Tissue culture process for Farfugium japonicum
Through specific tissue culture processes and steps, the problems of firefly Dawu Fengcao were solved and the color of unstable color during the culture process were solved, and efficient and stable plant proliferation and seedling rate were improved, meeting market demand.
Patent Information
- Application Number
- CN202411985880.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-31
- Publication Date
- 2025-04-11
AI Technical Summary
Firefly Dawu Fengcao is prone to mutation during the production and cultivation process and the plants are yellow and green, resulting in the production capacity not keeping up with market demand.
A firefly Dawu Fengcao tissue culture process is adopted, including explant treatment and disinfection, inoculation and culture, induction and induction, proliferation and expansion, seedlings for seedlings and rooting, and the stability and consistency of the plant is ensured through specific culture medium components and conditions control.
The efficient firefly Dawu Fengcao plant proliferation and stability control has been achieved, with a proliferation rate of up to 3.96-6.68, a seedling rate of up to 0.8-1.25, and a rooting rate of up to 100%. It can produce large quantities of plants that meet market demand.
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Figure CN119790982A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant tissue culture, in particular to a technology for the tissue culture of Schisandra chinensis. Background Art
[0002] Farfugium japonicum is also known as Bajiaowu, Huoxuelian, Jinboyu, Unicorn Lotus, Yiyelian, Damajiaoxiang, Damajiao, and Damajiao. It is a perennial herbaceous plant of the genus Asteraceae. Firefly Farfugium japonicum is one of them. Its Latin name is Farfugium japonicum'Aureomaculatum'. Its leaves are rounded at the tip, entire or with small teeth or palmately lobed, with a wide curved base. The young leaves are pure green, and then many yellow spots grow. Both sides are covered with grayish-white soft hairs when young, and then become hairless. Its shape is like Figure 1 shown.
[0003] Firefly clover grows under forests, valleys and grass in low-altitude areas. It likes semi-shade and humid environments. It is cold-resistant and can overwinter in the open in the south of the Yangtze River. It is afraid of direct sunlight. It has good adaptability to soil and is preferably planted in fertile, loose and well-drained black soil. Firefly clover is distributed in Hubei, Hunan, Guangxi, Guangdong, Fujian, Zhejiang and Taiwan in China. It is also cultivated in some botanical gardens and families at home and abroad. However, firefly clover is prone to mutation (except for spots, which are variant traits) and yellowish and greenish plants during the production and cultivation process. The production capacity always fails to keep up with market demand, which restricts the production and development of firefly clover. Summary of the invention
[0004] The purpose of the present invention is to provide a firefly wormwood tissue culture process to solve the problems of easy variation and yellowish or greenish plants in the process of culturing firefly wormwood in the prior art.
[0005] In order to achieve the above object, the present invention adopts the following technical solutions:
[0006] A firefly wormwood tissue culture process, the process comprising:
[0007] (1) Explant processing and disinfection;
[0008] (2) explant inoculation culture to germinate lateral buds, the inoculation medium composition is MS, BA 1-2 mg / L, NAA 0.01 mg / L, sucrose 30 g / L and carrageenan 7 g / L, pH 6.2;
[0009] (3) cutting off the lateral buds and inoculating them into a start-up induction medium to obtain adventitious bud masses, wherein the start-up induction medium comprises MS, BA 1-2 mg / L, NAA 0.1-0.3 mg / L, sucrose 30 g / L and carrageenan 7 g / L, and the pH is 6.2;
[0010] (4) The adventitious bud masses were inoculated into a proliferation and propagation medium for proliferation and propagation. The components of the proliferation and propagation medium were MS, BA 2-3 mg / L, NAA 0.1-0.3 mg / L, sucrose 30 g / L, carrageenan 7 g / L, and pH 6.2;
[0011] (5) cutting the propagated seedlings and inoculating them into a seedling medium for cultivation, and then rooting them after reaching the seedling standard. The seedling medium for cultivation is composed of MS, 0.1 mg / L NAA, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2, or MS, 1.0 g / L AC, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2, or 1 / 2 MS, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2;
[0012] (6) The seedlings that have reached the seedling standard are inoculated into a rooting medium to make them reach the rooting standard. The rooting medium composition is: MS, NAA 0.1 mg / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2, or MS, AC 0.1 g / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2.
[0013] Furthermore, the explant treatment process includes: selecting a mother plant that is healthy, pest-free, and genetically pure, making its stems and leaves dry, cutting off the shortened stems or side buds at the bottom of the plant, removing the shortened stems, leaves, and roots, cleaning the surface soil and impurities, cutting off the older part of the stem, ring-cutting to remove the petioles and pulling out the hairs inside the petioles, cutting the cluster buds into single buds, and grading them according to the degree of tenderness, and then wiping the explants with alcohol cotton pads.
[0014] Furthermore, the explant disinfection process includes: disinfection with 75% alcohol for 30 seconds, rinsing with sterile water, disinfection with 0.1% mercuric chloride for 20-40 minutes, and rinsing with sterile water.
[0015] Furthermore, the inoculation and culturing process of step (2) is as follows: after disinfection, the stem explants are cut off from the damaged parts of 0.2 cm at both ends, cut into small segments of 1.5 cm, and inserted into the inoculation culture medium with the morphological upper end facing upwards, and cultured for 21 days at 24-26°C, 3000LX, 12h light / 12h dark conditions.
[0016] Furthermore, in the process of step (3), the newly grown lateral buds are cut off, inoculated into the induction medium, and cultured for 38-42 days at 24-26°C, 3000LX, 12h light / 12h dark.
[0017] Furthermore, the process of step (4) is as follows: the clumps after initiation of induction culture are inoculated into a proliferation and expansion medium, cultured at 24-26°C, 3000LX, 12h light / 12h dark, with a transfer cycle of 30 days, and cultured once or twice or more.
[0018] Furthermore, in the step (5), the bud clusters obtained by proliferation and expansion are cut into small clusters of 2-3 buds with a plant height of 2.0-2.5 cm, transferred to seedling growth medium, cultured under 24-26°C, 3000LX, 12h light / 12h dark conditions, and the transfer cycle is 43-48 days.
[0019] Furthermore, in the process of step (6): the roots of the transitional seedling clusters are cut off, and individual plants with a plant height of 3.0-3.5 cm are selected and inoculated into a rooting medium, and cultured for 13 days at 24-26°C, 3000LX, 12h light / 12h dark conditions.
[0020] Furthermore, the seedling standard is that the plant height is 3.0-3.5 cm and the leaves have obvious spots.
[0021] Furthermore, the rooting standard is a plant height of 3.0-4.0 cm, a crown width of 3.0-4 cm, 4-6 leaves, a leaf length of 0.6-1.0 cm, a leaf width of 1.0-1.5 cm, 2-3 roots, a root length of 1.5-2 cm, no excessive growth, no variation, no curling of leaves, and a single plant with obvious leaf mottle.
[0022] The advantages of the invention are: the proliferation rate is as high as 3.96-6.68; there is no variation, the stability and consistency of the variety can be maintained, the seedling rate is as high as 0.8-1.25, the rooting rate is as high as 100%, and a large number of firefly giant wufeng grass plants can be produced to meet market demand. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:
[0024] Figure 1 It is a photo of the firefly Da Wufeng grass;
[0025] Figure 2 It is the mother stock drying state;
[0026] Figure 3 is a photograph of explant pretreatment;
[0027] Figure 4 It is a photo of the detailed processing of the explant;
[0028] Figure 5 This is a photo of the explant after disinfection;
[0029] Figure 6 These are photos of the explant status before and after inoculation and culture;
[0030] Figure 7 This is a photo of cutting side buds;
[0031] Figure 8 It is the photos of the status before and after the induction;
[0032] Fig. 9 The left picture is a photo of the inoculation of the multiplication and propagation culture, the middle picture is a photo of the multiplication and propagation culture 30 days later, and the right picture is a photo of the multiplication and propagation culture 35 days later;
[0033] Fig.10 This is a photo of the cut of the mass obtained by proliferation and expansion culture;
[0034] Fig.11 This is a photo of the cutting before inoculation of strong seedlings and seedling cultivation;
[0035] Fig.12 These are photos of the strong seedlings after inoculation and cultivation;
[0036] Fig.13 This is a photo of the cutting before inoculation of the rooting culture;
[0037] Fig.14 These are photos of the inoculation and culture period when the rooting medium hormones were MS and NAA 0.1 mg / L;
[0038] Fig.15 These are photos of the inoculation and culture period when the rooting medium hormones were MS and AC 0.1 g / L;
[0039] Fig.16 It is the rooting standard diagram;
[0040] Fig.17 is a photograph of the inoculation and culture status of treatment 1 in Example 3;
[0041] Fig.18 is a photograph of the inoculation and culture status of treatment 2 in Example 3;
[0042] Fig.19 This is a photo of the state after the first multiplication, expansion, inoculation and cultivation of Treatment 1-3 in Example 4;
[0043] Fig. 20 This is a photograph of the state of treatment 1-3 after the second proliferation and expansion culture in Example 4;
[0044] Fig.21 This is a photo of the inoculation and culture status of Treatment 1 in Example 5;
[0045] Fig. 22 This is a photo of the inoculation and culture status of treatment 2 in Example 5;
[0046] Fig.23 This is a photo of the inoculation and culture status of treatment 3 in Example 5;
[0047] Fig.24 This is a photo of the inoculation and culture status of treatment 4 in Example 5;
[0048] Fig.25 This is a photo of the inoculation and culture status of Treatment 1 of Example 6;
[0049] Fig.26 This is a photo of the inoculation and culture status of Treatment 2 of Example 6;
[0050] Fig. 27 This is a photo of the inoculation and culture status of treatment 3 of Example 6;
[0051] Fig.28 This is a photo of the inoculation and culture status of treatment 4 of Example 6;
[0052] Fig.29 is a photograph of the inoculation and culture status of treatment 1 of Example 7;
[0053] Fig.30 This is a photo of the inoculation and culture status of Treatment 2 of Example 7;
[0054] Fig.31 This is a photo of the inoculation and culture status of treatment 3 of Example 7;
[0055] Fig.32 This is a photo of the inoculation and culture status of treatment 4 of Example 7;
[0056] Fig.33 This is a photo of the inoculation and culture status of Treatment 5 of Example 7. DETAILED DESCRIPTION
[0057] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.
[0058] Embodiment 1
[0059] STG0 maternal pretreatment
[0060] ① Requirements for the mother plant: a strong, pest-free mother plant with pure genetic traits;
[0061] ② Treatment method: Place in a dry and ventilated place to dry the substrate, do not water, prevent rain, keep the stems and leaves dry, such as Figure 2 shown.
[0062] Sterilization and inoculation of STG1 explants
[0063] Explant pretreatment:
[0064] a) Time: Sunny day
[0065] b) Processing tools: scalpel or scissors
[0066] c) Treatment method: Use a knife to cut off the shortened stems or lateral buds at the bottom of the plant, remove the leaves and roots, and use a paper towel to wipe off the soil and impurities on the surface. Figure 3 shown.
[0067] Explant fine processing:
[0068] a) Processing tools: alcohol cotton or clean paper towel
[0069] b) Treatment method: Use a knife to cut off the older part of the stem, girdle the petiole and remove the hair inside the petiole, cut the cluster bud into single buds, and grade them according to their tenderness. Be careful not to cut the main part of the stem, and then wipe the explant with an alcohol cotton pad. Figure 4 shown.
[0070] Explant sterilization:
[0071] a) Preparation before disinfection: Open the clean bench and prepare sterile water
[0072] b) Processing tools: 75% alcohol, 0.1% mercuric chloride, 7-8 bottles of sterile water, sterilized wide-mouth bottles, bottles for waste mercury
[0073] c) Explant cutting method: Place the entire stem in a sterile bottle for sterilization
[0074] Disinfection method: 75% alcohol disinfection for 30 seconds, sterile water rinse 2-3 times, 0.1% mercuric chloride disinfection for 20-40 minutes, and then rinse with sterile water 5-6 times. Figure 5 As shown, the disinfection success rate is 37.5%-50%.
[0075] Explant inoculation:
[0076] a) Processing tools: culture medium, sterilized inoculation tools
[0077] b) Treatment method: Cut off the injured part of the stem explants at both ends, about 0.2 cm, cut into small pieces of about 1.5 cm, and insert them into the culture medium with the upper end facing upwards. Figure 6 As shown on the left
[0078] c) Inoculation medium: MS, BA 1-2 mg / L, NAA 0.01 mg / L, sucrose 30 g / L and carrageenan 7 g / L, pH 6.2,
[0079] d) Culture conditions: 24-26°C, 3000LX, 12h light / 12h dark
[0080] Note: After one week, remove those contaminated by fungi and bacteria; check and observe the growth of explants frequently. If the explants turn brown, it means that the disinfection time is too long and should be shortened.
[0081] STG2 starts the induction period
[0082] Inoculate and culture for 21 days. Figure 6 As shown in the right picture, side buds have grown; Figure 7 As shown, the newly grown lateral buds were cut off and inoculated into the start-up induction medium for further induction. After about 40 days of induction, the adventitious bud induction rate was 66.7%.
[0083] a) Induction medium composition: MS, BA2 mg / L, NAA 0.1 mg / L, sucrose 30 g / L and carrageenan 7 g / L, pH 6.2, 1 ball / bottle
[0084] b) Culture conditions: 24-26°C, 3000LX, 12h light / 12h dark
[0085] c) Transfer cycle: 38-42 days.
[0086] like Figure 8 The left picture shows the state of starting induction culture inoculation. Figure 8 The right figure shows the status on the 41st day after induction.
[0087] STG3 proliferation and expansion period
[0088] The clumps after induction culture are inoculated into the proliferation and expansion medium for proliferation and expansion culture, such as Fig. 9 As shown in the left figure,
[0089] a) Proliferation and expansion medium: MS, BA2 mg / L, NAA 0.2 mg / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2, 8-10 pellets / bottle
[0090] b) Culture conditions: 24-26°C, 3000LX, 12h light / 12h dark
[0091] c) Transfer cycle: 30 days
[0092] like Fig. 9 As shown in the middle figure, after culture, the proliferation clusters have dense buds, most of the leaves are small and no spots can be observed, and spots can be seen on a small number of larger leaves. The proliferation rate is 3.0-4.1, and the mutation rate is 0%.
[0093] If the culture time exceeds 1 month, the proliferating mass will vitrify. Fig. 9 As shown in the right figure, after 35 days of proliferation and expansion culture, the proliferated seedlings showed a certain degree of vitrification.
[0094] When it is necessary to continue to multiply and expand, Fig.10 As shown, the large leaves of the proliferation cluster are cut off, and small clusters of 4-5 buds are cut and then inoculated into the proliferation and expansion medium for cultivation.
[0095] STG4 seedling stage
[0096] like Fig.11 As shown, the bud clusters obtained by proliferation and expansion are cut into small clusters of 2-3 buds with a plant height of 2.0-2.5 cm, and transferred to seedling growth medium.
[0097] a) Seedling medium: MS, NAA 0.1mg / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2, or MS, AC1.0g / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2,
[0098] b) Culture conditions: 24-26°C, 3000LX, 12h light / 12h dark
[0099] c) Transfer cycle: 43-48 days
[0100] Seedling standard: plant height is 3.0-3.5cm, and leaves have obvious spots. Fig.12 The two pictures above are the inoculation status of two strong seedlings culture medium, and the two pictures below are the status of 46 days of culture. The seedlings can be formed after one transition, and the spots on the leaves can be clearly seen after 45 days of culture. The seedling rate is 0.8-1.25, and the proliferation rate is 0.1-0.3.
[0101] STG5 rooting seedling stage
[0102] Cutting method: Fig.13 As shown, the transition seedling mass is cut to remove the root system, and the individual plants with a plant height of 3.0-3.5 cm are selected. The cut seedlings are inoculated into a rooting medium for rooting culture.
[0103] a) Rooting medium: MS, NAA 0.1 mg / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2, or MS, AC 0.1 g / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2,
[0104] b) Culture conditions: 24-26°C, 3000LX, 12h light / 12h dark
[0105] Firefly giant wufeng grass takes root quickly. It starts to take root after 6 days of cultivation and the rooting rate is 100% after 13 days of cultivation.
[0106] The rooting medium is MS, NAA 0.1mg / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2. Fig.14 The upper picture shows the inoculation status, and the lower picture shows the 13-day culture status;
[0107] The rooting medium is MS, AC 0.1g / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2. Fig.15 The picture above is a photo of the inoculation status, and the picture below is the status after 13 days of cultivation.
[0108] like Fig.16 As shown, the rooting standard is plant height 3.0-4.0cm, crown width 3.0-4cm, 4-6 leaves, leaf length 0.6-1.0cm, leaf width 1.0-1.5cm, 2-3 roots, root length 1.5-2cm, no excessive growth, no mutation, no curling of leaves, and single plants with obvious leaf mottle.
[0109] Embodiment 2
[0110] This embodiment provides different explant disinfection tests. Except for the differences shown in Tables 1 and 2, the rest refer to Example 1, which is as follows:
[0111] Table 1
[0112]
[0113] Table 2
[0114]
[0115]
[0116] Only callus grew in treatments 1-3, and no callus or new buds grew in treatments 4-9. This variety was easily reverted to its ancestral home using callus induction, indicating that petioles, leaves, and petals were not suitable as explants.
[0117] Embodiment 3
[0118] This embodiment provides different startup induction tests. Except for the differences shown in Table 3, the other reference examples are as follows:
[0119] Table 3
[0120]
[0121] The culture medium in Table 3 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0122] The inoculation status of treatment 1 is as follows Fig.17 As shown in the left figure, the state after culture is Fig.17 As shown in the picture on the right.
[0123] The inoculation status of treatment 2 is as follows Fig.18 As shown in the left figure, the state after culture is Fig.18 As shown in the picture on the right.
[0124] It can be seen that increasing the dosage of NAA is not conducive to the formation of adventitious buds. If the buds in treatment 2 are white, attention should be paid to the subsequent variation.
[0125] Embodiment 4
[0126] This example provides a proliferation and expansion test. Except for the differences shown in Table 4 and Table 5, the other reference examples are as follows:
[0127] Table 4
[0128]
[0129]
[0130] The culture medium in Table 4 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0131] like Fig.19 The picture above shows the inoculation status, and the three pictures below from left to right show the status after proliferation and expansion culture of treatments 1-3.
[0132] The proliferation rate of this multiplication and expansion culture is 2.7-4.1, the number is small, and the mutation rate is temporarily unknown. The leaves of treatments 1 and 2 are green and yellowish; the proliferation rate of treatment 3 is 4.1, the petiole is longer and the leaves have obvious spots. It is necessary to continue to culture for several generations to observe the leaf mutation.
[0133] The proliferation rates of the three treatments were good. For example, spots were visible in the proliferation seedling stage, and the variation rate needed to be statistically analyzed.
[0134] The seedling clusters obtained by multiplication and expansion shown in Table 4 were subjected to further multiplication and expansion culture as shown in Table 5. Table 5:
[0135]
[0136] The culture medium in Table 5 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0137] like Fig. 20 Shown from left to right are the states after culture of treatments 1-3.
[0138] The proliferation rate of this multiplication and expansion culture was 3.96-6.68, which was relatively high. The buds were densely crowded together, and the variant clusters could not be determined based on the leaf spots. The variation situation was temporarily unclear, and a small number of clusters needed to be cultured for transitional culture observation. The same culture medium was used for this proliferation, but the proliferation rate was quite different, which should be caused by the different culture medium for the last proliferation and expansion. The proliferation rate of treatment 3 was higher than that of treatment 2, indicating that increasing NAA was beneficial to the formation and growth of adventitious buds. The higher proliferation rate of treatment 3 than that of treatment 1 indicated that excessive BA and NAA would inhibit the formation of adventitious buds.
[0139] There are fewer leaves with spots in this proliferation, which may be due to the large number of adventitious buds. The leaves are tender and have not formed spots yet. The BA and NAA can be reduced to continue observing the leaf spots. The proliferation rate is above 3, and the proliferation effect is good, but the variation is unclear. The number of clumps is large, so it can be used for the next step of seedling cultivation and further observation.
[0140] Embodiment 5
[0141] This example provides a seedling cultivation test. Except for the differences shown in Tables 6, 7, and 8, the other reference examples are as follows:
[0142] Table 6
[0143]
[0144] The culture medium in Table 6 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0145] The mutation rate and seedling rate of the two treatments were both 0. The bud clusters obtained from the two treatments were dense and arched into huge clumps. This should be caused by the high hormone content in the proliferation and expansion culture medium and the presence of BA in the seedling culture medium. The leaf spots were few and uneven, and the mutation rate could not be determined for the time being.
[0146] The seedlings of the two treatments were cultured for the second time to grow strong seedlings. The culture medium and the effects are shown in Tables 7 and 8:
[0147] Table 7
[0148]
[0149] The culture medium in Table 7 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0150] Table 8
[0151]
[0152] The second seedling cultivation had a seedling rate of 0.8-1.06 and a proliferation rate of 0.2-0.3. All of them had roots growing out, indicating that roots were easily formed during the transition stage.
[0153] like Figure 21-24 They are the inoculation and culture states of treatments 1-4, with the left picture showing the inoculation state and the middle and right pictures showing the culture states.
[0154] Embodiment 6
[0155] This example provides a seedling cultivation test. Except for the differences shown in Table 9, the other reference examples are as follows:
[0156] Table 9
[0157]
[0158] The culture medium in Table 8 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0159] The proliferation rate is 0.08-0.6, the transition seedling rate is 0.4-0.87, and the transition seedling rate of treatments 2 and 3 is 0.6-0.8, which is higher. The appearance of leggy growth and dead leaves should be caused by too long a cultivation time. According to the pictures, the experiment can be terminated after 45 days of cultivation; Figure 25-28 They are treatments 1-4, from left to right: inoculation, 45-day culture, and 70-day culture.
[0160] Treatment 4 showed clump death and slow growth, with poor growth status, and uneven growth of seedlings in treatment 4;
[0161] The seedling rates of treatments 2 and 3 were similar and both were better than treatment 1, indicating that halving the macronutrients and adding NAA were beneficial to the transition to seedlings. The culture medium of treatments 2 and 3 can be used as a medium for seedlings.
[0162] Embodiment 7
[0163] This example provides a rooting culture test. Except for the differences shown in Table 10, the other reference examples are as follows:
[0164] Table 10
[0165]
[0166] The culture medium in Table 10 also includes 30 g / L sucrose, 7 g / L carrageenan, and a pH of 6.2.
[0167] Table 11
[0168]
[0169] The rooting rates of the five treatments were all 100%. White roots began to form 6 days after inoculation and the rooting rate reached 100% after 13 days of cultivation. The rooting effect was good, indicating that Firefly Big Wufeng Grass was easy to form roots.
[0170] There was little difference in plant height, crown width, number of leaves, leaf length and leaf width among the five treatments, but the number of roots in treatment 1 was the least, and the number of roots in treatments 2, 3, 4 and 6 was not much different, indicating that the addition of activated carbon and NAA was beneficial to the root formation of firefly clover.
[0171] The rooting medium for treatments 3, 4, and 6 was the same, but the roots in treatment 4 were the shortest, which should be due to the different transition medium.
[0172] Adding activated carbon and NAA is beneficial to the rooting of firefly clover. MS, AC 0.1g / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2 and MS, NAA 0.1mg / L, sucrose 30g / L, carrageenan 7g / L, pH 6.2 can be used as rooting culture media for firefly clover.
[0173] Figure 29-33 They are the inoculation and post-cultivation states of treatments 1-5, the top two figures are the inoculation states, and the bottom two figures are the post-cultivation states.
[0174] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.
Claims
1. A firefly wormwood tissue culture process, characterized in that: The process includes: (1) Explant processing and disinfection; (2) explant inoculation culture to germinate lateral buds, the inoculation medium composition is MS, BA 1-2 mg / L, NAA 0.01 mg / L, sucrose 30 g / L and carrageenan 7 g / L, pH 6.2; (3) cutting off the lateral buds and inoculating them into a start-up induction medium to obtain adventitious bud masses, wherein the start-up induction medium comprises MS, BA 1-2 mg / L, NAA 0.1-0.3 mg / L, sucrose 30 g / L and carrageenan 7 g / L, and the pH is 6.2; (4) The adventitious bud masses were inoculated into a proliferation and propagation medium for proliferation and propagation. The components of the proliferation and propagation medium were MS, BA 2-3 mg / L, NAA 0.1-0.3 mg / L, sucrose 30 g / L, carrageenan 7 g / L, and pH 6.2; (5) cutting the propagated seedlings and inoculating them into a seedling medium for cultivation, and then rooting them after reaching the seedling standard. The seedling medium for cultivation is composed of MS, 0.1 mg / L NAA, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2, or MS, 1.0 g / L AC, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2, or 1 / 2 MS, 30 g / L sucrose, 7 g / L carrageenan, pH 6.2; (6) The seedlings that have reached the seedling standard are inoculated into a rooting medium to make them reach the rooting standard. The rooting medium composition is: MS, NAA 0.1 mg / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.2, or MS, AC 0.1 g / L, sucrose 30 g / L, carrageenan 7 g / L, pH 6.
2.
2. The tissue culture process of Firefly sphaerocephala according to claim 1, characterized in that: The explant treatment process includes: selecting a mother plant that is healthy, pest-free, and genetically pure, making its stems and leaves dry, cutting off the shortened stems or side buds at the bottom of the plant, removing the shortened stems, leaves, and roots, cleaning the surface soil and impurities, cutting off the older part of the stem, ring-cutting to remove the petioles and pulling out the hair inside the petioles, cutting the cluster buds into single buds, and grading them according to their tenderness, and then wiping the explants with alcohol cotton pads.
3. A firefly wormwood tissue culture process according to claim 1 or 2, characterized in that: The explant disinfection process includes: disinfection with 75% alcohol for 30 seconds, rinsing with sterile water, disinfection with 0.1% mercuric chloride for 20-40 minutes, and rinsing with sterile water.
4. The tissue culture process of Firefly sphaerocephala according to claim 1, characterized in that: The inoculation and culturing process of step (2) is as follows: after disinfection, the stem explants are cut off from the injured parts of 0.2 cm at both ends, cut into 1.5 cm small segments, and inserted into the inoculation culture medium with the morphological upper end facing upwards, and cultured for 21 days under the conditions of 24-26°C, 3000LX, 12h light / 12h dark.
5. The tissue culture process of Firefly sphaerocephala according to claim 1, characterized in that: The process of step (3) is as follows: the newly grown lateral buds are cut off, inoculated into the induction medium, and cultured for 38-42 days at 24-26° C., 3000 LX, 12 h light / 12 h dark.
6. The tissue culture process of Firefly sphaerocephala according to claim 1, characterized in that: The process of step (4) is as follows: the mass after initiation of induction culture is inoculated into a proliferation and expansion medium, cultured at 24-26° C., 3000 LX, 12 h light / 12 h dark conditions, with a transfer cycle of 30 days, and cultured once or twice or more.
7. The tissue culture process of Firefly Globeworm according to claim 1, characterized in that: The process of step (5) is as follows: the bud clusters obtained by proliferation and expansion are cut into small clusters of 2-3 buds with a plant height of 2.0-2.5 cm, and transferred to a seedling medium for seedling growth, and cultured under the conditions of 24-26° C., 3000 LX, 12 h light / 12 h dark, with a transfer cycle of 43-48 days.
8. The tissue culture process of Firefly sphaerocephala according to claim 1, characterized in that: The process of step (6) is as follows: the roots of the transitional seedling clusters are cut off, and individual plants with a plant height of 3.0-3.5 cm are selected and inoculated into a rooting medium, and cultured for 13 days at 24-26° C., 3000 LX, 12 h light / 12 h dark conditions.
9. The tissue culture process of Firefly Globeworm according to claim 1, characterized in that: The seedling standard is that the plant height is 3.0-3.5 cm and the leaves have obvious spots.
10. The tissue culture process of Firefly Globeworm according to claim 1, characterized in that: The rooting standard is that the plant height is 3.0-4.0 cm, the crown width is 3.0-4 cm, the number of leaves is 4-6, the leaf length is 0.6-1.0 cm, the leaf width is 1.0-1.5 cm, the number of roots is 2-3, the root length is 1.5-2 cm, the plant does not grow too tall, does not mutate, does not curl leaves, and the leaves have obvious mottled patterns.