Tissue culture and rapid propagation method for Dendrobium aurantium and application thereof

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a dendrobium aurantium tissue culture and rapid propagation method and application thereof.The dendrobium aurantium tissue culture and rapid propagation method comprises the steps that healthy and uncracked dendrobium aurantium pods are selected, fruit clamp shells of the dendrobium aurantium pods are repeatedly wiped clean with degreasing cotton under the alcohol concentration of 75%, then the dendrobium aurantium pods are soaked and disinfected with alcohol of 75%, and disinfection continues to be conducted with mercury bichloride of 0.1%; the method comprises the following steps: cutting small mouths of disinfected fruit pods, clamping the fruit pods with tweezers, uniformly shaking off dendrobium seeds in the fruit pods into a primary culture medium to culture cluster buds, then inoculating the cluster buds into a subculture proliferation culture medium for proliferation, cutting robust buds obtained by proliferation culture into single buds, and transferring the single buds into a rooting culture medium for rooting culture. According to the method, the fruit pods are adopted as the explants, and a strict disinfection procedure is combined, so that the virus carrying rate is effectively reduced, and healthy and disease-free tissue culture seedlings are obtained; two subculture multiplication media are adopted, the multiplication rate of 3.6 times can be achieved, the rooting rate reaches 89% or above, and the root system is robust.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture rapid propagation method of Dendrobium auriculatum and an application thereof. Background Art

[0002] Dendrobium hookerianum Lindl. is a plant of the genus Dendrobium in the Orchidaceae family. It has a drooping stem, hard texture, cylindrical shape, unbranched, and multi-noded. The leaves are thin leathery, bi-row, alternate on the entire stem, and are ovate-lanceolate or oblong. There are 1 to several racemes, which grow laterally in the middle of the old stem with leaves, 4-10 cm long, and sparsely 2-7 flowers; the flowers are thin, golden yellow, and spread out; the petals are oblong, 2.4-3.5 cm long, 10-18 mm wide, nearly obtuse at the tip, with 7 veins, and entire margins; the lip is nearly round, 2-3 cm wide, with short claws at the base, embracing the column on both sides, with compound tassels on the edge, and densely covered with short hairs on the surface, and the flowering period is from July to September. It grows on rocks in valleys at an altitude of 1000-2300 meters or on tree trunks in mountain forests.

[0003] Dendrobium officinale is a perennial herb that likes shade. It likes to grow in a warm, humid environment with an annual rainfall of more than 1,000 mm and half shade and half sun. It grows best in subtropical deep mountains and old forests with an average temperature of more than 8°C in January. The suitable growth temperature is 15-28 degrees, and the suitable growth air humidity is more than 60%. It is not very strict on soil and fertilizer. In the wild, it grows on loose and thick bark or trunks, and some also grow in stone cracks. It belongs to the aerial root system, which mainly requires good root permeability. The substrate used should be ventilated, breathable and water-filtering. Under suitable temperature and humidity, it grows fast and has very strong survival ability. In late spring and early summer every year, the inflorescence emerges from the upper nodes of the biennial stems. After flowering, new buds grow from the stem base and develop into stems. It enters a dormant period in autumn and winter.

[0004] The cultivation of Dendrobium officinale has high horticultural value. At present, the main propagation methods are division propagation, bud propagation and cutting propagation. Division propagation: Select the densely growing plants, take them out of the pot after flowering, remove the old roots, cut them from the base of the clustered stems, and form a group with the main plant to form a new plant. Bud propagation: When there are small plants on the top or base of the plants that have been potted for more than 3 years, select small plants with 3-4 leaves, 2-3 roots, and a root length of 4-5 cm, cut them from the mother plant, treat the wounds with plant ash or 70% mancozeb, and plant the seedlings in the pot. Cutting propagation: Select the whole plant that has not bloomed and is relatively full, cut the branches into several sections, each with 2-3 nodes, apply plant ash or 70% mancozeb to treat the wounds, and insert the stems into a matrix mixed with moss and peat. Summary of the invention

[0005] The object of the present invention is to provide a tissue culture and rapid propagation method and application of Dendrobium wardianum in view of the problems existing in the non-existing technology.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a tissue culture and rapid propagation method of Dendrobium wardianum, the steps include:

[0007] (1) Selection and disinfection of explants: Select healthy and unopened fruit pods of Dendrobium wardianum. First, wipe the outer shell of the fruit pods repeatedly with absorbent cotton under 75% alcohol concentration, then soak and disinfect with 75% alcohol for 30 - 40 s, rinse with sterile water 3 - 4 times, continue to disinfect with 0.1% mercuric chloride for 7 - 9 min, and rinse with sterile water 8 - 9 times;

[0008] (2) Primary culture medium: Cut a small opening in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the primary culture medium, and place it under the conditions of light and dark alternation culture at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a single-day sunlight of 10 h for 45 - 60 days;

[0009] The composition of the primary culture medium is 1 / 2MS basal medium;

[0010] (3) Subculture and proliferation medium: Transfer the clustered buds obtained from the primary culture medium to the subculture and proliferation medium, and place it under the conditions of light and dark alternation culture at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a sunlight of 12 h for 45 - 60 days;

[0011] (4) Rooting culture: Cut the robust buds obtained from the proliferation culture into single buds, transfer them to the rooting medium, and place it under the conditions of light and dark alternation culture at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a single-day sunlight of 10 h for 45 - 60 days;

[0012] The composition of the rooting medium is: 1 / 2MS + 6 - BA 0.2 - 0.3 mg / mL + NAA 0.2 - 0.3 mg / mL + mashed potato 40.0 - 60.0 g / mL.

[0013] Furthermore, in step (3), the composition of the subculture and proliferation medium is: MS + 6 - BA 2.0 - 3.0 mg / mL + NAA 0.1 - 0.2 mg / mL + mashed banana 40.0 - 60.0 g / mL + mashed potato 40.0 - 60.0 g / mL + AC 0.2 - 0.3 g / mL.

[0014] Furthermore, in step (3), the composition of the subculture and proliferation medium is: MS + 6 - BA 2.0 mg / mL + NAA 0.1 mg / mL + mashed banana 50.0 g / mL + mashed potato 50.0 g / mL + AC 0.2 g / mL.

[0015] Furthermore, in step (3), the subculture multiplication medium comprises: MS + KT 2.0 - 3.0 mg / mL + NAA 0.1 - 0.2 mg / mL + banana puree 40.0 - 60.0 g / mL + potato puree 40.0 - 60.0 g / mL + AC 0.2 - 0.3 g / mL.

[0016] Furthermore, in step (3), the subculture multiplication medium comprises: MS + KT 2.0 mg / mL + NAA 0.1 mg / mL + banana puree 50.0 g / mL + potato puree 50.0 g / L + AC 0.2 g / mL.

[0017] Furthermore, in step (4), the rooting medium comprises: 1 / 2MS + 6 - BA 0.2 mg / mL + NAA 0.2 mg / mL + potato puree 50.0 g / mL.

[0018] The beneficial technical effects of the present invention are as follows:

[0019] 1. The disinfection method of the present invention uses 75% alcohol to clean the fruit clip, and the organic combination of 75% alcohol and 0.01% mercuric chloride solution can minimize the contamination rate without damaging the explant, improve the success rate of establishing the aseptic system of Dendrobium wardianum, significantly improve the multiplication coefficient of the tissue - cultured seedlings of Dendrobium wardianum, shorten the propagation cycle, and achieve efficient propagation by optimizing the culture medium formula and culture conditions.

[0020] 2. The present invention uses the fruit clip as the explant and combines with a strict disinfection procedure, effectively reducing the virus - carrying rate and obtaining healthy virus - free tissue - cultured seedlings, showing good virus - elimination effect.

[0021] 3. The present invention uses two subculture multiplication media, both of which can achieve a multiplication rate of 3.6 times.

[0022] 4. By adjusting the types and concentrations of growth regulators in the rooting medium, the present invention enables the rooting rate to reach over 89% and the roots to be robust.

[0023] 5. The present invention uses conventional culture medium components, simplifies the operation steps, reduces the production cost, is suitable for large - scale production. Secondly, the tissue - cultured seedlings can stably inherit the excellent traits of the female parent, ensuring the variety consistency. Description of the Drawings

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is the result diagram of the primary culture for 90 days in Example 1 of the present invention.

[0026] Figure 2 It is the result diagram of the subculture proliferation for 90 days in Example 1 of the present invention. Detailed implementation manners

[0027] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0028] Example 1

[0029] A tissue culture and rapid propagation method for Tremella aurantialba - Dendrobium officinale, the steps include:

[0030] (1) Selection and disinfection of explants: Select healthy and unopened fruit pods of Tremella aurantialba - Dendrobium officinale. First, wipe the outer shell of the fruit pod repeatedly with absorbent cotton under 75% alcohol concentration, then soak and disinfect it with 75% alcohol for 35 s, rinse it 4 times with sterile water, continue to disinfect it with 0.1% mercuric chloride for 8 min, and rinse it 9 times with sterile water.

[0031] (2) Primary culture medium: Cut a small opening in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the 1 / 2MS basal medium. Place it under the conditions of light - dark alternation culture at a temperature of 25 ± 2°C, a light intensity of 2500 Lux, and a single - day sunlight of 10 h for 50 days.

[0032] (3) Subculture proliferation medium: Transfer the clustered buds obtained from the primary culture medium to the subculture proliferation medium (the composition is: MS + 6 - BA 2.0 mg / mL + NAA 0.1 mg / mL + banana puree 50.0 g / mL + potato puree 50.0 g / mL + AC 0.2 g / mL), and conduct light - dark alternation culture at a temperature of 25 ± 2°C, a light intensity of 2500 Lux, and a sunlight of 12 h for 50 days;

[0033] (4)Rooting culture: Cut the robust buds obtained from the proliferation culture into single buds and transfer them to the rooting medium (composition: 1 / 2MS + 6-BA 0.2 mg / mL + NAA 0.2 mg / mL + mashed potato 50.0 g / mL). Incubate them under the conditions of temperature 25±2°C, light intensity 2500 Lux, and 10-hour single-daylight photoperiod with light-dark alternation for 50 days.

[0034] (5)The subsequent processes are all operated according to the conventional processes.

[0035] Example 2

[0036] A tissue culture and rapid propagation method for Dendrobium wardianum, the steps include:

[0037] (1)Explant selection and disinfection: Select healthy and unopened fruit pods of Dendrobium wardianum. First, wipe the outer shell of the fruit pod repeatedly with absorbent cotton under 75% alcohol concentration, then soak it in 75% alcohol for disinfection for 30 s, rinse it 3 times with sterile water, and continue to disinfect it with 0.1% mercuric chloride for 8 minutes and rinse it 8 times with sterile water.

[0038] (2)Primary medium: Make a small cut in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the 1 / 2MS basal medium. Incubate it under the conditions of temperature 25±2°C, light intensity 2000 Lux, and 10-hour single-daylight photoperiod with light-dark alternation for 45 days.

[0039] (3)Subculture proliferation medium: Transfer the clustered buds obtained from the primary medium to the subculture proliferation medium (MS + KT 2.0 mg / mL + NAA 0.1 mg / mL + mashed banana 50.0 g / mL + mashed potato 50.0 g / L + AC 0.2 g / mL). Incubate it under the conditions of temperature 25±2°C, light intensity 2500 Lux, and 12-hour daylight photoperiod with light-dark alternation for 45 days.

[0040] (4)Rooting culture: Cut the robust buds obtained from the proliferation culture into single buds and transfer them to the rooting medium (composition: 1 / 2MS + 6-BA 0.3 mg / mL + NAA 0.3 mg / mL + mashed potato 60.0 g / mL). Incubate them under the conditions of temperature 25±2°C, light intensity 2500 Lux, and 10-hour single-daylight photoperiod with light-dark alternation for 45 days.

[0041] (5)The subsequent processes are all operated according to the conventional processes.

[0042] Example 3

[0043] A tissue culture and rapid propagation method for Dendrobium wardianum, the steps include:

[0044] (1)Explant selection and disinfection: Select healthy and unopened fruit pods of Dendrobium wardianum. First, wipe the outer shell of the fruit pods repeatedly with absorbent cotton under 75% alcohol concentration, then soak and disinfect them in 75% alcohol for 30 s, rinse with sterile water 3 times, and continue to disinfect them with 0.1% mercuric chloride for 7 min and rinse with sterile water 8 times.

[0045] (2)Primary culture medium: Make a small cut in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the 1 / 2MS basal medium. Incubate it under the conditions of alternating light and darkness at a temperature of 25±2 °C, a light intensity of 2000 Lux, and a single-day light exposure of 10 h for 60 days. The culture results are as Figure 1 shown.

[0046] (3)Subculture proliferation medium: Transfer the clustered buds obtained from the primary culture medium to the subculture proliferation medium (composition: MS + 6-BA 3.0 mg / mL + NAA 0.2 mg / mL + banana puree 60.0 g / mL + potato puree 60.0 g / mL + AC 0.3 g / mL), and incubate it under the conditions of alternating light and darkness at a temperature of 25±2 °C, a light intensity of 2000 Lux, and a light exposure of 12 h for 60 days. The culture results are as Figure 2 shown.

[0047] (4)Rooting culture: Cut the strong and healthy buds obtained from the proliferation culture into single buds, and transfer them to the rooting medium (1 / 2MS + 6-BA 0.2 mg / mL + NAA 0.2 mg / mL + potato puree 40.0 g / mL), and incubate it under the conditions of alternating light and darkness at a temperature of 25±2 °C, a light intensity of 2000 Lux, and a single-day light exposure of 10 h for 60 days.

[0048] (5)The subsequent processes are all operated according to the conventional processes.

[0049] Example 4

[0050] A tissue culture and rapid propagation method for Dendrobium wardianum, the steps include:

[0051] (1)Explant selection and disinfection: Select healthy and unopened fruit pods of Dendrobium wardianum. First, wipe the outer shell of the fruit pods repeatedly with absorbent cotton under 75% alcohol concentration, then soak and disinfect them in 75% alcohol for 40 s, rinse with sterile water 4 times, and continue to disinfect them with 0.1% mercuric chloride for 9 min and rinse with sterile water 9 times.

[0052] (2)Primary culture medium: Make a small cut in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the 1 / 2MS basal medium. Incubate it under the conditions of alternating light and darkness at a temperature of 25±2 °C, a light intensity of 3000 Lux, and a single-day light exposure of 10 h for 55 days.

[0053] (3) Subculture multiplication medium: The clustered buds obtained from the primary medium were transferred to the subculture multiplication medium (MS + 6-BA 2.5 mg / mL + NAA 0.15 mg / mL + banana puree 60.0 g / mL + potato puree 60.0 g / mL + AC 0.25 g / mL), and cultured under the conditions of alternating light and darkness for 55 days at a temperature of 25 ± 2 °C, a light intensity of 3000 Lux, and a sunlight exposure of 12 h.

[0054] (4) Rooting culture: The robust buds obtained from the multiplication culture were cut into single buds and transferred to the rooting medium (1 / 2MS + 6-BA 0.25 mg / mL + NAA 0.25 mg / mL + potato puree 60.0 g / mL), and cultured under the conditions of alternating light and darkness for 55 days at a temperature of 25 ± 2 °C, a light intensity of 3000 Lux, and a single sunlight exposure of 10 h.

[0055] (5) The subsequent processes were all carried out according to the conventional processes.

[0056] Comparative Example 1

[0057] Select the fruit pods of Dendrobium wardianum treated in step (1) of Example 1 of the present invention as the explants, and operate according to Example 1 of the invention patent with the publication number CN118318734A and the name: Rapid Propagation Method and Application of Dendrobium nobile.

[0058] Comparative Example 2

[0059] Select the fruit pods of Dendrobium wardianum treated in step (1) of Example 1 of the present invention as the explants, and operate according to Example 1 of the invention patent with the publication number CN101336605A and the name: A Tissue Culture Technique and Cultivation Method of Dendrobium nobile.

[0060] Comparative Example 3

[0061] Select the fruit pods of Dendrobium wardianum treated in step (1) of Example 1 of the present invention as the explants, and culture according to the culture method disclosed in Example 1 of the invention patent with the publication number CN104429975A and the name: A Culture Medium for Tissue Culture Seedlings of Dendrobium officinale.

[0062] The disinfection contamination rate, disinfection mortality rate, primary induction rate, multiplication coefficient, and rooting rate were statistically analyzed for Examples 1-3 and Comparative Examples 1-3, and the results are shown in Table 1.

[0063] Table 1 Statistical Results

[0064] Disinfection contamination rate Disinfection mortality rate Primary induction rate Proliferation coefficient Rooting rate Example 1 5% 6% 90% 4.3 92% Example 2 8% 5% 89% 3.8 89% Example 3 8% 5% 90% 3.6 90% Comparative example 1 10% 8% 84% 2.5 85% Comparative example 2 8% 7% 83% 2.3 81% Comparative example 3 8% 9% 79% 2.1 78%

[0065] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced. Any modification or partial replacement without departing from the spirit and scope of the present invention shall be covered by the scope of the claims of the present invention.

Claims

1. A tissue culture and rapid propagation method for Dendrobium wardianum, characterized in that the steps Including: (1) Explant selection and disinfection: Select healthy and unopened fruit pods of Dendrobium wardianum. First, wipe the outer shell of the fruit pods repeatedly with absorbent cotton under 75% alcohol concentration, then soak and disinfect them in 75% alcohol for 30 - 40 s, rinse with sterile water 3 - 4 times, continue to disinfect with 0.1% mercuric chloride for 7 - 9 min, and rinse with sterile water 8 - 9 times; (2) Primary culture medium: Make a small cut in the disinfected fruit pod, hold the fruit pod with forceps, and evenly shake the Dendrobium seeds inside into the primary culture medium. Incubate under the conditions of light - dark alternation at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a single - day sunlight of 10 h for 45 - 60 days; The composition of the primary culture medium is 1 / 2MS basal medium; (3) Sub - culture proliferation medium: Transfer the cluster buds obtained from the primary culture medium to the sub - culture proliferation medium, and incubate under the conditions of light - dark alternation at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a sunlight of 12 h for 45 - 60 days; (4) Rooting culture: Cut the robust buds obtained from the proliferation culture into single buds, transfer them to the rooting medium, and incubate under the conditions of light - dark alternation at a temperature of 25 ± 2 °C, a light intensity of 2000 - 3000 Lux, and a single - day sunlight of 10 h for 45 - 60 days; The composition of the rooting medium is: 1 / 2MS + 6 - BA 0.2 - 0.3 mg / mL + NAA 0.2 - 0.3 mg / mL + mashed potato 40.0 - 60.0 g / mL.

2. The method according to claim 1, characterized in that, In step (3), the composition of the sub - culture proliferation medium is: MS + 6 - BA 2.0 - 3.0 mg / mL + NAA 0.1 - 0.2 mg / mL + mashed banana 40.0 - 60.0 g / mL + mashed potato 40.0 - 60.0 g / mL + AC 0.2 - 0.3 g / mL.

3. The method according to claim 2, wherein In step (3), the composition of the sub - culture proliferation medium is: MS + 6 - BA 2.0 mg / mL + NAA 0.1 mg / mL + mashed banana 50.0 g / mL + mashed potato 50.0 g / mL + AC 0.2 g / mL.

4. The method according to claim 1, characterized in that In step (3), the composition of the sub - culture proliferation medium is: MS + KT 2.0 - 3.0 mg / mL + NAA 0.1 - 0.2 mg / mL + mashed banana 40.0 - 60.0 g / mL + mashed potato 40.0 - 60.0 g / mL + AC 0.2 - 0.3 g / mL.

5. The method according to claim 4, wherein In step (3), the composition of the sub - culture proliferation medium is: MS + KT 2.0 mg / mL + NAA 0.1 mg / mL + mashed banana 50.0 g / mL + mashed potato 50.0 g / L + AC 0.2 g / mL.

6. The method according to claim 1, characterized in that In step (4), the composition of the rooting medium is: 1 / 2MS + 6 - BA 0.2 mg / mL + NAA 0.2 mg / mL + mashed potato 50.0 g / mL.

7. Application of the method according to claim 1 in the tissue culture rapid propagation of Dendrobium wardianum.

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