Method for optimizing tissue culture, rapid propagation and seedling culture of valerian neolastoma

Abstract

The invention discloses a method for optimizing tissue culture, rapid propagation and seedling culture of valerian neolastoma. The method comprises the steps of explant selection, explant disinfection, bud induction, bud proliferation, subculture, rooting culture, test-tube plantlet transplanting and the like, in the process, by optimizing a tissue culture scheme, a culture medium of the same formula is used during bud induction, bud proliferation and cluster bud subculture, the convention of'replacement of the culture medium in stages' in traditional tissue culture is broken, and the tissue culture efficiency is improved. The defects of high cost, high error and high variation of traditional tissue culture are overcome, the proliferation multiple can reach 4.0-5.0, and compared with the prior art, the efficiency is higher; the valerian anemone seedlings cultivated by the method are stable in hereditary character, maintain the characteristics of parents, have the advantages of invariance, low investment, high yield, short period and the like, and can realize large-scale seedling production.

Description

Technical Field

[0001] This invention belongs to the field of rapid propagation technology of tissue culture, specifically relating to an optimized method for rapid propagation of Vitex negundo tissue culture and its seedling cultivation. Background Technology

[0002] Angraecum Veitchii is a species of orchid belonging to the genus Angraecum in the family Orchidaceae. This genus comprises approximately 221 species, distributed in tropical Africa, South Africa, and the Indian Ocean islands, with Madagascar being the largest producer. Angraecum Veitchii was cultivated in 1899 by the British horticulturist John Veitch, who hybridized Angraecum sesquipedale (female parent) with Angraecum eburneum (male parent). In the same year, Angraecum Veitchii was first exhibited and officially registered by the Royal Horticultural Society of London. It is a large, artificially hybridized orchid, with 7-10 flowers per stem and 5-10 flower stalks per plant. The apple-green flowers are elegant and possess a graceful, flowing beauty. In Guangzhou's climate, Angraecum Veitchii typically produces flower stalks in October and blooms from December to March of the following year, making it a highly valued orchid for its festive blooms during the Spring Festival. Its leaves are alternate, leathery, stiff, and dark green, and when not in bloom, it can be appreciated as a foliage plant. In 2010, Chen Heming et al. published an article in *Plant Physiology Communications* on "Rapid In Vitro Propagation of Young Lateral Buds from the Stems of *Cymbidium vegetaceum*", but the propagation ratio was low (3.0). Therefore, to meet market demand, this invention further optimizes the rapid propagation and seedling cultivation method of *Cymbidium vegetaceum* through tissue culture, achieving a propagation ratio of 4.0-5.0, and enabling the production of a large number of test-tube seedlings in a shorter time, providing a more efficient method to meet market demand. Summary of the Invention

[0003] In view of this, the technical problem solved by the present invention is to provide an optimized method for rapid propagation and seedling production of Vichydra variegata through tissue culture. By using the method of clustered buds for tissue culture of Vichydra variegata, a large number of test-tube seedlings with stable genetic traits can be obtained, maintaining the characteristics of the parent plants. It has advantages such as invariability, low input, high output, and short cycle, providing a more efficient method for the production of Vichydra variegata seedlings.

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

[0005] A method for optimizing the rapid propagation and seedling cultivation of *Cymbidium goeringii* through tissue culture includes the following steps:

[0006] (1) Selection of explants: Select tender axillary buds between stem segments, and use segments with axillary buds as explants;

[0007] (2) Disinfection of explants: Cut a stem segment with 6-10 leaves, remove the leaves, disinfect the stem segment, dry the remaining water on the stem segment with sterile filter paper, peel off the outer covering with a sterilized blade, and cut the segment with axillary buds as the explant.

[0008] (3) Bud induction: The disinfected segments were inoculated into the induction medium and cultured until the axillary buds turned green and enlarged, becoming new buds; the formula of the induction medium was Flower Treasure No. 1 3.0g / L + adenine 2.0-3.0mg / L + sucrose 30g / L + agar 7g / L, pH value 5.5-5.8;

[0009] (4) Bud proliferation: Cut off the induced new buds and transfer them to the proliferation medium for 60-90 days to obtain clustered buds; The formula of the proliferation medium is Flower Treasure No. 1 3.0g / L + adenine 2.0-3.0mg / L + sucrose 30g / L + agar 7g / L, pH value 5.5-5.8;

[0010] (5) Subculture: The clustered shoots were transferred to the subculture medium to obtain subculture seedlings. The formula of the subculture medium was 3.0 g / L of Flower Treasure No. 1 + 2.0-3.0 mg / L of adenine + 30 g / L of sucrose + 7 g / L of agar, with a pH of 5.5-5.8.

[0011] (6) Rooting culture: Transfer a certain number of subcultured seedlings to a rooting medium for rooting culture. After 60-90 days of culture, rooted test-tube seedlings are obtained. The formula of the rooting medium is 3.0 g / L of Flower Treasure No. 1 + 0.1-0.3 mg / L of naphthaleneacetic acid + 30 g / L of sucrose + 7 g / L of agar, with a pH of 5.5-5.8.

[0012] (7) Transplanting of test-tube seedlings: When the test-tube seedlings have 3-5 leaves, 3.0-5.0cm in length, 0.5-1.0cm in width, 2.0-5.0cm in length and 2-4 roots, they are placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 10-20 days of hardening, they are transplanted out of the bottle.

[0013] Preferably, in step (2), the disinfection process is as follows: wipe the surface with alcohol on the clean bench and soak it in 75% alcohol for 1 minute, then sterilize it with 0.1% mercuric chloride solution for 15 minutes, and rinse it with sterile water 5 times.

[0014] Preferably, in step (3), the conditions for bud induction culture are: culture temperature 25-28℃, dark culture for 20 days in the early stage, and culture for 30 days in the later stage under light intensity of 2000-2500lx and light duration of 12h / d.

[0015] Preferably, in step (4), the conditions for the bud proliferation culture are: culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0016] Preferably, in step (5), the conditions for subculture are: culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0017] Preferably, in step (6), the conditions for rooting culture are: culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0018] Compared with existing technologies, the advantages of this invention are as follows: the method is easy to operate, has low production costs, does not pollute the environment, and can achieve large-scale seedling production; by optimizing the tissue culture program, the same formula culture medium is used in the induction of buds, the proliferation of buds, and the subculture of clustered buds, breaking the convention of "changing the culture medium at each stage" in traditional tissue culture, and overcoming the disadvantages of "high cost, high error, and high variation" in traditional tissue culture. Its proliferation ratio can reach 4.0-5.0, which is more efficient than existing technologies; the seedlings of Vichylea vegetaceae cultivated by this method have stable genetic traits, maintain the characteristics of the parent, and have advantages such as invariance, low input, high output, and short cycle. Detailed Implementation

[0019] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0020] Example 1

[0021] (1) Selection of explants: Select tender axillary buds between stem segments, and use segments with axillary buds as explants.

[0022] (2) Disinfection of explants: Cut stem segments with 6-10 leaves, remove the leaves, wipe the surface with alcohol on a clean bench and soak in 75% alcohol for 1 minute, then sterilize with 0.1% mercuric chloride solution for 15 minutes, rinse 5 times with sterile water, dry the remaining water on the stem segments with sterile filter paper, then peel off the outer covering with a sterilized blade, and cut the segments with axillary buds as explants.

[0023] (3) Bud induction: The sterilized segments were inoculated into the induction medium and cultured for 50 days. The axillary buds turned green and enlarged, becoming new buds, which could then be used for the next step of proliferation culture. The induction medium was Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 2.0 mg / L + Sucrose 30 g / L + Agar 7 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃. The initial culture was carried out in the dark for 20 days, and the subsequent culture was carried out under a light intensity of 2000-2500 lx and a light duration of 12 h / d for 30 days.

[0024] (4) Bud proliferation: The induced new buds are cut off and transferred to a proliferation medium for 60-90 days to obtain clustered buds. The proliferation medium is Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (adenine) 2.0 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.5-5.8. The proliferation ratio can reach 4.0. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d.

[0025] (5) Subculture: The clustered shoots were transferred to a subculture medium to obtain subculture seedlings. The subculture medium consisted of Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 2.0 mg / L + Sucrose 30 g / L + Agar 7 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0026] (6) Rooting culture: Transfer a certain number of subcultured seedlings to rooting medium for rooting culture. After 60-90 days of culture, rooted test-tube seedlings are obtained. The rooting medium is Hyponex (Flower Treasure No. 1) 3.0g / L + NAA (Naphthaleneacetic Acid) 0.1mg / L + sucrose 30g / L + agar 7g / L, pH 5.5-5.8, culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0027] (7) Transplanting of test-tube seedlings: When the test-tube seedlings reach 3-5 leaves, with a leaf length of 3.0-5.0 cm, a leaf width of 0.5-1.0 cm, a root length of 2.0-5.0 cm, and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 10-20 days of hardening, they can be transplanted from the bottle. Generally, spring (March-May) or autumn (October-November) is chosen for transplanting. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then, soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them, and let them dry. Then, plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. The survival rate of transplanted seedlings can reach over 95%.

[0028] Example 2

[0029] (1) Selection of explants: Select tender axillary buds between stem segments, and use segments with axillary buds as explants.

[0030] (2) Disinfection of explants: Cut stem segments with 6-10 leaves, remove the leaves, wipe the surface with alcohol on a clean bench and soak in 75% alcohol for 1 minute, then sterilize with 0.1% mercuric chloride solution for 15 minutes, rinse 5 times with sterile water, dry the remaining water on the stem segments with sterile filter paper, then peel off the outer covering with a sterilized blade, and cut the segments with axillary buds as explants.

[0031] (3) Bud induction: The sterilized segments were inoculated into the induction medium and cultured for 50 days. The axillary buds turned green and enlarged, becoming new buds, which could then be used for the next step of proliferation culture. The induction medium was Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 2.5 mg / L + Sucrose 30 g / L + Agar 7 g / L, pH 5.5-5.8. The culture temperature was 25-28℃. The initial culture was carried out in the dark for 20 days, and the subsequent culture was carried out under a light intensity of 2000-2500 lx and a light duration of 12 h / d for 30 days.

[0032] (4) Bud proliferation: The induced new buds are cut off and transferred to a proliferation medium for 60-90 days to obtain clustered buds. The proliferation medium is Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (adenine) 2.5 mg / L + sucrose 30 g / L + agar 7 g / L, pH 5.5-5.8. The proliferation ratio can reach 4.5. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d.

[0033] (5) Subculture: The clustered shoots were transferred to a subculture medium to obtain subculture seedlings. The subculture medium consisted of Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 2.5 mg / L + Sucrose 30 g / L + Agar 7 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0034] (6) Rooting culture: Transfer a certain number of subcultured seedlings to rooting medium for rooting culture. After 60-90 days of culture, rooted test-tube seedlings are obtained. The rooting medium is Hyponex (Flower Treasure No. 1) 3.0g / L + NAA (Naphthaleneacetic Acid) 0.2mg / L + sucrose 30g / L + agar 7g / L, pH 5.5-5.8, culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0035] (7) Transplanting of test-tube seedlings: When the test-tube seedlings reach 3-5 leaves, with a leaf length of 3.0-5.0 cm, a leaf width of 0.5-1.0 cm, a root length of 2.0-5.0 cm, and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 10-20 days of hardening, they can be transplanted from the bottle. Generally, spring (March-May) or autumn (October-November) is chosen for transplanting. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then, soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them, and let them dry. Then, plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. The survival rate of transplanted seedlings can reach over 95%.

[0036] Example 3

[0037] (1) Selection of explants: Select tender axillary buds between stem segments, and use segments with axillary buds as explants.

[0038] (2) Disinfection of explants: Cut stem segments with 6-10 leaves, remove the leaves, wipe the surface with alcohol on a clean bench and soak in 75% alcohol for 1 minute, then sterilize with 0.1% mercuric chloride solution for 15 minutes, rinse 5 times with sterile water, dry the remaining water on the stem segments with sterile filter paper, then peel off the outer covering with a sterilized blade, and cut the segments with axillary buds as explants.

[0039] (3) Bud induction: The sterilized segments were inoculated into the induction medium and cultured for 50 days. The axillary buds turned green and enlarged, becoming new buds, which could then be used for the next step of proliferation culture. The induction medium was Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 3.0 mg / L + Sucrose 30 g / L + Agar 7 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃. The initial culture was carried out in the dark for 20 days, and the subsequent culture was carried out under a light intensity of 2000-2500 lx and a light duration of 12 h / d for 30 days.

[0040] (4) Bud proliferation: The induced new buds are cut off and transferred to a proliferation medium for 60-90 days to obtain clustered buds. The proliferation medium is Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 3.0 mg / L + Sucrose 30 g / L + Agar 7 g / L, pH 5.5-5.8. The proliferation ratio can reach 5.0. The culture temperature is 25-28℃, the light intensity is 2000-2500 lx, and the photoperiod is 12 h / d.

[0041] (5) Subculture: The clustered shoots were transferred to a subculture medium to obtain subculture seedlings. The subculture medium consisted of Hyponex (Flower Treasure No. 1) 3.0 g / L + Ad (Adenine) 3.0 mg / L + Sucrose 30 g / L + Agar 7 g / L, with a pH of 5.5-5.8. The culture temperature was 25-28℃, the light intensity was 2000-2500 lx, and the photoperiod was 12 h / d.

[0042] (6) Rooting culture: Transfer a certain number of subcultured seedlings to rooting medium for rooting culture. After 60-90 days of culture, rooted test-tube seedlings are obtained. The rooting medium is Hyponex (Flower Treasure No. 1) 3.0g / L + NAA (Naphthaleneacetic Acid) 0.3mg / L + sucrose 30g / L + agar 7g / L, pH 5.5-5.8, culture temperature 25-28℃, light intensity 2000-2500lx, and light duration 12h / d.

[0043] (7) Transplanting of test-tube seedlings: When the test-tube seedlings reach 3-5 leaves, with a leaf length of 3.0-5.0 cm, a leaf width of 0.5-1.0 cm, a root length of 2.0-5.0 cm, and 2-4 roots, they can be placed in a greenhouse with natural light diffusion for hardening and acclimatization. After 10-20 days of hardening, they can be transplanted from the bottle. Generally, spring (March-May) or autumn (October-November) is chosen for transplanting. When the seedlings emerge, use tweezers to remove them from the bottle and clean the culture medium from the roots. Then, soak them in a 0.01% potassium permanganate solution for 3 minutes, remove them, and let them dry. Then, plant them in plastic cups with sphagnum moss. Keep the greenhouse well-ventilated, maintain a humidity of 60-80%, and keep the temperature above 15℃. The survival rate of transplanted seedlings can reach over 95%.

[0044] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for optimizing the rapid propagation of Vetchwind orchid tissue culture and its seedling raising, characterized by, The method comprises the following steps: (1) selection of explants: selecting tender axillary buds of internodes of stems, and taking the internodes with axillary buds as explants; (2) sterilization of explants: cutting stems with 6-10 leaves, cutting off the leaves, sterilizing, absorbing residual moisture on the stems with sterile filter paper, and then peeling off the outer covering with a sterilized blade to cut the internodes with axillary buds as explants; (3) bud induction: inoculating the sterilized internodes into an induction medium for culture until the axillary buds turn green and grow into new buds; the induction medium is composed of 3.0 g / L of Hua Bao No. 1, 2.0-3.0 mg / L of adenine, 30 g / L of sucrose and 7 g / L of agar, and has a pH value of 5.5-5.8; (4) bud proliferation: cutting the induced new buds and transferring them into a proliferation medium for culture for 60-90 days to obtain clustered buds; the proliferation medium is composed of 3.0 g / L of Hua Bao No. 1, 2.0-3.0 mg / L of adenine, 30 g / L of sucrose and 7 g / L of agar, and has a pH value of 5.5-5.8; (5) subculture: transferring the clustered buds into a subculture medium for culture to obtain subculture seedlings; the subculture medium is composed of 3.0 g / L of Hua Bao No. 1, 2.0-3.0 mg / L of adenine, 30 g / L of sucrose and 7 g / L of agar, and has a pH value of 5.5-5.8; (6) rooting culture: transferring seedlings subcultured to a certain amount into a rooting culture medium for rooting culture to obtain rooted test-tube seedlings after 60-90 days of culture; the rooting culture medium is composed of 3.0 g / L of Hua Bao No. 1, 0.1-0.3 mg / L of naphthalene acetic acid, 30 g / L of sucrose and 7 g / L of agar, and has a pH value of 5.5-5.8; (7) transplantation of test-tube seedlings: when the test-tube seedlings have 3-5 leaves with a length of 3.0-5.0 cm, a width of 0.5-1.0 cm, roots with a length of 2.0-5.0 cm and a number of 2-4, they are placed into a greenhouse with natural light scattering for acclimatization, and are transplanted after 10-20 days of acclimatization.

2. The method of claim 1, wherein, In step (2), the sterilization treatment is as follows: the surface is cleaned with alcohol on an ultra-clean workbench, and is soaked in 75% alcohol for 1 minute, and then is sterilized with 0.1% mercury solution for 15 minutes, and is washed with sterile water for 5 times.

3. The method of claim 1, wherein, In step (3), the bud induction culture is carried out at a temperature of 25-28℃, in the dark for 20 days in the early stage, and under light intensity of 2000-2500 lx for 12 h / d in the later stage.

4. The method of claim 1, wherein, In step (4), the bud proliferation culture is carried out at a temperature of 25-28℃, under light intensity of 2000-2500 lx, and for 12 h / d.

5. The method of claim 1, wherein, In step (5), the subculture is carried out at a temperature of 25-28℃, under light intensity of 2000-2500 lx, and for 12 h / d.

6. The method of claim 1, wherein, In step (6), the rooting culture is carried out at a temperature of 25-28℃, under light intensity of 2000-2500 lx, and for 12 h / d.