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

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

A kind of separation breeding method of dendrobium orchid pure color flower technical field

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.

Background technique

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. 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.

Contents of the invention

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.

Technical scheme of the present invention is realized like this:

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.

To further illustrate, add 1-2 drops of Tween-80 to the 0.1% HgCl ₂ solution.

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;

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.

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 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 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. 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;

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.

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) 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) 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) 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) 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.

Detailed ways

In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

Example 1

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 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;

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 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;

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.

Example 2

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 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;

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 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;

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.

Example 3

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 light intensity 1500Lux, temperature 26°C, and dark culture temperature are both 26°C;

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. 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;

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.

Comparative example 1

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.

Comparative example 2

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.

Comparative 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.

1. Determination of separation of dendrobium flower pure color flower strains

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.

Table 1

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.

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.

Example 4

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, temperature 26°C, and dark culture temperature are 26°C;

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 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. 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;

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.

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%.

Example 5

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; the light culture conditions are light intensity 1500Lux, temperature 26°C, and dark culture temperature 26°C;

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. 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;

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.

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%.

Comparative example 4

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%.

2. Single experiment of the present invention

(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.

Table 2

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) 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.

table 3

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) 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.

Table 4

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) 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.

table 5

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) 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.

Table 6

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) 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.

Table 7

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) 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%.

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.

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. A method for separating and breeding dendrobium pure color flowers, characterized in that: comprising the following steps:

   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 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;

   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 protocorm differentiation: the protocorm is inoculated in a mass in the differentiation medium for solid-state culture to obtain seedlings;

   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;

   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. 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. 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. 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. 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. 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. 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. 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. 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.