Sterile sowing and rapid propagation method for anoectochilus mollissima

Abstract

The invention discloses a sterile sowing and rapid propagation method for anoectochilus mollissima, relates to the technical field of biology, and solves the technical problems that in the prior art, anoectochilus mollissima is difficult to germinate under natural conditions, and the resource reserves of anoectochilus mollissima cannot meet the sustainable utilization requirement. The method specifically comprises the following steps: collecting plump and non-cracked capsules of anoectochilus formosanus, cleaning, and sterilizing by using alcohol and mercury bichloride in sequence; the disinfected fruit pods are split, and seeds are evenly sown on a germination culture medium to be subjected to non-symbiotic germination culture; about 0-6 weeks after sowing, seed embryos expand and break through seed coats to form transparent and whiter protocorms; 6-10 weeks later, the number of epidermal hairs of the protocorm is increased to about 20, the protocorm is gradually differentiated, and yellow green budlets are formed at the upper end of morphology; after 10-18 weeks, buds gradually grow to form bud seedlings with leaves, and the seedling rate is about 30%; transferring the seedlings into a seedling strengthening and rooting culture medium for culturing; robust and big seedlings are obtained after 4-6 months, and a large number of sterile tissue culture seedlings of the anoectochilus formosanus are obtained through the seeds.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for rapid propagation of golden thread orchid aseptically sowed. Background Art

[0002] Plants of the genus Anoectochilus Blume in the Orchidaceae family have long been used as Chinese herbal medicines. Due to factors such as high habitat requirements and human excavation, the amount of wild germplasm resources has been reduced year by year. At present, all species of this genus are listed as national second-level key protected wild plants. A. medogensis HZTian & Y.Jin is a new species of the genus Anoectochilus Blume in the Orchidaceae family published in 2019. It is currently only found in Medog County, Tibet Autonomous Region. It has potential medicinal value and also has high ornamental value due to the spotted leaves. However, the number of wild plants is scarce and the distribution points are scattered, and its resource reserves are insufficient to meet the needs of sustainable utilization.

[0003] Aseptic sowing is an effective method for rapid and large-scale propagation of orchids. Although there have been several studies on aseptic sowing and rapid propagation techniques for other species of the genus Rhizoma, such as Rhizoma Cibotii and Rhizoma Cibotii, as an orchid, Rhizoma Cibotii Medog has small seeds, large numbers and no endosperm, and is difficult to germinate under natural conditions. There are currently no reports on aseptic sowing and rapid propagation of Rhizoma Cibotii Medog. Summary of the invention

[0004] In view of the shortcomings and deficiencies in the prior art, the present invention provides a method for rapid propagation of Golden thread orchid in Medog by aseptic sowing, which can be used for field return and industrial application.

[0005] To achieve the above purpose, the present invention is implemented by the following technical scheme: The method for aseptic sowing and rapid propagation of Golden Line Orchid in Medog provided by the present invention comprises the following steps:

[0006] (1) Artificial pollination and fruit selection: Artificial pollination was performed on the golden thread orchid in the wild, and then full and unbroken fruit pods were collected for sowing;

[0007] (2) Fruit pod disinfection: Use the full fruit pods of Medog golden thread orchid as explants, wash them and disinfect them with alcohol and mercuric chloride in turn;

[0008] (3) Seed germination culture: Split the sterilized pods and evenly sow the seeds on the germination medium for non-symbiotic germination culture. 0 to 6 weeks after sowing, the embryo swells and breaks through the seed coat to form a transparent white protocorm. 6 to 10 weeks later, the number of epidermal hairs on the protocorm increases to 15 to 25 and gradually differentiates, forming a yellow-green bud at the top. 10 to 18 weeks later, the bud gradually forms a leafy seedling.

[0009] (4) Rooting: The seedlings are transferred to a rooting medium for cultivation;

[0010] (5) Hardening and transplanting: The materials after the seedlings have taken root for 4 to 6 months are used for hardening and transplanting.

[0011] Furthermore, in step (1), the fruit pods are plump and unopened 60 to 80 days after pollination.

[0012] Furthermore, in step (2), the specific steps of disinfection are: cleaning the dust and dirt on the surface of the fruit pod with a small brush under running water, soaking it in 75% alcohol in a clean bench for 20 to 30 seconds after drying, rinsing it with sterile water, soaking it in a 0.1% mass percent concentration of mercuric chloride solution for 8 to 15 minutes after drying, rinsing it with sterile water after disinfection is completed; and pressing the fruit below the liquid surface with tweezers during the whole process.

[0013] Furthermore, in step (3), the germination medium is 1 / 2MS+NAA 0.3-1 mg / L+6-BA 0.01-0.1 mg / L+sucrose or white sugar 20 g / L+agar powder 8 g / L, and the pH value is 5.4-5.9.

[0014] Furthermore, in step (4), the rooting and seedling strengthening culture medium is 1 / 2MS + NAA 0.5-2 mg / L + 6-BA 0.1-0.5 mg / L + activated carbon 0-0.5 g / L + sucrose or white sugar 20-30 g / L + agar powder 8 g / L, and the pH value is 5.4-5.9.

[0015] Furthermore, the culture conditions in step (3) and step (4) are: using LED lamps with a light quality ratio of seven white and one red, a lighting time of 12h / d, a light intensity of 1200-1500Lux, and a culture temperature of 23-25°C.

[0016] The present invention provides a method for rapid propagation of Medog golden thread orchid by aseptic sowing. It has the following beneficial effects:

[0017] The aseptic sowing and rapid propagation method of Goldenrod Medog disclosed by the present invention is the first to explore a method for obtaining a large number of aseptic tissue culture seedlings of Goldenrod Medog through seeds, thus realizing a feasible aseptic germination and rapid propagation method of Goldenrod Medog. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Seed germination, differentiation and seedling growth of the golden thread orchid of Medog prepared in Example 1 of the present invention;

[0019] Figure 2 The present invention provides a robust bottle seedling of Goldenrod cymbidium in Medog prepared in Example 1 of the present invention.

[0020] Figure 3 The present invention is a comparative bar graph of the germination rate, differentiation rate and seedling rate of the golden thread orchid seeds prepared in Example 1 of the present invention and Comparative Examples 1 and 2. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0022] Example 1

[0023] The method for rapid propagation of golden thread orchid aseptic sowing of the present invention comprises the following steps:

[0024] (1) Artificial pollination and fruit selection: Artificial pollination was carried out on the wild Rhizoma Cinerariae Motuo, and full and uncracked fruit pods were collected 80 days after pollination for sowing.

[0025] (2) Fruit pod disinfection: Clean the dust and dirt on the surface of the fruit pod with a small brush under running water. After drying, soak it in 75% alcohol in a clean bench for 30 seconds, rinse it once with sterile water, dry it, and soak it in 0.1% mercuric chloride solution for 10 minutes. After disinfection, rinse it with sterile water three times and dry it. Use tweezers to press the fruit below the liquid surface during the whole process.

[0026] (3) Seed germination culture: Split the sterilized pods and evenly sow the seeds on the germination medium for non-symbiotic germination culture. The formula is 1 / 2MS + 6-BA 0.05 mg / L + NAA 0.3 mg / L + sucrose 20 g / L + agar powder 8 g / L, pH 5.8, light time 12 h / d, light intensity 1200-1500 Lux, culture temperature 25 ° C when illuminated, 23 ° C when not illuminated; Figure 1-Figure 2 As shown, about 0 to 6 weeks after sowing, the embryo swells and breaks through the seed coat to form a transparent, slightly whitish protocorm, with a seed germination rate of about 60%; 6 to 10 weeks, the number of epidermal hairs on the protocorm increases to about 20 and gradually differentiates, forming a yellow-green bud at the upper end morphologically, with a differentiation rate of about 60%; 10 to 18 weeks, the buds gradually grow to form seedlings with leaves, with a seedling rate of about 30%, and the seedlings have emerald green leaves and slender rhizomes.

[0027] Considering the germination rate, differentiation rate and seedling rate comprehensively, the above parameters are most conducive to the germination, differentiation and growth of golden thread orchid seeds into seedlings.

[0028] (4) Rooting of seedlings: Seedlings that have grown for 36 weeks on the germination medium and have 4 leaves of uniform growth are transferred to the rooting medium for cultivation. The formula is 1 / 2MS + 6-BA 0.1mg / L + NAA 1mg / L + activated carbon 0.5g / L + sucrose 20g / L + agar powder 8g / L, pH 5.8; the lighting time is 12h / d, the light intensity is 1200-1500Lux, and the culture temperature is 25℃ when illuminated and 23℃ when not illuminated. A comprehensive score is given to multiple indicators of rooting and seedling strengthening results.

[0029] First, the data is normalized. The data to be normalized is represented by C, and the minimum and maximum values ​​of the data to be normalized are represented by C min and C max Indicates that C new is the normalized data, C new =(CC min ) / (C min -C max ). Then, each indicator is weighted, and the comprehensive score = 100*(C new单株重 *0.4+C new根数量 *0.1+C new根长 *0.1+C new叶 *0.1+C new株高 *0.3), as shown in Table 1, the comprehensive score of rooting and seedling strengthening in Example 1 was 95.95, and the effect was the best.

[0030] Table 1 Results of orthogonal test on rooting and seedling growth of golden thread orchid in Medog for 6 months

[0031]

[0032] (5) Hardening and transplanting: Figure 2 As shown, the material after rooting and growing strong for 4 to 6 months is used for seedling hardening and transplanting.

[0033] Example 2

[0034] The method for rapid propagation of golden thread orchid aseptic sowing of the present invention comprises the following steps:

[0035] (1), (2), (3), (4) and (5) in Example 1 were repeated, except that the ratio of hormones and additives in the seedling rooting medium in step (4) of Example 2 was: 6-BA 0.3 mg / L + NAA 1.5 mg / L + activated carbon 0.5 g / L, and the other components and culture conditions were the same as those in Example 1. The comprehensive score of the seedling rooting result of Example 2 was 93.74, and the seedling rooting effect was second, which could also meet the growth needs of Modog golden thread orchid.

[0036] Example 3

[0037] The method for rapid propagation of golden thread orchid aseptic sowing of the present invention comprises the following steps:

[0038] (1), (2), (3), (4) and (5) in Example 1 were repeated, except that the ratio of hormones and additives in the seedling rooting medium in step (4) of Example 3 was: NAA 0.5 mg / L + banana 50 g / L + activated carbon 1 g / L, and the other ingredients and culture conditions were the same as in Example 1.

[0039] As shown in Table 2, an orthogonal test was carried out using 1 / 2MS medium + sucrose 20 g / L + agar powder 8 g / L + activated carbon 1 g / L as the basic components, 6-BA, NAA and organic additives were added, and the test was carried out according to the same indicators as in Example 1 and Example 2 after 3 months. The results show that Example 3 has the best effect in this group of experiments, with a comprehensive score of 89.54, which is slightly lower than that of Example 2. However, since this is data for 3 months, its rooting and seedling strengthening effect is still unknown compared with that of Example 1 and Example 2. In short, Example 3 can also meet the growth needs of Medog Golden Line Orchid.

[0040] Table 2 Results of orthogonal test on rooting and seedling growth of golden thread orchid in Medog for 3 months

[0041]

[0042] Comparative Example 1-Comparative Example 2

[0043] Comparative Example 1-Comparative Example 2 is the same as the specific method for rapid propagation of aseptic sowing of Golden Line Orchid in Medog as in Example 1, except that the 1 / 2MS medium in the germination medium formula in step (3) is replaced with KC medium and MS medium, respectively. After cultivation, the germination rate, differentiation rate and seedling rate of Golden Line Orchid in Medog in Comparative Example 1 are approximately 40%, 50% and 1%, respectively, and the germination rate, differentiation rate and seedling rate of Golden Line Orchid in Medog in Comparative Example 2 are approximately 70%, 60% and 18%, respectively. Figure 3 As shown. The germination rates of seeds treated with 1 / 2MS and MS medium were relatively high, with no significant difference, while the germination rate of seeds treated with KC medium was lower; there was no significant difference in the differentiation of Jinxianlan in Medog between 1 / 2MS and MS medium; but the seedling rate of 1 / 2MS medium treatment was the highest, with significant difference from the other two treatments. Considering the germination rate, differentiation rate and seedling rate, 1 / 2MS was the basic medium most conducive to the germination, differentiation and growth of Jinxianlan seeds into seedlings.

[0044] In summary, the aseptic sowing and rapid propagation method of Golden thread orchid in Medog of the present invention is scientific and reasonable, and the comprehensive score of strong seedlings and rooting results is high. It is the first time to explore a large number of aseptic tissue culture seedlings of Golden thread orchid in Medog through seeds, realizing a feasible aseptic germination and rapid propagation method of Golden thread orchid in Medog.

[0045] The above are only embodiments of the present invention. For example, in step (3), the germination medium is 1 / 2MS + NAA 0.3-1mg / L + 6-BA 0.01-0.1mg / L + sucrose or white sugar 20g / L + agar powder 8g / L, and the pH value is 5.4-5.9; in step (4), the rooting and seedling strengthening medium is 1 / 2MS + NAA 0.5-2mg / L + 6-BA 0.1-0.5mg / L + activated carbon 0-0.5g / L + sucrose or white sugar 20-30g / L + agar powder 8g / L, and the pH value is 5.4-5.9, both of which can realize the aseptic sowing and rapid propagation method of Medog golden thread orchid of the present invention.

[0046] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for rapid propagation of golden thread orchid by aseptic sowing, characterized in that: The following steps are involved: (1) Artificial pollination and fruit selection: Artificial pollination was performed on the golden thread orchid in the wild, and then full and unbroken fruit pods were collected for sowing; (2) Fruit pod disinfection: Use the full fruit pods of Medog golden thread orchid as explants, wash them and disinfect them with alcohol and mercuric chloride in turn; (3) Seed germination culture: Split the sterilized pods and evenly sow the seeds on the germination medium for non-symbiotic germination culture. 0 to 6 weeks after sowing, the embryo swells and breaks through the seed coat to form a transparent white protocorm. 6 to 10 weeks later, the number of epidermal hairs on the protocorm increases to 15 to 25 and gradually differentiates, forming a yellow-green bud at the top. 10 to 18 weeks later, the bud gradually forms a leafy seedling. (4) Rooting: The seedlings are transferred to a rooting medium for cultivation; (5) Hardening and transplanting: The materials after the seedlings have taken root for 4 to 6 months are used for hardening and transplanting.

2. The method for rapid propagation of golden thread orchid aseptic sowing according to claim 1, characterized in that: In step (1), the fruit pods are full and unopened 60 to 80 days after pollination.

3. The method for rapid propagation of golden thread orchid aseptic sowing according to claim 1, characterized in that: In step (2), the specific steps of disinfection are: cleaning the dust and dirt on the surface of the fruit pod with a small brush under running water, soaking it in 75% alcohol in a clean bench for 20 to 30 seconds after drying, rinsing it with sterile water, soaking it in a 0.1% mercuric chloride solution by mass concentration for 8 to 15 minutes after drying, rinsing it with sterile water after disinfection is completed, and drying it; using tweezers to press the fruit below the liquid surface during the whole process.

4. The method for rapid propagation of golden thread orchid aseptic sowing according to claim 1, characterized in that: In step (3), the germination medium is 1 / 2MS+NAA 0.3mg / L+6-BA 0.05mg / L+sucrose or white sugar 20g / L+agar powder 8g / L, and the pH value is 5.4-5.

9.

5. The method for rapid propagation of golden thread orchid aseptic sowing according to claim 1, characterized in that: In step (4), the rooting and seedling strengthening culture medium is 1 / 2MS+NAA 0-1.5mg / L+6-BA 0-0.5mg / L+activated carbon 0.1-1g / L+sucrose or white sugar 20-30g / L+agar powder 8g / L, and the pH value is 5.4-5.

9.

6. The method for rapid propagation of golden thread orchid aseptic sowing according to claim 1, characterized in that: The culture conditions in step (3), step (4) and step (5) are: using LED lamps with a light quality ratio of seven white and one red, a lighting time of 12h / d, a light intensity of 1200-1500Lux, and a culture temperature of 23-25°C.

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