Method for culturing tissue culture seedlings of small angel viridae

Abstract

The invention relates to the field of plant tissue culture, and discloses a method for culturing tissue culture seedlings of small angel viridae, which comprises the following steps: S1, explant selection and primary culture; s2, carrying out subculture multiplication culture; s3, rooting culture; s4, domesticating and transplanting; in the primary culture stage, MS is used as a basic culture medium, a proper amount of 6-BA, NAA, sucrose and agar are added, and induction of cluster buds is promoted; in the subculture multiplication culture stage, an optimal multiplication culture medium is screened through an orthogonal test, so that the multiplication coefficient is improved; in the rooting stage, key factors such as inorganic salt concentration, NAA and banana puree in MS are optimized, and the number and quality of root systems are improved; in the domestication transplanting stage, the optimal proportion of river sand, coco coir and peat soil is researched, and the transplanting survival rate and the tissue culture seedling quality are effectively improved. By means of the method, efficient propagation of the small angel vitis vinifera and large-scale production of high-quality seedlings are achieved, and important technical support is provided for industrial popularization of the plant.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for culturing angelica philodendron tissue culture seedlings. Background Art

[0002] Philodendron cv.Xanadu is an evergreen plant of the genus Philodendron in the family Araceae. It has a unique leaf shape and is evergreen all year round. It has a high ornamental value and is a common indoor potted plant and garden plant. The plant is native to Brazil and is adapted to high temperature and high humidity environments. It has a certain degree of cold resistance, but is easily affected by long-term low temperatures in winter. Due to the growing market demand for Philodendron, its seedling propagation method directly affects production efficiency and seedling quality.

[0003] At present, Angelica Philodendron mainly relies on division for propagation. However, division has a low propagation coefficient and a long cycle, which makes it difficult to meet the market demand for high-quality seedlings. In addition, during division propagation, due to improper operation or poor control of environmental conditions, problems such as uneven growth of seedlings, poor root development and low survival rate often occur, which is particularly evident in large-scale planting production.

[0004] In order to solve these problems, plant tissue culture technology has gradually been applied to the rapid propagation of plant seedlings. Although tissue culture methods have been used in the propagation of other plants to a certain extent, there are few studies on the tissue culture of Angelica dahurica. The existing technology has not formed a systematic solution for the formulation of culture medium, the selection of culture conditions, and the optimization of culture technology at different stages. In particular, there are still obvious technical deficiencies in key links such as explant sterilization and induction in the primary culture stage, the increase in the proliferation rate of subculture, the root system development in the rooting stage, and the improvement of the survival rate in the domestication and transplanting stage. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a method for culturing tissue culture seedlings of Angelica dahurica, which solves the problems of low reproduction efficiency, poor seedling quality and low transplant survival rate in the existing reproduction of Angelica dahurica seedlings.

[0006] To achieve the above objectives, the present invention is implemented through the following technical scheme: a method for culturing angelica philodendron tissue culture seedlings, comprising the following steps:

[0007] S1. Explant selection and primary culture: Select healthy angel philodendron tubers as explants, sterilize them under sterile conditions, and inoculate the explants in a culture medium, wherein the culture medium is MS culture medium, 6-BA 1.0-2.0 mg / L, NAA 0.05-0.15 mg / L, sucrose 20-30 g / L and agar 6-8 g / L are added, the pH is adjusted to 5.8-6.0, the culture temperature is 23-27°C, the light intensity is 2000-4000 Lx, and the photoperiod is 14-16 h / d;

[0008] S2, secondary proliferation culture: the clustered buds obtained from the primary culture were cut into clumps of 3 small buds, and inoculated into a proliferation medium, wherein the proliferation medium is MS medium, supplemented with 6-BA 1.0-2.0 mg / L, KT 0.5-1.5 mg / L, NAA 0.05-0.15 mg / L, sucrose 20-30 g / L and agar 6-8 g / L, and the pH is adjusted to 5.8-6.0;

[0009] S3, rooting culture: cutting the seedlings of subculture proliferation culture into individual plants, inoculating them into rooting medium, wherein the rooting medium is 1 / 4-3 / 4 MS medium, supplemented with NAA 0.5-1.5 mg / L, banana puree 50-150 g / L, sucrose 15-25 g / L and agar 4-6 g / L, and the pH is adjusted to 5.8-6.0;

[0010] S4, acclimatization and transplantation: the tissue culture seedlings obtained by the rooting culture are hardened, and after gradually adapting to the external environment in the natural environment, the seedlings are transplanted into a sterilized substrate, which is a mixture of peat soil and coconut bran.

[0011] Preferably, the explant sterilization treatment comprises the following steps:

[0012] 1) Sterilize with 75% alcohol solution for 30-60 seconds;

[0013] 2) Sterilize with 0.1%-0.3% mercuric chloride solution or 1%-2% sodium hypochlorite solution for 5-10 minutes;

[0014] 3) Rinse 3-6 times with sterile water.

[0015] Preferably, the temperature of the primary culture, secondary proliferation culture and rooting culture is room temperature, the photoperiod is 14-16h / d, and the light intensity is 2500-3500Lx.

[0016] Preferably, the proliferation cycle of the subculture proliferation culture is 20-30 days, and the proliferation coefficient is 3.5-5.0.

[0017] Preferably, the rooting culture has a culture time of 25-35 days, a rooting rate of 75%-90%, an average number of roots of 2-5, and an average root length of 2.5-4.0 cm.

[0018] Preferably, the transplanting matrix sterilization method comprises:

[0019] Soak the substrate in 500-600 times carbendazim solution for 3-6 minutes;

[0020] When transplanting, the moisture content of the substrate should be maintained at 30%-50%.

[0021] Preferably, the management during the acclimatization and transplanting process includes:

[0022] Shade treatment is carried out in the early stage of transplanting, and the light intensity is controlled at 1500-2000Lx;

[0023] Spray water on the leaves 2-3 times a day, and gradually increase the light intensity to more than 3000Lx 1-2 weeks after transplanting.

[0024] Preferably, the volume ratio of peat soil to coconut husk in the matrix is ​​2:1.

[0025] The present invention provides a method for cultivating angelica philodendron tissue culture seedlings. It has the following beneficial effects:

[0026] 1. The present invention adopts plant tissue culture technology and realizes the rapid propagation of Philodendron cherubim by optimizing the technical parameters of primary culture, secondary proliferation culture, rooting culture and domestication and transplantation. Compared with traditional division propagation, the propagation coefficient is greatly improved, the seedling raising cycle is shortened, and the large demand for seedlings in the market is effectively met.

[0027] 2. The present invention significantly improves the induction rate of clustered buds, the rooting rate and the overall growth quality of seedlings by optimizing the culture medium components at each culture stage. The cultured tissue culture seedlings have a well-developed root system and strong growth of the aboveground parts, laying a good foundation for subsequent transplanting.

[0028] 3. In the process of domestication and transplantation, the present invention adopts scientific and reasonable substrate ratios and domestication management measures, optimizes the use ratio of river sand, coconut bran and peat soil, and stably maintains the transplantation survival rate at above 90%. Among them, the survival rate of the best substrate ratio group reaches 100%, which significantly improves the success rate of seedling cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the angel philodendron tuber is selected for the present invention;

[0030] Figure 2 This is a schematic diagram of the tuber disinfection process of the present invention;

[0031] Figure 3 This is a schematic diagram of the clustered adventitious buds obtained after the induction culture of the present invention;

[0032] Figure 4 This is a schematic diagram of the clustered buds after subculture of the present invention;

[0033] Figure 5 This is a schematic diagram of tissue culture seedlings obtained after rooting culture of the present invention;

[0034] Figure 6 This is a schematic diagram of the root growth of tissue culture seedlings after rooting culture of the present invention;

[0035] Figure 7 It is a side view of the tissue culture seedling after root culture of the present invention;

[0036] Figure 8 It is a top view of the growth state of the tissue culture seedlings after hardening of the present invention;

[0037] Fig. 9 This is a schematic diagram of cleaning the tissue culture seedlings after hardening of the present invention;

[0038] Fig.10 This is a schematic diagram of the transplanted Philodendron cherubim tissue culture seedlings of the present invention;

[0039] Fig.11 A schematic diagram showing a comparison of fresh weights of seven matrix ratios in an embodiment of the present invention;

[0040] Fig.12 Schematic diagram of the average chlorophyll content of seven substrate ratios in the embodiments of the present invention. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the specification of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Please see attached Figure 1 -Attached Fig.10 The present invention provides a method for culturing angelica philodendron tissue culture seedlings, comprising the following steps:

[0043] S1. Explant selection and primary culture: Select healthy angelica philodendron tubers as explants, sterilize them under aseptic conditions, and inoculate the explants into culture medium;

[0044] S2, secondary proliferation culture: the clustered buds obtained from the primary culture were cut into clumps of three small buds and inoculated into proliferation medium;

[0045] S3, rooting culture: cutting the subcultured seedlings into individual plants and inoculating them into rooting medium;

[0046] S4. Acclimation and transplantation: The tissue culture seedlings obtained by rooting culture are hardened and gradually adapted to the external environment under natural conditions. Then, the seedlings are transplanted into sterilized substrates.

[0047] Step S1 is intended to provide sterile materials for the cultivation of Philodendron cherubim, and to induce the formation of buds or callus through appropriate culture medium and conditions, thereby laying a foundation for subsequent subculture proliferation culture.

[0048] In this embodiment, healthy and disease-free angelica philodendron tubers are selected as explants. The explants are preferably collected during the vigorous growth period of the plant (such as spring or early summer) to ensure cell activity and induction effect.

[0049] As an option, the size of the explant should be controlled at about 1-2 cm, and the cut surface of the petiole should be kept smooth to reduce mechanical damage to the cells. At the same time, to reduce the risk of contamination, it should be handled as soon as possible after collection and avoid contamination during collection and transportation. Figure 1 As shown, a schematic diagram of the use of the angelica philodendron tuber in the present invention is shown.

[0050] Specifically, in terms of explant sterilization, the present invention adopts a step-by-step sterilization method:

[0051] Exemplarily, the surface of the explant is first rinsed with clean water to remove obvious dirt and attachments.

[0052] Afterwards, the explants were immersed in a 75% (volume fraction) alcohol solution for 30-60 seconds for preliminary sterilization.

[0053] Then, soak the explants in a 0.1%-0.3% mercuric chloride solution or a 1%-2% sodium hypochlorite solution for 8-10 minutes to achieve deep sterilization. Figure 2 As shown, a schematic diagram of the tuber disinfection treatment of the present invention is shown.

[0054] After sterilization, rinse with sterile distilled water 3-6 times, 1 minute each time, to completely remove the residual sterilization solution.

[0055] It should be noted that the concentration and immersion time of the mercuric chloride or sodium hypochlorite solution can be appropriately adjusted according to the specific properties of the explants. For example, for relatively young tubers, a lower concentration of sterilization solution can be used to avoid chemical damage to the tissue during the sterilization process.

[0056] In a possible implementation, the preparation of the culture medium is crucial. In this embodiment, the primary culture medium is based on MS (Murashige and Skoog) culture medium, and the following components are added:

[0057] 6-BA (6-benzylaminoadenine) 1.0-2.0 mg / L, mainly used to promote bud differentiation and cell proliferation;

[0058] NAA (1-naphthylacetic acid) 0.05-0.15 mg / L, used to regulate cell division and improve the induction efficiency of callus tissue;

[0059] Sucrose 20-30 g / L, as the main carbon source required for explant growth;

[0060] Agar 6-8g / L is used to solidify the culture medium and provide a stable supporting environment.

[0061] It is understood that the pH value of the culture medium has a significant effect on the growth and differentiation of the explants. In this embodiment, the pH value of the culture medium is adjusted to 5.8-6.0 to maintain a suitable physiological environment. After the preparation is completed, the culture medium is placed in an autoclave at 121° C. for sterilization for 20 minutes.

[0062] It should be further noted that the ratio of hormones in the culture medium can be adjusted according to the type of explant and the growth stage. For example, in some embodiments, during the subculture proliferation culture, appropriately increasing the concentration of 6-BA (e.g., 1.5-2.0 mg / L) can induce bud differentiation faster.

[0063] In one possible implementation, the inoculated explants are placed in culture conditions as follows:

[0064] Temperature: 23-27℃, suitable growth range for Angelica dahurica;

[0065] Photoperiod: 14-16 hours / day, light intensity 2000-4000Lx to simulate the natural light environment and promote bud differentiation.

[0066] It should be noted that it is recommended to keep the culture away from light for 3-5 days in the early stage of culture to promote the recovery of the explants. After that, the light intensity and time should be gradually increased to induce the formation of buds or callus tissue.

[0067] Exemplarily, the primary culture time is 20-25 days, and significant growth of buds or callus can usually be observed. If the explants are browned or contaminated, they should be removed in time and the relevant parameters should be recorded.

[0068] As an improvement measure, an appropriate amount of antioxidants (such as ascorbic acid or activated carbon) can be added to the culture medium to reduce the browning of the explants. For example, the addition of 0.1 g / L ascorbic acid can significantly increase the survival rate of the explants.

[0069] The purpose of step S2 is to promote the rapid differentiation and proliferation of clustered buds through appropriate culture medium and culture conditions, thereby improving the reproduction efficiency and providing sufficient bud material for subsequent rooting culture.

[0070] In this embodiment, the clustered buds formed in the primary culture stage are cut into uniform-sized clumps, each of which contains 3 small buds to ensure the consistency of the proliferation effect. As an option, a sterile scalpel or a scalpel can be used for cutting, and mechanical damage should be avoided as much as possible during cutting.

[0071] Specifically, after the cluster buds are cut, they are inoculated into a subculture proliferation medium. The medium is based on MS (Murashigeand Skoog) medium and supplemented with the following ingredients:

[0072] 6-BA (6-benzylaminoadenine) 1.0-2.0 mg / L, used to promote rapid differentiation of buds;

[0073] KT (kinetin) 0.5-1.5 mg / L, used to coordinate with 6-BA to regulate the differentiation and balanced growth of buds;

[0074] NAA (1-naphthylacetic acid) 0.05-0.15 mg / L, the main function is to inhibit excessive callus formation and maintain cell division balance;

[0075] Sucrose 20-30 g / L, as the main carbon source for explant growth;

[0076] Agar 6-8g / L provides a stable supporting environment.

[0077] It is understandable that the ratio of hormones is one of the key technical parameters of proliferation culture. 4 )) got the optimal ratio, namely 6-BA 1.5mg / L, KT 0.5mg / L, NAA 0.1mg / L. Under this combination, the proliferation coefficient reached 4.17, showing a significant proliferation effect.

[0078] In a possible implementation, the pH value of the culture medium is adjusted to 5.8-6.0 and used after high pressure sterilization (121° C., 20 minutes). It should be noted that the culture medium needs to be cooled to room temperature after sterilization before inoculation to avoid damage to the clustered buds caused by high temperature.

[0079] The inoculated culture flasks were placed in the following culture conditions:

[0080] Temperature: 25±2℃, suitable for cell division and differentiation of Angelica philodendron;

[0081] Photoperiod: 16 hours / day, light intensity 3000Lx, used to promote photosynthesis and support the growth of cluster buds;

[0082] Culture period: 20-30 days. The specific culture time is determined by monitoring the differentiation and proliferation of buds.

[0083] It should be further noted that browning or uneven bud differentiation may occur during the subculture process. As an improvement measure, 0.1-0.5 g / L ascorbic acid or 0.1-0.2% activated carbon can be added to the culture medium to reduce browning and promote uniform growth of buds.

[0084] Exemplarily, in some embodiments, the proliferation efficiency and quality of the clustered shoots are significantly affected by the composition of the culture medium. For example:

[0085] When the sucrose concentration was 20 g / L, the proliferation rate of clustered buds was faster, but the buds were relatively weak.

[0086] When the sucrose concentration was 30 g / L, the quality of the sprouts was better, but the proliferation coefficient was slightly reduced;

[0087] Taking all factors into consideration, the optimal range of sucrose concentration is 25-30 g / L.

[0088] It should be noted that the ratio of 6-BA to KT in the culture medium has a synergistic effect on the differentiation and proliferation of buds. For example, the bud differentiation efficiency is highest when 6-BA 1.5 mg / L and KT 0.5 mg / L are combined, while when the 6-BA concentration is higher than 2.0 mg / L, the differentiation rate of buds decreases and some buds become deformed.

[0089] As an option, in this embodiment, during the culture process, the differentiation of buds is observed every 5-7 days, and the number, length, color change and other indicators of buds are recorded. At the end of the culture period, the clustered buds can usually differentiate into 4-6 new buds, and the proliferation coefficient of each bottle reaches 3.5-5.0. Figure 3 and 4 As shown, a schematic diagram of the clustered adventitious buds obtained after the induced culture of the present invention and the clustered buds after subculture is respectively shown.

[0090] For step S3, its main purpose is to promote the formation of a strong root system of the seedlings through optimized culture medium formula and environmental conditions, thereby laying the foundation for subsequent transplanting.

[0091] In this embodiment, the sprouts obtained by the subculture proliferation culture are cut into individual plants. As an option, old leaves, residual leaves and brown tissues at the base can be removed during cutting to ensure that the sprouts are healthy and can efficiently take root. The cut individual plants are rinsed with sterile water to remove residual culture medium and avoid contamination.

[0092] Specifically, the rooting medium is based on 1 / 4-3 / 4 MS medium and adds the following ingredients:

[0093] NAA (1-naphthylacetic acid): 0.5-1.5 mg / L, used to promote root formation and extension;

[0094] Banana puree: 50-150g / L, as a natural additive to provide additional nutrients and hormones;

[0095] Sucrose: 15-25g / L, providing energy source;

[0096] Agar: 4-6g / L, used as a solidifying agent to ensure the stability of the culture medium.

[0097] It is understandable that NAA, as a key hormone in the rooting stage, has a concentration that directly affects the number and quality of the root system. In one possible implementation, when the concentration of NAA is between 0.5 and 1.0 mg / L, the number of roots is large and evenly distributed; when the concentration is higher than 1.5 mg / L, the number of roots decreases and is prone to overgrowth or deformity.

[0098] It should be noted that banana puree, as a natural additive in the culture medium, can not only provide the nutrients required for rooting, but also play a significant hormone-assisted role. In this embodiment, the addition amount of banana puree is 50-150 g / L, wherein 100 g / L has the best effect, which helps to form a thick and evenly distributed root system.

[0099] Exemplarily, the pH value of the culture medium is adjusted to 5.8-6.0 to maintain a suitable physiological environment. The culture medium is sterilized by high pressure sterilization (121° C., 20 minutes), and a single sprout is inoculated after cooling to room temperature.

[0100] The inoculated culture flasks were placed in the following culture conditions:

[0101] Temperature: 25±2℃, which is the best temperature range for rooting of Angelica dahurica;

[0102] Photoperiod: 16 hours / day, light intensity 3000Lx, to promote balanced growth of roots and sprouts;

[0103] Cultivation time: 25-35 days, adjust the cultivation time according to the specific situation of the seedlings.

[0104] It should be further explained that, during the rooting culture process, the number, length and thickness of the roots are important indicators for evaluating the effect. The optimized formula of the present invention can achieve the following effects within the culture cycle:

[0105] Average number of roots: 2-5;

[0106] Average root length: 2.5-4.0cm;

[0107] Root morphology: thick, white, and without deformity.

[0108] like Figure 5 , 6 7 and 7 show the tissue culture seedlings obtained after rooting culture, the root growth of the tissue culture seedlings, and the side view of the tissue culture seedlings, respectively.

[0109] In some embodiments, in order to further improve the rooting effect, an appropriate amount of antioxidants (such as 0.1-0.2 g / L ascorbic acid) can be added to the culture medium to reduce the browning of the seedlings and maintain the health of the root system. In addition, by adjusting the light intensity and temperature, the quality of the root system can be further optimized. For example, when the light intensity is low (2500Lx), the number of roots increases but the length decreases; when the light intensity is high (3500Lx), the root length increases but the distribution is slightly sparse.

[0110] The purpose of step S4 is to gradually adapt the tissue culture seedlings to the external environment through reasonable seedling hardening and transplanting techniques, establish stable root function and photosynthesis capacity, and thus improve the transplant survival rate.

[0111] It should be noted that since tissue culture seedlings are in a high-humidity and sterile environment for a long time, their root absorption capacity and the protective effect of the leaf cuticle are weak. Therefore, they need to be fully domesticated when transplanted, and appropriate substrates and management measures must be selected to ensure seedling quality and growth performance.

[0112] In this embodiment, the angelica philodendron tissue culture seedlings obtained in the rooting culture stage are subjected to a domestication operation. As an option, the tissue culture seedlings can be domesticated under natural light conditions to gradually adapt to the external environment.

[0113] Specifically, the seedling hardening operation includes the following steps:

[0114] Move the tissue culture seedlings together with the culture bottles to the seedling acclimatization room, where the temperature is maintained at 25±2°C and the humidity is maintained at 60%-70%.

[0115] In the early stage of hardening (1-3 days), only slightly open the culture bottle cap to maintain a high humidity inside and prevent the leaves from losing water quickly. Then, gradually increase the angle and time of opening the cap every 1-2 days until the bottle cap is completely removed on the 7th day.

[0116] It should be noted that strong direct sunlight should be avoided during the seedling hardening process to prevent the leaves from drying up due to excessive transpiration. It is understandable that moderate shading (light intensity of about 1500-2000Lx) helps the gradual adaptation of tissue culture seedlings.

[0117] In a possible implementation, after the acclimatization is completed, the tissue culture seedlings are taken out from the culture medium, and the roots are washed with sterile water to remove the attached culture medium residues. Exemplarily, to avoid damaging the root system and the sprouts, a soft brush can be used to gently brush off the stubborn residues.

[0118] The cleaned tissue culture seedlings need to be properly dried. As an option, you can use sterile gauze to absorb excess moisture and place the tissue culture seedlings in a cool and ventilated place for 1-2 hours to further reduce surface humidity and reduce the risk of infection during transplantation.

[0119] Specifically, the substrate used for transplanting is a mixture of peat soil and coconut husk, and the volume ratio thereof is 1:2 or 2:1. It should be noted that different substrate ratios have a significant effect on the survival rate and growth performance of tissue culture seedlings.

[0120] In a possible implementation, the roots of the tissue culture seedlings are spread out during transplanting, and the planting depth is 0.5-1.5 cm to ensure that the roots are fully in contact with the substrate. For example, the substrate is lightly compacted after transplanting to stabilize the roots while avoiding excessive compaction that may reduce air permeability.

[0121] The management measures after transplanting directly determine the survival rate of tissue culture seedlings. In this embodiment, the following techniques are used for refined management:

[0122] Spray "planting water" immediately after transplanting to keep the substrate moist, which will help the roots quickly restore their absorption function.

[0123] In the early stage of transplanting (the first 7 days), the seedbed is shaded and the light intensity is controlled at 1500-2000Lx to reduce leaf transpiration.

[0124] Spray water on the leaves 2-3 times every morning and evening, but avoid excessive spraying to cause water accumulation; the principle of watering the substrate is "don't water unless it's dry, and water thoroughly when you do."

[0125] It should be noted that after 7 days, the light intensity can be gradually increased and eventually restored to more than 3000Lx to promote normal photosynthesis of tissue culture seedlings. At the same time, an appropriate amount of slow-release fertilizer (such as nitrogen, phosphorus and potassium compound fertilizer) can be added to the substrate to support later growth.

[0126] like Figure 8 , 9 10 , respectively show a top view of the growth state of the tissue culture seedlings after hardening of the present invention, a schematic diagram of tissue culture seedling cleaning, and a schematic diagram of the tissue culture seedlings of Angelica dahurica after transplantation.

[0127] In order to better understand the present invention, the above method is described in detail below in conjunction with specific embodiments.

[0128] Embodiment 1:

[0129] In this embodiment, the clustered buds of Philodendron cherubim were induced by treating and culturing the explants of the excellent mother plant.

[0130] Tubers were collected from excellent angel philodendron mother plants as explants. First, the tubers were rinsed under running water to remove dust and impurities attached to the surface. Then, the tubers were soaked in a 0.5% detergent solution for 5 minutes and stirred continuously to further remove impurities and oil stains on the surface. After washing, they were rinsed again with running water for 8 hours to ensure a clean surface.

[0131] The tuber explants were sterilized on a clean bench. The specific steps are as follows:

[0132] 1. Soak the tubers in 75% alcohol for 30 seconds, shaking gently to kill microorganisms attached to the surface. Then rinse with sterile water three times to completely remove alcohol residue.

[0133] 2. Soak the tubers in a 0.1% mercuric chloride (HgCl2) solution for 10 minutes, with gentle shaking, to kill deep-seated pathogenic microorganisms. After soaking, rinse with sterile water 5 times, 1 minute each time, to ensure that the mercuric chloride residue is completely removed.

[0134] 3. Gently dry the surface of the tuber with sterile filter paper. Under sterile conditions, cut the tuber into small pieces of about 1 cm × 1 cm, making sure the cut surface is flat to reduce tissue damage.

[0135] The cut tuber explants were inoculated in the primary culture medium. The culture medium formula was: MS (Murashige and Skoog) medium, 6-BA (6-benzylaminoadenine) 1mg / L, NAA (1-naphthaleneacetic acid) 0.1mg / L, agar 7g / L, sucrose 30g / L, pH adjusted to 5.8. The culture medium was autoclaved at 121°C for 20 minutes and cooled for use.

[0136] The inoculated culture bottles were placed in a culture room, and the culture conditions were: temperature 25±2°C, photoperiod 16 hours / day, and light intensity 3000Lx.

[0137] Embodiment 2:

[0138] In this example, the culture medium formula and culture conditions were optimized to perform subculture proliferation culture on the clustered shoots of Philodendron cherubim, so as to obtain a large number of healthy tissue culture seedlings.

[0139] The clustered buds obtained from the primary culture were cut into uniform-sized clumps, each containing 3 small buds, and inoculated into the proliferation medium. During inoculation, one clump was inoculated per bottle, and the height of the buds was about 2 cm to ensure the consistency of the proliferation conditions. The culture medium was based on MS, agar 7g / L, and the pH was adjusted to 5.8-6.0.

[0140] To optimize the proliferation effect, the experiment adopted a 4-factor 3-level orthogonal design L9 (3 4 ), the experimental factors and levels are shown in Table 1, including the optimization ranges of the following components:

[0141] 6-BA (6-benzylaminoadenine): 1.0-2.0 mg / L, used to promote bud differentiation and cell proliferation;

[0142] KT (kinetin): 0.5-1.5 mg / L, assists bud differentiation and balanced growth;

[0143] NAA (1-naphthylacetic acid): 0.05-0.15 mg / L, used to regulate cell division;

[0144] Sucrose: 20-30 g / L, provides the carbon source required by the culture medium.

[0145] Table 1 Proliferation culture of Philodendron cherubim L9 (3 4 )Orthogonal experimental factors and levels

[0146]

[0147] The inoculated tissue culture seedlings were placed in a culture room under the following culture conditions: temperature 25±2°C, photoperiod 16 hours / day, and light intensity 3000Lx. During the culture process, the proliferation of clustered buds was recorded every 5 days.

[0148] The test results show that different culture medium components have a significant effect on the proliferation coefficient of the clustered shoots of Little Angel Philodendron. The specific data are shown in Table 2, where the range analysis results of the test factors on the proliferation effect are: 6-BA> sucrose> KT> NAA, indicating that 6-BA has the greatest effect on the proliferation effect of Little Angel Philodendron, while NAA has the least effect.

[0149] Table 2 Analysis of the range of proliferation coefficients of different proliferation culture media

[0150]

[0151]

[0152] Note: The numbers in brackets represent factor levels, the same below.

[0153] From the test results, it can be seen that the culture medium formula of group 4 is the optimal combination, namely:

[0154] MS+1.5mg / L 6-BA+0.5mg / L KT+0.1mg / L NAA+30g / L sucrose+7g / L agar, the proliferation coefficient reached the highest value of 4.17.

[0155] Under the optimal culture medium conditions, the clustered buds showed a good proliferation state, the buds were strong and bright green, and the number of differentiated buds was significantly higher than that of other treatment groups. Compared with other formulas (such as Group 7, Group 8, and Group 9), this group not only had the highest proliferation coefficient, but also had good growth consistency of clustered buds, avoiding incomplete or deformed bud differentiation caused by unbalanced culture medium.

[0156] Further analysis showed that 6-BA, as the main factor, showed a trend of increasing first and then decreasing in its concentration on the proliferation coefficient. When the concentration of 6-BA was 1.5 mg / L, the differentiation rate of clustered buds was the fastest and the proliferation effect was the best; when the concentration increased to 2.0 mg / L, the proliferation coefficient decreased significantly (such as group 7, the proliferation coefficient was only 0.47), which may be because the high concentration of 6-BA inhibited cell division. In addition, an appropriate amount of sucrose (30 g / L) can significantly enhance the growth vitality of seedlings and has a synergistic effect with 6-BA.

[0157] Embodiment 3:

[0158] On the clean bench, the tissue culture seedlings of Little Angel Philodendron were processed, the residual leaves, old leaves and browned and aged tissues at the base were cut off, and the tissue culture seedlings were divided into single plants. The divided tissue culture seedlings were inoculated in 9 different rooting culture media (medium composition: sucrose 20g / L, agar powder 5g / L, pH 5.8-6.0) in an orthogonal experimental design under sterile conditions, 3 plants were inoculated in each bottle, and 20 bottles were set for each treatment.

[0159] The inoculated tissue culture seedlings were placed in a culture room under the conditions of 25±2℃, 3000Lx light intensity, and 16 hours / day photoperiod. The rooting of the tissue culture seedlings was observed every 30 days, including the number of roots, root length, plant height and other parameters, and detailed records and statistical analysis were performed.

[0160] Experimental factors and culture medium formulation

[0161] The composition of the rooting medium was based on L9(3 4 )Orthogonal experimental design was used for optimization, and the specific factors and levels are shown in Table 3.

[0162] Table 3 Angel Philodendron rooting medium L9 (3 4 )Orthogonal experimental factors and levels

[0163]

[0164] Experimental factors include:

[0165] MS medium concentration (1 / 4MS, 1 / 2MS, 3 / 4MS);

[0166] NAA (1-naphthylacetic acid) concentration (1.0 mg / L, 2.0 mg / L, 3.0 mg / L);

[0167] 6-BA concentration (0, 0.5 mg / L, 1.0 mg / L);

[0168] Banana puree content (50g / L, 100g / L, 150g / L).

[0169] Test results and analysis

[0170] According to the test results, there are significant differences in rooting coefficients and root growth conditions between different culture media. The statistics and range analysis of rooting coefficients are shown in Table 4, and the data on root growth characteristics are shown in Table 5.

[0171] Table 4 Analysis of the rooting coefficient range of different rooting media

[0172]

[0173]

[0174] Table 5 Effects of different rooting media on root growth

[0175]

[0176] From the data in Table 2 and Table 3, we can get:

[0177] The best rooting medium was treatment 1, which had the formula of 1 / 4MS+1.0mg / L NAA+0mg / L6-BA+50g / L banana puree. Under this condition, the average number of roots of the tissue culture seedlings reached 3.46, the rooting rate was 80%, and the average root length was 3.89cm.

[0178] With the increase of inorganic salt concentration in MS, the rooting number showed a downward trend; with the increase of 6-BA concentration and banana puree content, the rooting coefficient showed a trend of first decreasing and then increasing, and first increasing and then decreasing.

[0179] The results of range analysis showed that the concentration of inorganic salts in MS had the greatest impact on the rooting effect, while 6-BA had the least impact. The order of the influence of different factors on the rooting coefficient was: MS>NAA>banana puree>6-BA.

[0180] Effects of different culture media on root growth

[0181] Treatment 1: The tissue culture seedlings took root the fastest, with a strong root system, an average root length of 3.89 cm, an average root number of 3.46, emerald green leaves, and strong plants.

[0182] Treatment 2: The tissue culture seedlings grew well, the plants were large and strong, the average root length was 2.56cm, the root system was thick, and the leaves were slender.

[0183] Treatment 3: The root system is more abundant with root hairs, but the root length is shorter (1.24 cm), the plant is short, and the leaves are few and yellow.

[0184] Treatment 4: Rooting was slow, with an average root length of 0.93 cm and a thick root system, but the plant was overall short, new leaves grew slowly, and some old leaves turned yellow.

[0185] Treatment 5: The roots grew faster, with an average root length of 0.97 cm, but some roots had callus tissue. The plants were strong and the leaves were green and large.

[0186] Treatment 6: The root system was relatively thick, with an average root length of 0.81 cm, and the plant stem was thick, but the number of roots was small.

[0187] Treatment 7: The root system is thick, with an average root length of 2.04 cm, the plant is strong, but the number of roots is small.

[0188] Treatment 8: The root system was thin, with an average root length of only 0.36 cm, and more callus tissue appeared on the plants.

[0189] Treatment 9: The root system was the worst, with a root length of only 0.19 cm, short roots and few in number, and the plant stems were thin and weak.

[0190] Summary of results

[0191] The best culture medium formula: 1 / 4MS+1.0mg / L NAA+0mg / L 6-BA+50g / L banana puree. Under this formula, the average root length, root number and rooting rate of tissue culture seedlings all reached the best.

[0192] Key factors for rooting effect: MS medium concentration has the greatest impact on rooting effect, followed by NAA concentration and banana puree content, and 6-BA has the least effect.

[0193] Root morphology: The roots of the tissue culture seedlings under the best medium were thick, moderate in length, with a large number of roots, strong plants and good growth consistency. The roots of the tissue culture seedlings treated with the best medium were significantly better than those of the other treatment groups.

[0194] By means of the rooting culture method of the present invention, angelica philodendron tissue culture seedlings with good root morphology and high rooting rate can be efficiently obtained, thus providing high-quality materials for subsequent transplanting and commercial production.

[0195] Embodiment 4:

[0196] This example designs seven substrate ratios for the tissue culture seedlings of Philodendron cherubim, and conducts a comprehensive study on the effects of its acclimatization and transplantation. The experiment screens out suitable substrate ratio combinations by analyzing indicators such as survival rate, fresh weight, chlorophyll content, root growth, leaf number, and plant height, providing a scientific basis for subsequent transplantation.

[0197] Materials and steps

[0198] 1. Hardening of seedlings

[0199] Move the angel philodendron tissue culture seedlings into the seedling hardening room and place them in the natural environment for 7 days. During this period, gradually open the caps of the tissue culture bottles to allow the tissue culture seedlings to gradually adapt to the external environment.

[0200] 2. Cleaning the seedlings

[0201] Take out the tissue culture seedlings after hardening, wash them carefully with clean water to remove the culture medium remaining at the roots to avoid damaging the seedlings.

[0202] 3. Matrix Preparation

[0203] The matrix is ​​prepared according to different ratios. The matrix combination includes:

[0204] A: River sand;

[0205] B: Coconut bran;

[0206] C: peat soil;

[0207] D: river sand: coconut bran = 1:1;

[0208] E: river sand: coconut bran = 1:2;

[0209] F: peat soil: coconut bran = 1:2;

[0210] G: Peat soil: coconut coir = 2:1.

[0211] Soak the substrate with 500 times carbendazim wettable powder solution for 5 minutes, then take it out and dry it for later use.

[0212] 4. Transplantation operation

[0213] Transplant the tissue culture seedlings into the prepared substrate according to the root length, and control the planting depth at 0.5-1.5cm to allow the roots to stretch.

[0214] 5. Management after transplanting

[0215] Spray transplanting water to accelerate rooting;

[0216] Spray the leaves with clean water every morning and evening to prevent them from becoming dehydrated due to excessive transpiration;

[0217] Keep the seedbed moist, but follow the principle of "don't water unless it's dry, and water thoroughly when you do";

[0218] Appropriate shading is required in the early stage, and the light should be gradually increased after 7 days.

[0219] 6. Data Recording

[0220] The survival rate was calculated 10 days after transplanting;

[0221] Rooting was counted after 15 days;

[0222] Thereafter, fresh weight, chlorophyll content, number of roots, root length, number of leaves, and plant height were recorded every 15 days.

[0223] Test results and analysis

[0224] 1. Survival rate analysis

[0225] Among the seven substrates, the lowest survival rate was in group F (90%), followed by group C (93.33%), and the survival rates of the other groups were all 100%. Root damage during seedling washing was the main cause of death.

[0226] 2. Comparison of fresh weight and chlorophyll content

[0227] Fresh weight: from large to small: G (169.58g) > C (164.91g) > E (141.96g) > B (129.61g) > D (114.32g) > F (110.75g) > A (78.95g). Group G had the heaviest fresh weight, almost twice that of Group A, indicating that the ratio of peat soil and coconut bran of 2:1 was most beneficial to plant growth.

[0228] Chlorophyll content: from high to low: G (107.80) > E (81.37) > C (79.96) > D (77.07) > B (71.93) > A (67.40) > F (59.05). Group G was significantly higher than other groups.

[0229] Test results

[0230] The experiment was conducted from March 17 to June 2, and the seven substrate ratios had a significant effect on the survival rate and growth of the Angel Philodendron tissue culture seedlings. The results are as follows:

[0231] 1. Survival rate, fresh weight and chlorophyll content

[0232] The effects of different substrate ratios on survival rate, fresh weight and chlorophyll content are shown in Table 6.

[0233] Survival rate: Group G (peat soil: coconut husk = 2:1) performed best, reaching 100%; Group F (peat soil: coconut husk = 1:2) was the lowest, at 90%.

[0234] Fresh weight: Group G had the largest weight, which was 169.58g; Group A (river sand) had the smallest weight, which was only 78.95g.

[0235] Chlorophyll content: Group G had the highest content, which was 107.8; Group F had the lowest content, which was 59.05.

[0236] Table 6 Effects of substrate ratio on survival rate, fresh weight and chlorophyll content of Philodendron cherubim after transplantation

[0237]

[0238] Fig.11 The fresh weight comparison of seven matrix ratios is shown. The fresh weight of each group is from large to small: G>C>E>B>D>F>A. The fresh weight of group G is 169.58g, while that of group A is only 78.95, a difference of more than 2 times. The values ​​of group C and group G are very close. The differences between group C and group E, group E and group B, group B and group D, and group D and group F are all between 10 and 20, and the data of these groups do not fluctuate much.

[0239] Fig.12 The average chlorophyll content of the seven substrate ratios is shown. The chlorophyll content of group G is the highest, at 107.8. Except for group G and group F, the chlorophyll content of the other four groups is similar, with little fluctuation. The lowest value is group F, at 59.05. The order of chlorophyll content is: G>E>C>D>B>A>F.

[0240] 2. Number of roots, root length, number of leaves and plant height

[0241] As shown in Table 7, group D (river sand: coconut bran = 1:1) had the best rooting effect, with an average root number of 9.67 and an average root length of 3.19 cm; group G had the best growth of the aboveground part of the plants, with the number of leaves and plant height reaching 15.1 and 12.11 respectively.

[0242] Table 7 Average values ​​and growth values ​​of root number, root length, leaf number and plant height of seven substrate ratios

[0243]

[0244]

[0245] Conclusion

[0246] Optimal matrix ratio:

[0247] Rooting stage: Group D (river sand: coconut bran = 1:1).

[0248] Aboveground growth stage: Group G (peat soil: coconut bran = 2:1).

[0249] Experimental analysis:

[0250] The G group substrate has the best air permeability and water retention, which helps the roots and above-ground parts grow in coordination.

[0251] The river sand substrate (Group A) has poor water retention and is easily compacted, which restricts root growth.

[0252] The optimal substrate ratio screened out in this experiment provides important technical support for subsequent domestication, transplanting and large-scale production.

[0253] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for cultivating tissue culture seedlings of Angelica dahurica, characterized in that: The following steps are involved: S1. Explant selection and primary culture: Select healthy angel philodendron tubers as explants, sterilize them under sterile conditions, and inoculate the explants in a culture medium, wherein the culture medium is MS culture medium, 6-BA 1.0-2.0 mg / L, NAA 0.05-0.15 mg / L, sucrose 20-30 g / L and agar 6-8 g / L are added, the pH is adjusted to 5.8-6.0, the culture temperature is 23-27°C, the light intensity is 2000-4000 Lx, and the photoperiod is 14-16 h / d; S2, secondary proliferation culture: the clustered buds obtained from the primary culture were cut into clumps of 3 small buds, and inoculated into a proliferation medium, wherein the proliferation medium is MS medium, supplemented with 6-BA 1.0-2.0 mg / L, KT 0.5-1.5 mg / L, NAA 0.05-0.15 mg / L, sucrose 20-30 g / L and agar 6-8 g / L, and the pH is adjusted to 5.8-6.0; S3, rooting culture: cutting the seedlings of subculture proliferation culture into individual plants, inoculating them into rooting medium, wherein the rooting medium is 1 / 4-3 / 4 MS medium, supplemented with NAA 0.5-1.5 mg / L, banana puree 50-150 g / L, sucrose 15-25 g / L and agar 4-6 g / L, and the pH is adjusted to 5.8-6.0; S4, acclimatization and transplantation: the tissue culture seedlings obtained by the rooting culture are hardened, and after gradually adapting to the external environment in the natural environment, the seedlings are transplanted into a sterilized substrate, which is a mixture of peat soil and coconut bran.

2. A method for cultivating angelica philodendron tissue culture seedlings according to claim 1, characterized in that: The explant sterilization process comprises the following steps: 1) Sterilize with 75% alcohol solution for 30-60 seconds; 2) Sterilize with 0.1%-0.3% mercuric chloride solution or 1%-2% sodium hypochlorite solution for 5-10 minutes; 3) Rinse 3-6 times with sterile water.

3. A method for cultivating angelica philodendron tissue culture seedlings according to claim 1, characterized in that: The temperature of the primary culture, secondary proliferation culture and rooting culture is room temperature, the photoperiod is 14-16h / d, and the light intensity is 2500-3500Lx.

4. A method for cultivating angelica philodendron tissue culture seedlings according to claim 1, characterized in that: The proliferation period of the subculture proliferation culture is 20-30 days, and the proliferation coefficient is 3.5-5.

0.

5. A method for cultivating angelica philodendron tissue culture seedlings according to claim 1, characterized in that: The rooting culture has a cultivation time of 25-35 days, a rooting rate of 75%-90%, an average number of roots of 2-5, and an average root length of 2.5-4.0 cm.

6. A method for cultivating tissue culture seedlings of Angelica dahurica according to claim 1, characterized in that: The transplanting matrix sterilization method comprises: Soak the substrate in 500-600 times carbendazim solution for 3-6 minutes; When transplanting, the moisture content of the substrate should be maintained at 30%-50%.

7. A method for cultivating tissue culture seedlings of Philodendron cherubim according to claim 1, characterized in that: The management during the domestication and transplanting process includes: Shade treatment is carried out in the early stage of transplanting, and the light intensity is controlled at 1500-2000Lx; Spray water on the leaves 2-3 times a day, and gradually increase the light intensity to more than 3000Lx 1-2 weeks after transplanting.

8. The method for cultivating tissue culture seedlings of Philodendron cherubim according to claim 1, characterized in that: The volume ratio of peat soil to coconut husk in the matrix is ​​2:1.

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