Aseptic sowing and rapid propagation method for anoectochilus graligeri

Abstract

The invention discloses a sterile sowing and rapid propagation method for Glilian anoectochilus formosanus, and relates to the technical field of biology.The method specifically comprises the steps that plump and non-cracked fruit pods of the Glilian anoectochilus formosanus are collected, cleaned and disinfected; the disinfected fruit pods are split, and seeds are evenly sown on a germination culture medium to be cultured; seed embryos expand to break through seed coats 0-2 weeks after sowing, white protocorms are formed within 6 weeks, the germination rate is higher than 80%, yellow-green budlets are gradually differentiated and formed within 6-10 weeks, and the buds gradually expand to form leaves within 10-18 weeks; seedlings with three leaves about 20 weeks after sowing are transferred into a primary culture medium, and primary robust big seedlings with the plant height of 8-15 cm can be obtained after 6 months of culture; and carrying out subculture by using the primary big seedlings. A large number of sterile tissue culture bottle seedlings with high genetic diversity and richness can be rapidly obtained by utilizing the capsules of the anoectochilus graciliatus, and a germplasm resource library is provided for field regression and subsequent industrial application.

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 by aseptic sowing. Background Art

[0002] All species of Anoectochilus Blume in the Orchidaceae family are classified as Class II protected wild plants in my country. The plants of this genus have potential medicinal value and are also highly ornamental due to the spotted patterns on their leaves. They are commonly known as "gold thread lotus" in the market, but their wild resource reserves cannot meet the demand for sustainable utilization. China has discovered more than 20 species of Anoectochilus Blume plants, of which only a limited number have been developed and utilized, mainly Anoectochilus roxburghii (Wall.) Lindl., and many species have not yet been developed and utilized on a large scale.

[0003] The tissue culture of "Golden Thread Lotus" usually uses stem segments as explants to establish a sterile propagation system. This method is difficult to disinfect and has a high contamination rate. For example, in the Chinese patent with application number CN202411175052 and the name "A method for tissue culture of Pingbian Golden Thread Lotus", wild Pingbian Golden Thread Lotus is used as an explant, and the culture medium contamination rate is 30% to 50%. There is also a method of propagating "Golden Thread Lotus" by sterile sowing. Compared with using stem segments of wild plants as explants, using mature and uncracked fruit pods for sterile sowing and disinfection is less difficult, and it is easier to establish a sterile propagation system, and quickly obtain a large number of primary sterile seedlings with rich genetic diversity. For example, in the Chinese patent with application number CN201510212064 and title “A method for tissue culture and rapid seedling raising of Zhejiang golden thread orchid seeds”, after sowing, Zhejiang golden thread orchid must undergo germination culture, robust culture of sterile seedlings, cluster bud proliferation, and then rooting and seedling growth. In the Chinese patent with application number 202310490422 and title “A non-symbiotic germination culture medium and breeding method for seeds of plants of the same genus as golden thread orchid”, after the seeds germinate into protocorms, they need to undergo a subculture culture, and then proliferation culture and seedling growth and rooting culture, which involves many steps.

[0004] Anoectochilus griffithii Hook.f. has the characteristics of tall and strong plants and red flowers. It blooms in summer (August), which is different from the golden thread orchid (usually from November to January). Cultivating this species is beneficial to the breeding of new and superior varieties of "Golden Thread Lotus". There is currently no report on the sterile sowing and propagation of Anoectochilus griffithii. Summary of the invention

[0005] In view of the shortcomings and deficiencies in the prior art, the present invention provides a method for achieving rapid propagation of Gree Golden Line Orchid by aseptic sowing, and provides a germplasm resource library for field return and subsequent industrial application.

[0006] To achieve the above purpose, the present invention is implemented by the following technical scheme: The aseptic sowing and rapid propagation method of Golden Line Orchid provided by the present invention comprises the following steps:

[0007] (1) Pod selection and disinfection: Collect the full and unbroken pods of the golden thread orchid 60 to 120 days after pollination, and disinfect them with disinfectant I and disinfectant II in sequence after pretreatment;

[0008] (2) Seed germination: longitudinally split the sterilized pods and evenly sow the seeds on a germination medium with a formula of 1 / 2MS + activated carbon 0-1g / L + NAA 0-0.3mg / L + 6-BA 0-0.05mg / L + potato 0-50g / L + sucrose 15-20g / L + agar powder 6-8g / L, pH 5.5-5.8. The potatoes are peeled and weighed, and then crushed into a paste with a wall breaker. The culture medium is then prepared with other ingredients and cultured under suitable conditions. The embryo swells and breaks through the seed coat 0-2 weeks after sowing. The seeds germinate to form white protocorms within 6 weeks. The germination rate is higher than 80% in the 6th week. Yellow-green buds are gradually differentiated to form in 6-10 weeks. The buds gradually open into leaves in 10-18 weeks.

[0009] (3) Primary culture: The seedlings are transferred to the primary culture medium, the formula of which is 1 / 2MS + NAA 0-2 mg / L + 6-BA 0-0.1 mg / L + activated carbon 0.2-1 g / L + banana puree 0-100 g / L + sucrose or white sugar 20-30 g / L + agar powder 6-8 g / L, pH 5.6-6.0, and cultured under suitable conditions. After 4-6 months, primary bottle seedlings with a plant height of 8-15 cm are obtained for subculture;

[0010] (4) Subculture: excise the leaves and roots of the first generation of healthy bottle seedlings of Golden Line Orchid, cut the stems into stem segments with a length of 1 to 2 cm and one node, and inoculate them flatly into the subculture medium, the formula of which is 1 / 2MS + NAA 0 to 1 mg / L + activated carbon 0.2 to 1 g / L + banana puree 50 to 100 g / L + sucrose or white sugar 15 to 20 g / L + agar powder 7 to 8 g / L, pH 5.6 to 5.8, and small buds will gradually grow from the stem nodes in 2 to 3 weeks, and mature seedlings with a plant height of 8 to 15 cm will be obtained in 4 to 6 months; repeat step (4) for subculture to obtain healthy bottle seedlings for cultivation.

[0011] Preferably, in step (1), disinfectant I is 70% to 75% alcohol, and disinfectant II is 0.1% by mass concentration of mercuric chloride or 2% to 5% by mass concentration of sodium hypochlorite solution;

[0012] The specific steps of disinfection are: use a blade to remove the residual floral structure on the fruit pod, soak the fruit pod in water added with washing powder or detergent, gently scrub it with a soft brush, and then pack it with gauze or a non-woven bag, rinse it under running water for 10 minutes or more to remove the dirt on the surface of the fruit pod, and then put it into a sterile bottle and transfer it to the clean bench, then soak it in alcohol in the clean bench for 20 to 30 seconds, rinse it with sterile water at least once, and then soak it in 0.1% mercuric chloride solution or 2% to 5% sodium hypochlorite solution for 8 to 15 minutes. After the disinfection is completed, rinse it with sterile water at least 3 times, and dry it for use; keep the fruit below the liquid level during the disinfection process.

[0013] Preferably, the culture conditions in step (2), step (3) and step (4) are: using a lamp 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 20-25°C.

[0014] The present invention provides a method for rapid propagation of Golden Line Orchid by aseptic sowing. It has the following beneficial effects:

[0015] Compared with the existing literature, the aseptic sowing and rapid propagation method of Golden Rosmarinus officinalis of the present invention adopts a culture medium formula and a breeding method that can advance the germination time of Golden Rosmarinus officinalis seeds to 2 weeks, has low difficulty in sterilizing the selected explants, has a simple culture medium formula, and each component of the culture medium is easily available, thus saving costs.

[0016] At the same time, the browning problem during seed germination is avoided. The seeds can directly germinate, differentiate and develop into seedlings with expanded leaves on the germination medium, which simplifies the operation steps and reduces the contamination that may occur when changing the culture medium. It is possible to quickly obtain a large number of sterile tissue culture bottle seedlings with high genetic diversity using the fruit pods of Gree Golden Line Orchid, providing a germplasm resource library for wild return and subsequent industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The germination state of the Golden Line Orchid prepared in Example 1 of the present invention 2 weeks after aseptic sowing;

[0018] Figure 2 The germination, differentiation and seedling growth conditions of the golden thread orchid prepared in Example 1 of the present invention;

[0019] Figure 3 The primary and secondary robust bottle seedlings of Golden Line Orchid prepared in Example 1 of the present invention;

[0020] Figure 4 The growth status of the golden thread orchid seedlings prepared in Example 2 of the present invention;

[0021] Figure 5 The first-generation and second-generation robust bottle seedlings of Goldenrod lily prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] Example 1

[0024] The present invention explores the effects of different basic culture media on the germination of golden thread orchid seeds, and the effects of different hormone and additive combinations on the rooting and strong seedlings of golden thread orchid.

[0025] The method for rapid propagation of golden thread orchid by aseptic sowing of the present invention comprises the following steps:

[0026] (1) Fruit pod selection and disinfection: The fruit pods of the golden thread orchid that are full and not cracked are collected about 60 days after pollination for sowing. The surface of the fruit pods is cleaned with a small brush under running water. After drying, the fruit pods are immersed in 75% alcohol for 30 seconds in a clean bench, washed once with sterile water, dried, and then immersed in 0.1% mercuric chloride for 10 minutes. The fruit is pressed below the liquid surface with tweezers during the whole process. After disinfection, the fruit pods are washed three times with sterile water and dried;

[0027] (2) Seed germination: The sterilized pods were split open and the seeds were evenly sown on the germination medium for non-symbiotic germination culture. The germination medium formula was 1 / 2MS + NAA 0.3mg / L + 6-BA 0.05mg / L + sucrose 20g / L + agar powder 8g / L, pH 5.8. The control germination medium was 1 / 2MS replaced by MS and KC. The other components were the same as above. The culture conditions were light time 12h / d, light intensity 1200-1500Lux, culture temperature 25℃ when illuminated and 23℃ when not illuminated. 0-2 weeks after sowing, the embryo gradually expanded and broke through the seed coat. Figure 1As shown, white protocorms are formed within 6 weeks. The germination rate is counted at the 6th week. Three dishes are counted for each culture medium, and 100 seeds are counted for each dish. The germination rate = (number of germinated seeds / total number of observed seeds) × 100%. The germination standard is that the embryo swells to form a white protocorm, and the germination rate is about 80%. From 6 to 10 weeks, the protocorms differentiate into yellow-green buds. The differentiation rate is counted at the 10th week. Three dishes are counted for each culture medium, and 60 protocorms are counted for each dish. The differentiation rate = (number of differentiated protocorms / number of observed protocorms) × 100%. The differentiation standard is the number of protocorms. Morphologically, small buds differentiated from the upper end and turned green, with a differentiation rate of about 60%; from 10 to 18 weeks, the leaves of the seedlings gradually unfolded, and the seedling rate was counted at the 18th week. Three dishes were counted for each culture medium, and the seedling rate = (green leaf area / plant material area) × 100%. The seedling standard was the seedlings that sprouted into open or relatively open green leaves. Since the plants (>100 plants) on each dish of culture medium grew very densely, it was impossible to record the seedlings without opening the culture dish, so the green leaf rate was used instead of the seedling rate, in order to detect the effect of different culture media on leaf growth; Figure 2 As shown, the seedling rate was the highest in the 1 / 2MS medium treatment, with the leaves of the seedlings tender green and stretched, the rhizomes slender, and the browning degree was light. The leaves of the seedlings in the control group treated with the MS medium were swept, the rhizomes were thick and short, and the browning degree was medium. Almost no seedlings grew on the control group KC medium, the protocorm growth was not obvious, and the browning was serious. It can be seen that 1 / 2MS is the most suitable basic medium for the germination of Golden Line Orchid among the above three mediums.

[0028] (3) Primary culture: 20 weeks after sowing, seedlings with three leaves and uniform growth were transferred to primary culture medium for culture; three factors (NAA, 6-BA, culture medium additives) were selected, each with three levels, for a total of nine experimental combinations (Table 1); the remaining culture medium components were 1 / 2MS + sucrose 20 g / L + agar powder 8 g / L, pH 5.8; nine bottles were inoculated in each combination, and four seedlings were inoculated in each bottle; the illumination time was 12 h / d, the illumination intensity was 1200-1500 Lux, and the culture temperature was 25°C when illuminated and 23°C when not illuminated. After six months, three bottles were randomly selected from each experimental treatment, and the number of seedlings in each bottle was measured to comprehensively score the rooting and seedling strengthening effect; first, the data were normalized, and the normalized data was represented by C, and the minimum and maximum values ​​of the normalized data were represented by C, C, and C, respectively. 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 Single plant weight*0.4+C new Number of roots*0.1+Cnew Root length*0.1+C new Leaf length*0.1+C new Plant height*0.3), C new =(CC min ) / (C min -C max ), among which M4 had the highest comprehensive score of 95.77, and the primary generation of strong and robust seedlings of Gree Golden Line Orchid prepared by it had the best primary culture effect. Figure 3 As shown; M5's comprehensive score is second, 95.52;

[0029] Table 1 Rooting and seedling strengthening effect of primary culture of Gree Golden Line Orchid

[0030]

[0031]

[0032] (4) Subculture: excise the leaves and roots of the first generation of healthy sterile bottle seedlings of Golden Line Orchid, cut the stem into a stem segment with a length of 1 to 2 cm and a node, and inoculate it flatly into the subculture medium, the formula of which is 1 / 2MS + sucrose 20 g / L + agar powder 8 g / L + activated carbon 0.2 g / L + banana puree 100 g / L + NAA 0.3 mg / L, pH 5.8, light duration 12 h / d, light intensity 1200 to 1500 Lux, culture temperature 20 to 25 ° C, after 4 to 6 months, healthy bottle seedlings with a plant height of more than 10 cm, thick stems, and extended leaves can be obtained, and step (4) is repeated for subculture to obtain healthy bottle seedlings. Figure 3 shown.

[0033] Example 2

[0034] The present invention mainly explores the effects of different organic additives and hormone combinations on the germination of golden thread orchid seeds.

[0035] The method for rapid propagation of golden thread orchid by aseptic sowing of the present invention comprises the following steps:

[0036] (1) Selection and disinfection of fruit pods: The fruit pods of the golden thread orchid that are full and not cracked are collected about 90 days after pollination for sowing. The remaining floral structures on the fruit pods are removed with a blade, and the fruit pods are soaked in detergent water, gently scrubbed with a toothbrush, and then packed in a non-woven bag. The fruit pods are rinsed under running water for 20 minutes, and then placed in a sterile bottle and transferred to a clean bench. Subsequently, they are soaked in 75% alcohol in the clean bench for 30 seconds, rinsed once with sterile water, and then soaked in 2% sodium hypochlorite solution for 10 minutes. During the soaking process, the bottle containing the disinfectant is gently shaken from time to time to ensure that the fruit pods are fully in contact with the disinfectant. After the disinfection is completed, they are rinsed with sterile water for 3 times, and the water is absorbed with sterile filter paper for later use.

[0037] (2) Seed germination: Cut the sterilized pods longitudinally, hold them with tweezers, tap the tweezers on the edge of the culture dish or culture bottle, and evenly spread the seeds on the germination medium for culture. Use 1 / 2MS as the basic medium, add 20 g / L sucrose, 1 g / L activated carbon and 7-8 g / L agar powder, set different hormones and organic additives (Table 2), pH 5.5-5.6, use a light quality ratio of seven white and one red lamp in the culture room, the lighting time is 12 h / d, the light intensity is 1200-1500 Lux, and the temperature is 20-25 °C;

[0038] Table 2 Comparison of 9 formulations in the seed germination experiment of Gree Golden Line Orchid

[0039]

[0040] The growth status of seedlings after 6 months under the 9 formulations Figure 4 As shown, it can be seen that adding 50g / L banana, 0.3mg / L6-BA, or 0.5mg / L 6-BA is not conducive to the germination of Gree Golden Line Orchid seeds. Formulas 1 and 2 performed best, with seedlings growing neatly, leaves spreading, and dark green leaves. Therefore, 1 / 2MS+20g / L sucrose+1g / L activated carbon+8g / L agar powder, pH 5.5-5.6, or adding 0.3mg / L NAA and 50g / L mashed potato on this basis is a more suitable culture medium formula for Gree Golden Line Orchid seed germination.

[0041] (3) Primary culture: The primary culture uses a hormone-free formula, specifically 1 / 2MS + sucrose 20g / L + agar powder 7g / L + activated carbon 0.5g / L + banana puree 100g / L. The culture conditions are the same as step (2). After 4 to 6 months, sterile and strong primary seedlings are obtained for subculture. Figure 5 ;

[0042] (4) Subculture: excise the leaves and roots of the first-generation sterile bottle seedlings of Golden Line Orchid, cut the stems into stem segments with a length of 1 to 2 cm and one node, and inoculate them flatly into the subculture medium, the formula of which is 1 / 2MS + sucrose 20 g / L + agar powder 7 g / L + activated carbon 0.5 g / L + banana puree 100 g / L + NAA 0.5 mg / L, pH 5.6 to 5.8, and the culture conditions are the same as step (2). After 4 to 6 months, strong subculture seedlings are obtained. Figure 5 .

[0043] Example 2 uses sodium hypochlorite solution instead of the 0.1% mass percentage concentration of mercuric chloride in Example 1, which has good effect, is almost pollution-free, reduces the danger of the experimental process, and can reduce the harm to the human body and the environment; Figure 2 and Figure 4As shown, the addition of 1 g / L activated carbon in the germination medium solved the browning problem of the seedlings after germination in Example 1.

[0044] In summary, the aseptic sowing and rapid propagation method of Gree Golden Line Orchid of the present invention is the first to explore a aseptic germination and rapid propagation method of Gree Golden Line Orchid. The fruit pods of Gree Golden Line Orchid are used to quickly obtain a large number of sterile tissue culture bottle seedlings with high genetic diversity, providing a germplasm resource library for wild reintroduction and subsequent industrial applications.

[0045] 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 Line Orchid by aseptic sowing, characterized in that: The following steps are involved: (1) Fruit pod selection and disinfection: Collect fruit pods that are full and unbroken 60 to 120 days after pollination, wash them, and disinfect them with disinfectant I and disinfectant II in turn; (2) Seed germination: longitudinally cut open the sterilized pods and evenly sow the seeds on the germination medium with the formula of 1 / 2MS + NAA 0-0.3mg / L + 6-BA 0-0.05mg / L + activated carbon 0-1g / L + potato 0-50g / L + sucrose or white sugar 15-20g / L + agar powder 6-8g / L, pH 5.5-5.8; (3) Primary culture: The seedlings are transferred to the primary culture medium, the formula of which is 1 / 2MS + NAA 0-2 mg / L + 6-BA 0-0.1 mg / L + activated carbon 0.2-1 g / L + banana puree 0-100 g / L + sucrose or white sugar 20-30 g / L + agar powder 6-8 g / L, pH 5.6-6.0, and cultured under suitable conditions. After 4-6 months, primary bottle seedlings with a plant height of 8-15 cm are obtained for subculture; (4) Subculture: remove the leaves and roots of the primary seedlings, cut the stems into small segments with a length of 1 to 2 cm and one node, and place them flatly in a subculture medium with a formula of 1 / 2MS + sugarcane + NAA 0 to 1 mg / L + activated carbon 0.2 to 1 g / L + sucrose or white sugar 15 to 20 g / L + agar powder 6 to 8 g / L + banana puree 50 to 100 g / L, pH 5.6 to 6.

0. After 4 to 6 months, healthy seedlings with a plant height of 8 to 15 cm are obtained; repeat step (4) for subculture to obtain healthy seedlings for cultivation.

2. The method for rapid propagation of Golden Line Orchid by aseptic sowing according to claim 1, characterized in that: In step (1), disinfectant I is 70% to 75% alcohol, and disinfectant II is 0.1% by mass concentration of mercuric chloride (HgCl2) or 2% to 5% by mass concentration of sodium hypochlorite (NaClO) solution.

3. The method for rapid propagation of Golden Line Orchid by aseptic sowing according to claim 1, characterized in that: The culture conditions in steps (2), (3) and (4) are: using a lamp 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 20-25°C.

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