High-inductivity explant preparation and primary induction culture method of endangered plant golden camellia

Abstract

The invention discloses a high-inductivity explant preparation and primary induction culture method of endangered plant camellia nitidissima, which comprises the following steps: using a nutrient solution to promote a stem segment of a camellia nitidissima seed seedling to shoot for multiple times as an explant source, sterilizing, and inoculating into an optimized primary induction budding culture medium and an initial bud domestication culture medium for primary induction culture. According to the method disclosed by the invention, during explant acquisition and primary induction culture, a principle that a stem section of the golden camellia can be subjected to multi-time tip extraction is utilized, so that abundant explant materials can be obtained, the induction rate is remarkably improved, a large number of culture materials can be obtained in a short time, a material foundation is laid for further tissue culture breeding application research, economic characters of an explant stock plant can be traced, and the method has a wide application prospect. The method has important significance for promoting the development of golden camellia industry.

Description

Technical Field

[0001] The invention belongs to the technical field of camellia chrysantha tissue culture seedling raising, and in particular relates to a method for preparing camellia chrysantha explants with a high induction rate and a primary induction culture method. Background Art

[0002] Camellia nitidissima Chi is a plant of the Camellia genus of the Theaceae family. It is an evergreen shrub to a small tree. Its flowers are golden yellow and dazzling. It is called the "superstar" among flowers, the "queen of the tea family" and the "giant panda" of the plant world. According to the "Red List of Endangered Species of the World Conservation Union" (IUCN), Camellia nitidissima Chi is listed as a critically endangered species (CR) and a national first-class protected plant. It is a precious and rare ornamental plant and germplasm resource in the world. Camellia nitidissima Chi combines ornamental value, nutritional value and medicinal value. Not only does Camellia nitidissima Chi have bright colors and high ornamental value, its flowers, leaves and fruits can be used as medicine and health drinks, and have the effects of lowering cholesterol, lowering blood pressure, inhibiting tumors, inhibiting bacteria, and anti-aging. It has important medicinal and nutritional value, broad market development and utilization prospects, and has received widespread attention.

[0003] As the value of Camellia chrysantha is continuously developed and utilized, the artificial planting area of ​​Camellia chrysantha is expanding year by year, and the demand for Camellia chrysantha seedlings is increasing rapidly. Traditional seedling cultivation methods can no longer meet market demand. The breeding techniques of Camellia chrysantha include seed breeding, cutting breeding, grafting breeding, tissue culture rapid propagation and hybrid breeding. However, due to the low fruiting rate, germination rate and emergence rate of seed breeding; cutting and grafting are easy to carry the mother plant's own viruses, the survival rate and rooting rate are low, and the expansion is relatively slow due to the yield of the mother tree spikes; hybrid breeding has a low survival rate and other technical difficulties, Camellia chrysantha still faces the problem of seedling supply and demand. Tissue culture rapid propagation has the advantages of good consistency and controllable conditions. It is an effective way to mass produce seedlings and the key to alleviating the problem of insufficient supply of Camellia chrysantha seedlings. In recent years, tissue culture rapid propagation has been increasingly used in Camellia chrysantha. However, there are still various problems in the tissue culture of Camellia chrysantha, including incomplete explant sterilization, high contamination rate, low success rate of primary induction, low coefficient of tissue culture and subculture proliferation, and difficulty in rooting, which seriously restrict the rapid development of the Camellia chrysantha industry. In the prior art, aseptic sowing of seeds is often used as the source of explants, and the induction rate is relatively low, and the induction time of budding is long. Some technologies also use stem tips, leaves and semi-lignified stem segments as the source of explants, which generally have low sterilization success rate, small number of explant sources, difficulty in obtaining and seasonal limitations, and are difficult to use sustainably. In the process of plant tissue culture research and application, once there is a lack of abundant sterile explants, it is difficult to smoothly carry out subsequent application research. Even if clean and sterile explants can be obtained, if the explant vitality is not high, it is also an extremely thorny problem to induce budding. Therefore, overcoming the problems of limited explant source materials, difficulty in sustainable use and low sterilization success rate, and improving the success rate of primary induction at the same time, is a crucial link in breeding endangered plant Camellia chrysantha seedlings using tissue culture, and is also a technical problem that needs to be solved urgently. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing explants and primary induction culture of Camellia chrysantha, which has a rich source of explants, is easy to obtain and has a high induction rate. In order to solve the above technical problems, the technical scheme of the present invention is: using nutrient solution to promote the multiple sprouting of the seed seedlings of the endangered plant Camellia chrysantha as the source of explants, obtaining explants after sterilization, and then preparing exclusive primary induction budding medium and initial bud acclimation medium for them, and by controlling light culture, thereby improving its induction rate, forming a method for preparing explants and primary induction culture with a high induction rate of the endangered plant Camellia chrysantha.

[0005] The present invention is achieved in that:

[0006] A method for preparing high-induction rate explants and primary induction culture of the endangered plant Camellia chrysantha, the main operating steps are as follows:

[0007] (1) Explant acquisition: Select the endangered plant Camellia chrysantha and collect mature and healthy seeds of the same year. After germination in sand storage, place them in a substrate cup and carry out conventional seedling management. When the seedlings are ≥6 cm tall, cut the upper stem segment as the explant and keep the lower stem segment ≥3.5 cm. After cutting off the upper stem segment, spray nutrient solution and continue with conventional seedling management. When the buds grow again to a height of ≥6 cm, cut the upper stem segment again and repeat the operation;

[0008] (2) Explant sterilization: After removing the leaves from the upper stem segments, trim them into 1.5-3 cm long segments, clean them with a detergent, and then soak them in a 75% alcohol solution for 0.5-1 min. Rinse them with sterile water for 3-5 times. On a clean bench, select an ultrasonic cleaning cup with a blue light function and add a sterilant. Place the explant in the cup and perform ultrasonic cleaning for 5-10 min. Repeat this process. Rinse with sterile water for 5 times. Use sterile filter paper to absorb the moisture. Cut off 0.2-0.5 cm from each end of the stem segment.

[0009] (3) Culture medium preparation: Two culture media were used for primary induction culture. The formula of the primary bud induction culture medium was: 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 formula of the initial bud acclimation culture medium was: 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 the culture medium was prepared according to the formula, it was boiled for 5 min, the pH value was adjusted to 5.8-6.0, and the culture medium was dispensed into culture containers;

[0010] (4) Primary induction culture: The sterilized explants were vertically inserted into the primary bud induction medium. When the initial buds grew to ≥ 2 cm, they were cut off and inserted into the initial bud acclimation medium. The original explants were inserted back into the primary bud induction medium. The temperature of the culture room was controlled at about 25 ± 3 °C. After 7 to 10 days of weak light culture, they were switched to normal light culture.

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

[0012] Preferably, the conventional seedling management in step (1) is to place the seedlings in a ventilated outdoor seedling greenhouse with a shade net. Depending on the climate, when the temperature is too low, a small arch shed is built above the seedlings and covered with plastic film for insulation. Water is sprayed 1 to 2 times every 2 to 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) consists of 0.05% tea tree essential oil + 0.1% HgCl2 solution + 0.01% Tween-20.

[0015] Preferably, the modified WPM and modified MS culture medium in step (3) are 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 of 5.3 cm. 50 to 80 mL of culture medium is added to each bottle and sterilized under high pressure at 121° C. for 20 min.

[0018] Preferably, the weak light culture in step (4) is carried out by covering the culture container with two layers of newspapers under natural light conditions without turning on the lights, and the normal light culture is carried out by maintaining the light intensity at 2000-3000LX and 12-14 hours of light per day.

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

[0020] The beneficial effects of the present invention compared with the prior art are:

[0021] (1) The present invention utilizes the principle that the stem segments of Camellia chrysantha can sprout multiple times during the acquisition of explants and the primary induction culture. When the explants are acquired, seedlings are selected that are cultivated after the seeds are stored in sand for germination. After the upper stem segments are cut, part of the bud base is retained, which optimizes the height of the stem segments retained after cutting them off, and a specific nutrient solution is prepared for them to promote the multiple sprouting of the stem segments. The cycle can be as long as 1 year, and abundant explant materials can be obtained, which significantly improves the seed utilization rate. The obtained explant materials have strong vitality, high degree of juvenility, and are easy to induce sprouting. After the primary induction sprouting, the initial buds are cut off and part of the buds are retained on the explants. The original explants are inserted into the specific primary induction sprouting culture medium prepared by screening again, and sprouts can be sprouted again, which significantly improves the induction rate. The induction rate can reach 96.5%, and a large amount of culture materials can be obtained in a short period of time, laying a material foundation for further tissue culture and breeding application research.

[0022] (2) In the prior art, mature stem segments and seeds are mostly used as explant sources. Direct use of mature stem segments as explant materials has a high degree of lignification, significantly reduced induction rate, and a high contamination rate, making it difficult to obtain sterile and viable explant materials; direct use of seeds as explants has a short utilization period, low utilization times and low induction rate, and at the same time, the future economic traits of the seeds used cannot be known. The explant material selected in the present invention is the stem segment of the seedling germinated from the seed, which has relatively tender tissue, low degree of lignification, high sterilization success rate, controllable contamination rate below 5%, and high initial bud induction rate. In addition, the seedlings used as the source of explants can be cultivated into large trees without cutting the explants. While using them as explants for tissue culture, various economic traits of the mother plant can also be tracked, which is of great significance for targeted regulation of breeding and has obvious advantages over the prior art.

[0023] (3) The nutrient solution used in the explant acquisition stage of the present invention is obtained through a large number of experimental screenings. The nutrient solution uses KH2PO3 and NH4NO3 containing a large amount of mineral elements N, P, and K required for plant growth, and also uses plant growth regulators NAA and GGR. While ensuring sufficient nutrition for plant growth, the plant growth regulators are used to promote the stem segments to sprout again.

[0024] (5) The present invention selects the explants and initial buds to be cut, and retains some young stem segments and buds on the original seedlings and explants, making full use of the more mature tissues for nutrient transport to ensure sufficient nutrition while utilizing the young tissues with more developed meristems for re-merization, thereby promoting rapid germination.

[0025] (6) The present invention sets two culture media for primary culture in the primary culture stage. The primary bud induction culture medium is based on the MS culture medium with relatively complete mineral elements. The initial bud domestication stage is based on the WPM culture medium with lower mineral elements. By fine-tuning the content of KH2PO4 and the content of CaCl2, and supplemented with plant growth regulators suitable for inducing budding of Camellia chrysantha, the degree of lignification of stem segments can be reduced, cell division can be promoted, and thus growth can be promoted, so that the bud induction effect of Camellia chrysantha is ideal, the initial buds after domestication are in good condition, and there is no yellowing and falling leaves. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 These are photos of seed seedlings derived from explants in Example 1.

[0027] Figure 2 This is a photo of the seedling sprouting again after the upper stem segment was cut off in Example 1.

[0028] Figure 3 , 4These are photos of the explants in Example 1 being induced to sprout on the primary sprout induction medium.

[0029] Figure 5 , 6 This is a photograph of the initial buds of Example 1 being acclimated and cultured on the initial bud acclimation medium.

[0030] Figure 7 This is a photo of the seedlings dying and not sprouting after the upper stem segment was cut off in Comparative Example 1.

[0031] Figure 8 , 9 These are photos of Comparative Example 2, which used mature stem segments and seeds as explant materials for induction.

[0032] Fig.10 , 11 This is a photo of comparative example 3 showing a large amount of callus tissue growing at the bottom after induction using only the primary bud induction medium.

[0033] Fig.12 These are photos of comparative example 4 in which the initial buds showed yellowing and falling leaves in the primary induction without using the improved culture medium. DETAILED DESCRIPTION

[0034] The following embodiments can enable those skilled in the art to more fully understand the present invention. The described embodiments are part of the present invention, not all of the embodiments, and do not limit the present invention in any way. However, those skilled in the art should understand that various changes can be made to the embodiments in form and detail without departing from the scope defined by the claims of the present invention.

[0035] In the following examples, the compositions of Modified MS and MS, WPM and Modified WPM media are:

[0036]

[0037]

[0038] Example 1

[0039] (1) Explant acquisition: Mature and healthy seeds of the endangered plant Camellia chrysantha were collected in the current year and stored in sand for germination. The seeds were placed in a cup with a diameter of 11 cm and a height of 15 cm, and the matrix was red heart soil: fine sand: peat soil with a volume ratio of 8:1:1. Conventional seedling management was carried out, that is, the seedlings were placed in an outdoor seedling greenhouse with a shade net and ventilation. Depending on the climate, when the temperature was too low, a small arch shed was built above the seedlings and covered with plastic film for insulation. Water was sprayed 1 to 2 times every 2 to 3 days, and compound fertilizer with N:P:K = 15:10:20 was applied once a month. When the seedling height is ≥6cm, cut the upper stem segment as the explant, keep the lower stem segment ≥3.5cm, and spray the nutrient solution after cutting off the upper stem segment. The nutrient solution is 0.5mg / mL NAA+0.6mg / mL GGR+50mg / L KH2PO3+800mg / L NH4NO3. Continue with conventional seedling management. When the buds grow to a height of ≥6cm again, cut the upper stem segment again and repeat the operation.

[0040] (2) Explant sterilization: After removing the leaves from the cut upper stem segment, trim it into 3 cm long segments, scrub it with a detergent, and then soak it in a 75% alcohol solution for 0.5 min. Rinse it with sterile water for 3 to 5 times. On a clean bench, select an ultrasonic cleaning cup with a blue light function and add a sterilant. The sterilant is composed of 0.5% tea tree essential oil + 0.1% HgCl2 solution + 0.01% Tween-20. Place the explant in the ultrasonic cleaning cup and wash it for 10 min. Repeat once, wash it with sterile water for 5 times, and use sterile filter paper to absorb the moisture. Cut off 0.2 cm from each end of the stem segment.

[0041] (3) Culture medium preparation: Two culture media were used for primary induction culture. The formula of the primary induction budding culture medium was: modified MS + 2-IP 2.0 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 acclimation culture medium was: modified WPM + 2-IP 0.5 mg / L + NAA 0.8 mg / L + GA3 0.1 mg / L + sucrose 30 g / L + agar 4.5 g / L. After the culture medium was prepared according to the formula, it was boiled for 5 min, the pH value was adjusted to 5.8-6.0, and the culture containers were divided into 350 mL glass bottles with a height of 11 cm, a diameter of 7.6 cm, and a mouth of 5.3 cm. 50-80 mL of culture medium was added to each bottle and sterilized at 121 °C for 20 min.

[0042] (4) Primary induction culture: The sterilized explants were vertically inserted into the primary induction budding medium. When the initial buds grew to ≥2 cm, they were cut off and inserted into the initial bud acclimation medium. The initial buds were cut off in such a way that 0.2 to 0.5 cm of the bud base was retained on the explants. The original explants were inserted back into the primary induction budding medium. The temperature in the culture room was controlled at about 25±3°C. After 7 days of weak light culture, normal light culture was used. Weak light culture was carried out by covering the culture container with 2 layers of newspaper under natural light without turning on the lights. Normal light culture was carried out by maintaining the light intensity at 2000 to 3000 LX for 12 to 14 hours per day.

[0043] Example 2

[0044] (1) Explant acquisition: Mature and healthy seeds of the endangered plant Camellia chrysantha were collected in the current year and stored in sand for germination. The seeds were placed in a cup with a diameter of 11 cm and a height of 15 cm, and the matrix was red heart soil: fine sand: peat soil with a volume ratio of 8:1:1. Conventional seedling management was carried out, that is, the seedlings were placed in an outdoor seedling greenhouse with a shade net and ventilation. Depending on the climate, when the temperature was too low, a small arch shed was built above the seedlings and covered with plastic film for insulation. Water was sprayed 1 to 2 times every 2 to 3 days, and compound fertilizer with N:P:K = 15:10:20 was applied once a month. When the seedling height is ≥6cm, cut the upper stem segment as the explant, keep the lower stem segment ≥3.5cm, and spray the nutrient solution after cutting off the upper stem segment. The nutrient solution is 1.0mg / mL NAA+0.3mg / mL GGR+80mg / L KH2PO3+500mg / L NH4NO3. Continue with the conventional seedling management. When the buds grow to a height of ≥6cm again, cut the upper stem segment again and repeat the operation.

[0045] (2) Explant sterilization: After removing the leaves from the cut upper stem segment, trim it into 1.5 cm long stem segments, scrub it with a detergent, and then soak it in a 75% alcohol solution for 0.5 min. Rinse it with sterile water for 3 to 5 times. On a clean bench, select an ultrasonic cleaning cup with a blue light function and add a sterilant. The sterilant consists of 0.5% tea tree essential oil + 0.1% HgCl2 solution + 0.01% Tween-20. Place the explant in the ultrasonic cleaning cup and perform ultrasonic cleaning for 5 min. Repeat this process. Rinse it with sterile water for 5 times. Use sterile filter paper to absorb the moisture. Cut off 0.2 cm from each end of the stem segment.

[0046] (3) Culture medium preparation: Two culture media were used for primary induction culture. The formula of the primary induction budding 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 acclimation 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 culture medium was prepared according to the formula, it was boiled for 5 min, the pH value was adjusted to 5.8-6.0, and the culture containers were divided into 350 mL glass bottles with a height of 11 cm, a diameter of 7.6 cm, and a mouth of 5.3 cm. 50-80 mL of culture medium was added to each bottle and sterilized at 121°C for 20 min.

[0047] (4) Primary induction culture: The sterilized explants were vertically inserted into the primary induction budding medium. When the initial buds grew to ≥2 cm, they were cut off and inserted into the initial bud acclimation medium. The initial buds were cut off in such a way that 0.2 to 0.5 cm of the bud base was retained on the explants. The original explants were inserted back into the primary induction budding medium. The temperature in the culture room was controlled at about 25±3°C. After 10 days of weak light culture, normal light culture was used. Weak light culture was carried out by covering the culture container with two layers of newspapers under natural light without turning on the lights. Normal light culture was carried out by maintaining the light intensity at 2000 to 3000 LX for 12 to 14 hours per day.

[0048] Example 3

[0049] (1) Explant acquisition: Mature and healthy seeds of the endangered plant Camellia chrysantha were collected in the current year and stored in sand for germination. The seeds were placed in a cup with a diameter of 11 cm and a height of 15 cm, and the matrix was red heart soil: fine sand: peat soil with a volume ratio of 8:1:1. Conventional seedling management was carried out, that is, the seedlings were placed in an outdoor seedling greenhouse with a shade net and ventilation. Depending on the climate, when the temperature was too low, a small arch shed was built above the seedlings and covered with plastic film for insulation. Water was sprayed 1 to 2 times every 2 to 3 days, and compound fertilizer with N:P:K = 15:10:20 was applied once a month. When the seedling height is ≥6cm, cut the upper stem segment as the explant, keep the lower stem segment ≥3.5cm, spray the nutrient solution after cutting off the upper stem segment, the nutrient solution is 0.8mg / mL NAA+0.5mg / mL GGR+60mg / L KH2PO3+600mg / L NH4NO3, continue with the conventional seedling management, wait for the bud to grow to a height of ≥6cm again, cut the upper stem segment again and repeat the operation;

[0050] (2) Explant sterilization: After removing the leaves from the upper stem segment, trim it into 2.5 cm long segments, scrub it with a detergent, and then soak it in a 75% alcohol solution for 1 min. Rinse it with sterile water for 3 to 5 times. On a clean bench, select an ultrasonic cleaning cup with a blue light function and add a sterilant. The sterilant consists of 0.5% tea tree essential oil + 0.1% HgCl2 solution + 0.01% Tween-20. Place the explant in the ultrasonic cleaning cup and perform ultrasonic cleaning for 10 min. Repeat this process. Rinse it with sterile water for 5 times. Use sterile filter paper to absorb the moisture. Cut off 0.5 cm from each end of the stem segment.

[0051] (3) Culture medium preparation: Two culture media were used for primary induction culture. The formula of the primary bud induction culture medium 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 formula of the initial bud acclimation culture medium 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 boiled for 5 min, the pH value was adjusted to 5.8-6.0, and the culture containers were divided into 350 mL glass bottles with a height of 11 cm, a diameter of 7.6 cm, and a mouth of 5.3 cm. 50-80 mL of culture medium was added to each bottle and sterilized at 121°C for 20 min.

[0052] (4) Primary induction culture: The sterilized explants were vertically inserted into the primary induction budding medium. When the initial buds grew to ≥2 cm, they were cut off and inserted into the initial bud acclimation medium. The initial buds were cut off in such a way that 0.2 to 0.5 cm of the bud base was retained on the explants. The original explants were inserted back into the primary induction budding medium. The temperature in the culture room was controlled at about 25±3°C. After 7 days of weak light culture, normal light culture was used. Weak light culture was carried out by covering the culture container with 2 layers of newspaper under natural light without turning on the lights. Normal light culture was carried out by maintaining the light intensity at 2000 to 3000 LX for 12 to 14 hours per day.

[0053] The inventors have conducted a large number of experiments for comparative studies to verify the effects of the present invention. The following are some comparative experimental examples that can illustrate the problem.

[0054] Comparative Example 1

[0055] The steps of this comparative example are the same as those of Example 1, except that in step (1), when obtaining the explants, the upper stem segments are cut off without spraying nutrient solution, and conventional seedlings are directly sprayed with water.

[0056] Comparative Example 2

[0057] The steps of this comparative example are the same as those of Example 1, except that in step (1), when obtaining the explants, semi-lignified stem segments (Comparative Example 1-A) and seeds (Comparative Example 1-B) of mature Camellia chrysantha plants are selected as explants.

[0058] Comparative Example 3

[0059] The steps of this comparative example are the same as those of Example 1, except that in step (4) the primary induction culture, only one culture medium is selected, that is, only the primary induction budding medium (modified MS + 2-IP 2.0 mg / L + NAA 1.6 mg / L + IBA 0.5 mg / L + sucrose 30 g / L + agar 4.5 g / 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] Comparative Example 4

[0061] The steps of this comparative example are the same as those of Example 1, except that in step (2), when preparing the culture medium, the primary bud induction culture medium is MS, i.e., MS + 2-IP 2.0 mg / L + NAA 1.6 mg / L + IBA 0.5 mg / L + sucrose 30 g / L + agar 4.5 g / L; the initial bud acclimation culture medium is WPM, i.e., WPM + 2-IP 0.5 mg / L + NAA 0.8 mg / L + GA3 0.1 mg / L + sucrose 30 g / L + agar 4.5 g / L.

[0062] Comparative Example 5

[0063] The steps of this comparative example are the same as those of Example 1, except that in step (3), when preparing the culture medium, the culture medium formula of comparative example 5-A adopts the best initial bud induction culture medium formula disclosed in Chinese patent CN 107135948 B "A method for cultivating tissue culture seedlings of Camellia chrysantha tender branches under sunlight conditions" (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 culture medium formula of comparative example 5-B adopts the best initial bud induction culture medium formula disclosed in Chinese patent CN 106818493 B "Yunnan Camellia chrysantha 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 treatment methods of Examples 1-3 of the present invention and Comparative Examples 1-5 were respectively used to prepare explants and conduct primary induction culture on Camellia chrysantha. The test results were observed and statistically compared. The main calculation formulas were as follows: contamination rate (%) = number of contaminated explants / number of inoculated explants × 100; brown death rate (%) = number of sprouted explants / number of inoculated explants × 100; induction rate (%) = number of sprouted explants / number of inoculated explants × 100; plant mortality rate = number of dead plants / number of planted plants × 100; plant sprouting rate = number of plants that sprouted again / number of planted plants × 100; callus rate (%) = number of callus explants / number of inoculated explants × 100.

[0066] Table 1: Primary induction culture results of the examples

[0067]

[0068] The explant sterilization and primary induction effects of the three embodiments are shown in Table 1. As can be seen from Table 1, there is no obvious difference in the initial bud state of the three embodiments, but Embodiment 1 has the highest initial bud induction rate, the lowest contamination rate, the shortest germination start time, and the lowest brown death rate, and is the optimal embodiment.

[0069] Table 2: Growth of explant-derived plants in Example 1 and Comparative Example 1

[0070]

[0071] Comparative Example 1 mainly investigates whether nutrient solution treatment can promote the acquisition of more explants. A rich source of explants is the primary key step in tissue culture and rapid propagation, and a rich source of explants can greatly increase the probability of successful tissue culture. As shown in Table 2, in Comparative Example 2, after cutting off the first batch of seedling stems, the nutrient solution was not sprayed, and watering management was performed directly. The plant mortality rate reached 20.4%, the sprouting rate was 74.9%, the sprouting time was 55d, and the number of sprouts was only 1. Compared with Example 1, the plant mortality rate increased by 18.3%, the sprouting rate decreased by 21.5%, the sprouting time was delayed by 15d, and the number of sprouts was less than that of Example 1. It can be seen that the preferred nutrient solution of the present invention can not only promote sprouting and avoid plant death, but also shorten 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. At the same time, plant growth regulators NAA and GGR are used to quickly replenish nutrients after the plant stem segments are cut, thereby reducing the death of the plant due to lack of nutrition in the vulnerable stage. The nutrient solution ratio obtained by the present invention through a large number of experimental screenings can fully meet the nutrition of Camellia chrysantha seedlings after the stem segments are cut off, and while ensuring the nutrition required for growth, plant growth regulators are used to promote the stem segments to sprout again, enriching the source material of explants.

[0072] Table 3: Primary induction effects of Example 1 and Comparative Example 2

[0073]

[0074] Comparative Example 2 mainly investigates the effects of different types of materials as explants on primary induction culture. As shown in Table 3, when comparative example 2-A selects semi-lignified stem segments of mature plants as explant materials, the contamination rate reaches 15.4%, the brown death rate reaches 24.3%, and the induction rate is 62.4%. Compared with Example 1, the contamination rate and the brown death rate increase by 11.2% and 23.1%, respectively, and the induction rate decreases by 30.1%. Since the internodes of mature stem segments are long, most of them contain only one internode when cut into 3-5 cm, and can only induce one bud at the position of the stem node. Most of the internodes of seedling stem segments are short, and 3-5 cm usually contain multiple stem nodes, and multiple buds can be induced at multiple stem node positions at the same time, such as Figure 1 , 2 It can be seen. Comparative Example 2-B selects mature seeds as explants, and the contamination rate is 4.6%, which is not much different from Example 1, but the brown death rate reaches 8.9%, and the germination initiation time reaches 60d, which is 40d later than Example 1. The induction rate is only 48.9%, which is significantly lower than Example 1, and the primary induction effect is significantly worse than Example 1. Analysis of the reasons: The mature stem segment tissue is more mature and has been in contact with the natural environment for a long time. The bacteria it carries gradually accumulates, making it difficult to sterilize successfully. The mature seeds are wrapped in the seed coat to protect the seeds from the sterilizer, and the contamination rate and brown death rate are lower than those of mature stem segments, but the germination initiation time is longer and the induction rate is still significantly lower than that of Example 1. In summary, the preferred seedling stem segments of the present invention are used as explants for explant preparation, and the sterilization success rate and induction rate are high.

[0075] Table 4: Primary induction effects of Example 1 and Comparative Example 3

[0076]

[0077] Comparative Example 3 mainly investigates whether not using the acclimation medium affects the induction effect. As shown in Table 4, in the primary culture process of Comparative Example 3, the medium was not changed, and the primary induction budding medium was always used. The callus rate was 84.3%, the number of buds was low, less than 1 bud, the induced buds had larger leaves, the bottom callus was larger and gradually wrapped the main stem, and the callus was serious. The buds of Example 1 that were replaced with the acclimation medium can induce multiple buds, the leaves are relatively small, the absorption surface is moderate, the buds are young and tender, which is conducive to further proliferation and differentiation. Compared with Example 1, the bud state of Comparative Example 3 is significantly worse, and the bottom callus is serious. The analysis reason may be that the use of richer nutrition and higher concentration of plant growth regulators in the primary induction and promotion of budding of Camellia chrysantha can promote its rapid budding, but the use of higher plant growth regulators after budding will cause it to callus rapidly, and the larger callus gradually affects the absorption of nutrients, thereby affecting further budding. In the primary bud induction stage, the present invention selects a nutrient-rich improved MS culture medium and a relatively high concentration of plant growth regulators. After bud induction, the new buds are transferred to a modified WPM culture medium with reduced growth regulator concentrations and mineral elements as an acclimation culture medium, which can promote rapid budding while avoiding callus, thereby inducing multiple buds and significantly improving the induction effect.

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

[0079] Examples Number of buds Initial bud state Example 1 2.8 It induces multiple buds, the leaves are of moderate size, the absorption surface is of moderate size, and the buds are young and tender. Comparative Example 4 1.2 Most of them induce one bud, with yellow top and fallen leaves.

[0080] Comparative Example 4 mainly investigates the effect of the amount of mineral elements used in the basic culture medium on the primary induction effect. As shown in Table 5, the amount of mineral elements used in the basic culture medium not only affects the number of buds, but also affects the state of the initial buds. The number of buds in Comparative Example 4 is only 1.2, and the induced buds are prone to yellowing and falling leaves. For this reason, the present invention screens the basic culture medium, and by fine-tuning the content of KH2PO4 to a low level and the content of CaCl2 to a high level, it is found that the initial bud state is significantly improved. The reason for this may be that KH2PO4 is rich in K + , K + It can significantly improve the plant's absorption and utilization of nitrogen, promote protein synthesis, promote photosynthesis, and improve the plant's resistance to stress; CaCl2 can enhance the plant's calcium intake, promote plant growth, prevent plant wilting, and increase leaf chlorophyll content and antioxidant enzyme activity, and enhance active oxygen scavenging ability. Adjusting their usage to a concentration more suitable for Camellia chrysantha growth improves the state of the initial buds.

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

[0082]

[0083] Comparative Example 5 mainly examines whether the technology of the present invention has advantages over the prior art. The relatively mature culture medium formulas in the prior art were selected for the primary induction culture of Camellia chrysantha. As shown in Table 6, the induction rate of Example 1 was 1.7% and 6.2% higher than that of Comparative Examples 5-A and 5-B (94.8% and 90.3%), and the budding initiation time was shortened by 17d and 14d, and the primary induction effect had obvious advantages.

[0084] The above analysis shows that the explants prepared by the method of Example 1 have abundant explant source materials, which can be used sustainably, and the prepared explants have low contamination rate and browning rate, the highest induction rate and number of buds, and the best induction effect. This also shows that the explants prepared by the method formed by a series of innovative operations of the present invention have low contamination rate and browning rate, high induction rate, abundant explant sources, and can induce multiple robust initial buds in a short time, which can lay a material foundation for further tissue culture and breeding application research, and is of great significance for promoting the development of the Camellia chrysantha industry.

Claims

1. A method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha, characterized in that: Including explant acquisition, explant sterilization, culture medium preparation and primary induction culture, the main operation steps are as follows: (1) Explant acquisition: Select the endangered plant Camellia chrysantha and collect mature and healthy seeds of the same year. After germination in sand storage, place them in a substrate cup and carry out conventional seedling management. When the seedlings are ≥6 cm tall, cut the upper stem segment as the explant and keep the lower stem segment ≥3.5 cm. After cutting off the upper stem segment, spray nutrient solution and continue with conventional seedling management. When the buds grow again to a height of ≥6 cm, cut the upper stem segment again and repeat the operation; (2) Explant sterilization: After removing the leaves from the upper stem segments, trim them into 1.5-3 cm long segments, clean them with a detergent, and then soak them in a 75% alcohol solution for 0.5-1 min. Rinse them with sterile water for 3-5 times. On a clean bench, select an ultrasonic cleaning cup with a blue light function and add a sterilant. Place the explant in the cup and perform ultrasonic cleaning for 5-10 min. Repeat this process. Rinse with sterile water for 5 times. Use sterile filter paper to absorb the moisture. Cut off 0.2-0.5 cm from each end of the stem segment. (3) Culture medium preparation: Two culture media were used for primary induction culture. The formula of the primary bud induction culture medium was: 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 formula of the initial bud acclimation culture medium was: 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 the culture medium was prepared according to the formula, it was boiled for 5 min, the pH value was adjusted to 5.8-6.0, and the culture medium was dispensed into culture containers; (4) Primary induction culture: The sterilized explants were vertically inserted into the primary bud induction medium. When the initial buds grew to ≥ 2 cm, they were cut off and inserted into the initial bud acclimation medium. The original explants were inserted back into the primary bud induction medium. The temperature of the culture room was controlled at about 25 ± 3 °C. After 7 to 10 days of weak light culture, they were switched to normal light culture.

2. The method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha according to claim 1, characterized in that: The specification of the substrate cup in step (1) is 11 cm in diameter × 15 cm in height, and the volume ratio of the substrate is red heart soil: fine sand: peat soil is 8:1:

1.

3. The method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha according to claim 1, characterized in that: The conventional seedling management in step (1) is to place the seedlings in a ventilated outdoor seedling greenhouse with a shade net. Depending on the climate, when the temperature is too low, a small arch shed is built above the seedlings to cover them with plastic film for insulation. Water is sprayed 1 to 2 times every 2 to 3 days, and compound fertilizer with N:P:K=15:10:20 is applied once a month.

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

6. The method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha according to claim 1, characterized in that: The modified WPM and modified MS culture media described in step (3) are composed of:

7. The method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha according to claim 1, characterized in that: 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 of 5.3 cm. 50 to 80 mL of culture medium is added to each bottle and sterilized at 121° C. for 20 min.

8. The method for preparing high induction rate explants and primary induction culture of the endangered plant Camellia chrysantha according to claim 1, characterized in that: The weak light culture in step (4) is to cover the culture container with two layers of newspapers under natural light conditions without turning on the lights, and the normal light culture is to maintain the light intensity at 2000-3000LX and light for 12-14 hours a day.

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

Images