Tissue culture and rapid propagation method of eriodictyon lanceolatum

Abstract

The invention discloses a tissue culture and rapid propagation method of eriocaulon lanceolarium, and belongs to the technical field of plant rapid propagation. The tissue culture and rapid propagation method comprises the following steps: (1) axillary bud induction culture: taking down axillary buds of eupatorium japonicum, sterilizing and inoculating the axillary buds to a bud induction culture medium MS + 6-BA 0.1-0.5 mg / L + NAA 0.1-0.5 mg / L for bud induction; (2) subculture multiplication culture: cutting off buds, transferring the buds into a multiplication culture medium MS + 6-BA (0.1-0.5 mg / L) + NAA (0.1-1.0 mg / L), and carrying out subculture to obtain cluster buds; (3) rooting culture: the cluster buds are cut and inoculated to a rooting culture medium with 0.3-0.7 mg / L of 1 / 2MS + NAA for rooting culture, and tissue culture seedlings are obtained. According to the method, the culture time of the peanut roots of the eriocaulon lanceolatum is obviously shortened, meanwhile, bottle seedling hardening and domestication are conducted, the survival rate reaches 91%, and conversion of the eriocaulon lanceolatum from the bottle seedlings to the field is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant rapid propagation, and in particular relates to a tissue culture rapid propagation method of hairy medicinal flowers. Background Art

[0002] Bostrychanthera deflexa Benth. is an erect herb of the genus Bostrychanthera in the family Lamiaceae. It is endemic to my country and is mainly produced in Fujian, Guangxi, Jiangxi, Taiwan, Guangdong, Guizhou, Sichuan and other places. It is commonly found in provinces and regions where the She ethnic group is distributed. It likes to grow in subtropical climate zones, under forests at an altitude of 500 to 1,300 meters or in moist places in mountain valleys. Bostrychanthera deflexa Benth. is cool in nature, pungent and bitter in taste, and belongs to the lung meridian. The whole plant is used as medicine, and its effects are mainly to release heat, clear away heat and detoxify, and to promote blood circulation and stop bleeding. It is often used to treat colds, traumatic injuries, etc.

[0003] The medicinal use of Hairy Medicinal Flower is only recorded in the pharmacopoeia of ethnic minority areas. Currently, it is mainly collected from the wild. After consulting the literature, it was found that there are few reports on the artificial cultivation of Hairy Medicinal Flower and the complete tissue culture rapid propagation technology. Summary of the invention

[0004] The present invention provides a tissue culture and rapid propagation method for hairy medicinal flowers, and the tissue culture and rapid propagation method comprises the following steps:

[0005] (1) Axillary bud induction culture: The stem segments with axillary buds of the hairy medicinal flower were used as explants. The axillary buds were removed and disinfected. The buds were then inoculated into bud induction medium MS + 6-BA 0.1-0.5 mg / L + NAA 0.1-0.5 mg / L for bud induction. After inoculation, the buds were cultured in a tissue culture room at a temperature of (25±2)°C and a light intensity of 10-14 h / d for 43-47 days.

[0006] (2) Subculture proliferation culture: the buds obtained in step (1) were cut off and transferred into proliferation medium MS + 6-BA 0.1-0.5 mg / L + NAA 0.1-1.0 mg / L for subculture. After inoculation, they were placed in a tissue culture room at a temperature of (25±2)°C and a light intensity of 10-14 h / d for 30-35 days to obtain clustered buds;

[0007] (3) Rooting culture: The clustered buds obtained in step (2) are cut and inoculated into a rooting medium of 1 / 2MS + NAA 0.3-0.7 mg / L for rooting culture. After inoculation, the buds are first cultured in total darkness at 28-30°C for 4-6 days, and then cultured at 28-30°C for 34-38 days with 10-14 hours of light per day to obtain tissue culture seedlings.

[0008] Preferably, the concentration of 6-BA in the bud induction medium in step (1) is 0.2 mg / L.

[0009] More preferably, the concentration of NAA in the bud induction medium in step (1) is 0.2 mg / L.

[0010] More preferably, the illumination culture in step (1) is carried out for 45 days.

[0011] More preferably, the concentration of 6-BA in the proliferation medium in step (2) is 0.1 mg / L.

[0012] More preferably, the concentration of NAA in the proliferation medium in step (2) is 0.5 mg / L.

[0013] More preferably, the illumination culture is performed for 32 days in step (2).

[0014] More preferably, the concentration of NAA in the rooting medium in step (3) is 0.5 mg / L.

[0015] More preferably, the illumination intensity in step (3) is 32001x.

[0016] More preferably, in step (3), the cells are cultured in darkness for 5 days, followed by light culture for 36 days.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The invention significantly shortens the root culture time of the hairy medicinal flower, and simultaneously performs bottle seedling hardening and domestication, with a survival rate of 91%, thus realizing the transformation of the hairy medicinal flower from bottle seedlings to the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a picture of the axillary bud induction culture in Example 1 after 45 days.

[0020] Figure 2 This is a picture of the cells in Example 1 after 32 days of subculture.

[0021] Figure 3 This is a picture taken after 41 days of rooting culture in Example 1. DETAILED DESCRIPTION

[0022] Example 1

[0023] This embodiment provides a method for tissue culture and rapid propagation of gentiana scabra, which comprises the following steps:

[0024] Step (1), axillary bud induction culture: using the stem segment of the hairy medicinal flower with axillary buds as the explant, cutting off the leaves, removing the axillary buds, shaking and washing with running water for 1 hour, and then transferring to a super workbench for disinfection, first disinfecting with 75% ethanol for 30 seconds, then washing with sterile water 4 times, and then disinfecting with 0.1% mercuric chloride solution for 15 minutes, washing with sterile water 5 times, and then wiping off the water droplets on the surface with sterile filter paper, and then inoculating into bud induction medium MS+6-BA 0.2mg / L+NAA 0.2mg / L for bud induction, and after inoculation, placing in a tissue culture room at a temperature of (25±2)°C, a light intensity of 1500lx, and a light intensity of 12h / d for 45 days, the results are as follows Figure 1 As shown, the bud induction rate reached 96%;

[0025] Step (2), subculture proliferation culture: the buds with normal growth and obvious stem and leaf structures obtained in step (1) were cut off and transferred to proliferation medium MS + 6-BA 0.1mg / L + NAA 0.5mg / L for subculture. After inoculation, they were placed in a tissue culture room at a temperature of (25±2)°C, a light intensity of 1500lx, and a light intensity of 12h / d for 32 days to obtain clustered buds. The proliferation coefficient of the clustered buds was 3.4. The results are as follows Figure 2 As shown; this step can be repeated to cut the buds for multiple times for subculture proliferation culture to obtain more clustered buds;

[0026] Step (3), rooting culture: The clustered buds obtained in step (2) with a height of about 3.8 cm, good growth, and no browning, vitrification, and bleaching were cut and inoculated on a rooting medium 1 / 2MS + NAA 0.5 mg / L for rooting culture. After inoculation, they were first cultured in total darkness at 29°C for 5 days, and then illuminated for 12 hours a day, with a light intensity of 32001x and a culture temperature of 29°C for 36 days. The results are as follows: Figure 3 As shown, the induced rooting rate reached 100%, the average plant height was 6.37 cm, and the average root length was 2.62 cm.

[0027] Step (4), seedling hardening and transplanting: remove the bottle cap of the well-grown and strong rooted test tube seedlings with a height of about 7 cm obtained in step (3) and place them under natural light for hardening for 8 days, then take out the test tube seedlings from the culture bottle, wash off the root culture medium, plant them in a matrix mixed with nutrient soil and vermiculite in a ratio of 3:1, place them in a light culture box for cultivation, water the seedlings with 1 / 2MS macronutrient solution every day to maintain humidity, and transplant them to the field after the seedlings survive, with a survival rate of 91%.

[0028] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for rapid propagation of hairy medicinal flowers through tissue culture, characterized in that: The tissue culture rapid propagation method comprises the following steps: (1) Axillary bud induction culture: The stem segments of the hairy medicinal flowers with axillary buds were used as explants. The axillary buds were removed and disinfected. They were then inoculated into bud induction medium MS + 6-BA 0.1-0.5 mg / L + NAA 0.1-0.5 mg / L for bud induction. After inoculation, they were placed in a tissue culture room at a temperature of (25±2)°C and a light intensity of 10-14 h / d for 43-47 days. (2) Subculture proliferation culture: the buds obtained in step (1) were cut off and transferred into proliferation medium MS + 6-BA 0.1-0.5 mg / L + NAA 0.1-1.0 mg / L for subculture. After inoculation, they were placed in a tissue culture room at a temperature of (25±2)°C and a light intensity of 10-14 h / d for 30-35 days to obtain clustered buds; (3) Rooting culture: The clustered buds obtained in step (2) are cut and inoculated into a rooting medium of 1 / 2MS + NAA 0.3-0.7 mg / L for rooting culture. After inoculation, the buds are first cultured in total darkness at 28-30°C for 4-6 days, and then cultured at 28-30°C for 34-38 days with 10-14 hours of light per day to obtain tissue culture seedlings.

2. The method for rapid propagation of hairy medicinal flowers according to claim 1, characterized in that: The concentration of 6-BA in the bud induction medium in step (1) is 0.2 mg / L.

3. The method for rapid propagation of hairy medicinal flowers according to claim 2, characterized in that: The concentration of NAA in the bud induction medium in step (1) is 0.2 mg / L.

4. The method for rapid propagation of hairy medicinal flowers according to claim 3, characterized in that: The light culture in step (1) is carried out for 45 days.

5. The method for rapid propagation of hairy medicinal flowers according to claim 4, characterized in that: The concentration of 6-BA in the proliferation medium in step (2) is 0.1 mg / L.

6. The method for rapid propagation of hairy medicinal flowers according to claim 5, characterized in that: The concentration of NAA in the proliferation medium in step (2) is 0.5 mg / L.

7. The method for rapid propagation of hairy medicinal flowers according to claim 6, characterized in that: In the step (2), the culture is carried out under illumination for 32 days.

8. The method for rapid propagation of hairy medicinal flowers according to claim 7, characterized in that: The concentration of NAA in the rooting medium in step (3) is 0.5 mg / L.

9. The method for rapid propagation of hairy medicinal flowers according to claim 8, characterized in that: The illumination intensity in step (3) is 32001x.

10. The method for rapid propagation of hairy medicinal flowers according to claim 9, characterized in that: In the step (3), the cells were cultured in darkness for 5 days and then cultured in light for 36 days.

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