表2。 (1) Disinfection of lateral buds: the lateral buds of Dendrobium orchid were sterilized with 0.1% HgCl 2 at different times, and the disinfection effects are shown in Table 2.
表2Table 2
Figure PCTCN2021141023-appb-000002
Figure PCTCN2021141023-appb-000002
不同消毒时间效果不同,处理时间越长,污染率越低,但褐化率(消毒死亡率)增加。最好的处理是0.1%HgCl 2溶液(加1滴吐温80)消毒10min,污 染率仅为6%,褐化率(消毒死亡率)为10%。 Different disinfection times have different effects. The longer the treatment time, the lower the pollution rate, but the browning rate (disinfection mortality rate) increases. The best treatment is 0.1% HgCl 2 solution (plus 1 drop of Tween 80) for disinfection for 10 minutes, the pollution rate is only 6%, and the browning rate (disinfection mortality rate) is 10%.
(2)原球茎诱导:原球茎诱导培养基采用1/2MS附加不同浓度组合的BA和NAA,对原球茎诱导的影响,结果如表3。(2) Protocorm induction: The protocorm induction medium was supplemented with 1/2 MS with different concentrations of BA and NAA, the effect on protocorm induction, the results are shown in Table 3.
表3table 3
Figure PCTCN2021141023-appb-000003
Figure PCTCN2021141023-appb-000003
诱导结果表明,不同组合的BA和NAA均可诱导成芽,但产芽数和产芽率不同。其中,1/2MS+BA 3mg/L+NAA 0.2mg/L原球茎诱导效果最好,产芽率为2.72%,几乎所有接种的侧芽都能诱导出2~3个新芽。The induction results showed that different combinations of BA and NAA could induce bud formation, but the number and percentage of buds produced were different. Among them, 1/2MS+BA 3mg/L+NAA 0.2mg/L protocorm had the best induction effect, the germination rate was 2.72%, almost all inoculated lateral buds could induce 2-3 new buds.
(3)原球茎增殖:原球茎增殖培养基采用与原球茎诱导培养基相同的激素组合(BA 3mg/L+NAA 0.2mg/L),并试验有机附加物香蕉和土豆对原球茎增殖的影响,结果如表4。(3) Protocorm proliferation: The protocorm proliferation medium uses the same hormone combination as the protocorm induction medium (BA 3mg/L+NAA 0.2mg/L), and the effects of organic additives banana and potato on protocorm proliferation are tested , the results are shown in Table 4.
表4Table 4
Figure PCTCN2021141023-appb-000004
Figure PCTCN2021141023-appb-000004
结果表明,加入香蕉利于原球茎的增殖,土豆利于原球茎的生长,产生的原球茎健壮饱满。1/2MS+BA 3mg/L+NAA 0.2mg/L+香蕉30g/L+土豆20g/L增殖效果最好,原球茎增殖较多,原球茎颗粒较大、饱满,活力非常强,增殖过程中不易分化产生小芽,原球茎和新产生的小芽为浅绿色。The results showed that adding bananas was beneficial to the proliferation of protocorms, potatoes were beneficial to the growth of protocorms, and the resulting protocorms were strong and plump. 1/2MS+BA 3mg/L+NAA 0.2mg/L+banana 30g/L+potato 20g/L has the best proliferation effect, protocorm proliferates more, protocorm particles are larger, full, very strong vitality, difficult to differentiate during the proliferation process Small buds are produced, and the protocorms and newly produced small buds are light green.
(4)原球茎分化:研究不同浓度的BA对石斛兰原球茎分化的影响,结果如表5。(4) Protocorm differentiation: The effects of different concentrations of BA on the differentiation of Dendrobium orchid protocorms were studied, and the results are shown in Table 5.
表5table 5
Figure PCTCN2021141023-appb-000005
Figure PCTCN2021141023-appb-000005
结果表明,原球茎分化培养基在原球茎增殖培养基基础上,如果只保留细 胞分裂素BA,则会降低原球茎分化。The results showed that protocorm differentiation medium would reduce protocorm differentiation if only cytokinin BA was retained on the basis of protocorm proliferation medium.
(5)原球茎分化:研究不同浓度的NAA对石斛兰原球茎分化的影响,结果如表6。(5) Protocorm differentiation: The effects of different concentrations of NAA on the differentiation of Dendrobium orchid protocorms were studied, and the results are shown in Table 6.
表6Table 6
Figure PCTCN2021141023-appb-000006
Figure PCTCN2021141023-appb-000006
结果表明,1/2MS基本培养基附加0~0.2mg/L NAA均可分化至少1个芽出来,1/2MS+NAA 0.1mg/L分化效果最好,分化成苗率达2.24。前期试验表明,培养基附加香蕉30g/L+土豆20g/L可使原球茎变大变绿,分化成苗的原球茎增多,分化的苗株较大、较壮。因此,分化培养基采用1/2MS+NAA 0.1mg/L+30g/L+土豆20g/L,效果最佳。The results showed that 1/2MS basic medium plus 0-0.2mg/L NAA could differentiate at least one bud, and 1/2MS+NAA 0.1mg/L had the best differentiation effect, and the rate of differentiation reached 2.24. Preliminary experiments showed that adding 30g/L of bananas and 20g/L of potatoes to the medium could make the protocorms bigger and greener, and the number of protocorms differentiated into seedlings increased, and the differentiated seedlings were larger and stronger. Therefore, the differentiation medium uses 1/2MS+NAA 0.1mg/L+30g/L+potato 20g/L, the effect is the best.
(6)壮苗生根培养:当原球茎分化出小苗,经壮苗培养50d,挑苗株在2cm以上的移入生根培养基进行生根培养,试验不同组合的IBA与NAA对生根的影响,结果如表7。(6) rooting culture of strong seedlings: when the protocorm differentiates into small seedlings, after strong seedlings are cultivated for 50 days, the seedlings are picked and moved into the rooting medium above 2cm to carry out rooting culture, and the impact of different combinations of IBA and NAA on rooting is tested. The results are as follows: Table 7.
表7Table 7
Figure PCTCN2021141023-appb-000007
Figure PCTCN2021141023-appb-000007
Figure PCTCN2021141023-appb-000008
Figure PCTCN2021141023-appb-000008
结果表明,1/2MS+活性炭15g/L+IBA 0.2mg/L+NAA 0.2mg/L生根最好,生根率可达100%,且生根数量适中,长短适中,有利于后期移栽成活。前期试验表明,生根培养基中附加香蕉30g/L和土豆20g/L可在促进生根的同时促进壮苗,不仅更易出根,且植株叶片更厚、更宽,产生的根更粗、更壮。因此,1/2MS+香蕉30g/L+土豆20g/L+活性炭15g/L+IBA 0.2mg/L+NAA 0.2mg/L生根培养效果最好。The results showed that 1/2MS+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L had the best rooting rate, the rooting rate could reach 100%, and the number of roots was moderate and the length was moderate, which was conducive to the later transplanting and survival. Preliminary experiments have shown that adding 30g/L of banana and 20g/L of potato to the rooting medium can not only promote rooting but also promote strong seedlings. Not only are the roots easier to emerge, but the leaves of the plants are thicker and wider, and the roots produced are thicker and stronger. . Therefore, 1/2MS+banana 30g/L+potato 20g/L+activated carbon 15g/L+IBA 0.2mg/L+NAA 0.2mg/L has the best rooting effect.
(7)选取壮苗(株高5~8cm,4条根以上,根长约2cm以上)进行移栽,移栽成活率可达100%。(7) Select strong seedlings (plant height 5-8cm, more than 4 roots, root length more than about 2cm) to transplant, and the transplanting survival rate can reach 100%.
综上所述,本发明提供的石斛兰纯色花的分离育种方法,以石斛兰侧芽茎段为外植体,通过科学配制了各个培养基成分、利用原球茎增殖阶段采用液体悬浮培养和切掉原球茎新长出的芽的处理方式,控制培养光照和培养时间,实现了石斛兰花色嵌合体中纯色花的更好地稳定快速分离。In summary, the separation and breeding method of Dendrobium orchid solid-color flowers provided by the present invention uses the stem section of Dendrobium orchid lateral buds as explants, scientifically prepares each medium component, utilizes the protocorm proliferation stage to adopt liquid suspension culture and cuts off The processing method of newly grown buds from the protocorm, the control of the cultivation light and the cultivation time, have realized better, stable and rapid separation of the solid-colored flowers in the dendrobium orchid color chimera.
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (9)

  1. 一种石斛兰纯色花的分离育种方法,其特征在于:包括以下步骤:A method for separating and breeding dendrobium pure color flowers, characterized in that: comprising the following steps:
    S1外植体选择:以石斛兰侧芽茎段为外植体,侧芽消毒;S1 explant selection: the stem section of the lateral bud of Dendrobium orchid is used as the explant, and the lateral bud is sterilized;
    S2原球茎诱导:将消毒后的侧芽接种在配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L的诱导培养基上进行第一次固态培养;当新生腋芽长至0.8~1.2cm,叶片2~3片后,切下新芽,剥掉叶片,去掉顶芽,取腋芽,重新接入同配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L的新诱导培养基上进行第二次固态培养,得原球茎;S2 protocorm induction: Inoculate the sterilized lateral buds on the induction medium with the formula of 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L for the first solid culture; when the new axillary buds grow to 0.8 ~1.2cm, after 2~3 pieces of leaves, cut off the new shoots, peel off the leaves, remove the terminal buds, take the axillary buds, re-insert the same formula as 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L Carry out the second solid-state culture on the new induction medium of the above, and obtain the protocorm;
    S3原球茎增殖:切掉原球茎的顶部芽,接种至配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+香蕉20~40g/L+土豆10~30g/L的增殖培养基中,液体悬浮培养,每3~7d,切掉原球茎新长出的芽,接入同配方为1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+香蕉20~40g/L+土豆10~30g/L的新增殖培养基培养,得原球茎成团;Proliferation of S3 protocorms: cut off the top buds of protocorms and inoculate them into the multiplication culture with the formula of 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+banana 20~40g/L+potato 10~30g/L Medium, liquid suspension culture, every 3~7d, cut off the newly grown buds of the protocorm, insert the same formula as 1/2MS+BA 1~4mg/L+NAA 0.1~0.2mg/L+banana 20~40g/ L+Potato 10~30g/L new proliferation medium culture, get the protocorm into agglomerate;
    S4原球茎分化:将原球茎成团接种在分化培养基中固态培养,得小苗;S4 protocorm differentiation: the protocorm is inoculated in a mass in the differentiation medium for solid-state culture to obtain seedlings;
    S5壮苗生根培养:将小苗经壮苗培养基培养,得苗株,移入生根培养基,得生根苗;S5 rooting cultivation of strong seedlings: the seedlings are cultivated on the strong seedling medium to obtain seedlings, and then transferred to the rooting medium to obtain rooting seedlings;
    S6优良后代单株选择:将生根苗移栽,淘汰嵌合体杂色花植株,保留纯色花或单色花后代。Single plant selection of excellent offspring of S6: transplant the rooted seedlings, eliminate chimera plants with variegated flowers, and keep the offspring with pure or monochromatic flowers.
  2. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于:所述的S1步骤中,侧芽消毒方法为自来水冲洗干净,稀释的洗洁精浸泡5~15min,自来水冲洗干净,用体积浓度70%酒精浸泡50~70s,然后无菌水冲洗4~6次,在超净台上除去叶片及膜质叶鞘,转入质量浓度0.1%HgCl 2溶液消毒4~12min,无菌水冲洗4~6次,用无菌纱布或无菌滤纸吸干水。 The method for separating and breeding dendrobium pure color flowers according to claim 1, characterized in that: in the step S1, the lateral bud disinfection method is to rinse with tap water, soak in diluted detergent for 5-15min, rinse with tap water, and use Soak in 70% alcohol for 50-70 seconds, then rinse with sterile water for 4-6 times, remove leaves and membranous leaf sheaths on an ultra-clean bench, transfer to 0.1% HgCl 2 solution for disinfection for 4-12 minutes, rinse with sterile water 4 to 6 times, dry the water with sterile gauze or sterile filter paper.
  3. 根据权利要求2所述的石斛兰纯色花的分离育种方法,其特征在于:所述的质量浓度0.1%HgCl 2溶液加入1-2滴吐温-80。 The method for separating and breeding the solid-colored flowers of Dendrobium orchid according to claim 2, characterized in that: 1-2 drops of Tween-80 are added to the 0.1% HgCl solution with a mass concentration.
  4. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于:所述的S2步骤中,第一次固态培养,培养45~55d,其中,每天光照培养10~14h,黑暗培养10~14h;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度为24~28℃。The method for separating and breeding dendrobium pure color flowers according to claim 1, characterized in that: in the step S2, the first solid-state culture is carried out for 45 to 55 days, wherein, the light is cultivated for 10 to 14 hours every day, and the dark is cultivated for 10 hours. ~14h; light culture conditions are light intensity 1300~1700Lux, temperature 24~28°C, and dark culture temperature 24~28°C.
  5. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于:所 述的S2步骤中,第二次固态培养反复培养2-3次,每次第二次固态培养25~35d;每次第二次固态培养包括全黑暗培养9~11d后,每天光照培养10~14h,黑暗培养10~14h;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度为24~28℃。The method for separating and breeding dendrobium pure-color flowers according to claim 1, characterized in that: in the step S2, the second solid-state culture is repeated for 2-3 times, and the second solid-state culture is 25-35 days each time; Each second solid-state culture includes 9-11 days of full-dark culture, 10-14 hours of light culture every day, and 10-14 hours of dark culture. ~28°C.
  6. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于,所述的S3步骤中,挑选原球茎含有5~15个原球茎颗粒进行接种,在24~28℃、100~120rpm液体悬浮培养,共培养4~6个周期,每个周期为25~35d。The method for separation and breeding of Dendrobium solid color flowers according to claim 1, characterized in that, in the step S3, the selected protocorm contains 5 to 15 protocorm particles for inoculation, at 24 to 28°C, 100 to 120rpm Liquid suspension culture, co-cultivation for 4 to 6 cycles, each cycle is 25 to 35 days.
  7. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于,所述的S4步骤中,分化培养基为NAA0~0.2mg/L+香蕉20~40g/L+土豆10~30g/L;固态培养,培养35~45d,其中,包括全黑暗培养6~8d后,每天光照培养10~14h,黑暗培养10~14h;光照培养条件为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度为24~28℃。The method for separating and breeding dendrobium solid color flowers according to claim 1, wherein in the step S4, the differentiation medium is NAA0~0.2mg/L+banana 20~40g/L+potato 10~30g/L; Solid-state culture, culture for 35-45 days, including 6-8 days of total darkness culture, 10-14 hours of light culture every day, and 10-14 hours of dark culture; light culture conditions are light intensity 1300-1700Lux, temperature 24-28 ℃, dark culture The temperature is 24-28°C.
  8. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于,所述的S5步骤中,壮苗培养基为1/2MS+香蕉20~40g/L+土豆10~30g/L+活性炭10~20g/L,生根培养基为1/2MS+香蕉20~40g/L+土豆10~30g/L+活性炭10~20g/L+IBA 0~0.4mg/L+NAA 0~0.4mg/L;壮苗培养45~55d,其中,每天光照培养10~14h,黑暗培养10~14h;生根培养85~95d,其中,包括全黑暗培养6~8d后,每天光照培养10~14h,黑暗培养10~14h;光照培养条件均为光照强度1300~1700Lux,温度24~28℃,黑暗培养温度均为24~28℃。The method for separation and breeding of Dendrobium solid color flowers according to claim 1, characterized in that, in the step S5, the strong seedling medium is 1/2MS+banana 20~40g/L+potato 10~30g/L+activated carbon 10~ 20g/L, the rooting medium is 1/2MS+banana 20~40g/L+potato 10~30g/L+activated carbon 10~20g/L+IBA 0~0.4mg/L+NAA 0~0.4mg/L; strong seedling culture 45 ~55 days, of which, the light culture is 10-14 hours per day, and the dark culture is 10-14 hours; the rooting culture is 85-95 days, including 6-8 days after the total darkness culture, the light culture is 10-14 hours every day, and the dark culture is 10-14 hours; light culture The conditions are all light intensity of 1300-1700Lux, temperature of 24-28°C, and dark cultivation temperature of 24-28°C.
  9. 根据权利要求1所述的石斛兰纯色花的分离育种方法,其特征在于,所述的S6步骤中,待生根苗长至5~8cm,根支4条以上,根长2cm以上时,开始移栽。The method for separation and breeding of dendrobium solid color flowers according to claim 1, characterized in that, in the step S6, when the rooted seedlings grow to 5-8 cm, there are more than 4 root branches, and the root length is more than 2 cm, start to move plant.
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