A method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods

Abstract

The invention discloses a method for cultivating Atractylodes lancea tissue culture seedlings by combining in-bottle and out-bottle methods, which includes the steps of inducing adventitious buds of Atractylodes lancea explants, subculture and proliferation culture of Atractylodes lancea, rooting of subculture tissue culture seedlings outside the bottle, and transplanting of tissue culture seedlings. The invention further optimizes the adventitious bud induction, proliferation, adventitious bud induction medium, subculture and proliferation medium, and the matrix for cutting rooting of Atractylodes lancea through a large number of experiments, and for the first time combines the in-bottle and out-bottle rooting technologies to induce rooting of subculture buds of Atractylodes lancea, which can save production costs, shorten the seedling raising cycle, and improve the survival rate of transplanting, and provides a new method for obtaining Atractylodes lancea tissue culture seedlings, which serves the actual production of Atractylodes lancea seedling raising, further accelerates the process of factory-based seedling raising of Atractylodes lancea, and has important application value.

Description

Technical Field

[0001] The invention relates to a method for raising seedlings of traditional Chinese medicine, and in particular to a method for raising seedlings of Atractylodes lancea tissue by combining in-bottle and out-bottle methods. Background Art

[0002] Atractylodes lancea (Thunb.) DC. is a perennial herbaceous plant of the Asteraceae family. Its rhizome is used as a medicine, which has the effects of drying dampness and strengthening the spleen, dispelling wind and cold, and improving eyesight. The Atractylodes produced in Maoshan, Jiangsu Province and its surrounding areas has the best quality, so it is called Atractylodes lancea. Due to the low natural reproduction rate of wild Atractylodes lancea, coupled with unreasonable human excavation and habitat destruction, the resources of wild Atractylodes lancea are on the verge of exhaustion. At present, the Atractylodes lancea on the market are mainly artificially cultivated products, but the seedling emergence rate of Atractylodes lancea is low, the emergence is slow, and the emergence is uneven. The root and stem reproduction causes the Atractylodes lancea to degenerate from generation to generation and the quality decreases. Therefore, the use of tissue culture technology to realize the factory-based seedling cultivation of Atractylodes lancea can alleviate the market demand for Atractylodes lancea seedlings. Plant tissue culture has the characteristics of high reproduction coefficient, genetic stability and no restrictions on natural conditions. It can obtain a large number of seedlings in a relatively short time, making up for the disadvantages in traditional cultivation, which is of great significance for the breeding of Atractylodes lancea.

[0003] Traditionally, the rooting of Atractylodes lancea tissue culture seedlings is carried out in bottles, and rooting culture is crucial to the survival rate of seedlings after transplanting. However, according to previous studies, the root structure of Atractylodes lancea produced by rooting in the culture medium bottle is imperfect, no root hairs are formed, and the plants are more likely to die during transplanting and acclimatization, which limits the development of Atractylodes lancea tissue culture factory seedling cultivation. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art. Through a large number of experiments, the adventitious bud induction, proliferation, adventitious bud induction culture medium, subculture proliferation culture medium and matrix for cutting rooting of Atractylodes lancea are further optimized. For the first time, the rooting induction of subculture buds of Atractylodes lancea is carried out by combining the in-bottle and out-of-bottle rooting techniques, which can save production costs, shorten the seedling raising cycle, and improve the survival rate of transplanting. It provides a new method for obtaining Atractylodes lancea tissue culture seedlings, serves the actual production of Atractylodes lancea seedlings, and further accelerates the process of factory-based seedling raising of Atractylodes lancea, and has important application value.

[0005] Technical solution: In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0006] A method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods, comprising the following steps;

[0007] (1) Explant selection and sterilization

[0008] The seeds of Atractylodes lancea were collected from Maoshan, Jurong, Jiangsu, cleaned and soaked in clean water, and then the soaked seeds were disinfected with a certain concentration of ethanol, disinfected with mercuric chloride, and rinsed with sterile water; then the seeds were inoculated in MS culture medium, and adventitious buds were induced with sterile seedlings of 12 to 15 days old as explants;

[0009] (2) Adventitious bud induction of Atractylodes lancea explants

[0010] Select the robust sterile seedlings of Atractylodes macrocephala seedlings with an age of 12 to 15 days, cut the hypocotyls, cut the hypocotyls in a cross-section, cut off the roots, leaves and cotyledons, and only keep the hypocotyls, and inoculate the cut hypocotyls in an adventitious bud induction medium to induce adventitious buds of Atractylodes macrocephala explants;

[0011] (3) Subculture and proliferation of Atractylodes macrocephala

[0012] When the adventitious buds of Atractylodes lancea are induced to grow to 2.0-2.5 cm, they are planted vertically into a subculture proliferation medium for subculture proliferation culture to obtain subculture clustered buds of Atractylodes lancea;

[0013] (4) Rooting of Atractylodes macrocephala seedlings in vitro

[0014] (4.1) Hardening of seedlings

[0015] Select the successive generation clustered bud seedlings with a seedling height of ≥3.0 cm and a leaf number of ≥3 in step (3), move them to an artificial climate chamber for seedling hardening, turn the bottle cap halfway open to continue hardening, and then open the bottle cap fully for hardening before cutting;

[0016] (4.2) Cutting of subculture seedlings

[0017] Use tweezers to gently remove the subcultured buds, clean the remaining matrix with water, separate the individual buds with scissors, remove the brown callus tissue and disinfect it;

[0018] (4.3) Disinfection of Subculture Seedlings

[0019] After the callus tissue at the base of the stem of the single bud seedling in step (4.2) is partially removed, the single bud seedling is immersed in potassium permanganate, taken out, rinsed and rooted;

[0020] (4.4) Rooting of cuttings of successive seedlings

[0021] Treat the 1-2 cm base of the seedling stem with NAA, then poke a small hole in the middle of the substrate in the plug tray and plant the successive seedlings in at a depth of about 1-1.5 cm. Press the soil to make it firm, water it to set the roots, and keep the leaves moist.

[0022] (5) Transplantation of tissue culture seedlings

[0023] When the roots of the tissue culture seedlings grow to cover the substrate and the rooting outside the bottle is completed, they are then transplanted into a regular substrate seedling pot.

[0024] As a preferred embodiment, the present invention provides a method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods, comprising the following steps:

[0025] (1) Explant selection and sterilization

[0026] The seeds of Atractylodes lancea were collected from Maoshan, Jurong, Jiangsu, cleaned and soaked in clean water, disinfected with 75% ethanol for 30 seconds, disinfected with 0.1% mercuric chloride for 15 minutes, and rinsed with sterile water for 4 to 5 times, each time for no less than 3 minutes; then the seeds were inoculated in MS culture medium, and adventitious bud induction was carried out with 15-day-old sterile seedlings as explants;

[0027] (2) Adventitious bud induction of Atractylodes lancea explants

[0028] Select the robust sterile seedlings of Atractylodes macrocephala seedlings at 15 days of age, cut the hypocotyls, cut the hypocotyls in a cross-section, cut off the roots, leaves and cotyledons, and only keep the hypocotyls, and inoculate the cut hypocotyls in an adventitious bud induction medium to induce adventitious buds of Atractylodes macrocephala explants;

[0029] (3) Subculture and proliferation of Atractylodes macrocephala

[0030] When the adventitious buds of Atractylodes lancea grow to about 2.0 cm, they are planted vertically into the subculture proliferation medium for subculture proliferation culture;

[0031] (4) Rooting of Atractylodes macrocephala seedlings in vitro

[0032] (4.1) Hardening of seedlings

[0033] Select the successive clump sprouts with a seedling height of ≥3.0 cm and a leaf number of ≥3 in step (3), move them to an artificial climate chamber for seedling hardening for 3 days, turn the bottle cap halfway open to continue seedling hardening for 1 day, then fully open the bottle cap to harden the seedling for 2 days before cutting;

[0034] (4.2) Cutting of subculture seedlings

[0035] Use tweezers to gently remove the subcultured buds, clean the remaining matrix with water, separate the individual buds with scissors, remove the brown callus tissue and disinfect it;

[0036] (4.3) Disinfection of Subculture Seedlings

[0037] After the callus tissue at the base of the stem of the single bud seedling in step (4.2) is partially removed, the single bud seedling is placed in 0.05% potassium permanganate and soaked for 60 seconds, taken out and rinsed clean for rooting treatment;

[0038] (4.4) Rooting of cuttings of successive seedlings

[0039] Treat 1-2 cm of the base of the seedling stem with 0.1 mg / mL NAA, then poke a small hole about 2.0 cm deep in the middle of the plug tray, plant the subculture seedlings in it, and plant them at a depth of about 1-1.5 cm. Press the soil to make it firm, water it to fix the roots, and keep the leaves moist.

[0040] (5) Transplantation of tissue culture seedlings

[0041] When the roots of the tissue culture seedlings grow to cover the substrate and the rooting outside the bottle is completed, they are then transplanted into a regular substrate seedling pot.

[0042] As a preferred embodiment, in the above-mentioned method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods, the formula of the adventitious bud induction medium in step (2) is: MS medium + 6-BA 1.0mg / L + NAA 0.15mg / L + sucrose 30g / L, the agar content in the medium is 0.7%, and the pH is 5.8-6.0. The culture environment temperature is 25±1°C, the light condition is 6000lx, the light time is 12h / d, and the humidity is about 70%.

[0043] As a preferred embodiment, the above-mentioned method for cultivating Atractylodes macrocephala tissue culture seedlings by combining in-bottle and out-bottle methods, the formula of the subculture proliferation medium in step (3) is: MS medium + 6-BA 1.0mg / L + NAA 0.5mg / L + sucrose 50g / L, the agar content in the medium is 0.7%, the pH is 5.8-6.0. The culture environment temperature is 25±1°C, the light condition is 6000lx, the light time is 12h / d, and the humidity is about 70%.

[0044] As a preferred embodiment, in the above-mentioned method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-of-bottle methods, the substrate for cutting rooting in step (4) is prepared by the following method:

[0045] Cutting rooting medium: Mix peat, perlite and vermiculite in a volume ratio of 3:1:1, put them into a plastic hole tray, and water the medium thoroughly with clean water.

[0046] The vermiculite is prepared by the following method:

[0047] Weigh 1.00 g of chitosan and place it in a beaker containing 200 mL of 2% acetic acid aqueous solution, and mechanically stir at 60° C. for 2 h to obtain a uniform and transparent chitosan acetic acid aqueous solution;

[0048] The vermiculite was crushed into 80 meshes, and the expanded vermiculite was added into the chitosan acetic acid aqueous solution at a solid-liquid ratio of 1:10; the mixture was stirred at a constant temperature of 60°C for 6 hours to obtain a chitosan-modified vermiculite dispersion; after aging for 12 hours, the dispersion was separated by suction filtration, washed, and dried at 60°C to obtain the chitosan-modified vermiculite.

[0049] Beneficial effects: The method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods provided by the present invention has the following advantages over the prior art:

[0050] According to the characteristics of Atractylodes macrocephala seeds, the cutting method of the hypocotyl mainly includes whether the apical bud is removed and the retention of the cotyledons. These cutting methods are mainly based on the considerations of the apical dominance of the explant, the endogenous hormones in the cotyledons and the convenience of practical operation. The present invention screens the cutting methods of the hypocotyl for the first time. Studies have shown that the cutting method of "cross-cutting, cutting off the roots, leaves and cotyledons, and only retaining the hypocotyl" has the highest adventitious bud induction rate, with a maximum value of 5; it is better than the cutting methods of retaining the cotyledons and longitudinal cutting, and the average proliferation coefficient is 0.37 higher than that of retaining the cotyledons. It may be because the length and surface area of ​​the wound increase when a certain size of cotyledons are removed, thereby increasing the probability of adventitious buds. As for the fact that the effect of longitudinal cutting is much lower than that of cross-cutting, it may be because when the explant is cut, it is difficult to evenly divide the growth point between the cotyledon and the hypocotyl into two, thus causing the failure of adventitious bud induction of part of the explants.

[0051] In the process of plant tissue culture, appropriate light intensity is necessary and has a great impact on the growth and development of the culture. Different plant test tube seedlings have different requirements and responses to light intensity. This experiment unexpectedly found that 6000lx is more suitable as the light condition for inducing adventitious buds of Atractylodes lancea. According to previous literature research and investigation, scholars have defaulted 2000-2500lx as the optimal condition for inducing adventitious buds of Atractylodes lancea. However, in this experiment, with the increase of light intensity, the adventitious bud induction proliferation coefficient increased, reaching the maximum value at 6000lx, and the adventitious bud induction proliferation coefficient decreased when it exceeded 6000lx.

[0052] In the study of the proliferation culture of Atractylodes macrocephala, previous studies used the proliferation coefficient / proliferation rate as the main or only measurement index. However, combined with current research and production, a single evaluation index can only be used to measure the effect of the culture medium on the proliferation coefficient, and it cannot guarantee the provision of high-quality tissue culture seedlings for production. If large-scale seedling production is to be achieved, while pursuing a high proliferation coefficient, the uniformity of the agronomic traits of the tissue culture seedlings should also be investigated in combination with the actual needs of production, and a proliferation induction culture medium that can induce subculture seedlings with good quality, high uniformity of agronomic traits, and high reproduction coefficient should be screened. This study found that the culture medium MS+6-BA 1.0mg / L+NAA 0.5mg / L+sucrose 50g / L can meet the above requirements. Its proliferation coefficient is 12.96±1.55, and the highest proliferation induction number of a single bud is 15, which have reached a relatively high level. In addition, the uniformity of agronomic traits such as plant height, stem thickness, number of effective leaves, and fresh weight is high, which can provide tissue culture seedlings with excellent agronomic traits and uniformity for factory seedling cultivation. In order to realize large-scale factory seedling cultivation of Atractylodes lancea, although rooting in a bottle is a more commonly used method in the tissue culture process, rooting in a test tube still has problems such as weak root development, low transplant survival rate, long seedling production cycle, and high cost, which seriously restricts the factory production of tissue culture seedlings. The in vitro induction rooting culture technology can simultaneously complete the two stages of rooting and domestication, which can promote the root growth of seedlings to the greatest extent, improve the transplant survival rate, and greatly reduce the cost of seedling cultivation. For Atractylodes lancea, the time for rooting in a bottle is 26 to 28 days, plus the time for transplanting and domestication of 24 to 28 days, the total duration is about 50 to 56 days, and the present invention uses rooting outside the bottle to combine rooting with seedling hardening and transplanting, which only takes 36 to 42 days. In contrast, rooting and seedling hardening and transplanting outside the bottle can be shortened by about 14 days.

[0053] At the same time, the distance between the roots of Atractylodes lancea is small, and the creases and gaps in the middle are easy to trap the gelatinous culture medium, which is difficult to clean. It is difficult to wash the tissue culture seedlings, and the roots are easily damaged. The residual culture medium between the roots is not completely removed, which is very easy to breed bacteria during the transplanting and acclimation process, increasing the infection rate. If the root system is damaged due to improper operation during the cleaning process, the survival rate of the plant is extremely low. In the rooting outside the bottle, the callus tissue at the base of the seedling stem that is in contact with the culture medium will be partially removed before transplanting. When transplanting, only the culture medium adhering to the bottom of the callus tissue needs to be peeled off and simply cleaned. It is easy to operate, takes a short time, avoids the risk of re-infection of rooting culture, and the subculture seedlings have not yet formed a root system, which greatly reduces the difficulty of washing cutting rooting seedlings.

[0054] In particular, in the process of hardening and rooting the seedlings, the invention selects the successive bud seedlings with "seedling height ≥3.0cm and leaf number ≥3" for the best overall growth in rooting outside the bottle. Under standardized operation and management, the infection rate can reach 0, the survival rate is as high as 98%, the seedlings are upright, the leaves are dark green and leathery, the seedlings are of good quality, and the later transplanting is facilitated, thereby further creating conditions for realizing the factoryization of tissue culture seedlings of the endangered medicinal plant Atractylodes lancea. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 These are pictures of adventitious buds produced by different cutting methods of the hypocotyl.

[0056] Figure 2 This is a bar graph showing the effect of different light intensities on adventitious bud induction.

[0057] Figure 3 These are the growth diagrams of single cluster adventitious buds of Atractylodes lancea on different culture media (A. Culture medium No. 1; B. Culture medium No. 2; C. Culture medium No. 6).

[0058] Figure 4 The morphological characteristics of the aboveground parts of Atractylodes macrocephala at different stages of its external rooting. (A. March 1; B. March 13; C. March 22; D. April 7)

[0059] Figure 5 The root morphology characteristics of Atractylodes macrocephala at different stages of its external rooting. (A. March 13; B. March 22; C. April 7)

[0060] Figure 6 These are pictures of the successive generations of Atractylodes lancea seedlings at different growth times.

[0061] Figure 7 Rooting status inside and outside the bottle (A. Rooting inside the bottle; B. Rooting outside the bottle). DETAILED DESCRIPTION

[0062] The present invention is further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0063] Example 1

[0064] 1 Materials and methods

[0065] 1.1 Test materials

[0066] The seeds of Atractylodes lancea were collected from Maoshan, Jurong, Jiangsu, cleaned and impurities removed, and then soaked in clean water. The soaked seeds were disinfected with 75% ethanol for 30 seconds, disinfected with 0.1% mercuric chloride for 15 minutes, and rinsed with sterile water for 4 to 5 times, each time for no less than 3 minutes; then the seeds were inoculated into MS culture medium.

[0067] 1.2 Culture medium

[0068] 1.2.1 Atractylodes macrocephala adventitious shoot induction medium

[0069] The formula of adventitious bud induction medium is MS medium + 6-BA 1.0mg / L + NAA 0.15g / L + sucrose 30g / L, agar content of 0.7%, pH of 5.8-6.0, culture temperature of (25±1)℃, light duration of 12h / d, and humidity of about 70%.

[0070] 1.2.2 Atractylodes macrocephala proliferation culture medium

[0071] The basic culture medium for subculture proliferation is MS, the agar content in the culture medium is 0.7%, the pH is 5.8-6.0, the culture temperature is (25±1)℃, the light condition is 6000lx, the light time is 12h / d, and the humidity is about 70%.

[0072] 1.2.3 Basic medium for rooting of Atractylodes lancea in bottles

[0073] The rooting medium is 1 / 2MS. The sucrose content in the medium is 3%, the agar content is 0.7%, and the pH is 5.8-6.0. The culture temperature is (25±1)℃, the light condition is 6000lx, the light time is 12h / d, and the humidity is about 70%.

[0074] 1.2.4 The substrate for rooting cuttings was prepared by the following method:

[0075] Cutting rooting medium: Mix peat, perlite and vermiculite in a volume ratio of 3:1:1, put them into a plastic hole tray, and water the medium thoroughly with clean water.

[0076] The vermiculite is prepared by the following method:

[0077] Weigh 1.00 g of chitosan and place it in a beaker containing 200 mL of 2% acetic acid aqueous solution, and mechanically stir at 60° C. for 2 h to obtain a uniform and transparent chitosan acetic acid aqueous solution;

[0078] The vermiculite was crushed into 80 meshes, and the expanded vermiculite was added into the chitosan acetic acid aqueous solution at a solid-liquid ratio of 1:10; the mixture was stirred at a constant temperature of 60°C for 6 hours to obtain a chitosan-modified vermiculite dispersion; after aging for 12 hours, the dispersion was separated by suction filtration, washed, and dried at 60°C to obtain the chitosan-modified vermiculite.

[0079] 1.3 Study on the induction of adventitious buds of Atractylodes lancea

[0080] 1.3.1 Optimization of hypocotyl cutting method

[0081] The 15-day-old sterile seedlings of Atractylodes lancea were used as explants, and the hypocotyls were cut. The hypocotyl cutting methods were divided into 3 treatments: A: cross-cut, cutting off the roots and leaves, retaining the complete cotyledons and hypocotyls; B: longitudinal cutting, cutting off the roots and leaves, retaining the complete cotyledons and hypocotyls; C: cross-cut, cutting off the roots, leaves and cotyledons, retaining only the hypocotyls, as shown in Table 1. The morphological lower end of the explant was vertically inserted into the culture medium. Each treatment was inoculated with 7 bottles, and each bottle had 4 explants. After 4 weeks of culture, the adventitious bud induction rate and proliferation coefficient were counted.

[0082] Table 1 Different cutting methods of hypocotyl of Atractylodes lancea

[0083] Different cutting methods Text description A Cut horizontally, cut off the roots and leaves, and keep the cotyledons and hypocotyls intact B Cut longitudinally, remove roots and leaves, and keep intact cotyledons and hypocotyls C Cut horizontally, remove roots, leaves and cotyledons, and keep only the hypocotyl .

[0084] 1.3.2 Optimization of light intensity for adventitious bud induction

[0085] Cut hypocotyls with only the hypocotyls retained were used as explants, and the light intensity of the light incubator was 1000lx, 2000lx, 3000lx, 4000lx, 5000lx, 6000lx, and 7000lx. Seven bottles were inoculated for each treatment, with four explants in each bottle. After 4 weeks of culture, the adventitious bud induction rate and proliferation coefficient were counted.

[0086] 1.4 Optimization of the proliferation culture medium of Atractylodes macrocephala

[0087] 1.4.1 Orthogonal experimental design of Atractylodes macrocephala proliferation medium

[0088] Adventitious buds of about 2.0 cm induced by hypocotyls were selected and planted vertically into the proliferation medium. Different concentrations of 6-BA, NAA, and sucrose were added to the proliferation medium to fully explore the effects of the three plant growth regulators on the proliferation of Atractylodes lancea. The orthogonal table was referenced to L9(3 4 ), the fourth column is blank, as shown in Table 2. There are 9 treatments in total, each treatment has 8 bottles replicated, and each bottle is inoculated with 3 single buds. After inoculation, the morphological characteristics and agronomic traits of the buds were observed and counted.

[0089] Table 2 Proliferation medium factor level table

[0090] factor 6-BA concentration mg / L NAA concentration mg / L Sucrose concentration g / L Level 1 1.0 0.1 30 Level 2 2.0 0.5 40 Level 3 3.0 1.0 50 .

[0091] 1.4.2 Observation of morphological characteristics

[0092] Observe the overall state of the Atractylodes macrocephala tissue culture seedlings, leaf color, etc., and calculate its proliferation coefficient. The length from the leaf axil to the top of the bud is the bud growth, and the bud growth of more than 1.0 cm is an effective bud.

[0093] 1.4.3 Growth index determination

[0094] Three clumps of rootless seedlings were selected for each treatment group, the agar remaining at the stem base was washed, the callus tissue was excised, and the seedlings were cut into single buds for later use. The plant height, stem diameter, number of effective leaves, and fresh weight of each single bud were recorded.

[0095] Plant height: the vertical height from the junction of the plant stem and callus to the top of the plant, measured with a ruler, with the detection value accurate to 0.1cm; stem diameter: the diameter of the junction of the plant stem and callus, measured with a vernier caliper, with the detection value accurate to 0.01mm; number of effective leaves: count the leaves with leaf width and leaf length greater than 2mm; plant fresh weight is measured using a balance, accurate to 0.0001g.

[0096] 1.4.4 Calculation of uniformity

[0097] The inverse of the coefficient of variation was used as a measure of uniformity.

[0098] 1.5 Study on the rooting of Atractylodes macrocephala subculture seedlings outside the bottle

[0099] 1.5.1 Screening of exogenous rooting culture medium

[0100] The experiment used peat, perlite and vermiculite as test materials. The dosage of each matrix is ​​shown in Table 3. The mixed matrix was loaded into a 50-hole (high-legged) plug tray, and the subculture seedlings with the same state were selected and planted in the matrix. Each treatment was repeated 3 times, and the survival rate of each treatment was observed and counted.

[0101] Table 3 Bottle exogenous rooting culture matrix ratio

[0102]

[0103] 1.5.2 Rooting of Subculture Seedlings

[0104] (1) Rooting in a bottle

[0105] The subculture seedlings were cut into individual plants and inoculated into rooting medium. After 26 to 28 days of rooting in the bottle, the seedlings were hardened and acclimatized.

[0106] Select test tube seedlings with a height of about 4 cm and 4 to 5 new roots, place them in an artificial climate chamber and harden them for 7 to 8 days, take out the tissue culture seedlings, wash the culture medium at the roots and transplant them into the culture matrix. The transplanting matrix is ​​mixed with peat: perlite: vermiculite in a volume ratio of 3:1:1.

[0107] (2) Rooting outside the bottle

[0108] ①Seedling domestication

[0109] The subgeneration clustered seedlings were moved to the artificial climate chamber for hardening for 3 days, the bottle cap was half-opened and continued to harden for 1 day, and then the bottle cap was fully opened for hardening for 2 days before cuttings.

[0110] ② Cutting of successive sprouts

[0111] Use tweezers to gently remove the subcultured clustered seedlings, clean the remaining matrix with clean water, separate the individual seedlings with scissors, and proceed to the next step after removing the browned callus tissue.

[0112] ③Disinfection of subculture seedlings

[0113] After partially removing the callus tissue at the base of the stem of the single-bud seedling, the single-bud seedling was immersed in 0.05% potassium permanganate for 60 seconds, and then taken out and rinsed in clean water.

[0114] ④ Rooting of cuttings of successive seedlings

[0115] Treat 1-2 cm of the base of the seedling stem with 0.1 mg / mL NAA, then poke a small hole about 2 cm deep in the middle of the hole in the plug tray, and then poke a small hole about 2.0 cm deep in the middle of the substrate of the plug tray, plant the successive seedlings in, the planting depth is about 1-1.5 cm, press the soil to compact it, water it to fix the roots, and keep the leaves moist;

[0116] 1.5.3 Study on the rooting of subcultured seedlings at different proliferation times

[0117] The subcultured seedlings were divided into four time periods according to the culture time of Atractylodes lancea tissue culture seedlings in the proliferation medium. The subcultured seedlings of each stage were cut into 50-hole (high-foot) plug tray substrates (see Table 3). Each treatment group was sown in one plug tray, with one seedling per hole, and repeated three times. The rooting rate was counted 30 days after cutting.

[0118] 1.5.4 Comparative study of rooting outside and inside the bottle

[0119] Using the rooting in the bottle as the control, the treated subculture seedlings were cut into 50-hole (high-leg) plug tray substrates, with one plug tray sown for each treatment and one seedling per hole repeated three times. The rooting rate and survival rate were calculated 30 days after cutting.

[0120] 1.6 Data Processing

[0121] Induction rate = number of explants induced with adventitious buds / total number of explants inoculated × 100%.

[0122] The proliferation coefficient of adventitious bud induction = the number of proliferated adventitious buds / the total number of inoculated explants × 100%.

[0123] Proliferation coefficient of proliferation culture = number of effective buds proliferated / total number of explants inoculated × 100%.

[0124] The formula for calculating the inverse coefficient of variation is as follows:

[0125] 1 / CV=S / X

[0126] Where: CV is the coefficient of variation, S is the standard deviation, and X is the sample mean. The reciprocal value of the coefficient of variation is a method of indicating the uniformity of field growth of cultivated crops. It has no unit, and the larger the value, the higher the uniformity, that is, the more uniform.

[0127] Rooting rate = number of rooted seedlings / total number of seedlings from cuttings × 100%.

[0128] Contamination rate = number of infected seedlings / total number of seedlings grafted × 100%.

[0129] Survival rate = number of survivals / number of transplants × 100%.

[0130] Excel 2013 and SPSS 19.0 software were used for data analysis.

[0131] 2 Results and analysis

[0132] 2.1 Induction of adventitious buds of Atractylodes lancea

[0133] 2.1.1 Effects of different hypocotyl cutting methods on adventitious bud induction

[0134] As shown in Table 4, both explant cutting methods A and C can achieve 100% induction of hypocotyls, while cutting method B has a lower induction rate of only 70%. In terms of proliferation coefficient, cutting method C has the highest average proliferation coefficient of 3.80. Although it is not significantly different from cutting method A, cutting method C is more convenient in practice. Figure 1 Taking all factors into consideration, the cutting method of "cross-cutting, cutting off roots, leaves and cotyledons, and retaining only the hypocotyl" is recommended as the best cutting method for the hypocotyl of sterile seedlings in the tissue culture production practice of Atractylodes lancea.

[0135] Table 4 Effects of different hypocotyl cutting methods on adventitious bud induction

[0136] Different ways of cutting embryonic axis Induction rate / % Proliferation coefficient A 100a 3.43a B 70b 1.60b C 100a 3.80a

[0137] [Note] Different letters indicate significant differences using LSD analysis (P < 0.05), and the same letters indicate no significant differences.

[0138] 2.1.2 Effects of different light intensity on adventitious bud induction

[0139] like Figure 2As shown, the selection of different light intensities has a significant effect on the proliferation coefficient of adventitious buds of Atractylodes lancea. Within a certain range, as the light intensity increases, the proliferation coefficient of Atractylodes lancea also has an increasing trend. When it reaches 6000lx, the proliferation coefficient of adventitious buds of Atractylodes lancea is the highest, and the average proliferation coefficient is 4.36, among which the highest proliferation coefficient of a single hypocotyl is 7. When the light intensity is 7000lx, the proliferation coefficient of adventitious buds of Atractylodes lancea decreases, and the excessive light intensity inhibits the induction of adventitious buds of explants. Therefore, in the practice of Atractylodes lancea tissue culture production, 6000lx is preferably used as the light condition for adventitious bud induction.

[0140] 2.2 Optimization of the proliferation culture medium of Atractylodes macrocephala

[0141] 2.2.1 Effects of different culture media on adventitious bud proliferation

[0142] A preliminary comparison of the growth of the clump seedlings induced by each culture medium showed that the seedlings induced by culture mediums 1 to 6 grew vigorously, with upright and green leaves; the seedlings induced by culture mediums 7 to 9 grew weakly, and some leaves wilted.

[0143] As shown in Table 5, the analysis of the factors in the orthogonal table showed that the effect of each factor on the proliferation induction of adventitious buds of Atractylodes lancea was 6-BA>NAA>sucrose. The adventitious bud proliferation coefficient of medium No. 2 (6-BA 1.0mg / L+NAA 0.5mg / L+sucrose 50g / L) was the highest, 12.96±1.546, followed by formula No. 6 (6-BA 3.0mg / L+NAA 1.0mg / L+sucrose 30g / L) and formula No. 1 (6-BA 1.0mg / L+NAA 0.1mg / L+sucrose 30g / L). Among them, the single bud proliferation coefficient of medium No. 2 was the highest, 15.

[0144] Table 5 Effects of different formulas on the proliferation of adventitious buds of Atractylodes lancea

[0145]

[0146] 2.2.2 Effects of different proliferation media on adventitious bud growth indicators

[0147] In order to further screen out the proliferation culture medium with high propagation coefficient, high uniformity of various growth indicators and suitable for industrial promotion of tissue culture, the culture medium formulas with the top three proliferation coefficients were selected, medium 1 (8.00±1.02), medium 2 (12.96±

[0148] 1.55) and medium No. 6 (8.71±0.62). Three clusters of adventitious buds were randomly selected from each treatment, and their growth indices such as plant height (cm), stem diameter (mm), number of effective leaves, fresh weight (g) and their uniformity were measured.

[0149] As shown in Table 6, the uniformity of growth indicators such as stem diameter, number of effective leaves, and fresh weight of adventitious buds induced by Formula No. 2 was higher than that of Medium No. 1 and Medium No. 6, and its uniformity of plant height was relatively high, only slightly lower than that of Medium No. 6. Although Formula No. 1 had high average values ​​of indicators such as plant height (cm), stem diameter (mm), number of effective leaves, and fresh weight (g), its uniformity was the lowest, and the adventitious buds induced by Formula No. 1 were quite different, such as Figure 3 Considering the proliferation coefficients of the three culture media and the growth indicators of their adventitious buds, it is recommended to use medium No. 2 (MS+6-BA 1.0mg / L+NAA 0.5mg / L+sucrose 50g / L) in the production practice of Atractylodes lancea tissue culture, which has a high proliferation coefficient and high uniformity of the induced adventitious buds, which is conducive to practical operation.

[0150] Table 6 Effects of different proliferation culture media on adventitious bud growth indicators

[0151] .

[0152] 2.3 Study on the rooting of Atractylodes macrocephala seedlings in vitro

[0153] 2.3.1 Screening of exogenous rooting culture medium

[0154] Table 7 Effects of different seedling medium on the survival rate of subcultured seedlings of Atractylodes lancea

[0155] deal with Survival rate / % T1 77c T2 98a T3 85b

[0156] [Note] Different letters indicate significant differences using LSD analysis (P < 0.05), and the same letters indicate no significant differences.

[0157] As shown in Table 7, the survival rates of Atractylodes lancea tissue culture seedlings in different culture medium ratios vary greatly, among which T2 (peat: perlite: vermiculite) = 3:1:1 has the highest survival rate, which can reach 98%, which is significantly different from the survival rates of T1 and T3.

[0158] 2.3.2 Morphological changes of Atractylodes macrocephala seedlings during ex-bottle rooting

[0159] In this experiment, tissue culture seedlings subcultured in bottles for 40 days were used as materials to observe and record the morphological changes of rooting outside the bottle of subcultured Atractylodes lancea seedlings. Figure 4 According to the growth status of the underground part, the rooting of subculture seedlings outside the bottle can be divided into three important stages, such as Figure 5 As shown in Table 8, the first stage A: new leaves and new roots grow; the second stage B: small fibrous roots (root hairs) grow on the new roots; the third stage C: the roots wrap around the substrate. The completion of rooting outside the bottle is shown in Table 8.

[0160] Table 8 Morphological characteristics of Atractylodes macrocephala subculture seedlings at different stages of rooting in vitro

[0161]

[0162] 2.3.3 Screening of the best cutting time for subculture seedlings

[0163] As shown in Tables 9 and 10, the morphology of the succession seedlings of Atractylodes lancea at different growth times is different, and the morphology of the aboveground part of the tissue culture seedlings after cuttings and rooting is also different. The overall growth of the succession seedlings from 21 to 27 days is the worst, and the growth is slow after cuttings and rooting. The petioles of the succession seedlings from 43 to 49 days are longer and have a tendency to grow too long. After cuttings and rooting, the petioles of the tissue culture seedlings are long and tender, which is not conducive to field transplanting. The overall growth of the succession seedlings from 36 to 42 days is the best, with strong growth and no leggy growth. After cuttings and rooting, the tissue culture seedlings have upright plants, dark green and leathery leaves, and good seedling quality, which is conducive to later transplanting. Figure 6 shown.

[0164] As shown in Table 10, the rooting rate of subcultured seedlings was positively correlated with rooting outside the bottle within a certain range of proliferation time, and negatively correlated with the infection rate. Subcultured seedlings that were cultured for 21 to 27 days are more delicate and are more likely to die from water loss during the rooting process under the same humidity and temperature control conditions, resulting in a small number of rooted plants in the statistics. The rooting rate of subcultured seedlings that were cultured for 36 to 42 days was the highest, reaching 98%. Combined with the morphological characteristics, growth status and growth time of the plants, in the production practice of Atractylodes macrocephala tissue culture, 36 to 42 days of subcultured seedlings are the best materials for rooting outside the bottle of Atractylodes macrocephala.

[0165] Table 9 External morphology of Atractylodes macrocephala seedlings at different growth times

[0166]

[0167] Table 10 Effects of subcultured shoots of Atractylodes lancea at different growth times on rooting of cuttings outside the bottle

[0168]

[0169]

[0170] [Note] Different letters indicate significant differences using LSD analysis (P < 0.05), and the same letters indicate no significant differences.

[0171] 2.3.4 Comparative study of rooting outside and inside the bottle

[0172] As shown in Table 11, rooting in the bottle: the growth time of the subculture seedlings in the rooting medium is 26 to 28 days, and the rooting rate is 98%. The strong seedlings with a height of about 4 cm and 4 to 5 new roots are selected. The seedling hardening time is 7 to 8 days. After transplanting, they grow for 17 to 20 days. When the seedlings grow new leaves, they can be considered alive. The whole process of rooting, hardening and transplanting takes 50 to 56 days, and the survival rate is 91%.

[0173] Rooting outside the bottle is the direct cutting of successive seedlings, omitting the rooting step in the bottle. The seedling hardening time is 5 to 6 days, the rooting time is 31 to 36 days, and the rooting rate is 98%. The rooting of cuttings outside the bottle ends when the roots wrap the substrate. The whole process takes 36 to 42 days, and the survival rate is 98%, which is significantly different from rooting in the bottle.

[0174] Table 11 Effects of two rooting methods on the hardening and transplanting of Atractylodes macrocephala seedlings

[0175] Rooting Method Rooting rate / % Survival rate / % Rooting in a bottle 98a 91b Rooting outside the bottle 98a 98a

[0176] [Note] Different letters indicate significant differences using LSD analysis (P < 0.05), and the same letters indicate no significant differences.

Claims

1. A method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods, characterized in that: The steps include: (1) Explant selection and sterilization The seeds of Atractylodes lancea were collected from Maoshan, Jurong, Jiangsu, cleaned and soaked in clean water, disinfected with 75% ethanol, disinfected with mercuric chloride, and rinsed with sterile water; then the seeds were inoculated in MS medium, and adventitious buds were induced with sterile seedlings of 12-15 days old as explants; (2) Adventitious bud induction from Atractylodes macrocephala explants Select the robust sterile seedlings of Atractylodes macrocephala with an age of 12 to 15 days, cut the hypocotyls, cut the hypocotyls in a cross-section, cut off the roots, leaves and cotyledons, and only keep the hypocotyls, and inoculate the cut hypocotyls in the adventitious bud induction medium to induce adventitious buds of Atractylodes macrocephala explants; (3) Subculture and proliferation of Atractylodes macrocephala When the adventitious buds of Atractylodes lancea grow to 2.0-2.5 cm, they are planted vertically into the subculture proliferation medium for subculture proliferation culture to obtain subculture clustered buds of Atractylodes lancea. (4) Rooting of Atractylodes macrocephala seedlings in vitro (4.1) Hardening of seedlings Select the successive generation clustered seedlings with a seedling height of ≥3 cm and <5 cm and 5-7 leaves in step (3), move them to an artificial climate chamber for seedling hardening, turn the bottle cap halfway open to continue hardening, and then open the bottle cap fully to harden the seedlings before cutting; (4.2) Cutting of subculture seedlings Use tweezers to gently remove the subcultured buds, clean the remaining matrix with water, separate the individual buds with scissors, remove the brown callus tissue and disinfect it; (4.3) Disinfection of subculture seedlings After the callus tissue at the base of the stem of the single bud seedling in step (4.2) is partially removed, the single bud seedling is immersed in potassium permanganate, taken out, rinsed and rooted; (4.4) Rooting of cuttings of successive seedlings Treat the 1-2 cm base of the seedling stem with NAA, then poke a small hole in the middle of the substrate in the plug tray, plant the successive seedlings in, press the soil to the planting depth, water the roots, and keep the leaves moist; (5) Transplanting of tissue culture seedlings When the roots of the tissue culture seedlings grow to cover the substrate and the rooting outside the bottle is completed, they are then transplanted into a conventional substrate seedling pot; The formula of the adventitious bud induction medium in step (2) is: MS medium + 6-BA 1.0 mg / L + NAA 0.15 g / L + sucrose 30 g / L, the agar content in the medium is 0.7%; the light condition is 6000 lx, and the light time is 12 h / d; The formula of the subculture proliferation medium in step (3) is: MS medium + 6-BA 1.0 mg / L + NAA 0.5 mg / L + sucrose 50 g / L, and the agar content in the medium is 0.7%; The substrate for rooting the cuttings in step (4) is made of peat, perlite and vermiculite in a volume ratio of 3:1:1; The vermiculite is prepared by the following method: 1.00 g of chitosan was weighed and placed in a beaker containing 200 mL of 2% acetic acid aqueous solution, and mechanically stirred at 60 °C for 2 h to obtain a uniform and transparent chitosan acetic acid aqueous solution; Vermiculite crushed to 80 mesh, expanded vermiculite was added to chitosan acetic acid aqueous solution at a solid-liquid ratio of 1:10, and stirred at a constant temperature of 60°C for 2 hours until the chitosan was completely dissolved; stirred at a constant temperature of 60°C for 6 hours, aged for 12 hours, and then filtered and separated to obtain a chitosan-modified vermiculite dispersion, which was washed and dried at 60°C to obtain chitosan-modified vermiculite.

2. The method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods according to claim 1, characterized in that: The steps include: (1) Explant selection and sterilization The seeds of Atractylodes lancea were collected from Maoshan, Jurong, Jiangsu Province, cleaned and soaked in clean water, and then the seeds were disinfected with 75% ethanol for 30 seconds, 0.1% mercuric chloride for 15 minutes, and rinsed with sterile water for 4 to 5 times, each time for no less than 3 minutes; then the seeds were inoculated in MS medium, and adventitious bud induction was carried out with 15-day-old sterile seedlings as explants; (2) Adventitious bud induction from Atractylodes macrocephala explants Select the robust sterile seedlings of Atractylodes macrocephala at 15 days old, cut the hypocotyls, cut the hypocotyls in a cross-section, cut off the roots, leaves and cotyledons, and only keep the hypocotyls, and inoculate the cut hypocotyls in the adventitious bud induction medium to induce adventitious buds of Atractylodes macrocephala explants; (3) Subculture and proliferation of Atractylodes macrocephala When the adventitious shoots of Atractylodes lancea grow to 2.0 cm, they are planted vertically into the subculture proliferation medium for subculture proliferation culture to obtain subculture cluster shoots of Atractylodes lancea. (4) Rooting of Atractylodes macrocephala seedlings in vitro (4.1) Hardening of seedlings Select the successive generation clustered seedlings with seedling height ≥3 cm and <5 cm and 5-7 leaves in step (3), move them to an artificial climate chamber for 3 days of seedling hardening, turn the bottle cap halfway open and continue hardening for 1 day, then fully open the bottle cap and harden for 2 days before cutting; (4.2) Cutting of subculture seedlings Use tweezers to gently remove the subcultured buds, clean the remaining matrix with water, separate the individual buds with scissors, remove the brown callus tissue and disinfect it; (4.3) Disinfection of subculture seedlings After the callus tissue at the base of the stem of the single bud seedling in step (4.2) was removed, the single bud seedling was immersed in 0.05% potassium permanganate for 60 s, taken out and rinsed for rooting treatment; (4.4) Rooting of cuttings of successive seedlings Treat 1-2 cm of the base of the seedling stem with 0.1 mg / mL NAA, then poke a 2.0 cm deep hole in the middle of the substrate of the plug tray, plant the successive seedlings in it, and plant them at a depth of 1-1.5 cm. Press the soil to make it firm, water it to root it, and keep the leaves moist; (5) Transplanting of tissue culture seedlings When the roots of the tissue culture seedlings grow to cover the substrate and the rooting outside the bottle is completed, they are then transplanted into a regular substrate seedling pot.

3. The method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods according to claim 1 or 2, characterized in that: Step (2) The pH of the adventitious bud induction medium is 5.8-6.0; the culture environment temperature is 25 ± 1°C and the humidity is 70%.

4. The method for cultivating Atractylodes lancea tissue seedlings by combining in-bottle and out-bottle methods according to claim 1 or 2, characterized in that: Step (3) The pH of the subculture proliferation medium is 5.8-6.0; the culture environment temperature is 25±1°C, the light condition is 6000 lx, the light time is 12 h / d, and the humidity is 70%.

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