A method for preparing and primary inducing culture of high-induction rate explants of an endangered plant camellia nitidissima

Abstract

The application discloses a preparation and primary induction culture method of endangered plant Camellia nitidissima high-induction-rate explants, which utilizes a nutrient solution to promote the stems of Camellia nitidissima seedlings to sprout multiple times as the explant source, and after sterilization treatment, the stems are connected to the optimally selected primary induction bud culture medium and initial bud domestication culture medium to perform primary induction culture. The application utilizes the principle that the stems of Camellia nitidissima can sprout multiple times to obtain rich explant materials and significantly improve the induction rate, so that a large amount of culture materials can be obtained in a short period, which lays a material foundation for further tissue culture breeding and application research, and meanwhile, the economic traits of the explant mother plants can be tracked, which has important significance for promoting the development of the Camellia nitidissima industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of tissue culture and seedling raising of Camellia nitidissima, and particularly relates to a method for preparing and primary inducing culture of Camellia nitidissima explants with high induction rate. BACKGROUND

[0002] Camellia nitidissima Chi is a plant of Camellia nitidissima group of Camellia in Theaceae, and is a evergreen shrub to small tree. Its flowers are golden yellow and dazzling, and are known as the “super star” of flowers, the “queen of tea” and the “giant panda” in the plant kingdom. According to the Red List of Endangered Species of the World (IUCN), Camellia nitidissima is listed as a critically endangered species (CR) and is a national first-class protected plant. It is a rare and precious ornamental plant and germplasm resource in the world. Camellia nitidissima has ornamental value, nutritional value and medicinal value. The flowers, leaves and fruits of Camellia nitidissima can be used for medicine and health drinks, have the effects of lowering cholesterol, lowering blood pressure, inhibiting tumors, inhibiting bacteria and resisting aging, have important medicinal and nutritional values, have broad market development and utilization prospects, and have attracted widespread attention.

[0003] With the value of Camellia nitidissima being continuously developed and utilized, the artificial planting area of Camellia nitidissima is expanding year by year, and the demand for Camellia nitidissima seedlings is rapidly increasing. The traditional seedling raising method cannot meet the market demand. The propagation techniques of Camellia nitidissima include seed propagation, cutting propagation, grafting propagation, tissue culture and rapid propagation, and hybrid breeding. However, due to the problems such as low seed propagation rate, low germination rate and low seedling rate, the cutting and grafting are easy to carry viruses from the mother plant, the survival rate and rooting rate are low, the expansion is slow due to the influence of the yield of the mother tree, and the survival rate of hybrid breeding is low, Camellia nitidissima still faces the problem of seedling supply and demand. Tissue culture and rapid propagation have the advantages of good consistency and controllable conditions, and is an effective way for large-scale production of seedlings, and is the key to alleviate the problem of insufficient supply of Camellia nitidissima seedlings. In recent years, tissue culture and rapid propagation have been more and more applied to Camellia nitidissima. However, there are still various problems in the tissue culture of Camellia nitidissima at present, including incomplete sterilization of explants, high pollution rate, low success rate of primary induction, low proliferation coefficient of subculture, and difficulty in rooting, which seriously restricts the rapid development of Camellia nitidissima industry. In the existing technology, seed aseptic sowing is often used as the source of explants, and the induction rate is relatively low, and the induction of buds takes a long time. Some technologies use shoot tips, leaves and semi-lignified stem segments as the source of explants, and there are problems such as low sterilization success rate, small amount of explant source, difficult to obtain and seasonal limitation, which is difficult to be used continuously. In the process of plant tissue culture research and application, once there is a lack of aseptic explants, it is difficult to smoothly carry out subsequent application research. Even if clean and sterile explants can be obtained, it is extremely difficult to induce buds if the explants have low vitality. Therefore, it is very important to overcome the problems of limited source of explants, difficult to be used continuously and low sterilization success rate, and to improve the success rate of primary induction, which is a key link for using tissue culture to propagate endangered plant Camellia nitidissima seedlings and a technical problem to be solved urgently. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a Camellia nitidissima explant preparation and primary induction culture method with abundant and easy-to-obtain explants and high induction rate. To solve the above technical problems, the technical solution of the present application is to use the stem segments of the seed seedlings of the endangered plant Camellia nitidissima as the source of explants, and then obtain the explants after sterilization treatment, and then prepare the special primary induction bud culture medium and initial bud acclimation medium for them, and then control the light culture to improve the induction rate, thereby forming an endangered plant Camellia nitidissima high-induction-rate explant preparation and primary induction culture method.

[0005] The present application is implemented as follows:

[0006] A Camellia nitidissima high-induction-rate explant preparation and primary induction culture method, the main operation steps are as follows:

[0007] (1) Obtaining explants: Selecting the endangered plant Camellia nitidissima seedlings which were collected and stored in the sand for germination, and then placed in the substrate cup for conventional seedling management. When the height of the seedlings is greater than or equal to 6 cm, the upper stem segments are cut as explants, and the lower stem segments are greater than or equal to 3.5 cm. After cutting the upper stem segments, nutrient solution is sprayed, and the conventional seedling management is continued. When the germination grows to a height of greater than or equal to 6 cm again, the upper stem segments can be cut again for cyclic operation.

[0008] (2) Sterilization of explants: The cut upper stem segments are trimmed to 1.5-3 cm long after removing the leaves, then washed clean with a cleaning agent, soaked in 75% alcohol solution for 0.5-1 min, washed with sterile water for 3-5 times, placed on the clean bench, and then put into the ultrasonic cleaning cup with blue light function and sterilizing agent. The explants are ultrasonically cleaned for 5-10 min, repeated once, and then washed with sterile water for 5 times. The water is absorbed with sterilized filter paper, and the both ends of the stem segments are cut off 0.2-0.5 cm.

[0009] (3) Preparation of culture medium: Two kinds of culture media are selected for primary induction culture. The primary induction culture medium formula is: modified MS + 2-IP 1.0-2.0 mg / L + NAA 1.6-1.8 mg / L + IBA 0.5-1.5 mg / L + sucrose 30 g / L + agar 4.5 g / L. The initial bud acclimation medium formula is: modified WPM + 2-IP 0.5-1.0 mg / L + NAA 0.5-0.8 mg / L + GA3 0.1-0.2 mg / L + sucrose 30 g / L + agar 4.5 g / L. After preparing the culture medium according to the formula, it is continuously boiled for 5 min, the pH value is adjusted to 5.8-6.0, and then it is divided into culture containers.

[0010] (4) Primary induction culture: The sterilized explants are vertically inserted into the primary induction bud culture medium. When the initial buds grow to a length of greater than or equal to 2 cm, the initial buds are cut and inserted into the initial bud acclimation medium. The original explants are inserted back into the primary induction bud culture medium. The culture room temperature is controlled at about 25±3℃, and the culture is carried out under weak light for 7-10 days, and then under normal light.

[0011] Preferably, the substrate cup in step (1) has a specification of 11 cm in diameter and 15 cm in height, and the substrate is red heart soil:sand:peat soil in a volume ratio of 8:1:1.

[0012] Preferably, the conventional seedling management in step (1) is to place the seedlings in an outdoor seedling greenhouse with shading net and ventilation. According to the climate, a small arched shed is built above the seedlings to cover plastic film for heat preservation when the air temperature is too low. The seedlings are sprayed with water 1-2 times every 2-3 days, and compound fertilizer with N:P:K=15:10:20 is applied once a month.

[0013] Preferably, the nutrient solution in step (1) is 0.5-1.0 mg / mL NAA+0.3-0.6 mg / mL GGR+50-80 mg / L KH2PO3+500-800 mg / L NH4NO3.

[0014] Preferably, the sterilizing agent in step (2) is composed of 0.05% tea tree oil+0.1% HgCl2 solution+0.01% Tween-20.

[0015] Preferably, the modified WPM and modified MS medium in step (3) is composed of:

[0016]

[0017] Preferably, the culture container in step (3) is a 350 mL glass bottle with a height of 11 cm, a diameter of 7.6 cm, and a mouth diameter of 5.3 cm, and 50-80 mL of the medium is added into each bottle and sterilized at 121 ℃ for 20 min.

[0018] Preferably, the weak light culture in step (4) is performed by covering the culture container with 2 layers of newspapers in the absence of light, and the normal light culture is performed by maintaining the light intensity at 2000-3000 LX and illuminating for 12-14 h per day.

[0019] Preferably, the operation mode of cutting the initial bud in step (4) is to retain 0.2-0.5 cm of the bud base on the explant.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The present application utilizes the principle of multiple sprouting of the Camellia nitidissima stem segment in obtaining the explant and inducing the primary culture. The seedling cultivated after the seed sand storage germination is selected as the explant, the upper stem segment is cut, and part of the bud base is retained. The height of the stem segment after cutting is optimized, and a specific nutrient solution is prepared therefor to promote the multiple sprouting of the stem segment, which is recycled for a period of up to 1 year. Rich explant material can be obtained, the seed utilization rate is significantly improved, and the obtained explant material has strong activity, high juvenile degree, and is easy to induce the bud; after the bud is induced in the primary culture, the initial bud is cut, part of the bud body is retained on the explant, and the original explant is inserted into the specific primary culture medium for bud induction again, so that the bud is induced again, the induction rate is significantly improved, and the induction rate can reach 96.5%. A large amount of culture material can be obtained in a short period of time, which lays a material foundation for further tissue culture and application research.

[0022] (2) The prior art usually uses mature stem segments and seeds as explant sources, directly uses mature stem segments as explant materials, and has a high degree of lignification, a significantly reduced induction rate, a high contamination rate, and difficulty in obtaining sterile and viable explant materials. Directly using seeds as explants has a shorter utilization period, a lower utilization frequency and induction rate, and cannot obtain future economic traits of the seeds. The explant material selected in the present application is the stem segment of the seedling after germination, which is tender, has a low degree of lignification, a high sterilization success rate, a contamination rate of less than 5%, and a high initial bud induction rate. In addition, the seedling as the explant source can be cultivated into a large tree after not cutting the explant, and can track various economic traits of the mother plant while being used as an explant for tissue culture, which has important significance for targeted regulation and breeding, and has obvious advantages compared with the prior art.

[0023] (3) The nutrient solution used in the explant acquisition stage of the present application is obtained through a large number of experiments, and contains KH2PO3 and NH4NO3 containing a large number of mineral elements N, P and K required for plant growth, and plant growth regulators NAA and GGR. The sufficient nutrients required for plant growth are ensured, and the plant growth regulators are used to promote the stem segment to germinate again.

[0024] (5) The present application selects the cut explant and the initial bud, both of which retain part of the tender stem segment and the bud on the original seedling and the explant, fully utilize the relatively mature tissue for nutrient transport, ensure sufficient nutrients, and utilize the tender tissue with well-developed meristems for re-meristem, thereby promoting rapid bud emergence.

[0025] (6) The present application sets two kinds of culture media for primary culture in the primary culture stage. The initial bud induction medium is selected as MS medium based on various mineral elements, and the initial bud domestication stage is selected as WPM medium based on low mineral elements. By adjusting the content of low KH2PO4 and the content of high CaCl2, and adding suitable plant growth regulators for inducing bud of Camellia nitidissima, the degree of lignification of the stem segment can be reduced, cell division can be promoted, and the induction of bud of Camellia nitidissima can be ideal, so that the initial bud after domestication is in good condition without yellow leaf and leaf fall. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The photo of the seedling of the explant source of Example 1.

[0027] Figure 2 The photo of the seedling after cutting the upper stem segment of Example 1.

[0028] Figure 3 , 4Photo of Example 1 explants induced to sprout on primary induction sprouting medium.

[0029] Figure 5 , 6 Photo of Example 1 initial sprouts acclimatized on initial sprout acclimatization medium.

[0030] Figure 7 Photo of Comparative Example 1 seedlings died and did not sprout after cutting upper stem segments.

[0031] Figure 8 , 9 Photo of Comparative Example 2 using mature stem segments and seeds as explant materials for induction.

[0032] Figure 10 , 11 Photo of Comparative Example 3 using only primary induction sprouting medium for induction, a large amount of callus grew at the bottom.

[0033] Figure 12 Photo of Comparative Example 4 primary induction of initial sprouts using yellow leaves and leaf fall without using modified medium. DETAILED DESCRIPTION

[0034] The following examples can make those skilled in the art more fully understand the present application, the described examples are part of the embodiments of the present application, not all the embodiments, in any way limit the present application. But those skilled in the art should understand that various changes can be made in form and details, without departing from the scope defined by the claims of the present application.

[0035] In the following examples, the composition of the modified MS and MS, WPM and modified WPM medium is as follows:

[0036]

[0037]

[0038] Example 1

[0039] (1) Explant acquisition: Select mature and healthy seeds of the endangered plant Camellia chrysantha collected in the current year, and after germination by sand storage, place them in substrate cups with a diameter of 11cm and a height of 15cm. The substrate is red soil: fine sand: peat soil in a volume ratio of 8:1:1. Perform routine seedling management, that is, place the seedlings in an outdoor seedling greenhouse with shade netting and ventilation. Depending on the climate, if the temperature is too low, build a small arched shed above the seedlings and cover it with plastic film for insulation. Spray water 1-2 times every 2-3 days, and apply compound fertilizer with N:P:K = 15:10:20 once a month. Once the seedlings reach a height of ≥6cm, cut off the upper stem segments as explants, retaining the lower stem segments ≥3.5cm. After cutting off the upper stem segments, spray with a nutrient solution consisting of 0.5mg / mL NAA + 0.6mg / mL GGR + 50mg / L KH2PO3 + 800mg / L NH4NO3. Continue with routine seedling management until the sprouts grow to a height of ≥6cm again, then cut off the upper stem segments again and repeat the process.

[0040] (2) Sterilization of explants: After removing the leaves from the upper stem segments, trim them into 3cm long segments, clean them with detergent, and then soak them in 75% alcohol solution for 0.5min. Rinse them with sterile water 3-5 times. On the ultra-clean workbench, select an ultrasonic cleaning cup with blue light function and add sterilizing agent. The sterilizing agent consists of 0.5% tea tree oil + 0.1% HgCl2 solution + 0.01% Tween-20. Place the explants in it and ultrasonically clean them for 10min. Repeat once. Stir and clean them with sterile water 5 times. Blot dry with sterile filter paper and cut off 0.2cm from both ends of the stem segments.

[0041] (3) Culture medium preparation: Two culture media were used for the initial induction culture. The formula of the initial bud induction culture medium was: modified MS + 2-IP 2.0mg / L + NAA 1.6mg / L + IBA 0.5mg / L + sucrose 30g / L + agar 4.5g / L; the formula of the initial bud acclimatization culture medium was: modified WPM + 2-IP 0.5mg / L + NAA 0.8mg / L + GA3 0.1mg / L + sucrose 30g / L + agar 4.5g / L. After preparing the culture medium according to the formula, it was boiled for 5 minutes to adjust the pH value to 5.8-6.0. It was then dispensed into culture containers. The culture containers were 350mL glass bottles with a height of 11cm, a diameter of 7.6cm, and a mouth diameter of 5.3cm. 50-80mL of culture medium was added to each bottle and autoclaved at 121℃ for 20 minutes.

[0042] (4) Primary induction culture: the sterilized explants were vertically inserted into the primary induction of bud culture medium, and when the initial bud grew to ≥2 cm, the initial bud was cut and inserted into the initial bud acclimation medium. The operation mode for cutting the initial bud was to reserve 0.2-0.5 cm of the bud base on the explant, and the original explant was inserted back into the primary induction of bud culture medium. The culture room temperature was controlled at about 25±3℃, and after 7 days of weak light culture, normal light culture was changed, i.e. the weak light culture was under the condition of no light turning on, and 2 layers of newspaper were covered on the top of the culture container, and the normal light culture was under the condition of light intensity of 2000-3000 LX, and light culture for 12-14 hours per day.

[0043] Example 2

[0044] (1) Obtaining explants: the endangered plant Camellia nitidissima was selected, and the mature and healthy seeds collected in the current year were stored in a substrate cup with a diameter of 11 cm and a height of 15 cm, and the substrate was red heart soil: fine sand: peat soil with a volume ratio of 8:1:1. The seedlings were placed in an outdoor seedling raising greenhouse with shading net and ventilation, and a small arched shed was built above the seedlings to cover plastic film for heat preservation when the air temperature was too low. Water was sprayed 1-2 times every 2-3 days, and compound fertilizer with N:P:K=15:10:20 was applied once a month. When the seedlings were ≥6 cm high, the upper stem segments were cut as explants, and the lower stem segments were ≥3.5 cm. After cutting the upper stem segments, nutrient solution was sprayed, and the nutrient solution was 1.0 mg / mL NAA+0.3 mg / mL GGR+80 mg / L KH2PO3+500 mg / L NH4NO3. The routine seedling raising management was continued, and when the buds grew to ≥6 cm high again, the upper stem segments were cut again for cyclic operation;

[0045] (2) Explant sterilization: the cut upper stem segments were cut to 1.5 cm long after removing the leaves, washed clean with a cleaning agent, then soaked in 75% alcohol solution for 0.5 min, washed with sterile water for 3-5 times, and placed on an ultraclean workbench with a blue light function. An ultrasonic cleaning cup was added with sterilizing agent composed of 0.5% tea tree oil+0.1% HgCl2 solution+0.01% Tween-20, and the explants were placed in it for ultrasonic cleaning for 5 min, repeated once, and washed with sterile water for 5 times. The water was absorbed with sterile filter paper, and the stem segments were cut by 0.2 cm at both ends;

[0046] (3) Medium preparation: Two kinds of medium were selected for primary induction culture. The formula of the primary induction bud culture medium was: modified MS + 2-IP 1.0 mg / L + NAA 1.6 mg / L + IBA 0.5 mg / L + sucrose 30 g / L + agar 4.5 g / L. The formula of the initial bud domestication culture medium was: modified WPM + 2-IP 0.5 mg / L + NAA 0.8 mg / L + GA3 0.2 mg / L + sucrose 30 g / L + agar 4.5 g / L. After the medium was prepared according to the formula, it was continuously boiled for 5 min, the pH value was adjusted to 5.8-6.0, and it was then divided into culture containers. The culture containers were 350 mL glass bottles with a height of 11 cm, a diameter of 7.6 cm, and a mouth diameter of 5.3 cm. 50-80 mL of the medium was added to each bottle, and the bottles were autoclaved at 121°C for 20 min;

[0047] (4) Primary induction culture: The sterilized explants were vertically inserted into the primary induction bud culture medium. When the initial buds grew to ≥2 cm, the initial buds were cut and inserted into the initial bud domestication culture medium. The operation mode for cutting the initial buds was to reserve 0.2-0.5 cm of the bud base on the explants, and the original explants were inserted back into the primary induction bud culture medium. The culture room temperature was controlled at about 25±3°C. After 10 days of weak light culture, the culture was changed to normal light culture. The weak light culture was performed under the condition that the natural light was not turned on and 2 layers of newspapers were covered on the culture containers. The normal light culture was performed under the condition that the light intensity was 2000-3000 LX, and the light culture was performed for 12-14 h per day.

[0048] Example 3

[0049] (1) Obtaining explants: Selecting endangered plant Camellia nitidissima seedlings that were sown and germinated from sand storage of mature and healthy seeds in the year, and placing them in a substrate cup with a diameter of 11 cm and a height of 15 cm, and the substrate is red heart soil: fine sand: peat soil with a volume ratio of 8:1:1. The seedlings were managed in a conventional way, i.e. placing them in a outdoor nursery greenhouse with shading net and ventilation, according to the climate, when the temperature is too low, a small arched shed is built above the seedlings to cover plastic film for insulation, and the seedlings are sprayed with water 1-2 times every 2-3 days, and compound fertilizer with N:P:K=15:10:20 is applied once a month. When the seedlings are ≥6 cm tall, the upper stem segments are cut as explants, and the lower stem segments are ≥3.5 cm. After cutting the upper stem segments, a nutrient solution is sprayed, which is 0.8 mg / mL NAA + 0.5 mg / mL GGR + 60 mg / L KH2PO3 + 600 mg / L NH4NO3. The seedlings are continuously managed in a conventional way, and when the buds grow to ≥6 cm tall again, the upper stem segments are cut again for cyclic operation.

[0050] (2) Sterilization of explants: the cut upper stem segments, after cutting off the leaves, were trimmed to 2.5 cm long, washed clean with a cleaning agent, then soaked in 75% alcohol solution for 1 min, washed with sterile water for 3-5 times, on the clean bench, an ultrasonic cleaning cup with blue light function was selected, sterilizing agent was added, the sterilizing agent was composed of 0.5% tea tree oil + 0.1% HgCl2 solution + 0.01% Tween-20, the explants were placed in it for ultrasonic cleaning for 10 min, repeated once, washed with sterile water for 5 times, the water was absorbed with sterile filter paper, and the stem segments were cut off 0.5 cm at both ends respectively;

[0051] (3) Preparation of culture medium: two kinds of culture medium were selected for primary induction culture, the primary induction bud culture medium formula was: modified MS + 2-IP 1.5 mg / L + NAA 1.7 mg / L + IBA 1.2 mg / L + sucrose 30 g / L + agar 4.5 g / L; the initial bud domestication culture medium formula was: modified WPM + 2-IP 0.8 mg / L + NAA 0.6 mg / L + GA3 0.2 mg / L + sucrose 30 g / L + agar 4.5 g / L; after the culture medium was prepared according to the formula, it was continuously boiled for 5 min, the pH value was adjusted to 5.8-6.0, and was divided into culture containers, the culture containers were 350 mL glass bottles with a height of 11 cm, a diameter of 7.6 cm, and a mouth diameter of 5.3 cm, 50-80 mL of culture medium was added into each bottle, and was autoclaved at 121°C for 20 min;

[0052] (4) Primary induction culture: the sterilized explants were vertically inserted into the primary induction bud culture medium, when the initial buds grew to ≥2 cm, the initial buds were cut off and inserted into the initial bud domestication culture medium, the operation mode for cutting off the initial buds was to reserve 0.2-0.5 cm of the bud base on the explants, the original explants were inserted into the primary induction bud culture medium again, the culture room temperature was controlled at about 25±3°C, after 7 days of weak light culture, normal light culture was changed, the weak light culture was under the condition that the natural light was not turned on and 2 layers of newspapers were covered on the top of the culture container, and the normal light culture was light culture with a light intensity of 2000-3000 LX, and light illumination time of 12-14 h per day.

[0053] The inventors verified the effect of the application by a large number of experiments for comparative study, and the following are some comparative experimental examples which can explain the problem.

[0054] Comparative Example 1

[0055] The procedure of this comparative example is the same as that of Example 1, and the difference lies in that in step (1), after cutting off the upper stem segments, no nutrient solution is sprayed, and the seedlings are directly sprayed with water and conventionally cultivated.

[0056] Comparative Example 2

[0057] The counter example and the step process of example 1 are the same, the difference is that in step (1), the semi-lignified stem segments (counter example 1-A) and seeds (counter example 1-B) of Camellia nitidissima mature plants are selected as explants.

[0058] Counter example 3

[0059] The counter example and the step process of example 1 are the same, the difference is that in step (4), only one medium is selected for primary induction culture, i.e. only the primary induction bud culture medium (modified MS+2-IP 2.0mg / L+NAA 1.6mg / L+IBA 0.5mg / L+sucrose 30g / L+agar 4.5g / L) is used to culture explants and initial buds, and the initial bud acclimation medium is not used, and the initial bud acclimation culture is not performed.

[0060] Counter example 4

[0061] The counter example and the step process of example 1 are the same, the difference is that in step (2), the primary induction bud culture medium is selected as MS, i.e. MS+2-IP 2.0mg / L+NAA 1.6mg / L+IBA 0.5mg / L+sucrose 30g / L+agar 4.5g / L; the initial bud acclimation medium is selected as WPM, i.e. WPM+2-IP 0.5mg / L+NAA 0.8mg / L+GA3 0.1mg / L+sucrose 30g / L+agar 4.5g / L.

[0062] Counter example 5

[0063] The counter example and the step process of example 1 are the same, the difference is that in step (3), the counter example 5-A medium formula is selected as the best initial bud induction culture medium formula disclosed in Chinese patent CN 107135948 B “A method for cultivating Camellia nitidissima tender branch tissue culture seedlings under sunlight” (i.e. MS+Na2SO4 50mg / L+BA 2mg / L+NAA 1mg / L+vitamin C 10mg / L+hydrolyzed casein 600mg / L+sucrose 30g / L+agar 4.5g / L); the counter example 5-B medium formula is selected as the best initial bud induction culture medium formula disclosed in Chinese patent CN 106818493 B “Yunnan Camellia nitidissima tissue culture rapid propagation method” (i.e. MS+BA 2mg / L+IAA 0.5mg / L+sucrose 30g / L+agar 4.5g / L).

[0064] Implementation effect

[0065] The explants of Camellia nitidissima are prepared and primary induction culture is conducted by using the treatment methods of the examples 1-3 and the comparative examples 1-5, and the statistical test results are observed and compared, and the main calculation formulae are as follows: the pollution rate (%) = the number of polluted explants / the number of inoculated explants x 100; the brown death rate (%) = the number of germinated explants / the number of inoculated explants x 100; the induction rate (%) = the number of germinated explants / the number of inoculated explants x 100; the plant death rate = the number of dead plants / the number of planted plants x 100; the plant sprouting rate = the number of plants sprouting again / the number of planted plants x 100; the callus rate (%) = the number of callus explants / the number of inoculated explants x 100.

[0066] Table 1: primary induction culture effect of examples

[0067]

[0068] The explant sterilization and primary induction effects of the three examples are shown in Table 1. As shown in Table 1, the initial bud states of the three examples have no obvious difference, but the initial bud induction rate of example 1 is the highest, the pollution rate is the lowest, the germination starting time is the shortest, and the brown death rate is the lowest, so example 1 is the optimal example.

[0069] Table 2: plant growth of explant source of example 1 and comparative example 1

[0070]

[0071] The comparative example 1 mainly investigates whether the nutrient solution treatment can promote more explants. The rich source of explants is the primary key step of tissue culture and rapid propagation, and the rich source of explants can greatly increase the success rate of tissue culture. As shown in Table 2, the comparative example 2 does not spray the nutrient solution after cutting off the first batch of seedling stems, and directly manages the water, and the plant death rate is 20.4%, the sprouting rate is 74.9%, the sprouting time is 55d, and the number of sprouts is only one. Compared with example 1, the plant death rate is increased by 18.3%, the sprouting rate is reduced by 21.5%, the sprouting time is delayed by 15d, and the number of sprouts is less than that of example 1. Therefore, the preferred nutrient solution of the present application not only promotes sprouting and avoids plant death, but also shortens the sprouting time. The main reason is that KH2PO3 and NH4NO3 are used in the nutrient solution, which contains a large amount of mineral elements N, P and K required for plant growth, and plant growth regulators NAA and GGR are also used, which rapidly replenish nutrients after cutting off the stems of the plants, reduce the death of the plants due to lack of nutrients during the fragile stage, and the nutrient solution obtained by a large number of experiments in the present application fully meets the nutrients required by the Camellia nitidissima seedlings after cutting off the stems, and at the same time, ensures the nutrients required for growth, and also uses plant growth regulators to promote the stems to sprout again, thereby enriching the source of explant materials.

[0072] Table 3: primary induction effect of example 1 and comparative example 2

[0073]

[0074] From Table 3, in the comparative example 2-A, the semi-lignified stem segments of mature plants were selected as explant materials, the contamination rate was 15.4%, the browning rate was 24.3%, and the induction rate was 62.4%. Compared with the example 1, the contamination rate and the browning rate were increased by 11.2% and 23.1% respectively, and the induction rate was decreased by 30.1%. Since the internodes of the mature stem segments were long, 3-5 cm of the stem segments usually contained only one internode, and only one bud could be induced at the position of the stem node. The internodes of the seedling stem segments were short, 3-5 cm of the stem segments usually contained multiple stem nodes, and multiple buds could be induced at the positions of the multiple stem nodes. Figure 1 、 2 It can be seen that in the comparative example 2-B, the mature seeds were selected as explants, the contamination rate was 4.6%, which was not much different from that of the example 1, but the browning rate was 8.9%, the germination starting time was 60 days, which was delayed by 40 days compared with the example 1, and the induction rate was only 48.9%, which was significantly lower than that of the example 1. The primary induction effect was significantly poorer than that of the example 1. The reason was that the mature stem segments had high tissue maturity, and the bacteria carried by the mature stem segments gradually accumulated after being exposed to the natural environment for a long time, so it was difficult to achieve successful sterilization. The mature seeds could be protected from the damage of sterilizing agents due to the protection of the seed coat, so the contamination rate and the browning rate were lower than those of the mature stem segments. However, the germination starting time was longer, and the induction rate was still significantly lower than that of the example 1. In summary, the preferred seedling stem segments were used as explants for explant preparation in the present application, and the sterilization success rate and the induction rate were high.

[0075] Table 4: Primary induction effects of the example 1 and the comparative example 3

[0076]

[0077] Example 3 is mainly to investigate whether the use of acclimation medium affects the induction effect. As can be seen from Table 4, the callus rate of Example 3 is 84.3% during the primary culture process without changing the medium, and the number of buds is less than 1, the leaf of the induced bud is large, the bottom callus is large and gradually wraps the main stem, and the callus is serious. The buds of Example 1 can induce multiple buds after being changed to the acclimation medium, the leaf is relatively small, the size of the absorption surface is moderate, the bud body is tender, which is conducive to further proliferation and differentiation. Compared with Example 1, the state of the bud body of Example 3 is obviously deteriorated, and the bottom callus is serious. The analysis reason may be that the use of rich nutrients and high concentration of plant growth regulators during the primary induction of the explant of Camellia nitidissima can promote rapid budding, but the use of high concentration of plant growth regulators after budding can cause rapid callus, and the large callus gradually affects the absorption of nutrients, thereby affecting further budding. In the present application, the improved MS medium with rich nutrients and high concentration of plant growth regulators is selected during the primary induction of the explant, and the new buds are transferred to the improved WPM medium with reduced concentration of plant growth regulators and mineral elements as the acclimation medium after the induction of the buds, which can promote rapid budding and avoid callus, thereby inducing multiple buds and significantly improving the induction effect.

[0078] Table 5: Primary induction effect of Example 1 and Comparative Example 4

[0079] Example Number of sprouts Initial sprout state Example 1 2.8 Sprouts were induced, the leaf size was moderate, the size of the absorption surface was moderate, and the sprout body was tender. Comparative Example 4 1.2 Most induced 1 sprout, the sprout body was yellow-tipped, and the leaves fell off.

[0080] Comparative Example 4 mainly investigates the influence of the amount of mineral elements in the basic medium on the primary induction effect. As can be seen from Table 5, the amount of mineral elements in the basic medium not only affects the number of buds, but also affects the state of the initial bud. The number of buds of Comparative Example 4 is only 1.2, the induced bud body is prone to yellowing, and the leaves will also fall off. Therefore, the basic medium is selected in the present application, and the content of KH2PO4 is adjusted slightly lower and the content of CaCl2 is adjusted slightly higher, which can obviously improve the state of the initial bud. The analysis reason may be that KH2PO4 is rich in K + , K + can obviously improve the absorption and utilization of nitrogen by plants, promote the synthesis of proteins, also can promote photosynthesis, improve the stress resistance of plants; CaCl2 can enhance the intake of calcium by plants, promote plant growth, prevent plants from wilting, also can improve the content of chlorophyll in leaves and the activity of antioxidant enzymes, and enhance the ability of active oxygen scavenging, by adjusting the amount of use to a concentration suitable for the growth of Camellia nitidissima, thereby improving the state of the initial bud.

[0081] Table 6: Primary induction effect of Example 1 and Comparative Example 5

[0082]

[0083] The comparative example 5 mainly investigates whether the present application has advantages over the prior art. The relatively mature medium formula in the prior art is selected for the initial induction culture of Camellia nitidissima, and it can be seen from Table 6 that the induction rate of Example 1 is 1.7% and 6.2% higher than that of Comparative Examples 5-A and 5-B, which are 94.8% and 90.3%, respectively, and the germination start time is shortened by 17 days and 14 days, respectively, and the initial induction effect is obviously superior.

[0084] The above analysis shows that the explant prepared by the method of Example 1 has the advantages of rich source of explant material, low pollution rate and browning rate, high induction rate and the highest number of sprouts, and the induction effect is optimal. It also shows that the explant prepared by the method formed by a series of innovative operations has low pollution rate and browning rate, high induction rate, rich source of explant, and can induce multiple healthy initial buds in a short time, which can lay a material foundation for further tissue culture and application research, and has important significance for promoting the development of Camellia nitidissima industry.

Claims

1. A method for preparing explants with high induction rate and primary induction culture of the endangered plant Camellia chrysantha, characterized in that: The main operation steps include explant acquisition, explant sterilization, medium preparation and primary induction culture, and are as follows: (1) Explant acquisition: select the mature and healthy seeds collected from the endangered plant Camellia nitidissima in the current year, store them in a substrate cup, and perform conventional seedling management. When the seedlings are higher than or equal to 6 cm, cut the upper stem segments as explants, and reserve the lower stem segments higher than or equal to 3.5 cm. After cutting the upper stem segments, spray nutrient solution, and continue to perform conventional seedling management. When the sprouts grow to a height of 6 cm again, the upper stem segments can be cut again for cyclic operation; (2) Explant sterilization: cut the upper stem segments, remove the leaves, and trim them into 1.5-3 cm long stem segments. Then, clean them with a cleaning agent, immerse them in 75% alcohol solution for 0.5-1 min, wash them with sterile water for 3-5 times, and put them in an ultraclean workstation. Select an ultrasonic cleaning cup with blue light function, add sterilizing agent, put the explants into the ultrasonic cleaning cup, and ultrasonically clean them for 5-10 min. Repeat the operation once, wash them with sterile water for 5 times, dry them with sterilized filter paper, and cut 0.2-0.5 cm from both ends of the stem segments; (3) Medium preparation: two kinds of culture media are selected for primary induction culture. The primary induction culture medium formula is: modified MS+2-IP 1.0-2.0 mg / L+NAA 1.6-1.8 mg / L+IBA 0.5-1.5 mg / L+sucrose 30 g / L+agar 4.5 g / L. The initial bud acclimation medium formula is: modified WPM+2-IP 0.5-1.0 mg / L+NAA 0.5-0.8 mg / L+GA3 0.1-0.2 mg / L+sucrose 30 g / L+agar 4.5 g / L. After preparing the medium according to the formula, continuously boil it for 5 min, adjust the pH value to 5.8-6.0, and divide it into culture containers; (4) Primary induction culture: vertically insert the sterilized explants into the primary induction culture medium. When the initial buds grow to a height of 2 cm or more, cut the initial buds, insert them into the initial bud acclimation medium, and insert the original explants into the primary induction culture medium again. The culture room temperature is controlled at about 25±3 ℃, and the culture is performed under weak light for 7-10 days, and then under normal light; The modified WPM and modified MS culture media in step (3) are composed of: 。 2. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The substrate cup in step (1) has a specification of 11 cm in diameter and 15 cm in height, and the substrate is red heart soil:sand:peat soil in a volume ratio of 8:1:

1.

3. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The conventional seedling management in step (1) is to place the seedlings in an outdoor seedling raising greenhouse with shading net and ventilation. According to the climate, a small arched shed is built above the seedlings to cover plastic film for heat preservation when the air temperature is too low. Spray water 1-2 times every 2-3 days, and apply compound fertilizer with N:P:K=15:10:20 once a month.

4. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The nutrient solution in step (1) is 0.5-1.0 mg / mL NAA+0.3-0.6 mg / mL GGR+50-80 mg / L KH2PO3+500-800 mg / L NH4NO3.

5. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The sterilization agent of step (2) is composed of 0.05% tea tree oil + 0.1% HgCl2 solution + 0.01% Tween-20.

6. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The culture container of step (3) is a 350 mL glass bottle with a height of 11 cm, a diameter of 7.6 cm, and a mouth diameter of 5.3 cm, and 50-80 mL of culture medium is added to each bottle, which is sterilized at 121 ℃ for 20 min.

7. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The weak light culture of step (4) is achieved by covering the culture container with 2 layers of newspapers in the absence of light, and the normal light culture is achieved by maintaining a light intensity of 2000-3000 LX and light for 12-14 h per day.

8. The method for preparing and inducing primary culture of high induction rate explants of endangered plant Camellia pitardii according to claim 1, characterized in that: The operation mode of step (4) for cutting the initial bud is to reserve 0.2-0.5 cm of the base of the bud on the explant.

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