Rapid propagation method of Daqing oil palm tissue culture and its application

Abstract

The invention relates to the technical field of plant tissue culture, and is a method and application of rapid propagation of daqing tissue culture. The invention uses unlignified young stem segments as explants, sterilizes them with 75% alcohol and 0.1% mercuric chloride solution in sequence after cleaning, and establishes a rapid propagation technology system of daqing tissue culture through primary induction, secondary proliferation, strong bud culture and rooting culture, providing technical support for its production and reproduction, collection and preservation of wild germplasm resources, introduction and domestication, development and utilization. The invention controls the explant inoculation contamination rate within 15% and the mortality rate within 10% by controlling the explant collection time and sterilization time; the addition of polyvinyl pyrrolidone (PVP) during primary induction can effectively inhibit the browning of the explant; the primary induction rate is as high as 80% or more, the proliferation coefficient reaches about 11.6, and the rooting rate reaches more than 95% through hormone regulation; a certain concentration of coconut juice is added during strong bud and rooting culture to make the leaves more vivid green, the seedlings and roots more robust, and finally improve the survival rate of seedling hardening.

Description

Technical Field

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

[0002] Clerodendrum cyrtophyllum Turcz. is a deciduous shrub belonging to the genus Clerodendrum of the Verbenaceae family. The plant can reach a height of 10 meters; the young branches are covered with soft hairs; the leaves are elliptical or oblong-lanceolate, with acuminate or pointed tips, entire or crenate, glabrous on both sides or sparsely covered with soft hairs along the veins; the corymbose cymes are covered with yellow-brown fine hairs and glandular dots, the corolla is white, and the lobes are ovate; the drupes are spherical or obovate, blue-purple, and are covered by red persistent calyx; the flowering and fruiting period is from June to February of the following year. The leaves and roots of Clerodendrum cyrtophyllum Turcz. have the effects of clearing away heat and detoxifying, cooling blood and stopping bleeding, and can be used to treat exogenous febrile diseases, heat-induced thirst, sore throat, heat-toxic dysentery, acute enteritis, carbuncle swelling and poison, and traumatic bleeding. It is a traditional Chinese medicine. The young leaves of Clerodendrum cyrtophyllum Turcz. can be eaten fresh and are an important forest food resource. Because of its large inflorescence and red-purple fruits, it can be planted on forest edges, lawns, etc. It also has certain horticultural ornamental value.

[0003] Daqing is mainly distributed in East China, Guangxi Zhuang Autonomous Region, Guangdong, Guizhou, Yunnan and other provinces. It grows in plains, hills, mountain forests or valleys below 1,700 meters above sea level. It likes warm and humid environment and is suitable for growing in fertile soil with sufficient sunlight.

[0004] There are three ways to propagate the Daqing: one is to collect natural wild seedlings in the mountains for centralized planting, but due to limited wild resources, it is difficult to meet the needs of large-scale cultivation; the second is cutting propagation, but the survival rate is extremely low. Even if treated with ABT No. 1 rooting powder, the survival rate is only about 20%, which is difficult to promote and apply in production; the third is seed sowing and seedling raising. Seed propagation has a long flowering period, which leads to a longer seed maturity time, thus resulting in a long breeding period, and seed propagation cannot maintain the excellent traits of the mother plant. Summary of the invention

[0005] The purpose of the invention is to provide a tissue culture rapid propagation method and application of scutellaria baicalensis to solve the problems of low propagation rate and long propagation cycle in view of the problems existing in the existing propagation technology of scutellaria baicalensis.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for rapid propagation of Aglaonema dasyphylla tissue culture, comprising the following steps:

[0007] (1) Explant collection: After 2 to 3 consecutive sunny days, collect young and tender stem segments that are not lignified as explants after 10 a.m.;

[0008] (2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for sterilization for 20 to 30 seconds, rinsed with sterile water 2 to 3 times, immersed in 0.1% mercuric chloride solution and shaken for sterilization for 7 to 8 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture of the stem segments is absorbed with sterile filter paper.

[0009] (3) Primary induction: explants were cut into 1.0-2.0 cm segments with nodes and inoculated into primary induction medium according to polarity and cultured for 15-20 days;

[0010] The formula of the primary induction medium is: WPM medium + 6-BA 0.4-0.6 mg / L + NAA 0.01-0.02 mg / L + PVP 3.0-5.0 g / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L;

[0011] (4) Subculture proliferation: The primary induced clustered buds are cut into single buds and inoculated into subculture proliferation medium according to polarity and cultured for 25 to 35 days;

[0012] The formula of the subculture proliferation medium is: MS medium + 6-BA 1.0-1.2 mg / L + TDZ 0.01-0.02 mg / L + IBA 0.4-0.6 mg / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L;

[0013] (5) Strong bud culture: The clustered buds from subculture are divided into single buds and inoculated into strong bud culture medium according to polarity and cultured for 25 to 35 days;

[0014] The formula of the bud-strengthening culture medium is: MS culture medium + 6-BA 1.8-2.0 mg / L + coconut juice 45.0-55.0 mL / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L;

[0015] (6) Rooting culture: The single buds after strong bud culture are inoculated into rooting medium and cultured for 30 to 40 days;

[0016] The formula of the rooting medium is: 1 / 2 MS medium + NAA 0.5-0.7 mg / L + IBA 0.2-0.3 mg / L + coconut juice 45.0-55.0 mL / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L.

[0017] Furthermore, in steps (3), (4), (5) and (6), the culture conditions are: temperature 25±2°C, light intensity 2000-3000Lx, and light exposure of 10-14 h per day for alternating light and dark culture.

[0018] Furthermore, the formula of the primary induction medium is: WPM medium + 6-BA 0.5 mg / L + NAA0.01 mg / L + PVP 4.0 g / L + sucrose 30.0 g / L + agar 7.0 g / L.

[0019] Furthermore, the formula of the subculture proliferation medium is: MS medium + 6-BA 1.1 mg / L + TDZ0.01 mg / L + IBA 0.5 mg / L + sucrose 30.0 g / L + agar 7.0 g / L.

[0020] Furthermore, the formula of the bud-strengthening culture medium is: MS culture medium + 6-BA 1.9 mg / L + coconut juice 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L.

[0021] Furthermore, the formula of the rooting medium is: 1 / 2 MS medium + NAA 0.6 mg / L + IBA 0.3 mg / L + coconut milk 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L.

[0022] The beneficial technical effect of the present invention is: the present invention provides a method for tissue culture and rapid propagation of the medicinal plant Isatis indigofera, using non-lignified young stem segments as explants, through primary induction, secondary proliferation, bud culture and rooting culture, a Isatis indigofera tissue culture and rapid propagation technology system is established, providing technical support for its production and reproduction, as well as the collection, preservation, introduction and domestication, development and utilization of wild germplasm resources.

[0023] Daqing is particularly sensitive to sterilization. If the sterilization time is too long, it is very easy to cause the death of the explants. Even if the explants are not dead, they will be severely browned, resulting in the failure to successfully induce clustered buds. If the sterilization time is too short, it is very easy to cause contamination. The present invention controls the explant inoculation contamination rate within 15% by controlling the timing of explant collection and the sterilization time, while reducing the damage to the explants caused by sterilization, and controlling the mortality rate within 10%, thereby facilitating the induction of primary clustered buds.

[0024] Daqing is rich in phenolic substances, and the explants are very likely to turn brown along the incision. In the primary induction of the present invention, adding a certain concentration of polyvinyl pyrrolidone (PVP) to the culture medium can effectively inhibit the browning of the explants. In the present invention, adding a certain concentration of coconut juice during bud and root culture can make the leaves greener, the buds and roots stronger, and ultimately improve the survival rate of seedlings.

[0025] The primary induction rate of the invention is as high as over 80%, the proliferation coefficient reaches about 11.6, and the rooting rate reaches over 95%, which can realize the efficient reproduction of Daqing and is almost not restricted by factors such as season and climate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0027] Figure 1 It is the non-lignified large green young stem segment explant collected in sunny weather in Example 1.

[0028] Figure 2 The explants on the clean bench of Example 1 are sterilized.

[0029] Figure 3 This is the result of the primary induction culture in Example 1.

[0030] Figure 4 This is the result of the subculture proliferation culture in Example 1.

[0031] Figure 5 It is embodiment 1 strong bud cultivation result.

[0032] Figure 6 This is the rooting culture result of Example 1. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for rapid propagation of cycad tissue culture, comprising the following steps:

[0036] (1) Explant collection: After two consecutive sunny days, collect young stem segments that are not lignified as explants after 10 a.m., such as Figure 1 shown.

[0037] (2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for 20 seconds, rinsed twice with sterile water, immersed in 0.1% mercuric chloride solution and shaken for 7 minutes, and rinsed four times with sterile water. Figure 2 As shown, sterile filter paper was used to absorb the surface moisture of the stem segments.

[0038] (3) Primary induction: Explants were cut into 1.0 cm long segments with nodes and inoculated into primary induction medium (formula: WPM medium + 6-BA 0.4 mg / L + NAA 0.01 mg / L + PVP 3.0 g / L + sucrose 25.0 g / L + agar 6.5 g / L) according to polarity. The cells were placed in a light-dark alternation culture at 25 ± 2 °C, 2000 Lx, and 14 h of light per day for 15 days. The culture results were as follows: Figure 3 shown.

[0039] (4) Subculture: The clustered buds induced in the primary generation were cut into single buds and inoculated into subculture proliferation medium (formula: MS medium + 6-BA 1.0 mg / L + TDZ 0.01 mg / L + IBA 0.4 mg / L + sucrose 25.0 g / L + agar 6.5 g / L) according to polarity. The culture was placed at 25±2℃, light intensity 2000 Lx, and light intensity 14 h per day for 25 days with alternating light and dark. The culture results are as follows: Figure 4 shown.

[0040] (5) Strong bud culture: The clustered buds of subculture proliferation culture were divided into single buds and inoculated into strong bud culture medium (formula: MS medium + 6-BA 1.8 mg / L + coconut juice 45.0 mL / L + sucrose 25.0 g / L + agar 6.5 g / L) according to polarity. The medium was placed at 25±2℃, light intensity 2000 Lx, and light intensity 14 h per day for 25 days with alternating light and dark. The culture results were as follows: Figure 5 shown.

[0041] (6) Rooting culture: The single buds after bud culture were inoculated into rooting medium (formula: 1 / 2 MS medium + NAA 0.5 mg / L + IBA 0.2 mg / L + coconut juice 45.0 mL / L + sucrose 25.0 g / L + agar 6.5 g / L), placed in a temperature of 25±2℃, light intensity of 2000 Lx, and light intensity of 14 h per day for 30 days of light-dark alternation. The culture results are as follows: Figure 6 shown.

[0042] Example 2

[0043] A method for rapid propagation of cycad tissue culture, comprising the following steps:

[0044] (1) Explant collection: After 3 consecutive sunny days, young and tender stem segments that have not yet become lignified were collected after 10 a.m. as explants.

[0045] (2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for sterilization for 30 seconds, rinsed with sterile water three times, immersed in 0.1% mercuric chloride solution and shaken for sterilization for 8 minutes, rinsed with sterile water five times, and the surface moisture of the stem segments is absorbed with sterile filter paper.

[0046] (3) Primary induction: Explants were cut into 1.5 cm node segments and inoculated according to polarity into primary induction medium (formula: WPM medium + 6-BA 0.6 mg / L + NAA 0.02 mg / L + PVP 5.0 g / L + sucrose 35.0 g / L + agar 7.5 g / L), and cultured in alternating light and dark conditions at 25±2℃, 3000 Lx, and 14 h of light per day for 20 days.

[0047] (4) Subculture: The clustered buds induced in the primary generation were cut into single buds and inoculated into subculture proliferation medium (formula: MS medium + 6-BA 1.2 mg / L + TDZ 0.02 mg / L + IBA 0.6 mg / L + sucrose 35.0 g / L + agar 7.5 g / L) according to polarity. The culture was placed in a light-dark alternating medium at 25±2℃, 3000 Lx light intensity, and 14 h light per day for 35 days.

[0048] (5) Strong bud culture: The clustered buds of subculture proliferation culture were divided into single buds and inoculated into strong bud culture medium (formula: MS medium + 6-BA 2.0 mg / L + coconut juice 55.0 mL / L + sucrose 35.0 g / L + agar 7.5 g / L) according to polarity. The culture was placed in a temperature of 25±2℃, a light intensity of 3000 Lx, and a light intensity of 14 h per day for 35 days with alternating light and dark.

[0049] (6) Rooting culture: The single buds after strong bud culture were inoculated into rooting medium (formula: 1 / 2 MS medium + NAA 0.7 mg / L + IBA 0.3 mg / L + coconut juice 55.0 mL / L + sucrose 35.0 g / L + agar 7.5 g / L) and placed in a light-dark alternating culture medium at 25±2℃, light intensity 3000 Lx, and 14 h of light per day for 40 days.

[0050] Example 3

[0051] A method for rapid propagation of cycad tissue culture, comprising the following steps:

[0052] (1) Explant collection: After two consecutive sunny days, young, non-lignified stem segments were collected after 10 a.m. as explants.

[0053] (2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for sterilization for 25 seconds. They are then rinsed twice with sterile water. They are then immersed in 0.1% mercuric chloride solution and shaken for sterilization for 7 minutes. They are then rinsed five times with sterile water. The surface moisture of the stem segments is then absorbed with sterile filter paper.

[0054] (3) Primary induction: Explants were cut into 2.0 cm long segments with nodes and inoculated according to polarity into primary induction medium (formula: WPM medium + 6-BA 0.5 mg / L + NAA 0.01 mg / L + PVP 4.0 g / L + sucrose 30.0 g / L + agar 7.0 g / L), and cultured in alternating light and dark conditions at 25±2℃, 2500 Lx, and 12 h of light per day for 17 days.

[0055] (4) Subculture: The clustered buds induced in the primary generation were cut into single buds and inoculated into subculture proliferation medium (formula: MS medium + 6-BA 1.1 mg / L + TDZ 0.01 mg / L + IBA 0.5 mg / L + sucrose 30.0 g / L + agar 7.0 g / L) according to polarity. The culture was placed in a light-dark alternation environment at 25±2℃, 2500 Lx light intensity, and 12 h of light per day for 30 days.

[0056] (5) Strong bud culture: The clustered buds of subculture proliferation culture were divided into single buds and inoculated into strong bud culture medium (formula: MS medium + 6-BA 1.9 mg / L + coconut juice 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L) according to polarity. The culture was placed in a temperature of 25±2℃, light intensity of 2500 Lx, and light intensity of 12 h per day for 30 days with alternating light and dark.

[0057] (6) Rooting culture: The single buds after strong bud culture were inoculated into rooting medium (formula: 1 / 2 MS medium + NAA 0.6 mg / L + IBA 0.3 mg / L + coconut juice 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L) and placed in a light-dark alternating culture medium at 25±2℃, light intensity 2500 Lx, and 12 h of light per day for 35 days.

[0058] Comparative Example 1

[0059] Use the end branches of the big green tree for cutting propagation and adopt conventional cutting management methods for management.

[0060] The disinfection contamination rate, disinfection mortality rate, cluster bud induction rate, proliferation coefficient and rooting rate of Examples 1-3 and Comparative Example 1 were statistically compared, and the results are shown in Table 1.

[0061] Table 1 Statistical comparison results

[0062] Disinfection contamination rate Disinfection mortality rate Primary generation bud induction rate Subculture proliferation coefficient Rooting rate Example 1 14.6% 8.7% 83.7% 10.9 95.8% Example 2 13.3% 9.1% 86.2% 11.2 96.2% Example 3 12.8% 7.7% 88.1% 11.8 97.4% Comparative Example 1 \ \ \ \ 20%

[0063] 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 skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for rapid propagation of Rhizoma Cibotii tissue culture, characterized in that the steps include: (1) Explant collection: After 2 to 3 consecutive sunny days, collect young and tender stem segments that are not lignified as explants after 10 a.m.; (2) Explant sterilization: After the leaves are cut off, the stem segments are cleaned and transferred to a clean bench. They are then immersed in 75% alcohol solution and shaken for sterilization for 20 to 30 seconds, rinsed with sterile water 2 to 3 times, immersed in 0.1% mercuric chloride solution and shaken for sterilization for 7 to 8 minutes, rinsed with sterile water 4 to 5 times, and the surface moisture of the stem segments is absorbed with sterile filter paper. (3) Primary induction: explants were cut into 1.0-2.0 cm segments with nodes and inoculated into primary induction medium according to polarity and cultured for 15-20 days; The formula of the primary induction medium is: WPM medium + 6-BA 0.4-0.6 mg / L + NAA 0.01-0.02 mg / L + PVP 3.0-5.0 g / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L; (4) Subculture proliferation: The primary induced clustered buds are cut into single buds and inoculated into subculture proliferation medium according to polarity and cultured for 25 to 35 days; The formula of the subculture proliferation medium is: MS medium + 6-BA 1.0-1.2 mg / L + TDZ 0.01-0.02 mg / L + IBA 0.4-0.6 mg / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L; (5) Strong bud culture: The clustered buds from subculture are divided into single buds and inoculated into strong bud culture medium according to polarity and cultured for 25 to 35 days; The formula of the bud-strengthening culture medium is: MS culture medium + 6-BA 1.8-2.0 mg / L + coconut juice 45.0-55.0 mL / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L; (6) Rooting culture: The single buds after strong bud culture are inoculated into rooting medium and cultured for 30 to 40 days; The formula of the rooting medium is: 1 / 2 MS medium + NAA 0.5-0.7 mg / L + IBA 0.2-0.3 mg / L + coconut juice 45.0-55.0 mL / L + sucrose 25.0-35.0 g / L + agar 6.5-7.5 g / L.

2. The method according to claim 1, characterized in that: In steps (3), (4), (5) and (6), the culture conditions are: temperature 25±2°C, light intensity 2000-3000 Lx, and light exposure for 10-14 h per day for alternating light and dark culture.

3. The method according to claim 1, characterized in that: The formula of the primary induction medium is: WPM medium + 6-BA 0.5 mg / L + NAA 0.01 mg / L + PVP 4.0 g / L + sucrose 30.0 g / L + agar 7.0 g / L.

4. The method according to claim 1, characterized in that: The formula of the subculture proliferation medium is: MS medium + 6-BA 1.1 mg / L + TDZ 0.01 mg / L + IBA 0.5 mg / L + sucrose 30.0 g / L + agar 7.0 g / L.

5. The method according to claim 1, characterized in that: The formula of the bud-strengthening culture medium is: MS culture medium + 6-BA 1.9 mg / L + coconut juice 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L.

6. The method according to claim 1, characterized in that: The formula of the rooting medium is: 1 / 2 MS medium + NAA 0.6 mg / L + IBA 0.3 mg / L + coconut milk 50.0 mL / L + sucrose 30.0 g / L + agar 7.0 g / L.

7. Use of the method according to any one of claims 1 to 6 in rapid propagation of Isatis indica tissue culture.

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