Clematis florida tissue culture method

Abstract

The invention discloses a clematis tissue culture method which comprises the following steps: (1) sterilizing a clematis explant, and inoculating the clematis explant to an axillary bud induction culture medium with the morphological lower end upward for culture; (2) inoculating the explant cultured in the step (1) to a hormone-free culture medium for culturing; (3) inoculating the explant cultured in the step (2) to a rooting culture medium for culturing; and (4) domesticating and transplanting the explants cultured in the step (3) to obtain clematis plants. By means of the tissue culture method, high-quality rapid rooting of the clematis explant can be achieved, meanwhile, a complete plant with a thick and strong root system can be effectively induced, weather conditions do not need to be considered, a large number of high-quality clematis seedlings can be produced within a short time, market requirements are met, and great benefits are brought to industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to a plant tissue culture method, in particular to a Clematis L. tissue culture method. BACKGROUND

[0002] Clematis L. is a collective term for plants of the genus Clematis L. in the Ranunculaceae family. It has a variety of flower types and bright colors, and is known as the "Queen of Flowering Vines" for its high ornamental value. In addition, Clematis L. also has various pharmacological activities such as antibacterial, antitumor, anti-inflammatory analgesic, and antiviral, and has attracted widespread attention in the medical field.

[0003] Currently, the main propagation methods of Clematis L. include sowing, layering and cutting, but these methods have obvious limitations in practical application. Sowing propagation is simple to operate, but has a high seed abortion rate and harsh germination conditions, resulting in generally low survival rate. Layering propagation can maintain the maternal traits, but requires high quality of branches and environmental humidity, has a long propagation cycle and limited efficiency. Cutting propagation is a common method of asexual reproduction, but it is technically difficult. For example, the cutting of 'Ru' and other varieties is extremely sensitive to substrate aeration and temperature. In summer, it is prone to rot due to high temperature and humidity, and in winter, it is slow to root due to low temperature, making it difficult to stably control the survival rate. These propagation difficulties seriously limit the large-scale production of Clematis L. and cannot meet the market demand for commercial seedling production with large quantities, uniform traits and controllable costs. SUMMARY

[0004] The purpose of the present application is to provide a Clematis L. tissue culture method that can quickly root.

[0005] The technical scheme of the present application is a Clematis L. tissue culture method, comprising the following steps:

[0006] (1) After the Clematis L. explant is disinfected, it is inoculated into the axillary bud induction medium with the lower end facing up for culture;

[0007] (2) The explant after step 1 is inoculated into the hormone-free medium for culture;

[0008] (3) The explant after step 2 is inoculated into the rooting medium for culture;

[0009] (4) The explant after step 3 is acclimated and transplanted to obtain Clematis L. plants.

[0010] Preferably, in step 1, the Clematis L. explant is a healthy and disease-free mother plant, the leaves are removed, and the 0.5-1 cm petiole is retained. The internode part is cut to obtain a 1-2.5 cm long semi-lignified stem segment of the current year.

[0011] More preferably, the clematis mother plant is the 'Rugu' clematis mother plant.

[0012] Preferably, in step 1, the disinfection step includes soaking in carbendazim solution, soaking in ethanol solution, and soaking in sodium hypochlorite solution. After each soaking disinfection, the explant is thoroughly rinsed with water, and finally, the damaged parts at both ends of the explant are removed.

[0013] Preferably, in step 1, the disinfection step includes soaking in carbendazim solution, soaking in ethanol solution, and soaking in sodium hypochlorite solution. After each soaking disinfection, the explant is thoroughly rinsed with water, and finally, the damaged parts at both ends of the explant are removed.

[0014] A further preferred method is to soak the stem segment in a 1% carbendazim solution for 12-15 minutes, then rinse it with running water for 1 hour. Next, transfer it to a clean bench, soak it in a 75% ethanol solution for 25-30 seconds, rinse it with sterile water 3 times, then soak it in a 2-5% sodium hypochlorite solution for 8-10 minutes with shaking, and finally rinse it with sterile water 5 times. After drying the surface moisture, cut off the damaged parts at both ends of the stem segment.

[0015] Preferably, in step 1, the axillary bud induction medium is MS medium with pH=5.6-6.0 containing 1-2 mg / L 6-benzylaminopurine, 0.1 mg / L 1-naphthaleneacetic acid, sucrose and agar; more preferably, the amount of sucrose added is 30 g / L and the amount of agar added is 6.0 g / L.

[0016] Preferably, the axillary buds of the budded stem segments begin to sprout after 6-8 days of culture in the induction medium, and after 1-2 weeks of continued culture until the plant height reaches 5-6 cm, step 2 of the culture is carried out.

[0017] Preferably, in step 2, the hormone-free culture medium is MS medium with pH=5.8-6.2 containing sucrose and agar; more preferably, the amount of sucrose added is 30 g / L, the amount of agar added is 6.0 g / L, and the culture time is 6-8 days.

[0018] Preferably, in step 3, the rooting medium is a 1 / 2 MS medium with pH=5.6-6.0 containing 1-3 mg / L indolebutyric acid, 0.2-0.4 mg / L 1-naphthaleneacetic acid, sucrose and agar; more preferably, the amount of sucrose added is 20-30 g / L and the amount of agar added is 6.0 g / L.

[0019] In a further preferred manner, the roots are cultured until they reach a length of 3-4 cm before proceeding to step 4.

[0020] Preferably, steps 1 and 3 are light culture, and step 2 is dark culture.

[0021] Further preferred conditions are: light intensity of 2000-2500 Lx, light duration of 14 h / d, temperature of 23-25℃, and relative humidity of 60%-80%; dark culture is carried out without light, and the temperature and humidity conditions are the same as those for light culture.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. Through the optimized tissue culture method, the clematis explants can be rapidly rooted and the root quality is high; 2. The method can effectively induce robust and complete plants without considering climatic conditions, and can produce a large number of high-quality clematis seedlings in a short time to meet market demand and bring great benefits to industrial production. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of different explant inoculation methods in Example 1;

[0024] Figure 2 This is a diagram showing root growth on day 35 of tissue culture in Example 1.

[0025] Figure 3 This is a diagram showing root growth on day 35 of tissue culture in Example 2;

[0026] Figure 4 This is a diagram showing root growth on day 35 of tissue culture in Example 3;

[0027] Figure 5 This is a diagram showing root growth on day 35 of tissue culture in Example 4;

[0028] Figure 6 These are images showing root growth on days 22 and 35 of tissue culture in Example 5.

[0029] Figure 7 This is a diagram showing the root growth of tissue culture on day 35 in Comparative Example 1. Detailed Implementation

[0030] The technical solution of the present invention will be further described below.

[0031] Example 1: Clematis tissue culture based on different inoculation methods of semi-lignified stem segment explants

[0032] (1) Selection of explants: In late April, select healthy and disease-free Clematis 'Rugu' mother plants, cut semi-lignified stem segments of the current year, remove the leaves and keep 0.5-1 cm petioles, cut stem segments with a length of 1-2.5 cm from the internodes, and use them as explants for later use.

[0033] (2) Material disinfection: The explants were soaked in 1% carbendazim solution for 15 min, then rinsed with running water for 1 h, and then transferred to a clean bench. They were soaked in 75% alcohol for 30 s, rinsed with sterile water 3 times, and then soaked in 5% sodium hypochlorite solution for 8 min with shaking. Finally, they were rinsed with sterile water 5 times, dried, and the damaged parts at both ends of the stem segment were cut off to obtain sterile explants.

[0034] (3) Primary culture: Aseptic explants were cultured separately according to... Figure 1 As shown, insert upright (morphologically, the lower end faces down). Figure 1 A) Inverted (morphologically, the lower end is facing upwards), Figure 1 B) or lay flat ( Figure 1 C) Inoculate into axillary bud induction medium, and culture conditions are: temperature 24±1℃, light intensity 2250±250 Lx, light duration 14 h / d, relative humidity 70±10%, culture until the stem segment with buds grows to a height of 5-6 cm. The axillary bud induction medium is MS medium with pH=5.8 supplemented with 2 mg / L 6-benzylaminopurine (6-BA), 0.1 mg / L 1-naphthaleneacetic acid (NAA), 30 g / L sucrose, and 6.0 g / L agar.

[0035] (4) Hormone-free transition culture: Explants from axillary buds were transferred to hormone-free transition culture medium and cultured in the dark for 7 days. The temperature and humidity conditions were the same as those of the primary culture. The hormone-free culture medium was MS medium with pH=6.0 and 30 g / L sucrose and 6.0 g / L agar.

[0036] (5) Rooting culture: After hormone-free transition culture, the aging tissue at the base of the explants was removed and they were inoculated into the rooting medium and cultured for 35 days. The rooting medium was a 1 / 2 MS medium with pH=5.8 containing 1 mg / L indolebutyric acid (IBA), 0.2 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar.

[0037] Rooting of explants induced by different inoculation methods, as follows Figure 2 As shown, this is day 35 of the rooting culture. Figure 2 A is the 'Rugu' clematis inoculated using the vertical insertion method (morphological lower end facing down). This inoculation method results in slow root growth, short roots, and only one root system. Figure 2 B is the 'Rugu' clematis inoculated by vertical upside-down insertion (morphological lower end facing up). The root length and number are greater than those inoculated by the upright insertion method, and the rooting time is earlier. Roots can be seen growing on the 12th day of rooting culture, and the average root length can reach 6 cm by the 35th day of rooting culture. Figure 2C represents 'Rugu' clematis inoculated horizontally. By the 35th day of rooting culture, the axillary buds were growing vigorously, but no roots were observed. Callus tissue formed at the point where the stem segment contacted the culture medium.

[0038] Example 2: Clematis tissue culture based on young stem segment explants

[0039] (1) Selection of explants: In late April, select healthy and disease-free Clematis 'Rugu' mother plants, cut young stem segments of the current year, remove leaves and keep 0.5-1 cm petioles, cut stem segments with a length of 1-2.5 cm from the internodes, and use them as explants for later use.

[0040] (2) Material disinfection: The explants were soaked in 1% carbendazim solution for 12 min, then rinsed with running water for 1 h, and then transferred to a clean bench. They were soaked in 75% alcohol for 25 s, rinsed with sterile water 3 times, and then soaked in 2% sodium hypochlorite solution for 8 min with shaking. Finally, they were rinsed with sterile water 5 times, and the damaged parts at both ends of the stem segment were cut off after drying to obtain sterile explants.

[0041] (3) Primary culture: Aseptic explants were inserted upright (morphologically downward) into axillary bud induction medium. The culture conditions were: temperature 24±1℃, light intensity 2250±250 Lx, light duration 14 h / d, and relative humidity 70±10%. The plants were cultured until the stem segments with buds reached a height of 5-6 cm. The axillary bud induction medium was MS medium with pH=5.8 containing 2 mg / L 6-benzylaminopurine (6-BA), 0.1 mg / L 1-naphthaleneacetic acid (NAA), 30 g / L sucrose, and 6.0 g / L agar.

[0042] (4) Hormone-free transition culture: Explants from axillary buds were transferred to hormone-free transition culture medium and cultured in the dark for 7 days. The temperature and humidity conditions were the same as those for light culture. The hormone-free culture medium was MS medium with pH=6.0 and 30 g / L sucrose and 6.0 g / L agar.

[0043] (5) Rooting culture: After hormone-free transition culture, the aging tissue at the base of the explants was removed and inoculated into the rooting medium, which was a 1 / 2 MS medium with pH=5.8 containing 3 mg / L indolebutyric acid (IBA), 0.4 mg / L NAA, 30 g / L sucrose and 6.0 g / L agar.

[0044] The rooting status of explants on day 35 of rooting culture is as follows: Figure 3 As shown, a large amount of callus tissue forms at the base of the stem segment of the tissue culture seedling. Although roots are induced, the induced roots are weak and difficult to acclimatize and transplant.

[0045] Example 3: Optimization of Clematis tissue culture based on semi-lignified stem segment explants 1

[0046] (1) Selection of explants: In early May, select healthy and disease-free Clematis 'Rugu' mother plants, cut young stem segments of the current year, remove leaves and keep 0.5-1 cm petioles, cut stem segments with a length of 1-2.5 cm from the internodes, and use them as explants for later use.

[0047] (2) Material disinfection: The explants were soaked in 1% carbendazim solution for 15 min, then rinsed with running water for 1 h, and then transferred to a clean bench. They were soaked in 75% alcohol for 30 s, rinsed with sterile water 3 times, and then soaked in 2% sodium hypochlorite solution for 10 min with shaking. Finally, they were rinsed with sterile water 5 times, the surface moisture was dried, and the damaged parts at both ends of the stem segment were cut off to obtain sterile explants.

[0048] (3) Primary culture: Aseptic explants were inoculated into axillary bud induction medium by upright insertion (morphological lower end facing down) or inverted insertion (morphological lower end facing up). The culture conditions were: temperature 24±1℃, light intensity 2250±250 Lx, light duration 14 h / d, and relative humidity 70±10%. The plants were cultured until the stem segments with buds reached a height of 5-6 cm. The axillary bud induction medium was MS medium with pH=5.8 supplemented with 1 mg / L 6-BA, 0.1 mg / L NAA, 30 g / L sucrose, and 6.0 g / L agar.

[0049] (4) Hormone-free transition culture: Explants from axillary buds were transferred to hormone-free transition culture medium and cultured in the dark for 7 days. The temperature and humidity conditions were the same as those for light culture. The hormone-free culture medium was MS medium with pH=6.0 and 30 g / L sucrose and 6.0 g / L agar.

[0050] (5) Rooting culture: After hormone-free transition culture, the aging tissue at the base of the explants was removed and inoculated into the rooting medium, which was a 1 / 2 MS medium with pH=5.8 containing 0.5 mg / L IBA, 20 g / L sucrose and 6.0 g / L agar.

[0051] The rooting status of explants on day 35 of rooting culture is as follows: Figure 4 As shown, where Figure 4 A is a 'Rugu' clematis inoculated using the straight cutting method. Figure 4 B is the 'Rugu' clematis inoculated by inverted insertion. The number of roots induced by explants of different inoculation methods is only 1-2, and the roots are thin, short (less than 1cm), grow slowly, and are difficult to acclimatize and transplant.

[0052] Example 4: Optimization of Clematis tissue culture based on semi-lignified stem segment explants 2

[0053] This embodiment refers to the method of embodiment 3, with the following difference:

[0054] In the initial culture stage, inoculation was carried out only by inverted insertion (morphological lower end facing upwards). After the initial culture, no hormone-free transition culture was carried out, and rooting culture was carried out directly. The rooting medium was 1 / 2 MS medium with pH=5.8, supplemented with 1.5 mg / L IBA, 0.2 mg / L NAA, 20 g / L sucrose and 6.0 g / L agar.

[0055] The rooting status on day 35 of rooting culture is as follows: Figure 5 As shown, under this clematis tissue culture method, the number of clematis roots is relatively large and the roots are relatively thick, but the average root length is only 2 cm.

[0056] Example 5: Optimization of the selection of acclimatization and transplanting time in Clematis tissue culture based on semi-lignified stem segment explants

[0057] Based on Example 1, explants were inoculated using an inverted method and subjected to primary culture, hormone-free transition culture, and rooting culture.

[0058] To determine the appropriate acclimatization and transplanting period for 'Rugu' clematis after rooting culture, tissue culture seedlings on the 22nd and 35th day of rooting culture were acclimatized and transplanted. The specific steps were as follows:

[0059] After rooting culture, the explants were moved to natural light for 3 days to harden off. Then, the caps of the culture bottles were opened and hardened off for another 3 days. After that, the seedlings were taken out, the culture medium was washed off the roots, and they were transplanted into a substrate made of peat moss, perlite and vermiculite in a ratio of 3:1:1. The substrate humidity was maintained at 65±5%, the air humidity at 82.5±2.5%, the temperature at 22.5±2.5℃, and the shading rate at 50%. After 12 days of recovery, they were transferred to normal management.

[0060] In this embodiment, the rooting time of the clematis tissue culture method is earlier, with roots appearing as early as 12 days, and the growth rate is also faster than in Example 4. The root condition on day 22 of rooting culture is as follows: Figure 6 As shown in Figure A, the average length has reached 3 cm; the root system condition on day 35 is as follows. Figure 6 As shown in Figure B, the average root length can reach 6 cm, but it can be observed that the root system turns brown and shows signs of aging. At the same time, it was found that the root system was too long when cultivated to 35 days, and transplanting at this time would easily cause the root system to break. In addition, due to the aging of the root system, the survival rate was only 72%, while the survival rate of transplanting after acclimatization at 22 days can reach 91%.

[0061] Comparative Example 1: Clematis tissue culture based on 1 / 2 WPM medium

[0062] This comparative example refers to the method of Example 3, the difference being:

[0063] The rooting medium was adjusted to a 1 / 2 WPM medium with pH=5.8 containing 1.5 mg / L IBA, 0.2 mg / L NAA, 20 g / L sucrose, and 6.0 g / L agar.

[0064] The rooting status on day 35 of rooting culture is as follows Figure 7 As shown, where Figure 7 A is a 'Rugu' clematis inoculated by the straight cutting method, with its stem segment swollen at the base; Figure 7 B is a 'Rugu' clematis inoculated by inverted insertion. A small amount of green callus tissue can be seen forming at the morphologically upward-facing lower end of the inoculated plant. Roots could not be induced whether the inoculation was upright or inverted.

Claims

1. A method for tissue culture of Clematis chinensis, characterized in that the steps are as follows: include: (1) After disinfection, the clematis explants were inoculated into axillary bud induction medium with the lower morphological end facing upward for culture; (2) The explants cultured in step 1 were inoculated into hormone-free medium for culture; (3) The explants cultured in step 2 were inoculated into rooting medium for culture; (4) The explants cultured in step 3 were acclimatized and transplanted to obtain clematis plants.

2. The method according to claim 1, characterized in that, In step 1, the clematis explant is a healthy clematis mother plant free from pests and diseases. Leaves are removed, leaving 0.5-1cm petioles, and semi-lignified stem segments of the current year with a length of 1-2.5 cm are cut from the internodes.

3. The method according to claim 1, characterized in that, In step 1, the disinfection steps include soaking in carbendazim solution, soaking in ethanol solution, and soaking in sodium hypochlorite solution. After each soaking disinfection, the explant is rinsed thoroughly with water, and finally the damaged parts at both ends are removed.

4. The method according to claim 1, characterized in that, In step 1, the axillary bud induction medium is MS medium with pH=5.6-6.0 containing 1-2 mg / L 6-benzylaminopurine, 0.1 mg / L 1-naphthaleneacetic acid, sucrose and agar.

5. The method according to claim 1, characterized in that, In step 1, after the plants reach a height of 5-6 cm, step 2 is carried out.

6. The method according to claim 1, characterized in that, In step 2, the hormone-free culture medium is MS medium with pH=5.8-6.2 containing sucrose and agar.

7. The method according to claim 1, characterized in that, In step 2, after culturing for 6-8 days, step 3 is carried out.

8. The method according to claim 1, characterized in that, In step 3, the rooting medium is a 1 / 2 MS medium with pH=5.6-6.0 containing 1-3 mg / L indolebutyric acid, 0.2-0.4 mg / L 1-naphthaleneacetic acid, sucrose and agar.

9. The method according to claim 1, characterized in that, In step 3, after the root system reaches a length of 3-4 cm, step 4 is carried out.

10. The method according to claim 1, characterized in that, Steps 1 and 3 are light culture, and step 2 is dark culture.

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