A culture combination and its application in rapid propagation of sedum lineare

Abstract

The present application belongs to the technical field of Eupatorium fortunei tissue culture, and particularly relates to a culture combination and application thereof in rapid propagation of Eupatorium fortunei. The present application improves the proliferation efficiency of Eupatorium fortunei callus and the subsequent rooting efficiency by reasonably regulating the components of callus induction medium, proliferation medium, cluster bud induction medium and rooting medium. By adding TDZ in the cluster bud induction medium, the differentiation of callus is improved, and the proliferation efficiency of callus is also significantly improved, which is conducive to the rapid propagation and survival of Eupatorium fortunei. The experiment shows that, after the technical scheme provided by the present application is adopted, the callus induction rate can reach 100%, and the emerald green callus has good growth; the cluster bud induction rate reaches 100% in 60 days; the rooting rate reaches 98.89% in 60 days, and the fibrous roots are more, which lays a foundation for subsequent realization of resource utilization of Eupatorium fortunei and other Millettia plants, establishment of tissue culture and rapid propagation system and factory production.

Description

Technical Field

[0001] The invention belongs to the technical field of Rehmannia glutinosa tissue culture, and particularly relates to a culture combination and application thereof in rapid propagation of Rehmannia glutinosa. Background Art

[0002] False blue ( Crotalaria ferruginea Grah., also known as yellow wild lily, is a perennial herb in the genus Crotalaria of the Leguminosae family. It contains multiple active ingredients. Its dried whole herb can nourish the kidneys and liver, relieve cough and asthma, and promote dampness and detoxification. It is used to treat symptoms such as tinnitus, deafness, dizziness, chronic cough with blood in sputum, nephritis, and boils. False dwarf bluegrass is primarily found in Guizhou, Yunnan, and Sichuan. It is a common folk medicine among the Bai, Yi, and Dong ethnic groups in these regions. It not only has high medicinal value but can also kill maggots and mosquito larvae and serve as a green manure, thus offering broad market prospects.

[0003] However, due to ecological damage, the number of Lycoris radiata has drastically decreased, and existing wild resources can no longer meet the continued large-scale demand. Currently, most research on Lycoris radiata focuses on its medicinal properties and chemical composition, such as the in vitro antibacterial activity of Lycoris radiata hay, and the anti-inflammatory and analgesic properties of Lycoris radiata with n-butanol extraction. However, research on tissue culture rapid propagation systems is limited, and there are currently no reports on the rapid establishment of Lycoris radiata tissue culture systems. Summary of the Invention

[0004] The purpose of the present invention is to provide a culture combination and its application in the rapid propagation of Crotalaria tinctoria. By providing a specific culture medium combination, the planting cycle is shortened and the tissue culture efficiency of Crotalaria genus plants including Crotalaria tinctoria is improved.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a culture medium combination, comprising a callus induction medium, a proliferation medium, a cluster bud induction medium and a rooting medium, wherein the callus induction culture uses MS medium as a basal medium and further comprises the following components: 0.5-1.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 1.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder;

[0007] The proliferation medium is based on MS medium and further includes the following components: 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.7-0.8 mg / L 2,4-D, 0.5 g / L PVP, 30 g / L sucrose and 5 g / L agar powder;

[0008] The cluster bud induction medium is based on MS medium and further includes the following components: 1.5 mg / L 6-BA, 0.1 mg / L NAA, 0.01-0.05 mg / L TDZ, 30 g / L sucrose and 5 g / L agar powder;

[0009] The rooting medium is based on 1 / 2 MS medium and further includes the following components: 1.0-1.5 mg / L IBA, 1.0-1.5 mg / L NAA, 30 g / L sucrose and 5 g / L agar powder;

[0010] The pH values ​​of the callus induction medium, the proliferation medium, the cluster bud induction medium and the rooting medium are respectively 5.5-7.5.

[0011] Preferably, the culture medium combination further comprises a sterile seedling culture medium;

[0012] The sterile seedling culture medium uses 1 / 2 MS culture medium as a basic culture medium and further includes the following components: 0.7-1.2 mg / L 6-BA, 0.3-0.7 mg / L NAA, 1.2-2.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder;

[0013] The cluster shoot induction medium in the culture medium combination does not contain PVP.

[0014] The present invention provides an application of the culture medium combination described in the above technical solution in constructing a tissue culture system for Crotalaria plants.

[0015] Preferably, the Crotalaria plant includes Crotalaria tinctoria.

[0016] The present invention provides a method for rapidly propagating Rehmannia glutinosa, which uses the culture medium combination described in the above technical solution, comprising the following steps:

[0017] inoculating the sterile explant into the callus induction medium for callus induction culture to obtain callus tissue after induction culture;

[0018] inoculating the callus tissue after the induction culture into the proliferation culture medium for proliferation culture to obtain the proliferated callus tissue;

[0019] inoculating the proliferated callus into the cluster bud induction medium for cluster bud induction culture to obtain tissue culture seedlings;

[0020] inoculating the tissue culture seedlings into the rooting medium for rooting culture to obtain regenerated seedlings;

[0021] The regenerated seedlings are transplanted and cultivated to obtain regenerated Rehmannia glutinosa.

[0022] Preferably, the method for preparing the sterile explant comprises: inoculating sterilized seeds into a sterile seedling culture medium for seed culture to obtain the sterile explant.

[0023] Preferably, the seed disinfection comprises: disinfecting the seeds with alcohol and mercuric chloride in sequence;

[0024] The volume percentage of the alcohol is 75%;

[0025] The mass percentage of the mercuric chloride is 0.1%.

[0026] Preferably, the sterile explants include sterile young leaves.

[0027] Preferably, the culture conditions for the callus induction culture, proliferation culture, cluster bud induction culture, rooting culture and seed culture respectively include: temperature of 21-25°C, light intensity of 2000-3000 lux, and illumination time of 12-18 h / d.

[0028] Preferably, the regenerated seedlings are transplanted and cultivated when the root length is 1-2 cm.

[0029] Beneficial effects:

[0030] The present invention provides a culture medium combination, comprising a callus induction culture medium, a proliferation culture medium, a cluster bud induction culture medium and a rooting culture medium, wherein the callus induction culture medium uses an MS culture medium as a basic culture medium and further comprises the following components: 0.5-1.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 1.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder; the proliferation culture medium uses an MS culture medium as a basic culture medium and further comprises the following components: 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.7-0.8 mg / L 2,4-D, 0.5 g / L PVP, 30 g / L sucrose and 5 g / L agar powder. The invention relates to a method for preparing a callus induction medium comprising: ... By rationally regulating the components of each culture medium, the efficiency of callus expansion growth and subsequent rooting of Crotalaria plants, including Pseudo-bluebells, was improved; by adding TDZ to the cluster bud induction medium, not only the differentiation of callus tissue was improved, but also the proliferation efficiency of callus tissue was significantly improved; it is beneficial to the rapid reproduction and survival of Crotalaria plants, including Pseudo-bluebells.

[0031] Based on the above technical advantages, the present invention also provides a method for rapid propagation of false blue, using the culture medium combination described in the above technical solution, comprising the following steps: inoculating a sterile explant into the callus induction medium for callus induction culture to obtain callus tissue after induction culture; inoculating the callus tissue after induction culture into the proliferation medium for proliferation culture to obtain proliferated callus tissue; inoculating the proliferated callus tissue into the cluster bud induction medium for cluster bud induction culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into the rooting medium for rooting culture to obtain regenerated seedlings; transplanting the regenerated seedlings to obtain regenerated false blue. Experiments have shown that after adopting the technical solution provided by the present invention, the callus induction rate of false blue can reach 100%, and the callus is emerald green and grows well; the cluster bud 60d induction rate reaches 100%; the 60d rooting rate reaches 98.89%, and there are many fibrous roots. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0033] Figure 1 The sterile seedlings obtained by culture in Example 1;

[0034] Figure 2 The growth potential of the callus in Examples 1 to 5 and Comparative Examples 1 to 3;

[0035] Figure 3 The results are as follows:

[0036] Figure 4 The growth potential of the buds in Example 1, Example 7 and Comparative Examples 11 to 13;

[0037] Figure 5 The clustered buds in Example 1 bloom in the bottle;

[0038] Figure 6 The clustered buds in Example 1 took root autonomously in the bottle and had poor growth potential;

[0039] Figure 7 In Example 1, the clustered buds took root autonomously in the bottle and grew well;

[0040] Figure 8 Rooting of tissue culture seedlings in Example 1, Example 8 and Comparative Example 14;

[0041] Figure 9 The growth of the regenerated seedlings when transplanted using the solution in Comparative Example 15;

[0042] Figure 10 The growth of the regenerated seedlings after 1 day of transplanting using the scheme in Comparative Example 15;

[0043] Figure 11 The growth of the regenerated seedlings after 7 days of transplanting using the scheme in Comparative Example 15;

[0044] Figure 12 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Comparative Example 15;

[0045] Figure 13 The growth of the regenerated seedlings when transplanted using the solution in Comparative Example 16;

[0046] Figure 14 The growth of the regenerated seedlings after 1 day of transplanting using the scheme in Comparative Example 16;

[0047] Figure 15 The growth of the regenerated seedlings after 7 days of transplanting using the scheme in Comparative Example 16;

[0048] Figure 16 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Comparative Example 16;

[0049] Figure 17 The growth of the regenerated seedlings after 1 day of transplanting using the scheme in Comparative Example 17;

[0050] Figure 18 The growth of the regenerated seedlings after 7 days of transplanting using the scheme in Comparative Example 17;

[0051] Figure 19 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Comparative Example 17;

[0052] Figure 20 The growth of the regenerated seedlings after 7 days of transplanting using the scheme in Comparative Example 18;

[0053] Figure 21 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Comparative Example 18;

[0054] Figure 22 The growth of the regenerated seedlings 21 days after transplanting using the scheme in Comparative Example 18;

[0055] Figure 23 The growth of the regenerated seedlings after 7 days of transplanting using the scheme in Example 1;

[0056] Figure 24 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Example 1;

[0057] Figure 25 The growth of the regenerated seedlings 21 days after transplanting using the scheme in Example 1;

[0058] Figure 26 The growth of the regenerated seedlings 14 days after transplanting using the scheme in Comparative Example 19;

[0059] Figure 27 The growth of the regenerated seedlings 21 days after transplanting using the scheme in Comparative Example 19. DETAILED DESCRIPTION

[0060] The present invention provides a culture medium combination, comprising a callus induction medium, a proliferation medium, a cluster bud induction medium and a rooting medium. In the present invention, unless otherwise specified, all raw materials used are purchased conventionally.

[0061] In the present invention, the callus induction medium uses MS medium as the basal medium and further includes the following components: 0.5-1.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 1.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder; preferably, the callus induction medium uses MS medium as the basal medium and only contains the following components: 0.5-1.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 1.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder. In the present invention, the concentration of 6-BA in the callus induction medium is 0.5~1.0 mg / L, more preferably 0.8~1.0 mg / L, more preferably 1.0 mg / L; the concentration of NAA in the callus induction medium is 0.5~1.5 mg / L, more preferably 0.5~1.0 mg / L, more preferably 0.5 mg / L; the concentration of 2,4-D in the callus induction medium is 1.0 mg / L; the concentration of sucrose in the callus induction medium is 30 g / L; the concentration of agar powder in the callus induction medium is 5 g / L; the pH value of the callus induction medium is 5.5~7.5, more preferably 6.0~7.0, more preferably 6.0.

[0062] The proliferation culture medium of the present invention is based on MS culture medium and further includes the following components: 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.7-0.8 mg / L 2,4-D, 0.5 g / L PVP, 30 g / L sucrose and 5 g / L agar powder; preferably, the MS culture medium is used as the base culture medium and only contains the following components: 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.7-0.8 mg / L 2,4-D, 0.5 g / L PVP, 30 g / L sucrose and 5 g / L agar powder; the concentration of 6-BA in the proliferation medium is 0.5 mg / L; the concentration of NAA in the proliferation medium is 0.5 mg / L; the concentration of 2,4-D in the proliferation medium is 0.7~0.8 mg / L, more preferably 0.8 mg / L; the concentration of PVP in the proliferation medium is 0.5 g / L; the concentration of sucrose in the proliferation medium is 30 g / L; the concentration of agar powder in the proliferation medium is 5 g / L; the pH value of the proliferation medium is 5.5~7.5, more preferably 6.0~7.0, more preferably 6.0.

[0063] The cluster bud induction medium of the present invention is based on MS medium and further comprises the following components: 1.5 mg / L 6-BA, 0.1 mg / L NAA, 0.01-0.05 mg / L TDZ, 30 g / L sucrose and 5 g / L agar powder; preferably, the MS medium is used as the base medium and only contains the following components: 1.5 mg / L 6-BA, 0.1 mg / L NAA, 0.01-0.05 mg / L TDZ, 30 g / L sucrose and 5 g / L agar powder; the concentration of 6-BA in the cluster bud induction medium is 1.5 mg / L; the concentration of NAA in the cluster bud induction medium is 0.1 mg / L; the concentration of TDZ in the cluster bud induction medium is 0.01~0.05 mg / L, further preferably 0.03~0.05 mg / L, more preferably 0.05 mg / L; the concentration of sucrose in the cluster bud induction medium is 30 g / L; the concentration of agar powder in the cluster bud induction medium is 5 g / L; the cluster bud induction medium preferably does not contain PVP; the pH value of the cluster bud induction medium is 5.5~7.5, further preferably 6.0~7.0, more preferably 6.0.

[0064] The rooting medium of the present invention is based on 1 / 2MS medium and further comprises the following components: 1.0-1.5 mg / L IBA, 1.0-1.5 mg / L NAA, 30 g / L sucrose and 5 g / L agar powder; preferably, the 1 / 2MS medium is based on the medium and further comprises only the following components: 1.0-1.5 mg / L IBA, 1.0-1.5 mg / L NAA, 30 g / L sucrose and 5 g / L agar powder; the concentration of IBA in the rooting medium is 1.0~1.5 mg / L, further preferably 1.2~1.5 mg / L, more preferably 1.5 mg / L; the concentration of NAA in the rooting medium is 1.0~1.5 mg / L, further preferably 1.2~1.5 mg / L, more preferably 1.5 mg / L; the concentration of sucrose in the rooting medium is 30 g / L; the concentration of agar powder in the rooting medium is 5 g / L; the pH value of the rooting medium is 5.5~7.5, further preferably 6.0~7.0, more preferably 6.0.

[0065] Based on the advantages of the above-mentioned culture medium combination, the present invention preferably combines the culture medium combination with any conventional sterile seedling culture medium in the art, and can quickly establish a breeding system for Crotalaria plants including Crotalaria spp.

[0066] In the present invention, the sterile seedling culture medium is based on 1 / 2MS culture medium and further includes the following components: 0.7~1.2mg / L 6-BA, 0.3~0.7mg / L NAA, 1.2~2.0mg / L 2,4-D, 30g / L sucrose and 5g / L agar powder; preferably, the sterile seedling culture medium is based on 1 / 2MS culture medium and only contains the following components: 0.7~1.2mg / L 6-BA, 0.3~0.7mg / L NAA, 1.2~2.0mg / L 2,4-D, 30g / L sucrose and 5g / L agar powder; the sterile seedling culture medium is preferably based on 1 / 2MS culture medium. The concentration of 6-BA in the sterile seedling culture medium is preferably 0.7~1.2 mg / L, more preferably 0.8~1.1 mg / L, more preferably 1.0 mg / L; the concentration of NAA in the sterile seedling culture medium is preferably 0.3~0.7 mg / L, more preferably 0.4~0.5 mg / L, more preferably 0.5 mg / L; the concentration of 2,4-D in the sterile seedling culture medium is preferably 1.2~2.0 mg / L, more preferably 1.4~1.7 mg / L, more preferably 1.5 mg / L; the concentration of sucrose in the sterile seedling culture medium is preferably 30 g / L; the concentration of agar powder in the sterile seedling culture medium is preferably 5 g / L.

[0067] The present invention also provides the use of the culture medium combination described in the above technical solution in constructing a tissue culture system for Crotalaria plants. The Crotalaria plants described in the present invention preferably include Rehmannia glutinosa. The above culture medium combination is advantageously used to improve the efficiency and survival rate of Rehmannia glutinosa tissue culture.

[0068] The present invention also provides a method for rapid propagation of Rehmannia glutinosa, which adopts the above-mentioned culture medium combination and comprises the following steps: inoculating a sterile explant into the callus induction culture medium for callus induction culture to obtain induced cultured callus tissue; inoculating the induced cultured callus tissue into the proliferation culture medium for proliferation culture to obtain proliferated callus tissue; inoculating the proliferated callus tissue into the cluster bud induction culture medium for cluster bud induction culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into the rooting culture medium for rooting culture to obtain regenerated seedlings; and transplanting and cultivating the regenerated seedlings to obtain regenerated Rehmannia glutinosa.

[0069] The present invention preferably cultivates sterile explants, and the sterile explants are preferably sterile young leaves; the preparation method of the sterile explants preferably includes: inoculating sterilized seeds into a sterile seedling culture medium for seed cultivation to obtain the sterile explants.

[0070] The present invention preferably rinses the seeds of the pseudo-indigofera to obtain rinsed seeds. In the present invention, the pseudo-indigofera seeds are preferably mature and plump seeds; the rinsing is preferably performed with running water; there is no special requirement for the rinsing method, as long as the seeds are rinsed clean.

[0071] After obtaining the washed seeds, the present invention preferably sterilizes the washed seeds with alcohol to obtain alcohol-sterilized seeds. In the present invention, the volume percentage of the alcohol is preferably 75%. There are no special requirements for the method of alcohol sterilization, and techniques well known in the art can be used.

[0072] After obtaining the alcohol-disinfected seeds, the present invention preferably mixes and soaks the alcohol-disinfected seeds with mercuric chloride to obtain mercuric chloride-disinfected seeds. In the present invention, the mass percentage of mercuric chloride is preferably 0.1%; the mixing and soaking time is preferably 8 minutes; and the mercuric chloride disinfection is preferably performed in a sterile environment. This facilitates more thorough disinfection of the seeds.

[0073] After obtaining the mercuric chloride-sterilized seeds, the present invention preferably washes the mercuric chloride-sterilized seeds with sterile water to obtain washed seeds. In the present invention, the washing is preferably performed 2 to 3 times. There are no particular requirements for the washing method, and techniques well known in the art can be used.

[0074] After obtaining the cleaned seeds, the present invention preferably inoculates the cleaned seeds onto a sterile seedling culture medium for seed culture to obtain sterile seedlings. The sterile seedling culture medium of the present invention has been defined and optimized in the above technical solution and will not be repeated here; the temperature of the seed culture is preferably 21-25°C, the light intensity is preferably 2000-3000 lux, more preferably 2000 lux, and the light duration is preferably 12-18 h / d, more preferably 12 h / d; the time of the seed culture is not particularly limited and ends when the sterile seedlings grow to 7-8 cm and the leaves are 3-4 cm long.

[0075] After obtaining the sterile seedlings, the present invention preferably cuts the young leaves from the sterile seedlings to obtain sterile explants. The method and size of the cutting are not particularly specified and can be performed using techniques well known in the art. In a specific embodiment of the present invention, the young leaves are cut to obtain sterile explants measuring 0.5 cm x 0.5 cm. Furthermore, directly sterilizing the pseudo-indigofera to obtain sterile explants is also within the scope of the present invention.

[0076] After obtaining the sterile explant, the present invention inoculates the sterile explant into a callus induction medium for callus induction culture to obtain callus tissue after induction culture. The callus induction medium of the present invention has been defined and optimized in the above technical solution and will not be repeated here. The temperature of the callus induction culture is preferably 21-25°C, the light intensity is preferably 2000-3000 lux, more preferably 2000 lux, and the light duration is preferably 12-18 hours / day, more preferably 12 hours / day. The callus induction culture duration is preferably 30-45 days, at which time the callus tissue is visible as green, significantly expanded, and growing well.

[0077] After obtaining the induced callus, the present invention inoculates the induced callus into a proliferation culture medium for proliferation culture to obtain proliferated callus. The proliferation culture medium of the present invention has been defined and optimized in the above technical solution and will not be repeated here. The temperature of the proliferation culture is preferably 21-25°C, the light intensity is preferably 2000-3000 lux, more preferably 2000 lux, and the illumination time is preferably 12-18 hours / day, more preferably 12 hours / day. The proliferation culture time is preferably 20-30 days.

[0078] After obtaining the proliferated callus, the present invention inoculates the proliferated callus into the cluster bud induction medium for cluster bud induction culture to obtain tissue culture seedlings. The cluster bud induction medium of the present invention has been defined and optimized in the above technical solution and will not be repeated here; the temperature for the cluster bud induction culture is preferably 21-25°C, the light intensity is preferably 2000-3000 lux, more preferably 2000 lux, and the light duration is preferably 12-18 h / d, more preferably 12 h / d; the time for the cluster bud induction culture is preferably 80-100 d, more preferably 90 d. This is conducive to inducing differentiation to obtain a larger number of cluster buds, and the cluster buds grow well and have a uniform plant shape.

[0079] After obtaining the tissue culture seedlings, the present invention inoculates the tissue culture seedlings into a rooting medium for rooting culture to obtain regenerated seedlings. The rooting medium of the present invention has been defined and optimized in the above technical solution and will not be repeated here. The temperature of the rooting culture is preferably 21-25°C, the light intensity is preferably 2000-3000 lux, more preferably 2000 lux, and the light duration is preferably 12-18 hours / day, more preferably 12 hours / day. The rooting culture duration is preferably 25-40 days, more preferably 30 days.

[0080] After obtaining the regenerated seedlings, the present invention transplants and cultivates the regenerated seedlings to obtain regenerated pseudo-indigofera. The present invention preferably transplants and cultivates the regenerated seedlings when the root length of the regenerated seedlings is 1 to 2 cm, and further preferably the regenerated seedlings whose root system is purple-white as a whole and whose root length is 1 to 2 cm; when transplanting, it is preferred to remove the old leaves on the regenerated seedlings before transplanting, and when transplanting, the callus part is completely buried but the stem part is not buried; the substrate during transplanting and cultivation is preferably an artificial substrate; the artificial substrate preferably includes vermiculite, organic soil and deep soil; the organic soil is preferably peat; the deep soil is preferably river sand; the volume ratio of the vermiculite, organic soil and deep soil is preferably 2:1:1; the sources of the peat and river sand are not limited, and conventional purchase is sufficient; the artificial substrate is preferably lightly compacted, covered with a film and small holes are poked in the film, so as to help maintain plant breathing;

[0081] The present invention provides a small amount of watering every day for 7 days before transplanting; the small amount of watering is preferably based on keeping the soil surface moist; after 7 days of transplanting, the hormone solution is preferably irrigated once every 2 days; the hormone solution uses water as a solvent and preferably includes 1.0 mg / L IBA and 1.0 mg / L NAA; the absence of wilting 20 days after transplanting indicates that the regenerated seedlings are alive and subsequent routine management is sufficient; the routine management preferably includes watering; the amount of water used and the method of application are not special, and techniques well known in the art can be used.

[0082] The present invention improves the expansion growth and subsequent rooting efficiency of the bluebell callus by rationally regulating the components of each culture medium. The addition of TDZ to the bud induction medium not only enhances callus differentiation but also significantly improves the growth of bud clusters, thus facilitating the rapid propagation and survival of Crotalaria plants, including bluebell. Experiments have shown that the technical solution provided by the present invention can achieve a callus induction rate of 100%, with emerald green calli and good growth. The bud cluster induction rate reached 100% within 60 days, and the rooting rate reached 98.89% within 60 days, with abundant fibrous roots.

[0083] To further illustrate the present invention, a culture combination provided by the present invention and its application in the rapid propagation of Rehmannia glutinosa are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Preparation before the experiment:

[0085] The experimental materials used in this study were mature seeds of Pseudo-Dryopteris tinctorius, and sterile seedlings were obtained to carry out tissue culture-related research.

[0086] The culture medium in different treatments was based on MS, in which 6-BA (6-benzylaminopurine), 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthylacetic acid), IBA (indolebutyric acid), TDZ (thiadiazole), and PVP (polyvinylpyrrolidone) were purchased conventionally. The culture medium for different treatments was sterilized in an autoclave (121°C) for 20 minutes before use.

[0087] The formulas used for data statistics and calculations during the experiment are as follows:

[0088] Contamination rate = number of contaminated individuals / number of inoculated individuals × 100%; germination rate = number of germinated individuals / number of inoculated individuals × 100%;

[0089] Induction rate = number of induced individuals / number of inoculated individuals × 100%; browning rate = number of browned individuals / number of inoculated individuals × 100%;

[0090] Average plant height = total plant height / total number of plants; average number of cluster buds = total number of cluster buds / total number of plants;

[0091] Rooting rate = number of roots / number of inoculations × 100%; average root length = total root length / number of inoculations;

[0092] Average number of taproots = total number of taproots / number of inoculated roots.

[0093] Example 1

[0094] A method for rapidly propagating dwarf blue, comprising the following steps:

[0095] 1) Obtaining sterile seedlings

[0096] Select mature and plump seeds of Pseudo-indigofera serrata, rinse them with running water, and disinfect the surface of the seeds with 75% alcohol by volume. Then move the seeds into a clean bench and treat them with 0.1% mercuric chloride by mass for 8 minutes for disinfection. Then wash them with sterile water 2-3 times, wipe dry the water on sterile filter paper, and inoculate the seeds into 1 / 2MS+6-BA 1.0mg / L+2,4-D 1.5mg / L+NAA 0.5mg / L culture medium (the culture medium contains 30g / L sucrose and 5g / L agar powder, and the pH value of the culture medium is 6.0). Incubate at 25℃ and 2000lux under light conditions for 12h per day to obtain sterile seedlings (see the growth of sterile seedlings for details). Figure 1 ), when the sterile seedlings grew to 7-8 cm in height and the leaves were 3-4 cm long (in this embodiment, the seeds began to sprout white buds on the 45th day and the seeds grew leaves on the 60th day), subsequent experiments were carried out.

[0097] 2) Callus induction

[0098] Tender leaves from the sterile seedlings obtained in step 1) were cut into 0.5 cm × 0.5 cm pieces and inoculated onto MS + 1.0 mg / L 6-BA + 0.5 mg / L NAA + 1.0 mg / L 2,4-D medium (the medium contained 30 g / L sucrose and 5 g / L agar powder, and the pH of the medium was 6.0). One to two cut tender leaves were inoculated into each bottle, and a total of 20 bottles were inoculated. Each bottle was a replicate, i.e., one treatment consisted of 20 parallel replicates. The callus tissue was induced under conditions of 25°C and 2000 lux light exposure for 12 h per day. The culture medium used in this treatment was designated as A1.

[0099] 3) Callus proliferation

[0100] The callus tissue successfully induced 30 days after step 2) was cut into small pieces of 0.5 cm × 0.5 cm, and inoculated on MS + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 0.8 mg / L 2,4-D + 0.5 g / L PVP medium (the medium contains 30 g / L sucrose and 5 g / L agar powder, and the medium has a pH of 6.0). One small piece was inoculated per bottle, and a total of 20 bottles were inoculated. Each bottle was a replicate, that is, one treatment with 20 parallel replicates. The experiments were carried out at 25°C and 2000 lux under light conditions for 12 h per day to obtain the proliferated callus tissue. The medium during this treatment was recorded as B9.

[0101] 4) Cluster bud induction

[0102] Take the callus tissue that has been proliferating for 20 days in step 3), cut it into small pieces of 0.5 cm × 0.5 cm, and inoculate it into MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 0.05 mg / L TDZ medium (the medium contains 30 g / L sucrose and 5 g / L agar powder, and the pH value of the medium is 6.0), 2 small pieces per bottle, and inoculate 20 bottles in total. Each bottle is a replicate, that is, one treatment with 20 parallel replicates. Under conditions of 25°C and 2000 lux light conditions for 12 h per day, tissue culture plantlets are obtained. The medium during this treatment is recorded as C3.

[0103] 5) Rooting culture

[0104] Take the 7-8 cm clustered buds differentiated from the tissue culture seedlings cultured for 30 days in step 4), retain the 2-3 smaller leaves at the top, and inoculate the tissue culture seedlings into 1 / 2MS + IBA 1.5 mg / L + NAA 1.5 mg / L medium (the medium also contains 30 g / L sucrose and 5 g / L agar powder, and the medium has a pH of 6.0), 2-3 clustered seedlings per bottle, and a total of 20 bottles are inoculated. Each bottle is a replicate, that is, one treatment with 20 parallel replicates. Under conditions of 25°C and 2000 lux light exposure for 12 h per day, regenerated seedlings are obtained. The culture medium during this treatment is recorded as D4.

[0105] 6) Transplantation and cultivation

[0106] The regenerated seedlings with a medium root length of 1-2 cm and a purple-white root system as a whole in step 5) were transplanted. The artificial substrate during transplantation was a volume ratio of vermiculite: organic soil (peat soil): deep soil (river sand) = 2:1:1. Excess old leaves were removed during transplantation, the callus was completely buried, and the stem was not buried. The soil was lightly compacted, covered with a film and small holes were poked in the film. Watered every day for 7 days to keep the regenerated seedlings moist. After 7 days, the hormone solution (the hormone solution is water as the solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA) was poured every 2 days. After 21 days of culture, watered once every 4 days to obtain the regenerated pseudo-indigo plant.

[0107] Example 2

[0108] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A2 medium to obtain induced callus tissue. The specific components of A2 medium are shown in Table 1.

[0109] Example 3

[0110] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A3 medium to obtain induced callus tissue. The specific components of A3 medium are shown in Table 1.

[0111] Example 4

[0112] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A5 medium to obtain induced callus tissue. The specific components of A5 medium are shown in Table 1.

[0113] Example 5

[0114] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A6 medium to obtain induced callus tissue. The specific components of A6 medium are shown in Table 1.

[0115] Comparative Example 1

[0116] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A4 medium to obtain induced callus tissue. The specific components of A4 medium are shown in Table 1.

[0117] Comparative Example 2

[0118] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A7 medium to obtain induced callus tissue. The specific components of A7 medium are shown in Table 1.

[0119] Comparative Example 3

[0120] The difference from Example 1 is that only steps 1) and 2) of Example 1 are included, and in step 2), the young leaves on the sterile seedlings are cut into pieces of 0.5 cm × 0.5 cm in size and inoculated on A8 medium to obtain induced callus tissue. The specific components of A8 medium are shown in Table 1.

[0121] Table 1 Different culture medium components for callus induction

[0122]

[0123] In the callus induction process of Examples 1 to 5 and Comparative Examples 1 to 3, the callus induction rate was counted at 30 days of culture, and the browning rate was counted at 45 days of culture. The specific results are shown in Tables 2 and Figure 2 (exist Figure 2 , A1 represents the growth potential of the callus in Example 1, A2 represents the growth potential of the callus in Example 2, A3 represents the growth potential of the callus in Example 3, A4 represents the growth potential of the callus in Comparative Example 1, A5 represents the growth potential of the callus in Example 4, A6 represents the growth potential of the callus in Example 5, A7 represents the growth potential of the callus in Comparative Example 2, and A8 represents the growth potential of the callus in Comparative Example 3).

[0124] Table 2 Effects of different treatments on callus induction

[0125]

[0126] From Table 2 and Figure 2 It can be seen that after culturing for 30 days in the culture medium supplemented with different concentrations of 6-BA, 2,4-D and NAA, the callus incision swelled and small yellow-green particles appeared. The callus grew rapidly and had good growth. At 45 days, the callus began to brown to varying degrees. In the MS medium, when the concentration of 2,4-D was kept constant at 1.0 mg / L, the addition of different concentrations of 6-BA and NAA had no obvious effect on the callus induction rate of Pseudo-indigo leaves, but the induction effects were different. When the 6-BA concentration remained unchanged, with the increase of NAA concentration, the callus swelling effect and growth showed a downward trend, and the browning rate showed an upward trend. When the NAA concentration was 2.0 mg / L, the callus induction rate was 98.33%, and the callus induction rates of the other combinations were all 100%. When NAA concentration remained constant, increasing 6-BA concentration resulted in a 100% callus induction rate. Callus expansion increased, while browning initially increased and then decreased. The browning rate was lowest at 0.5 mg / L and highest at 1.5 mg / L. At 2.0 mg / L, the callus exhibited a pale yellow-green color, unlike other concentrations, at 30 days, and turned green at 45 days. When NAA concentrations ranged from 0.5 to 1.5 mg / L and 6-BA concentrations from 0.5 to 1.0 mg / L, the callus exhibited a green color, significant expansion, and good growth. When 1.0 mg / L 6-BA and 0.5 mg / L NAA were added, the induction rate reached 100%, with the callus appearing emerald green, exhibiting the most significant expansion, low browning, and exhibiting the best growth. Therefore, the optimal culture medium for callus induction was MS+6-BA 1.0 mg / L+NAA 0.5 mg / L+2,4-D 1.0 mg / L.

[0127] Example 6

[0128] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B8 culture medium to obtain proliferated callus tissue. The specific components of B8 culture medium are shown in Table 3.

[0129] Comparative Example 4

[0130] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B1 culture medium to obtain proliferated callus tissue. The specific components of B1 culture medium are shown in Table 3.

[0131] Comparative Example 5

[0132] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B2 medium to obtain proliferated callus tissue. The specific components of B2 medium are shown in Table 3.

[0133] Comparative Example 6

[0134] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B3 culture medium to obtain proliferated callus tissue. The specific components of B3 culture medium are shown in Table 3.

[0135] Comparative Example 7

[0136] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B4 medium to obtain proliferated callus tissue. The specific components of B4 medium are shown in Table 3.

[0137] Comparative Example 8

[0138] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B5 culture medium to obtain proliferated callus tissue. The specific components of B5 culture medium are shown in Table 3.

[0139] Comparative Example 9

[0140] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B6 culture medium to obtain proliferated callus tissue. The specific components of B6 culture medium are shown in Table 3.

[0141] Comparative Example 10

[0142] The difference from Example 1 is that only steps 1) to 3) of Example 1 are included, and in step 3), the successfully induced callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on B7 culture medium to obtain proliferated callus tissue. The specific components of B7 culture medium are shown in Table 3.

[0143] Table 3 Different culture medium components for callus proliferation

[0144]

[0145] In the process of callus proliferation in Example 1, Example 6, and Comparative Examples 4 to 10, the proliferation coefficient was calculated at 20 days of proliferation, and the browning rate was calculated at 30 days of proliferation. The specific results are shown in Tables 4 and Figure 3 (exist Figure 3 In the table, B1 represents the growth potential of callus proliferation in Comparative Example 4, B2 represents the growth potential of callus proliferation in Comparative Example 5, B3 represents the growth potential of callus proliferation in Comparative Example 6, B4 represents the growth potential of callus proliferation in Comparative Example 7, B5 represents the growth potential of callus proliferation in Comparative Example 8, B6 represents the growth potential of callus proliferation in Comparative Example 9, B7 represents the growth potential of callus proliferation in Comparative Example 10, B8 represents the growth potential of callus proliferation in Example 6, and B9 represents the growth potential of callus proliferation in Example 1).

[0146] Table 4 Effects of different treatments on callus proliferation

[0147]

[0148] From Table 4 and Figure 3 The effects of different treatments on callus proliferation were significant. At 0 mg / L 2,4-D, callus expansion continued, but the browning rate was high, and callus expansion was average. At 0.5 mg / L 6-BA and NAA concentrations, the browning rate decreased with increasing 2,4-D concentrations. At 0.7-0.8 mg / L 2,4-D concentrations, callus tissue became yellow-green at 30 days and green at 45 days, exhibiting significant expansion, good growth, and low browning rate. Callus tissue grew rapidly, forming large callus masses by 20 days and beginning to brown rapidly at 30 days. When 0.8 mg / L 2,4-D was added, the lowest browning rate (11.6%) was achieved, with callus tissue appearing emerald green, expanding most significantly, and exhibiting the best growth. Although PVP significantly prevented callus browning in P. cerevisiae, it did have a certain inhibitory effect on its expansion rate. When the concentrations of 6-BA, 2,4-D, and NAA remained constant, the browning rate of the combination with PVP at 45 days was much lower than that of the combination without PVP, but the callus expansion rate of the combination without PVP was faster. Therefore, the optimal medium for callus proliferation is MS + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 0.8 mg / L 2,4-D + 0.5 g / L PVP.

[0149] Example 7

[0150] The difference from Example 1 is that only steps 1) to 4) of Example 1 are included, and in step 4), the proliferated callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on C2 medium to obtain tissue culture seedlings. The specific components of C2 medium are shown in Table 5.

[0151] Comparative Example 11

[0152] The difference from Example 1 is that only steps 1) to 4) of Example 1 are included, and in step 4), the proliferated callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on C1 culture medium to obtain tissue culture seedlings. The specific components of C1 culture medium are shown in Table 5.

[0153] Comparative Example 12

[0154] The difference from Example 1 is that only steps 1) to 4) of Example 1 are included, and in step 4), the proliferated callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on C4 medium to obtain tissue culture seedlings. The specific components of C4 medium are shown in Table 5.

[0155] Comparative Example 13

[0156] The difference from Example 1 is that only steps 1) to 4) of Example 1 are included, and in step 4), the proliferated callus tissue is cut into small pieces of 0.5 cm × 0.5 cm and inoculated on C5 culture medium to obtain tissue culture seedlings. The specific components of C5 culture medium are shown in Table 5.

[0157] Table 5 Different culture medium components for cluster bud induction

[0158]

[0159] In the process of inducing cluster buds in Example 1, Example 7, and Comparative Examples 11 to 13, the number of differentiated buds produced in each treatment at 60 days of induction culture was counted at 90 days. The specific results are shown in Tables 6 and Figure 4 (exist Figure 4 In the figure, C1 represents the growth potential of the clustered buds in Comparative Example 11, C2 represents the growth potential of the clustered buds in Example 7, C3 represents the growth potential of the clustered buds in Example 1, C4 represents the growth potential of the clustered buds in Comparative Example 12, and C5 represents the growth potential of the clustered buds in Comparative Example 13).

[0160] Table 6 Effects of different treatments on cluster bud differentiation

[0161]

[0162] From Table 6 and Figure 4It can be seen that calli of Pseudo-dementia indigofera begin to differentiate around day 45. Calli differentiation is successful at a TDZ concentration of 0 mg / L. Adding different TDZ concentrations to MS medium can induce callus differentiation, but the effects on differentiation are significantly different. At 6-BA concentrations of 1.5 mg / L and NAA concentrations of 0.1 mg / L, the differentiation rate initially increases and then decreases with increasing TDZ concentrations. At TDZ concentrations between 0.01 and 0.05 mg / L, calli begin to brown at day 30, becoming severely brown by day 60, with green buds emerging. Clustered buds then begin to differentiate, reaching a differentiation rate of 100% by day 90. Within the TDZ concentration range of 0.01 to 0.05 mg / L, callus differentiation significantly improves with increasing TDZ concentrations. A TDZ concentration of 0.05 mg / L induced the greatest number of clustered buds, with large, numerous, green leaves, robust stems, uniform internodes, and a uniform plant shape. Excessively high TDZ concentrations are detrimental to callus differentiation. When the TDZ concentration is greater than 1.0 mg / L, callus differentiates into small buds after 90 days, with a low differentiation rate and a long differentiation time. The buds after differentiation show average growth, resulting in short plants and sturdy stems. Therefore, the optimal medium for inducing clustered bud differentiation is MS + 1.5 mg / L 6-BA + 0.1 mg / L NAA + 0.05 mg / L TDZ.

[0163] In addition, during the induction of clustered buds, there are phenomena of flowering and autonomous rooting in the bottle, see Figures 5-7 (in Figure 5 What is shown is the phenomenon of flowering in a vase. Figure 6 and 7 Although some induced clustered shoots will spontaneously root and form complete plants after about 60 days, most of the roots at this time are dark red or brown, with a small number of long roots. Most of these clustered shoots will not survive after transplantation, indicating that their roots have little vitality and ability to absorb nutrients. A very small number of roots are white-purple, numerous, and long, and can successfully survive transplantation.

[0164] The three concentrations of TDZ used in the present invention (0 mg / L, 0.03 mg / L, and 0.05 mg / L) for bud induction all successfully induced in-bottle flowering in tissue culture seedlings. In the later stages of callus differentiation, the bud induction flourished and maintained strong growth. All seedlings treated with all three treatments gradually developed flower buds and formed buds around the same time, with a flowering period lasting up to 15 days. This indicates that cytokinin treatment is not essential for in-bottle flowering in Pseudo-Rehmannia glutinosa. While cytokinin treatment may have had some effect on flower bud differentiation in the two groups treated with TDZ, no significant difference was observed compared to those treated without TDZ. In subsequent year-long tissue culture experiments, no in-bottle flowering was observed during bud induction. However, subsequent callus-induced bud inductions shared a common characteristic: short, mediocre growth in the later stages, ultimately leading to complete death and loss of vitality. Therefore, the main factor affecting the flowering of the bluebell in the bottle is the physiological development stage of plant growth. In subsequent experiments, the bluebell buds in the vegetative growth process were in poor condition and died before entering the reproductive growth and development period. However, the buds in the several groups of materials that flowered in the bottle grew vigorously during the vegetative growth stage, successfully developed into the reproductive growth stage, differentiated into flower buds, and bloomed. This shows that the technical solution provided by the present invention is conducive to inducing bluebell. In other words, bluebell can be used as tissue culture micro-landscape material to increase ornamental value; secondly, it can provide bluebell seed material required for subsequent research; and it provides the possibility of hybridization and breeding new varieties and improving the production efficiency of fast-propagation commercial seedlings.

[0165] Example 8

[0166] The difference from Example 1 is that it only contains steps 1) to 5) of Example 1, and in step 5), the tissue culture seedlings are inoculated on the D2 culture medium to obtain regenerated seedlings. The specific components of the D2 culture medium are shown in Table 7.

[0167] Example 9

[0168] The difference from Example 1 is that only steps 1) to 5) of Example 1 are included, and in step 5), the tissue culture seedlings are inoculated on D3 culture medium to obtain regenerated seedlings. The specific components of D3 culture medium are shown in Table 7.

[0169] Comparative Example 14

[0170] The difference from Example 1 is that it only contains steps 1) to 5) of Example 1, and in step 5), the tissue culture seedlings are inoculated on the D1 culture medium to obtain regenerated seedlings. The specific components of the D1 culture medium are shown in Table 7.

[0171] Table 7 Different culture medium components for rooting culture

[0172]

[0173] Observe the rooting of the stem segments in Example 1, Example 8 and Comparative Example 14 respectively. Figure 8 (exist Figure 8 In the table, D1-1 represents the rooting of the stem segment of Comparative Example 14 observed from the side, D2-1 represents the rooting of the stem segment of Example 8 observed from the side, D2-2 represents the rooting of the stem segment of Example 8 observed from the bottom, D3-1 represents the rooting of the stem segment of Example 9 observed from the side, D3-2 represents the rooting of the stem segment of Example 9 observed from the bottom, D4-1 represents the rooting of the stem segment of Example 1 observed from the side, and D4-2 represents the rooting of the stem segment of Example 1 observed from the bottom), and the rooting rates of the different treatments were calculated at 60d. The results are shown in Table 8.

[0174] Table 8 Effects of different treatments on rooting

[0175]

[0176] From Table 8 and Figure 8 It can be seen that although 1 / 2MS culture medium with different concentrations of NAA and IBA can induce rooting of Pseudo-blue stem segments, the effects on root induction are significantly different; rooting cannot be induced in 1 / 2MS culture medium without hormone addition, and the growth of seedlings deteriorates after 30 days, the leaves turn yellow, and gradually die; with the increase of NAA and IBA concentrations, root length, root number and rooting rate show an upward trend; when the NAA and IBA concentrations are 1.0~1.5 mg / L, the rooting rate is high, the average number of roots is large and thick, the average root length is long, the fibrous roots are many, the leaves are green, and the growth is good; among them, when 1.0 mg / L NAA and IBA are added, the rooting rate is as high as 100%, and when 1.5 mg / L NAA and IBA are added, the rooting rate is as high as 98.89% after 60 days, the average root length is 4 cm, the average number of main roots is 5, the fibrous roots are the most, the leaves are green, the growth is the best, and the subsequent transplanting survival rate is high. Therefore, the optimal culture medium for rooting is 1 / 2MS+IBA 1.5mg / L+NAA 1.5mg / L.

[0177] Comparative Example 15

[0178] The difference from Example 1 is that in step 6), when transplanting the regenerated seedlings, the callus is completely buried, the soil is compacted, the hole cover is covered, and the holes for breathing are retained. Water is applied once every 2 days and a hormone solution is applied once every 4 days; the hormone solution uses water as a solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA.

[0179] Comparative Example 16

[0180] The difference from Example 1 is that in step 6), when transplanting the regenerated seedlings, the callus is completely buried, the soil is compacted, the plug tray is covered with a lid, and the holes for breathing are retained. The hormone solution is water-based and also contains 1.0 mg / L IBA and 1.0 mg / L NAA.

[0181] Comparative Example 17

[0182] The difference from Example 1 is that in step 6), the root system of the regenerated seedlings is black (brown) as a whole (i.e., the root system activity is weak), the excessive old leaves of the regenerated seedlings are removed during transplanting, the callus is completely buried, the stem is not buried, the soil is slightly compacted, a film is attached and small holes are poked in the film, a small amount of water is applied every day before 7 days, and a hormone solution is applied every 2 days after 7 days; the hormone solution uses water as a solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA. After 21 days of cultivation, water is applied once every 4 days.

[0183] Comparative Example 18

[0184] The difference from Example 1 is that in step 6), the root system of the plant is white as a whole (i.e., the root system is more active), excessive old leaves are removed during transplanting, the callus is completely buried, the stem is not buried, the soil is slightly compacted, a film is attached and small holes are poked in the film, a small amount of water is applied every day before 7 days, and a hormone solution is applied every 2 days after 7 days; the hormone solution uses water as a solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA. After culturing for 21 days, water is applied once every 4 days.

[0185] Comparative Example 19

[0186] The difference from Example 1 is that in step 6), the root system of the plant is purple-white as a whole (the root system activity is lower than that of the white root system), excessive old leaves are removed during transplanting, the callus is completely buried, the stem part is not buried, the soil is slightly compacted, a film is attached and small holes are poked in the film, a small amount of water is applied every day before 7 days, and a hormone solution is applied every 2 days after 7 days; the hormone solution uses water as a solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA. After culturing for 21 days, water is applied once every 4 days.

[0187] The growth status of the regenerated seedlings of Example 1 and Comparative Examples 15-19 were observed after transplanting for 1 day, 7 days, 14 days and 21 days. Figures 9 to 27 .

[0188] Depend on Figures 9 to 27 It can be seen that during transplanting and cultivation, when the root system is black or dark purple-white, the transplanting survival rate of the regenerated seedlings is low and it is difficult to survive; and when the soil is too wet, it is not conducive to the transplanting survival of the regenerated seedlings of Pseudo-blue.

[0189] In summary, the culture medium combination provided by the present invention is conducive to improving the expansion growth and subsequent rooting efficiency of the false blue callus, significantly improving the proliferation efficiency of the callus, and is conducive to the rapid reproduction and survival of the false blue. Experiments have shown that after adopting the technical solution provided by the present invention, the callus induction rate can reach 100%, and the emerald green callus has good growth; the 60d induction rate of the clustered buds reaches 100%; the 60d rooting rate reaches 98.89%, and there are many fibrous roots, which lays the foundation for the subsequent realization of resource utilization of Crotalaria plants including false blue, the establishment of tissue culture rapid propagation system and factory production.

[0190] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A culture medium combination, characterized in that: The method comprises a callus induction medium, a proliferation medium, a cluster bud induction medium and a rooting medium, wherein the callus induction medium is composed of MS medium, 0.5-1.0 mg / L 6-BA, 0.5-1.5 mg / L NAA, 1.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder; The proliferation medium consists of MS medium, 0.5 mg / L 6-BA, 0.5 mg / L NAA, 0.7-0.8 mg / L 2,4-D, 0.5 g / L PVP, 30 g / L sucrose and 5 g / L agar powder; The cluster bud induction medium consists of MS medium, 1.5 mg / L 6-BA, 0.1 mg / L NAA, 0.01-0.05 mg / L TDZ, 30 g / L sucrose and 5 g / L agar powder; The rooting medium consists of 1 / 2 MS medium, 1.0-1.5 mg / L IBA, 1.0-1.5 mg / L NAA, 30 g / L sucrose and 5 g / L agar powder; The pH values ​​of the callus induction medium, the proliferation medium, the cluster bud induction medium and the rooting medium are respectively 5.5-7.

5.

2. The culture medium combination according to claim 1, characterized in that The culture medium combination also includes a sterile seedling culture medium; The sterile seedling culture medium consists of 1 / 2 MS culture medium, 0.7-1.2 mg / L 6-BA, 0.3-0.7 mg / L NAA, 1.2-2.0 mg / L 2,4-D, 30 g / L sucrose and 5 g / L agar powder; The cluster shoot induction medium in the culture medium combination does not contain PVP.

3. Use of the culture medium combination according to claim 1 or 2 in constructing a tissue culture system for Crotalaria plants; The Crotalaria plant is Crotalaria pseudo-indigofera.

4. A method for rapid propagation of Pseudo-indigofera var. The method of claim 1 comprises the following steps: Inoculating sterile explants into the callus induction medium for callus induction culture to obtain callus tissue after induction culture; the sterile explants are sterile young leaves; inoculating the callus tissue after the induction culture into the proliferation culture medium for proliferation culture to obtain the proliferated callus tissue; inoculating the proliferated callus into the cluster bud induction medium for cluster bud induction culture to obtain tissue culture seedlings; inoculating the tissue culture seedlings into the rooting medium for rooting culture to obtain regenerated seedlings; Transplanting and cultivating the regenerated seedlings to obtain regenerated Lycoris radiata; During the transplanting, excessive old leaves are removed, the callus is completely buried without burying the stem part, the soil is slightly compacted, covered with a film and small holes are poked in the film, water is applied every day before 7 days to keep the regenerated seedlings moist, and after 7 days, the hormone solution is applied every 2 days, and water is applied once every 4 days after culturing for 21 days; the hormone solution uses water as a solvent and also contains 1.0 mg / L IBA and 1.0 mg / L NAA.

5. The method according to claim 4, characterized in that The method for preparing the sterile explant comprises the following steps: inoculating sterilized seeds into a sterile seedling culture medium for seed culture to obtain the sterile explant.

6. The method according to claim 5, characterized in that The seed disinfection comprises: disinfecting the seeds with alcohol and mercuric chloride in sequence; The volume percentage of the alcohol is 75%; The mass percentage of the mercuric chloride is 0.1%.

7. The method according to claim 4 or 5, characterized in that The culture conditions for the callus induction culture, proliferation culture, cluster bud induction culture, rooting culture and seed culture respectively include: temperature of 21-25° C., light intensity of 2000-3000 lux, and light duration of 12-18 h / d.

8. The method according to claim 4, characterized in that The regenerated seedlings were transplanted and cultivated when the root length was 1-2 cm.

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