Callus induction, differentiation and plant regeneration method taking rhododendron petals as explants and application thereof

Abstract

The invention provides a callus induction, differentiation and plant regeneration method with rhododendron petals as explants and application of the callus induction, differentiation and plant regeneration method, and belongs to the technical field of tissue culture. S2, performing callus induction; s3, carrying out callus subculture; s4, adventitious bud differentiation; s5, adventitious root differentiation. In the method, the limiting conditions of each step and the components of the culture medium are specifically limited, and by adopting the culture medium and the related condition control method provided by the invention, the pollution rate of rhododendron petal tissue culture can be effectively reduced, the callus rate and differentiation rate of calluses can be improved, and the survival rate of regenerated plants can be further improved.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, in particular to a method for callus induction, differentiation and plant regeneration using azalea petals as explants and an application thereof. Background Art

[0002] Rhododendron is a general term for plants of the genus Rhododendron L. in the family Ericaceae. It is known as the "most beautiful flower" and has extremely high ornamental and ecological value. There are more than 1,200 species of Rhododendron in the world, and my country has the largest population of Rhododendron, with about 600 species. Except for the arid deserts in Ningxia and Xinjiang, Rhododendron is distributed all over China, with the most abundant resources in the Hengduan Mountains in the southwest and the Qinghai-Tibet Plateau. The classic propagation methods of Rhododendron include seed sowing, cutting, grafting, layering, etc. These methods are all restricted to varying degrees by the source of species, seed germination, the number of parents, etc., so overall the development of the Rhododendron industry is relatively slow. Plant tissue culture has the characteristics of high proliferation efficiency and fast reproduction speed, and can produce a large number of test tube seedlings in a shorter time and less space. The tissue culture of azalea generally uses leaves and stem segments as explants, but the surface of azalea leaves is covered with hairs, which carry many pathogens, making tissue culture disinfection difficult and resulting in a high contamination rate. At the same time, due to physiological reasons of azalea plants, the survival rate of azalea tissue culture is usually much lower than that of other varieties. Summary of the invention

[0003] The invention aims to provide a method for tissue culture of azalea with high survival rate, simplicity and low pollution rate.

[0004] To achieve the above object, the present invention provides a method for callus induction and rapid plant propagation using azalea petals as explants, which specifically comprises the following steps:

[0005] S1: Explant selection and sterilization

[0006] After treating the azalea flowers at the early stage at 2-4°C, take the azalea petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash with sterile water, soak in 6% sodium hypochlorite for 7-8 minutes, wash with sterile water, dry the surface of the petals, and remove the browned parts and the edges of the petals by hand tearing to induce wounds;

[0007] S2: Callus Induction

[0008] Inoculate the petals after the treatment in step S1 into a callus induction medium, and culture for 25 to 30 days at 25±2°C, 400 to 600 Lx, and 16 h / d of light, wherein the components and proportions of the callus induction medium are as follows: WPM medium, with additional final concentrations of 1.0 to 1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 to 0.2 mg / L NAA, and 0.1 to 0.2 mg / L TDZ;

[0009] S3: Callus subculture

[0010] Subculturing the callus tissue using a callus tissue subculture medium, subculturing once every 18 to 20 days, for a total of 2 times, the culture conditions are 25±2°C, 400 to 600 Lx, and 16 h / d of light, the composition and ratio of the callus tissue subculture medium are: WPM medium, with a final additional concentration of 1.0 to 1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 to 0.2 mg / L NAA, and 0.1 to 0.2 mg / L TDZ;

[0011] S4: Adventitious bud induction

[0012] The callus tissue was induced and cultured using an adventitious bud induction medium at 25±2°C, 2000-2200Lx, and 16h / d of light for 25-30 days. The composition and proportion of the adventitious bud induction medium were as follows: WPM medium, with a final additional concentration of 1.0-1.2g / L calcium nitrate, 30g / L sucrose, 7g / L agar, 0.2-0.25mg / L NAA, and 1.0-1.2mg / L ZT;

[0013] S5: Adventitious root differentiation

[0014] The callus tissue was induced and cultured using an adventitious root differentiation induction medium at 25±2°C, 2000-2200 Lx, and 16 h / d of light for 25-30 days. The composition and ratio of the adventitious root differentiation induction medium were as follows: WPM medium, with a final additional concentration of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.3-0.4 g / L activated carbon, 0.25-0.3 mg / L NAA, and 2.0-2.2 mg / L IBA.

[0015] Preferably, step S1 specifically includes the following steps: after treating the azalea in the early opening stage at 2-4°C, taking the outer petals, removing dust with running water, soaking the petals in 70-75% ethanol for 30 seconds, washing with sterile water twice, soaking in 6% sodium hypochlorite for 7-8 minutes, washing with sterile water three times, drying the surface moisture of the petals and removing the browned parts and the edges of the petals by hand tearing to induce wounds.

[0016] Furthermore, the second aspect of the present invention also provides a callus induction medium for azalea tissue culture, the components and proportions of the callus induction medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1-0.2 mg / L NAA, and 0.1-0.2 mg / L TDZ.

[0017] Furthermore, the third aspect of the present invention also provides a callus subculture medium for azalea tissue culture, the components and proportions of the callus subculture medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1-0.2 mg / L NAA, and 0.1-0.2 mg / L TDZ.

[0018] Furthermore, the fourth aspect of the present invention also provides an adventitious bud induction medium for azalea tissue culture, the components and proportions of the adventitious bud induction medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.2-0.25 mg / L NAA, and 1.0-1.2 mg / L ZT.

[0019] Furthermore, the fifth aspect of the present invention also provides an adventitious root differentiation induction medium for azalea tissue culture, the components and ratios of the adventitious root differentiation induction medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.3-0.4 g / L activated carbon, 0.25-0.3 mg / L NAA, and 2.0-2.2 mg / L IBA.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention establishes an azalea regeneration system by using azalea petals as explants. The callus induction and plant rapid propagation method using azalea petals as explants provided by the present invention can greatly improve the induction success rate and differentiation rate of azalea callus, thereby improving the survival rate of azalea regenerated plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention provides a picture of each step of callus induction, differentiation and plant regeneration using azalea petals as explants and the finished product aseptic seedlings. DETAILED DESCRIPTION

[0023] In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation method and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the scope of protection of the claims of the present invention.

[0024] It should be noted that the endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this article.

[0025] Unless otherwise defined, all terms, symbols and other scientific terms used herein are intended to have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. In some cases, terms with conventionally understood meanings are defined herein for the purpose of clarification or ease of reference, and such definitions herein should not be construed as indicating significant differences from conventional understandings in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents and instruments is carried out in accordance with the protocols and parameters given by the manufacturer.

[0026] As described in the background technology, due to the physiological characteristics of azalea itself, the survival rate of regenerated plants of azalea is usually much lower than that of other varieties. Based on this, the specific embodiment of the present invention provides a method for callus induction and rapid plant propagation using azalea petals as explants and its application. Specifically, the above-mentioned method for callus induction and rapid plant propagation using azalea petals as explants includes the following steps:

[0027] S1: Explant selection and sterilization

[0028] After treating the azalea flowers at the early stage at 2-4°C, take the azalea petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash twice with sterile water, soak in 6% sodium hypochlorite for 7-8 minutes, wash three times with sterile water, dry the surface of the petals, and remove the browned parts and the edges of the petals by hand tearing to induce wounds;

[0029] S2: Callus Induction

[0030] Inoculate the petals after the treatment in step S1 into a callus induction medium, and culture for 25 to 30 days at 25±2°C, 400 to 600 Lx, and 16 h / d of light, wherein the components and proportions of the callus induction medium are as follows: WPM medium, with additional final concentrations of 1.0 to 1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 to 0.2 mg / L NAA, and 0.1 to 0.2 mg / L TDZ;

[0031] S3: Callus subculture

[0032] Subculturing the callus tissue using a callus tissue subculture medium, subculturing once every 18 to 20 days, for a total of 2 times, the culture conditions are 25±2°C, 400-600Lx, and a light intensity of 16h / d, and the composition and ratio of the callus tissue subculture medium are: WPM medium, with a final additional concentration of 1.0-1.2g / L calcium nitrate, 30g / L sucrose, 7g / L agar, 0.1-0.2mg / L NAA, and 0.1-0.2mg / L TDZ;

[0033] S4: Adventitious bud induction

[0034] The callus tissue was induced and cultured using an adventitious bud induction medium at 25±2°C, 2000-2200Lx, and 16h / d of light for 25-30 days. The composition and proportion of the adventitious bud induction medium were as follows: WPM medium, with a final additional concentration of 1.0-1.2g / L calcium nitrate, 30g / L sucrose, 7g / L agar, 0.2-0.25mg / L NAA, and 1.0-1.2mg / L ZT;

[0035] S5: Adventitious root differentiation

[0036] The callus tissue was induced and cultured using an adventitious root differentiation induction medium at 25±2°C, 2000-2200 Lx, and 16 h / d of light for 25-30 days. The composition and ratio of the adventitious root differentiation induction medium were as follows: WPM medium, with a final additional concentration of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.3-0.4 g / L activated carbon, 0.25-0.3 mg / L NAA, and 2.0-2.2 mg / L IBA.

[0037] Figure 1 The following are photos of the various steps of the method for rapid plant propagation using azalea petals as explants, wherein: Figure 1 A in the figure is a flower explant. Figure 1 B in the figure is a photo of callus tissue induced by petals. Figure 1 C in the figure is a photo of callus subculture. Figure 1 D in the figure is a photo of callus adventitious bud differentiation. Figure 1 E in the figure is a photo of adventitious root differentiation. Figure 1 F in the figure is a photograph of the final sterile seedling.

[0038] The specific implementation mode of the present invention adopts a new culture medium, and strictly limits the wound initiation mode, callus induction, light intensity during subculture, and culture medium composition ratio, thereby improving the survival rate of azalea tissue culture.

[0039] The technical solution of the present invention is further described below through specific embodiments.

[0040] Example 1

[0041] Effects of Sodium Hypochlorite Disinfection Time on Contamination Rate and Browning Rate of Explants

[0042] The test material was an 8-year-old azalea with healthy growth and no pests and diseases. The flowers in the early opening stage were collected at 8 o'clock in the morning. After being placed at 4℃ for 24 hours, the outermost petals were taken, and the surface of the petals was washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in a clean bench, washed with sterile water twice, and soaked in 6% sodium hypochlorite for 2, 4, 6, 8, and 10 minutes, washed with sterile water three times, and the surface moisture of the petals was dried with sterile paper towels. The browned part and the edge of the petals were cut off with a knife to induce wounds. The petals were inoculated on WPM medium (without calcium nitrate) with the back facing down for callus induction. The medium was supplemented with 0.5g / L calcium nitrate, 30g / L sucrose, and 7g / L agar. One petal was inoculated in each bottle, and 10 bottles were inoculated in each treatment, and repeated 3 times. The culture temperature was set at 25℃, the light intensity was 400Lx, and the light time was 16h / d. The contamination rate and browning rate were calculated after 14 days of culture. Among them, contamination rate = (number of contaminated explants / number of inoculated explants) × 100%; browning rate = (number of browning explants / number of inoculated explants) × 100%. The relevant experimental results are shown in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] It can be seen from the data in Table 1 that when the sodium hypochlorite disinfection time is 8 and 10 minutes, the explant contamination rate is 0%. However, compared with the disinfection time of 10 minutes, the explant browning rate is lower when disinfected for 8 minutes. Therefore, the most preferred disinfection time of sodium hypochlorite is 8 minutes.

[0047] Example 2

[0048] Effects of different hormone formulas on callus induction

[0049] The basic medium for callus induction was WPM medium (without calcium nitrate), supplemented with 0.5 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, and hormones. The three hormone formulas shown in Table 2 were set.

[0050] Flowers in the early opening stage were collected at 8 a.m., and placed at 4°C for 24 hours. The outermost petals were taken, and the surface of the petals were rinsed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in a clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 8 minutes, and washed with sterile water three times. The surface moisture of the petals was absorbed with sterile paper towels, and the browned parts and the edges of the petals were cut off with a knife to induce wounds. The petals were inoculated with the back side down on the callus induction medium containing different hormone formulas. Ten bottles were inoculated for each treatment, and one explant was inoculated in each bottle, and repeated three times. The culture temperature was set at 25°C, the light intensity was 400Lx, and the light time was 16h / d. After 30 days of culture, the callus rate and browning rate were calculated. Among them, the callus rate = (the number of explants forming callus tissue / the number of inoculated explants) × 100%. The experimental results are shown in Tables 2 and Figure 1 shown.

[0051] Table 2

[0052]

[0053] From the data in Table 2, it can be seen that after culturing the explants with the three hormone formulas for 30 days, only the NT treatment group successfully induced callus tissue, the petal color became lighter, from rose red to white, the callus tissue was yellow-green, and it was relatively loose. Figure 1 As shown, the callus rate was 73%, and the explants in the DT and DNZ treatment groups all turned brown and died, which was not suitable for the induction of petal callus. Therefore, the most preferred hormone combination in the callus induction medium was 0.1 mg / L NAA+0.1 mg / L TDZ.

[0054] Example 3

[0055] Effects of wound induction methods on callus induction

[0056] The basic medium for callus induction was WPM medium (without calcium nitrate), supplemented with 0.5 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA, and 0.1 mg / L TDZ.

[0057] The flowers in the early opening period were collected at 8 o'clock in the morning. After being placed at 4℃ for 24 hours, the outermost petals were taken, and the surface of the petals was washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in the clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 6 minutes, washed with sterile water 3 times, and the surface moisture of the petals was dried with sterile paper towels. The browned parts and the edges of the petals were removed by tearing and cutting to induce wounds, and the petals were inoculated on the callus induction medium with the back of the petals facing down. Ten bottles were inoculated for each treatment, and one explant was inoculated in each bottle, and repeated 3 times. The culture temperature was set at 25℃, the light intensity was 400Lx, and the light time was 16h / d. After 30 days of culture, the healing rate and browning rate were counted. The experimental results are shown in Table 3.

[0058] Table 3

[0059] Wound Cause Cure rate (%) Browning rate (%) Tearing method 87 13 Knife cutting method 73 27

[0060] As shown in Table 3, the wounds induced by the tearing method and the knife cutting method can both successfully produce callus tissue in the azalea petals. However, compared with the knife cutting method, the tearing method has a higher callus rate and a lower browning rate, indicating that the tearing method is suitable for the induction of petal callus tissue.

[0061] Example 4

[0062] Effect of flowering period on callus induction

[0063] The basic medium for callus induction was WPM medium (without calcium nitrate), supplemented with 0.5 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA, and 0.1 mg / L TDZ.

[0064] Flowers in the bud, early opening and full bloom stages were collected at 8 a.m., and placed at 4°C for 24 h. The outermost petals were taken, and the petals were washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 s in a clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 6 min, washed with sterile water 3 times, and the surface moisture of the petals was dried with sterile paper towels. The browned parts and petal edges were removed by hand tearing to induce wounds. The petals were inoculated on the callus induction medium with the back facing down. Ten bottles were inoculated per treatment, and one explant was inoculated per bottle, and repeated 3 times. The culture temperature was set at 25°C, the light intensity was 400 Lx, and the light time was 16 h / d. The callus induction rate and browning rate were counted after 30 days of culture. The experimental results are shown in Table 4.

[0065] Table 4

[0066] Flowering period Cure rate (%) Browning rate (%) Bud stage 60 40 Initial stage 87 13 Blooming period 37 63

[0067] As shown in Table 4, compared with the petals in the initial opening stage, the callus rate of the petals in the bud stage and the full bloom stage is lower, while the browning rate is higher. This may be because the petals in the bud stage are too young and tender, and the high concentration of hormones will cause cell damage, while the cells in the full bloom stage have low omnipotence and are not easy to differentiate. The results show that the petals in the initial opening stage are suitable for the induction of callus tissue.

[0068] Example 5

[0069] Effects of different temperature pretreatments on callus induction

[0070] The basic medium for callus induction was WPM medium (without calcium nitrate), supplemented with 0.5 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA, and 0.1 mg / L TDZ.

[0071] At 8 o'clock in the morning, the flowers in the early opening period were collected and placed at different temperatures (4℃, 25℃, and 37℃) for 24 hours. The outermost petals were taken and the petals were washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in the clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 6 minutes, washed with sterile water three times, and the surface moisture of the petals was dried with sterile paper towels. The browned parts and the edges of the petals were removed by hand tearing to induce wounds. The petals were inoculated on the callus induction medium with the back facing down. Ten bottles were inoculated for each treatment, and one explant was inoculated in each bottle, and repeated three times. The culture temperature was set at 25℃, the light intensity was 400Lx, and the light time was 16h / d. After 30 days of culture, the callus induction rate and browning rate were counted. The experimental results are shown in Table 5.

[0072] Table 5

[0073] Temperature(℃) Cure rate (%) Browning rate (%) 4 87 13 25 53 47 37 40 60

[0074] As can be seen from Table 5, compared with the 4°C treatment, the petal callus rate after 25°C and 37°C treatments was lower and the browning rate was higher. This may be because low temperature treatment can improve the state of petal cells and increase the level of endogenous hormones, indicating that 4°C pretreatment is suitable for the induction of petal callus tissue.

[0075] Example 6

[0076] Effect of Calcium Nitrate Concentration on Callus Induction

[0077] The basic medium for callus induction was WPM medium (without calcium nitrate), supplemented with 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA, 0.1 mg / L TDZ and calcium nitrate. The concentration of calcium nitrate was set to 0.1 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, and 2.0 g / L.

[0078] At 8 o'clock in the morning, the flowers in the early opening stage were collected, and after being placed at 4℃ for 24 hours, the outermost petals were taken, and the surface of the petals was washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in the clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 6 minutes, washed with sterile water 3 times, and the surface moisture of the petals was dried with sterile paper towels. The browned parts and the edges of the petals were removed by hand tearing to induce wounds. The back of the petals was inoculated with different calcium nitrate concentrations on the callus induction medium. Ten bottles were inoculated for each treatment, and one explant was inoculated in each bottle, and repeated 3 times. The culture temperature was set at 25℃, the light intensity was 400Lx, and the light time was 16h / d. After 30 days of culture, the callus induction rate and browning rate were counted. The experimental results are shown in Table 6.

[0079] Table 6

[0080]

[0081]

[0082] As shown in Table 6, both too low and too high calcium nitrate concentrations were not conducive to the induction of petal callus. Among them, 1.0 g / L calcium nitrate was most suitable for the induction of petal callus, and the callus rate reached 100%.

[0083] Example 7

[0084] Effects of light on callus induction

[0085] The basic medium for callus induction was WPM medium (without calcium nitrate) supplemented with 1.0 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA and 0.1 mg / L TDZ.

[0086] At 8 o'clock in the morning, the flowers in the early opening period were collected, and after being placed at 4℃ for 24 hours, the outermost petals were taken, and the surface of the petals was washed with running water to remove dust. The petals were soaked in 75% ethanol for 30 seconds in the clean bench, washed with sterile water twice, soaked in 6% sodium hypochlorite for 6 minutes, washed with sterile water 3 times, and the surface moisture of the petals was dried with sterile paper towels. The browned parts and the edges of the petals were removed by hand tearing to induce wounds. The petals were inoculated on the callus induction medium with the back facing down. The culture temperature was set to 25℃, the light intensity was set to 400Lx and 0Lx, the light time was 16h / d, 10 bottles were inoculated for each treatment, and 1 explant was inoculated in each bottle, and repeated 3 times. After 30 days of culture, the callus induction rate and browning rate were counted, and the experimental results are shown in Table 7.

[0087] Table 7

[0088] Light intensity (Lx) Cure rate (%) Browning rate (%) 400 100 0 0 63 37

[0089] As shown in Table 7, compared with the light treatment, the petal callus rate (63%) of the shading treatment was lower and the browning rate (37%) was higher, which was different from the callus conditions of other varieties, indicating that light treatment was suitable for the induction of callus tissue of azalea petals.

[0090] Example 8

[0091] Callus subculture

[0092] The basic medium for callus subculture was WPM medium (without calcium nitrate) supplemented with 1.0 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 mg / L NAA and 0.1 mg / L TDZ.

[0093] Use a scalpel to cut off the callus tissue, transfer it to the callus tissue subculture medium, inoculate 6 pieces of callus tissue in each bottle, set the culture temperature to 25℃, the light intensity to 400Lx, the light time to 16h, subculture once every 20d, subculture twice, and calculate the callus tissue browning rate. After the callus tissue is subcultured twice, its morphology is relatively dense, yellow-green, and the browning rate is 0%.

[0094] Example 9

[0095] Induction of adventitious buds

[0096] The basic medium for adventitious bud induction was WPM medium, supplemented with 1.0 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, NAA, and ZT. Orthogonal experimental design was used to study the effect of hormone combination on adventitious bud induction. The callus tissue was transferred to the adventitious bud induction medium, 6 callus tissues were inoculated in each bottle, the culture temperature was set to 25 ° C, the light intensity was set to 2000 Lx, the light time was 16 h / d, and the browning rate and germination rate were counted after 30 days. The experimental results are shown in Table 8.

[0097] Table 8

[0098] serial number NAA ZT Germination rate (%) Browning rate (%) NZ1 0.1 1.0 0 0 NZ2 0.1 1.5 0 10 NZ3 0.1 2.0 0 23 NZ4 0.2 1.0 83 3 NZ5 0.2 1.5 50 17 NZ6 0.2 2.0 40 20 NZ7 0.3 1.0 23 3 NZ8 0.3 1.5 13 23 NZ9 0.3 2.0 13 37

[0099] It can be seen from Table 8 that when the hormone formula is 0.2-0.3 mg / L NAA + 1.0-2.0 mg / L ZT, the germination rate is relatively high. Among them, when the hormone formula is 0.2 mg / L NAA + 1.0 mg / L ZT, the germination rate reaches 83%, which is most suitable for bud differentiation.

[0100] Example 10

[0101] Effects of hormone formula on adventitious root differentiation

[0102] The basic medium for adventitious root differentiation is WPM medium, supplemented with 1.0g / L calcium nitrate, 30g / L sucrose, 7g / L agar, activated carbon (AC), NAA, and IBA. Orthogonal experimental design was used to study the effect of hormone combination on adventitious root differentiation. The adventitious buds and lower callus tissue were cut off and transferred to the adventitious root differentiation medium. Six adventitious buds were inoculated in each bottle. The culture temperature was set to 25°C, the light intensity was set to 2000Lx, and the light time was 16h / d. After 30 days, the browning rate and rooting rate were statistically analyzed. The experimental results are shown in Table 9.

[0103] Table 9

[0104] serial number IBA(mg / L) NAA(mg / L) AC(g / L) Rooting rate (%) Browning rate (%) INA1 0.5 0 0 0 17 INA2 0.5 0.25 0.3 0 10 INA3 0.5 0.5 0.6 0 3 INA4 1.0 0 0 0 27 INA5 1.0 0.25 0.3 0 20 INA6 1.0 0.5 0.6 0 13 INA7 2.0 0 0 63 13 INA8 2.0 0.25 0.3 80 7 INA9 2.0 0.5 0.6 53 40

[0105] It can be seen from Table 9 that when the formula of hormone and activated carbon is 0.3-0.6g / L activated carbon, 0.25-0.5mg / L NAA, and 1.0-2.0mg / L IBA, the rooting rate is relatively high. Among them, when the hormone formula is 2.0mg / L IBA+0.25mg / L NAA and 0.3g / L AC, the rooting rate is the highest, reaching 80%, which is most suitable for the differentiation of adventitious roots.

[0106] It can be seen from the above embodiments that the use of the culture medium and related condition control methods provided by the present invention can effectively reduce the contamination rate of azalea petal tissue culture, increase the callus initiation rate and differentiation rate, and further increase the survival rate of regenerated plants obtained by using azalea petals as explants.

[0107] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for callus induction, differentiation and plant regeneration using azalea petals as explants, characterized in that: The following steps are involved: S1: Explant selection and sterilization After treating the azalea flowers at the early stage at 2-4℃, take the azalea petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash with sterile water, soak in 6% sodium hypochlorite for 7-8 minutes, wash with sterile water, dry the surface of the petals, and remove the browned parts and the edges of the petals by hand tearing to induce wounds; S2: Callus Induction The petals after the treatment in step S1 are inoculated into a callus induction medium, and cultured for 25 to 30 days at 25±2°C, 400 to 600 Lx, and 16 h / d of light. The components and proportions of the callus induction medium are as follows: WPM medium, with additional final concentrations of 1.0 to 1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1 to 0.2 mg / L NAA, and 0.1 to 0.2 mg / L TDZ; S3: Callus subculture The callus tissue was subcultured using a callus tissue subculture medium, subcultured once every 18-20 days, and subcultured twice in total, the culture conditions were 25±2°C, 400-600 Lx, and a light intensity of 16h / d, and the composition and ratio of the callus tissue subculture medium were: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1-0.2 mg / L NAA, and 0.1-0.2 mg / L TDZ; S4: Adventitious bud differentiation The callus tissue was cultured using an adventitious bud differentiation medium at 25±2°C, 2000-2200 Lx, and 16 h / d of light for 25-30 days. The composition and ratio of the adventitious bud differentiation medium were as follows: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.2-0.25 mg / L NAA, and 1.0-1.2 mg / L ZT; S5: Adventitious root differentiation The callus tissue was cultured using an adventitious root differentiation medium at 25±2°C, 2000~2200 Lx, and 16h / d of light for 25~30 days. The composition and ratio of the adventitious root differentiation medium were as follows: WPM medium, with a final additional concentration of 1.0~1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.3~0.4 g / L activated carbon, 0.25~0.3 mg / L NAA, and 2.0~2.2 mg / L IBA.

2. The method according to claim 1, characterized in that The step S1 specifically includes the following steps: after treating the azalea in the early opening stage at 2-4°C, taking the outer petals, removing dust with running water, soaking the petals in 70-75% ethanol for 30 seconds, washing with sterile water twice, soaking in 6% sodium hypochlorite for 7-8 minutes, washing with sterile water three times, drying the surface moisture of the petals, and removing the browned parts and the edges of the petals by hand tearing to induce wounds.

3. The method according to claim 1, characterized in that In step S2, the wound is caused by cutting with a knife or tearing with a tear.

4. A callus induction medium for azalea tissue culture, characterized in that: The components and proportions of the callus induction medium are as follows: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1-0.2 mg / L NAA, and 0.1-0.2 mg / L TDZ.

5. A callus subculture medium for azalea tissue culture, characterized in that: The components and proportions of the callus subculture medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.1-0.2 mg / L NAA, and 0.1-0.2 mg / L TDZ.

6. A medium for adventitious bud differentiation for tissue culture of azalea, characterized in that: The composition and proportion of the adventitious bud differentiation medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.2-0.25 mg / L NAA, and 1.0-1.2 mg / L ZT.

7. An adventitious root differentiation medium for azalea tissue culture, characterized in that: The composition and proportion of the adventitious root differentiation medium are: WPM medium, with additional final concentrations of 1.0-1.2 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, 0.3-0.4 g / L activated carbon, 0.25-0.3 mg / L NAA, and 2.0-2.2 mg / L IBA.

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