A method for tissue culture of centaurea
By using tissue culture methods of cornflower leaves, including inducing callus and adventitious bud differentiation, devitrification, and rooting culture, the problem of instability in the cornflower regeneration system was solved, and an efficient regeneration system was established, laying the foundation for research on the synthesis and genetic transformation of bioactive substances.
Patent Information
- Application Number
- CN202410033090.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-09
- Publication Date
- 2025-11-28
- Estimated Expiration
- 2044-01-09
AI Technical Summary
The existing cornflower regeneration system is unstable and has poor reproducibility, which hinders the establishment of genetic transformation systems and the research on the synthesis of bioactive substances.
Using cornflower leaves as explants, an efficient tissue culture method was established through steps such as inducing callus and adventitious bud differentiation, devitrification culture, and rooting. The culture was carried out using MS and 1/2 MS media with specific formulations, including callus induction and adventitious bud differentiation medium, devitrification medium, and rooting medium.
A stable cornflower regeneration system was established, providing a foundation for the verification of key gene functions and the synthesis of bioactive substances, and improving the reproducibility and efficiency of the regeneration system.
Smart Images
Figure BDA0004656777740000061 
Figure BDA0004656777740000071 
Figure HDA0004656777980000011
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of plant tissue culture, in particular to a tissue culture method of Centaurea cyanus. BACKGROUND
[0002] Centaurea cyanus is a kind of medicinal and edible flowers, which is widely used in medicine, cosmetics and food industry. The flower extract contains a large amount of bioactive substances, which can play a role in anti-inflammatory and antioxidant function, and can be used as a source of edible pigment and dietary fiber in the future. The flower color is beautiful blue, which has important application value in cut flower and ground cover cultivation. In the field of blue flower research, Centaurea cyanus is a representative species of cyanin blue due to its unique coloring substances, and has extremely important scientific research value. Foreign researchers have preliminarily explored the regeneration system of Centaurea cyanus, but the system is not stable and has poor repeatability, which hinders the establishment of genetic transformation system. The key candidate genes obtained based on transcriptome analysis cannot be functionally analyzed in the original plant, which greatly hinders the research on the molecular mechanism of medicinal and edible ingredient synthesis and blue flower formation.
[0003] At present, the most stable gene function verification system is still the genetic transformation verification established in the original plant. The most commonly used is the tissue culture environment and transformation technology based on Agrobacterium infection, and the realization of this technology depends on the efficient regeneration system. The establishment of efficient tissue culture system of Centaurea cyanus can fundamentally solve the problem of Centaurea cyanus regeneration, lay a foundation for key gene function verification, and is beneficial to the in vitro synthesis and development and utilization of endogenous bioactive substances of Centaurea cyanus, and provides biological technology for flower color research. SUMMARY
[0004] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a tissue culture regeneration method of Centaurea cyanus.
[0005] To solve the above technical problems, the present application provides a tissue culture method of Centaurea cyanus, which comprises the following steps:
[0006] The leaf of Centaurea cyanus is used as an explant and inoculated on the induction and differentiation medium for callus induction and adventitious bud differentiation to produce callus and adventitious buds successively. The adventitious buds are inoculated on the de-glassification medium for de-glassification culture. The de-glassified adventitious buds are inoculated on the rooting medium for rooting culture to obtain Centaurea cyanus rooting seedlings.
[0007] In the above method, the induction and differentiation medium for callus induction and adventitious bud differentiation is a solid medium based on MS medium, and 6-BA, NAA and hydrolyzed casein are added to the medium to obtain a solid medium containing 3 mg / L of 6-BA, 0.5 mg / L of NAA and 100 mg / L of hydrolyzed casein.
[0008] In the method, the de-glassifying medium is MS solid medium.
[0009] In the method, the rooting medium is 1 / 2MS solid medium as a base medium, and IBA is added to the base medium to obtain a solid medium with IBA content of 0.8mg / L.
[0010] In the method, the MS solid medium is MS liquid medium as a base medium, and a coagulant is added to the base medium to obtain a solid medium with pH value of 5.8-6.0.
[0011] In the method, the MS liquid medium is a liquid medium obtained by adding water (solvent) and sucrose to MS basic medium salt with vitamins (Murashige & Skog Basal Medium with Vitamins (product of PhytoTech LABS company, product ID: M519)), and the content of the MS basic medium salt with vitamins in the MS liquid medium is 4.43g / L, and the content of sucrose is 30g / L.
[0012] In the method, the 1 / 2MS solid medium is 1 / 2MS liquid medium as a base medium, and a coagulant (the coagulant can be plant gel or agar, etc.) is added to the base medium to obtain a solid medium with pH value of 5.8-6.0. In the embodiment of the application, agar is selected as the coagulant, and the content of sucrose in the 1 / 2MS solid medium is 30g / L.
[0013] In the method, the 1 / 2MS solid medium is 1 / 2MS liquid medium as a base medium, and a coagulant is added to the base medium to obtain a solid medium with pH value of 5.8-6.0.
[0014] In the method, the solute (except water) of the 1 / 2MS liquid medium is the same as that of the MS liquid medium, the content of the macroelement is half of the corresponding solute content in the MS medium, and the content of the remaining solute is the same as that of the MS liquid medium.
[0015] In the method, the pH value of the callus induction and adventitious bud differentiation medium, the de-glassifying medium and the rooting medium is 5.8-6.0.
[0016] In the method, the explant is obtained from the scabious of sterile sowing and growing to 4 true leaves, and the first to third true leaves from the cotyledon.
[0017] In the method, the explant is a small disc leaf with a diameter of 0.5-0.8 cm.
[0018] In the method, the culture is carried out at a temperature of 20-22℃, a light intensity of 2000lx, and a light / dark cycle of 16h light and 8h dark per day.
[0019] In the method, the induction culture is carried out for 40-60 days.
[0020] In the method, the de-glassing culture is carried out for 30-40 days.
[0021] In the method, the rooting culture is carried out for 10-15 days.
[0022] The method for tissue culture of Centaurea also comprises hardening, transplanting and long-day cultivation.
[0023] The application also protects the use of the method for tissue culture of Centaurea in genetic transformation of Centaurea.
[0024] The application uses Centaurea leaves as explants, and obtains regenerated flowering plants after induction of callus and adventitious bud differentiation, de-glassing culture, rooting, hardening, transplanting and long-day cultivation, thereby establishing a regeneration system of Centaurea, and laying a foundation for further establishment and use of a genetic transformation system to analyze internal bioactive substances and flower color molecular mechanisms of Centaurea. BRIEF DESCRIPTION OF DRAWINGS
[0025] Figure 1 The figure shows the growth state of the Centaurea sterile seedling at the sampling time in Example 1 of the application, and the scale is 1 cm.
[0026] Figure 2 The figure shows the differentiation state of the Centaurea leaf in Example 1 of the application cultured in different media for 40 days, and the scale is 2 cm.
[0027] Figure 3 The figure shows the growth state and change process of the Centaurea adventitious bud in Example 1 of the application in different de-glassing culture media for 0-40 days, and the scale is 2 cm. The upper row shows the change process of the de-glassing culture on the MS medium, and the lower row shows the change process of the de-glassing culture on the MSC medium.
[0028] Figure 4 The figure shows the process of rooting, transplanting, hardening and flowering of the Centaurea after de-glassing in Example 1 of the application, and the scale is 1 cm. In the figure, Figure 4 A is the adventitious bud after de-glassing, Figure 4 B is the rooting of the adventitious bud, Figure 4 C is the small seedling after transplanting, Figure 4 D is the large seedling after hardening, Figure 4E of the plant is a flowering plant. DETAILED DESCRIPTION
[0029] The application will be further described in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.
[0030] The experimental methods in the following examples are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.
[0031] The MS solid medium in the following examples is a solid medium with a pH value of 5.8-6.0, which is obtained by adding a coagulant (the coagulant can be selected from plant gel or agar, etc.) to the MS liquid medium as the base medium. In the examples of the present application, agar is selected as the coagulant, and the content of agar in the MS solid medium is 7.0 g / L.
[0032] The MS liquid medium can be a liquid medium obtained by adding water (solvent) and sucrose to the MS basal medium with vitamins (Murashige & Skog Basal Medium with Vitamins (product of PhytoTech LABS company, product ID: M519)), and the content of the MS basal medium with vitamins in the MS liquid medium is 4.43 g / L, and the content of sucrose is 30 g / L.
[0033] The 1 / 2MS solid medium in the following examples is a solid medium with a pH value of 5.8-6.0, which is obtained by adding a coagulant (the coagulant can be selected from plant gel or agar, etc.) to the 1 / 2MS liquid medium as the base medium. In the examples of the present application, agar is selected as the coagulant, and the content of sucrose in the 1 / 2MS solid medium is 30 g / L.
[0034] The solutes (except water) of the 1 / 2MS liquid medium, the solvent (water) and the MS liquid medium are the same, and the content of the macroelement is half of the corresponding solute content in the MS medium, and the content of the remaining solutes is the same as that of the MS liquid medium.
[0035] Example 1
[0036] The present application provides a leaf regeneration system of Centaurea, which will be further described in conjunction with the specific embodiments:
[0037] The material used in the present application is the seed of Centaurea cyanus (LOT: N3130-1) purchased from Outsidepride Garden Store, USA.
[0038] The method for establishing the regeneration system of Centaurea cyanus leaf comprises the following steps:
[0039] A. Obtaining of the aseptic seedlings
[0040] After washing the seed of Centaurea cyanus, the following steps are performed for disinfection in the clean bench: 1%-2% sodium hypochlorite disinfection for 8 min; washing with sterile water for 6-8 times, 2 min each time. Shake the triangular cone bottle during the disinfection process to make the seed and liquid fully contact. After disinfection, the explant (seed) is inoculated into the MS solid medium to obtain the aseptic seedlings.
[0041] B. Culture of the aseptic seedlings
[0042] The obtained aseptic seedlings are cultured in the tissue culture room for 20-30 d to grow to 4 true leaves, and the picture is shown in Figure 1 .
[0043] The culture condition of the tissue culture room is that the temperature is 20-22℃, the light intensity is 2000 lx, and the light is on for 16 h and dark for 8 h per day.
[0044] C. Establishment of the regeneration system
[0045] 1) Callus induction and adventitious bud differentiation
[0046] Nine different callus induction and adventitious bud differentiation media, N11 medium, N12 medium, N13 medium, N21 medium, N22 medium, N23 medium, N31 medium, N32 medium and N33 medium, are set. The nine different callus induction and adventitious bud differentiation media are all based on MS solid medium, and equal amounts of hydrolyzed casein and different contents of 6-BA and NAA are added to the base medium to obtain the solid medium with the content of hydrolyzed casein being 100 mg / L, the content of 6-BA being 1.0-3.0 mg / L, the content of NAA being 0-1.5 mg / L, and the pH value being 5.8-6.0. See Table 1 for details: the content of 6-BA in the N11 medium is 1.0 mg / L, and the content of NAA is 0.5 mg / L; the content of 6-BA in the N12 medium is 1.0 mg / L, and the content of NAA is 1.0 mg / L; the content of 6-BA in the N13 medium is 1.0 mg / L, and the content of NAA is 1.5 mg / L; the content of 6-BA in the N21 medium is 2.0 mg / L, and the content of NAA is 0.5 mg / L; the content of 6-BA in the N22 medium is 2.0 mg / L, and the content of NAA is 1.0 mg / L; the content of 6-BA in the N23 medium is 2.0 mg / L, and the content of NAA is 1.5 mg / L; the content of 6-BA in the N31 medium is 3.0 mg / L, and the content of NAA is 0.5 mg / L; the content of 6-BA in the N32 medium is 3.0 mg / L, and the content of NAA is 1.0 mg / L; the content of 6-BA in the N33 medium is 3.0 mg / L, and the content of NAA is 1.5 mg / L.
[0047] Nine different callus induction and adventitious bud differentiation media were prepared, which were all based on MS solid medium, and equal amounts of hydrolyzed casein and different amounts of 6-BA and IAA were added to the base medium, to obtain a solid medium with a hydrolyzed casein content of 100 mg / L, a 6-BA content of 1.0-3.0 mg / L, an IAA content of 0-0.3 mg / L, and a pH value of 5.8-6.0. Table 1 shows the specific contents of the media: the I11 medium contains 1.0 mg / L of 6-BA and 0.1 mg / L of IAA; the I12 medium contains 1.0 mg / L of 6-BA and 0.2 mg / L of IAA; the I13 medium contains 1.0 mg / L of 6-BA and 0.3 mg / L of IAA; the I21 medium contains 2.0 mg / L of 6-BA and 0.1 mg / L of IAA; the I22 medium contains 2.0 mg / L of 6-BA and 0.2 mg / L of IAA; the I23 medium contains 2.0 mg / L of 6-BA and 0.3 mg / L of IAA; the I31 medium contains 3.0 mg / L of 6-BA and 0.1 mg / L of IAA; the I32 medium contains 3.0 mg / L of 6-BA and 0.2 mg / L of IAA; and the I33 medium contains 3.0 mg / L of 6-BA and 0.3 mg / L of IAA.
[0048] The callus induction and adventitious bud differentiation media of the 18 different formulations were prepared as described above, and the sterile puncher was used to punch 0.5 cm diameter discs from the 1st-3rd true leaves of the aseptic seedlings of Centaurea jacea grown to 4 true leaves in step B, which were inoculated onto the callus induction and adventitious bud differentiation media of the 18 different formulations (N11 medium and the like for 9 and I12 medium and the like for 9), with the edge cut of the leaf disc contacting the callus induction and adventitious bud differentiation medium. The callus and adventitious buds were obtained in succession in a tissue culture room at a temperature of 20-22°C, a light intensity of 2000 lx, and a light duration of 16 h / 8 h darkness. The medium of the same formulation was replaced every 15 days during the culture process, and the inoculation was started in a 100 ml conical flask containing the medium of the same formulation.
[0049] Each medium was repeated 3 times, and 7 explants were treated for each medium in each repetition.
[0050] The number of chirps in each group was counted at 20 days of culture, and the number of differentiations and brownings in each group was counted at 40 days. The chirp rate, differentiation rate, and browning rate, as well as their mean and standard deviation, were calculated. Duncan's test was performed using SPSS 19.0, and P < 0.05 and P < 0.01 were respectively.
[0051] The calculation formula is as follows:
[0052] Callus formation rate = (Number of explants that induced callus formation / Total number of uncontaminated explants in the same treatment) × 100%;
[0053] Browning rate = (Number of browned explants / Total number of uncontaminated explants in the same treatment) × 100%;
[0054] Differentiation rate = (Number of explants differentiating into adventitious shoots / Total number of uncontaminated explants in the same treatment) × 100%;
[0055] Table 1. Culture medium formulations and induction status for callus induction and adventitious shoot differentiation.
[0056]
[0057]
[0058] Note: Lowercase letters in the same column indicate significant differences at the 5% level, and uppercase letters indicate highly significant differences at the 1% level.
[0059] As shown in Table 1, different combinations of 6-BA and NAA are more conducive to callus induction and adventitious bud differentiation in cornflower leaves, with the highest differentiation rate observed in N31 medium.
[0060] At the same time Figure 2 It is known that different differentiation states occur when cultured in different media for the same amount of time. In N31 medium, adventitious shoots differentiate the fastest, and adventitious shoots can be obtained in a relatively short time. Therefore, N31 medium (MS + 100 mg / L hydrolyzed casein + 3.0 mg / L 6-BA + 0.5 mg / L NAA, i.e., MS solid medium as the basal medium, to which 6-BA, NAA, and hydrolyzed casein are added, resulting in a hydrolyzed casein content of 100 mg / L, a 6-BA content of 3.0 mg / L, a NAA content of 0.5 mg / L, and a pH of 5.8-6.0) is the optimal medium for the differentiation of callus and adventitious shoots in cornflower.
[0061] 2) Devitrification of adventitious buds
[0062] MS, MSC two de-vitrification medium for the de-vitrification culture of adventitious buds. Among them, MS medium is MS solid medium, MSC medium is MS solid medium as the basic medium, and activated carbon is added to the basic medium to obtain a solid medium with activated carbon content of 500 mg / L and pH value of 5.8-6.0. The medium is placed in a 240 ml volume glass culture bottle.
[0063] The vitrified adventitious buds grown to 0.8 cm in N31 medium in step 2) are inoculated into de-vitrification medium, and de-vitrification culture is carried out in a tissue culture room with temperature of 20-22℃, light intensity of 2000lx, and 16h light and 8h darkness per day.
[0064] Each medium is repeated 3 times, and 7 vitrified adventitious buds are treated for each medium in each repetition.
[0065] After 40 days of culture, the number of normal buds is counted, the de-vitrification rate is calculated, Duncan's test is performed using SPSS19.0, and P<0.05 and P<0.01.
[0066] The calculation formula is as follows:
[0067] De-vitrification rate = (de-vitrification normal bud number / non-contaminated vitrification seedling inoculation number in the same treatment) x 100%
[0068] The results show that MS medium and MSC medium can both restore vitrified buds, with de-vitrification rates of 64.00% (±4.46) and 57.5% (±3.15) respectively, as shown in Figure 3 , and there is no significant difference between the two media. Based on the consideration of drug and technical cost, MS medium is considered to be the optimal de-vitrification medium.
[0069] 3) Inducing adventitious roots
[0070] Five different formulations of rooting medium are set, G1 medium, G2 medium, G3 medium, G4 medium and G5 medium, all of which are 1 / 2MS solid medium as the basic medium, and IBA is added to the basic medium to obtain a solid medium with IBA content of 0-1.6 mg / L and pH value of 5.8-6.0. See Table 2 for details: the IBA content in G1 medium is 0 mg / L; the IBA content in G2 medium is 0.2 mg / L; the IBA content in G3 medium is 0.4 mg / L; the IBA content in G4 medium is 0.8 mg / L; the IBA content in G5 medium is 1.6 mg / L.
[0071] The normal adventitious shoots cultured on MS medium without glass were inoculated into rooting medium, and the rooting culture was carried out in a tissue culture room with temperature of 20-22℃, light intensity of 2000lx, and 16h light and 8h dark per day.
[0072] Each medium was repeated for 3 times, and 7 normal adventitious shoots were treated for each medium.
[0073] After 15d culture, the number of rooting shoots and roots was counted, and the rooting rate and average rooting number were calculated.
[0074] The calculation formula was as follows:
[0075] Rooting rate=(number of rooting shoots / total number of un-contaminated shoots in the same treatment) x 100%
[0076] Table 2 Rooting medium formula and rooting condition
[0077] Number IBA content (mg / L) Rooting rate (%) Average number of roots G1 0 20.00 3.33±0.47b G2 0.2 26.67 3.25±0.83b G3 0.4 13.33 3.50±0.5b G4 0.8 33.33 5.20±0.75a G5 1.6 13.33 3.00±0.00b
[0078] Note: The same column lowercase letters represent significant difference at 5% level. As shown in Table 2, the adventitious shoots can root in each rooting medium, the rooting rate reaches 33.33% in G4 medium, the average rooting number reaches 5.20 roots per shoot, the rooting time is shorter, and the root system grows well, see Figure 4 B. G4 medium (1 / 2MS + 0.8mg / L IBA, i.e. 1 / 2MS solid medium as basic medium, and IBA is added to the medium to obtain a solid medium with IBA content of 0.8mg / L and pH value of 5.8-6.0) is the optimal rooting medium for Centaurea moschata adventitious shoots.
[0079] 4) Seedling, transplanting and long-day cultivation: the adventitious shoots with good growth condition were transplanted into mixed substrate in an artificial climate room with temperature of 25℃, light intensity of 2000lx, and 16h light / 8h dark. The mixed substrate was nutrient soil: vermiculite = 1:1 (v:v), which was sterilized at 121℃ high temperature and high pressure, cooled and stirred uniformly, and filled into 9cm wide black square pots. The specific operation was as follows: in the artificial climate room, the sterile culture bottle was opened, the regenerated seedling base was gently clamped with tweezers, and then the base medium was washed clean with water after careful removal, and then transplanted into the moist mixed substrate. After transplanting, the surface was sprayed with water, and then covered with plastic wrap. After 5d, the plastic wrap was opened at one corner, and after 10d, it was completely removed. Watering was carried out every 3-4d, see Figure 4 C and Figure 4 D. In a long-day artificial climate room with temperature of 25℃, air humidity of 40-50%, light intensity of 2000lx, and 16h light / 8h dark, the long-day cultivation was carried out until flowering, and the flowering regenerated seedlings were obtained, see Figure 4 E.
[0080] In summary, the application constructs a regeneration method of the leaf of Centaurea, which is specifically as follows.
[0081] The leaf of Centaurea is used as an explant, inoculated into N31 medium as a callus induction and adventitious bud differentiation medium to be cultured, to generate callus and adventitious bud successively; the adventitious bud is inoculated into MS medium as a de-glassification medium to be de-glassified, and the normal adventitious bud after de-glassification is inoculated into G4 medium as a rooting medium to be rooted, to obtain the rooting seedling of Centaurea. The seedling is obtained through artificial climate room and long-daylight cultivation, and the regenerated seedling is flowered.
[0082] The application is described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, contents and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special embodiment, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art out of the range disclosed in the application. Some basic features can be applied according to the scope of the following attached claims.
Claims
1. A method for cornflower tissue culture, characterized in that, The method includes the following steps: Cornflower leaves were used as explants and inoculated onto callus induction and adventitious shoot differentiation medium for induction culture to produce callus and adventitious shoots; the adventitious shoots were inoculated onto devitrification medium for devitrification culture; and the devitrified adventitious shoots were inoculated onto rooting medium for rooting culture to obtain rooted cornflower seedlings. The induction medium for callus induction and adventitious shoot differentiation is based on MS solid medium, to which 6-BA, NAA and hydrolyzed casein are added to obtain a solid medium with 6-BA content of 3 mg / L, NAA content of 0.5 mg / L and hydrolyzed casein content of 100 mg / L. The devitrification medium is MS solid medium; The rooting medium is a solid medium with 1 / 2 MS solid medium as the base medium, to which IBA is added to obtain a solid medium with an IBA content of 0.8 mg / L. The pH values of the callus induction and adventitious shoot differentiation medium, the devitrification medium, and the rooting medium are all 5.8-6.0; The MS solid medium is a solid medium with a pH of 5.8-6.0 obtained by adding a solidifying agent to the MS liquid medium as the base medium; the MS liquid medium is a liquid medium obtained by adding water and sucrose to the MS basic medium salt containing vitamins, wherein the content of the MS basic medium salt containing vitamins in the MS liquid medium is 4.43 g / L and the content of sucrose is 30 g / L; the MS basic medium salt containing vitamins is M519.
2. The method according to claim 1, characterized in that, The explants were taken from the second and third true leaves of cornflower plants that had grown to four true leaves.
3. The method according to any one of claims 1-2, characterized in that, The culture was carried out at a temperature of 20-22℃, a light intensity of 2000 lx, and 16 hours of light and 8 hours of darkness per day.
4. The method according to any one of claims 1-2, characterized in that, The induction culture time is 40-60 days; the devitrification culture time is 30-40 days; and the rooting culture time is 10-15 days.
5. The method according to any one of claims 1-2, characterized in that, The method also includes transplanting and planting the cornflower rooted seedlings after hardening them off.
6. The application of the method according to any one of claims 1-5 in the genetic transformation of cornflower.