Tissue culture and rapid propagation seedling raising method for dendrobium officinale

Abstract

The invention discloses a dendrobium officinale tissue culture and rapid propagation seedling raising method, which belongs to the field of biotechnology, and comprises the following steps: S1, explant treatment: selecting a dendrobium officinale plant with good growth vigor and no obvious pest and disease damage, carrying out high temperature stress treatment at 40-45 DEG C for 6-8 days, then taking the tender stem of the dendrobium officinale plant subjected to high temperature stress treatment as the explant, and carrying out tissue culture and rapid propagation on the explant; directly transferring the explant to a low temperature of 2-6 DEG C for stress treatment for 8-12 hours; s2, callus induction: taking the explant treated in the step S1, disinfecting the explant, taking the explant without axillary buds, cutting the explant into small blocks, inoculating the small blocks to a callus induction culture medium, and carrying out dark culture for 12-15 days; the callus induction culture medium comprises 1.5 to 3 mg / L of MS + 2, 4-D, 2 to 3 mg / L of KT and 0.1 to 0.3 mg / L of NAA; s3, bud differentiation induction and rooting induction. According to the tissue culture and rapid propagation method for the dendrobium officinale, the browning rate of the callus during callus induction can be effectively reduced, and the tissue culture seedling raising efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapid propagation of Dendrobium officinale seedlings through tissue culture. Background Art

[0002] Dendrobium officinale ( Dendrobiumoffcinale ), also known as black knot grass, is an orchid family ( Orchidaceae )Dendrobium( Dendrobium ) Plant, Dendrobium officinale is a precious Chinese herbal medicine with extremely high health benefits. It is sweet and slightly cold in nature, enters the stomach and kidney meridians, benefits the stomach and produces body fluids, nourishes yin and clears heat from the mouth.

[0003] Dendrobium officinale has high requirements for its growth environment, and its natural propagation and cutting propagation are inefficient. Currently, rapid tissue culture is often used to obtain seedlings to quickly obtain Dendrobium officinale plants. However, browning of the callus tissue during rapid tissue culture propagation is common, reducing the efficiency of obtaining seedlings. Summary of the Invention

[0004] In order to overcome the problems existing in the prior art, the present invention proposes a method for rapid propagation of Dendrobium officinale seedlings through tissue culture.

[0005] To achieve the above object, the present invention is implemented through the following technical solutions: A method for rapid propagation of Dendrobium officinale seedlings by tissue culture, comprising the following steps: S1. Explant treatment: Select well-growing Dendrobium officinale plants without obvious diseases and insect pests, and subject them to high temperature stress at 40-45°C for 6-8 days. Then, take the young stems of the high temperature-stressed Dendrobium officinale plants as explants and directly transfer the explants to low temperature stress at 2-6°C for 8-12 hours. S2, callus induction: taking the explants treated in step S1, sterilizing the explants, cutting the explants without axillary buds into small pieces, and inoculating them into callus induction medium, and culturing them in the dark for 12-15 days; the callus induction medium contains: MS + 2,4-D 1.5-3 mg / L, KT 2-3 mg / L, NAA 0.1-0.3 mg / L; S3, induction of shoot differentiation and rooting.

[0006] Furthermore, the method of inducing bud differentiation is: cutting the expanded callus into small pieces and inoculating them into bud induction medium, the bud induction medium is: MS+ 6-BA0.8-1.2 mg / L, NAA0.4-0.8 mg / L, KT2.0-3.0 mg / L and sucrose 35 g / L, and culturing for 18-22 days under light conditions of 1000-1200 lux.

[0007] Furthermore, the rooting induction method is: separating clustered buds to obtain single buds, inoculating the single buds into a rooting medium, the rooting medium is: MS+NAA 1.5-3 mg / L, activated carbon 1-2 g / L and white sugar 20 g / L, the rooting culture light intensity is 10000 lux-12000 lux, and the light is on for 12-16 hours per day.

[0008] Furthermore, the explants were disinfected by soaking them in 70% ethanol solution for 30 seconds, rinsing them with sterile water for 30 seconds, and then disinfecting them with 40% sodium hypochlorite aqueous solution for 15 minutes.

[0009] Through the above technical solution, the present invention can achieve at least the following beneficial effects: The Dendrobium officinale tissue culture rapid propagation method of the present invention can effectively reduce the browning rate of callus tissue during callus induction and improve the efficiency of tissue culture seedlings. DETAILED DESCRIPTION

[0010] Unless otherwise stated, all materials and reagents used in the present invention are commercially available.

[0011] Example 1 A method for rapid propagation of Dendrobium officinale seedlings by tissue culture, comprising the following steps: S1, explant treatment: Select Dendrobium officinale plants with good growth and no obvious diseases and insect pests, and treat them under high temperature stress at 43°C for 7 days. Then, take the young stems of the Dendrobium officinale plants treated with high temperature stress as explants, and directly transfer the explants to low temperature stress treatment at 4°C for 10 hours; S2, inducing callus tissue: taking the explants treated in step S1, disinfecting the explants, cutting the explants without axillary buds into small pieces, and inoculating them into callus induction medium, and culturing them in the dark for 13 days; the callus induction medium contains: MS+2,4-D2.2 mg / L, KT 2.5 mg / L, NAA 0.2 mg / L; wherein the explant disinfection method is: soaking the explants in 70% ethanol solution for 30 seconds, rinsing with sterile water for 30 seconds, and then disinfecting them with 40% sodium hypochlorite aqueous solution for 15 minutes.

[0012] S3, induced shoot differentiation and rooting; The method for inducing bud differentiation is as follows: cut the expanded callus into small pieces and inoculate them into bud induction medium. The bud induction medium is: MS+ 6-BA1mg / L, NAA0.6mg / L, KT2.5mg / L and sucrose 35g / L, and culture them for 20 days under the condition of 1000lux light.

[0013] The method of inducing rooting is: separate the clustered buds to obtain single buds, and inoculate the single buds into the rooting medium. The rooting medium is: MS+NAA 2.3 mg / L, activated carbon 1.5 g / L and white sugar 20 g / L. The light intensity of the rooting culture is 11000 lux, and the light is on for 14 hours per day.

[0014] Example 2 A method for rapid propagation of Dendrobium officinale seedlings by tissue culture, comprising the following steps: S1, explant treatment: Select Dendrobium officinale plants with good growth and no obvious diseases and insect pests, and treat them under high temperature stress at 40°C for 6 days. Then, take the young stems of the Dendrobium officinale plants treated with high temperature stress as explants, and directly transfer the explants to low temperature stress treatment at 2°C for 8 hours; S2, inducing callus tissue: taking the explants treated in step S1, disinfecting the explants, cutting the explants without axillary buds into small pieces, and inoculating them into callus induction medium, and culturing them in the dark for 12 days; the callus induction medium contains: MS+2,4-D1.5 mg / L, KT2 mg / L, NAA0.1 mg / L; wherein the explants are disinfected by soaking the explants in 70% ethanol solution for 30 seconds, rinsing with sterile water for 30 seconds, and then disinfecting them with 40% sodium hypochlorite aqueous solution for 15 minutes.

[0015] S3, induced shoot differentiation and rooting; The method of inducing bud differentiation is as follows: the expanded callus is cut into small pieces and inoculated into a bud induction medium containing: MS + 6-BA 0.8 mg / L, NAA 0.4 mg / L, KT 2.0-3.0 mg / L and sucrose 35 g / L, and cultured under 1000-lux light conditions for 18 days; The method of inducing rooting is: separating clustered buds to obtain single buds, and inoculating the single buds into rooting medium. The rooting medium is: MS+NAA 1.5 mg / L, activated carbon 1 g / L and white sugar 20 g / L. The light intensity of rooting culture is 10000 lux, and the light is on for 12 hours per day.

[0016] Example 3 A method for rapid propagation of Dendrobium officinale seedlings by tissue culture, comprising the following steps: S1, explant treatment: Select well-growing Dendrobium officinale plants without obvious diseases and insect pests and subject them to high temperature stress at 45°C for 8 days. Then, take the young stems of the Dendrobium officinale plants that have been subjected to high temperature stress as explants and directly transfer the explants to low temperature stress at 6°C for 12 hours. S2, inducing callus tissue: taking the explants treated in step S1, disinfecting the explants, cutting the explants without axillary buds into small pieces, and inoculating them into callus induction medium, and culturing them in the dark for 15 days; the callus induction medium contains: MS+2,4-D 3mg / L, KT 3mg / L, NAA 0.0.3mg / L; wherein, the explant disinfection method is: soaking the explants in 70% ethanol solution for 30 seconds, rinsing with sterile water for 30 seconds, and then disinfecting them with 40% sodium hypochlorite aqueous solution for 15 minutes.

[0017] S3, induction of shoot differentiation and rooting.

[0018] The method for inducing bud differentiation is as follows: cut the expanded callus into small pieces and inoculate them into bud induction medium. The bud induction medium is: MS+ 6-BA1.2mg / L, NAA0.8mg / L, KT3.0mg / L and sucrose 35g / L, and culture them for 22 days under the condition of 1200lux light.

[0019] The method of inducing rooting is: separate the clustered buds to obtain single buds, and inoculate the single buds into the rooting medium. The rooting medium is: MS+NAA 3mg / L, activated carbon 2g / L and white sugar 20g / L. The light intensity of the rooting culture is 12000 lux, and the light is on for 16 hours per day.

[0020] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the explants are not subjected to high temperature stress treatment.

[0021] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the explants are not subjected to low temperature stress treatment. Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the explants were neither subjected to high temperature stress treatment nor to low temperature stress treatment.

[0022] Experimental example According to the technical solutions of Examples 1 to 3 and Comparative Examples 1 to 3, three replicates were set for each treatment, tissue culture was carried out to propagate Dendrobium officinale, and the browning rate of callus tissue was counted. The results are shown in Table 1 below.

[0023] Among them, callus browning rate = (number of browned explants / number of inoculated explants) * 100% Table 1 Statistical table of browning rate of callus tissue in each treatment As can be seen from Table 1 above, the Dendrobium officinale tissue culture seedling method of the present invention can effectively reduce the browning rate of callus tissue and indirectly improve the efficiency of tissue culture seedling.

[0024] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for rapid propagation of Dendrobium officinale seedlings through tissue culture, characterized in that: The following steps are included: S1. Explant treatment: Select well-growing Dendrobium officinale plants without obvious diseases and insect pests, and subject them to high temperature stress at 40-45°C for 6-8 days. Then, take the young stems of the high temperature-stressed Dendrobium officinale plants as explants and directly transfer the explants to low temperature stress at 2-6°C for 8-12 hours. S2, callus induction: taking the explants treated in step S1, sterilizing the explants, cutting the explants without axillary buds into small pieces, and inoculating them into callus induction medium, and culturing them in the dark for 12-15 days; the callus induction medium contains: MS + 2,4-D 1.5-3 mg / L, KT 2-3 mg / L, NAA 0.1-0.3 mg / L; S3, induction of shoot differentiation and rooting.

2. A method for rapid propagation of Dendrobium officinale seedlings through tissue culture according to claim 1, characterized in that: The method for inducing bud differentiation is as follows: cutting the expanded callus into small pieces and inoculating them into a bud induction medium containing: MS+6-BA0.8-1.2 mg / L, NAA0.4-0.8 mg / L, KT2.0-3.0 mg / L and sucrose 35 g / L, and culturing them for 18-22 days under the condition of 1000-1200 lux.

3. The method for rapid propagation of Dendrobium officinale seedlings through tissue culture according to claim 1, wherein: The rooting induction method comprises the following steps: separating clustered buds to obtain single buds, inoculating the single buds into a rooting medium containing 1.5-3 mg / L MS+NAA, 1-2 g / L activated carbon, and 20 g / L white sugar; and providing light intensity of 10,000 lux to 12,000 lux for 12-16 hours per day for the rooting culture.

4. The method for rapid propagation of Dendrobium officinale seedlings through tissue culture according to claim 1, wherein: The explant disinfection method is as follows: soak the explant in 70% ethanol solution for 30 seconds, rinse with sterile water for 30 seconds, and then disinfect with 40% sodium hypochlorite aqueous solution for 15 minutes.

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