A method for inducing callus, differentiating and regenerating plant from rhododendron petal as explant and application thereof

Abstract

The application provides a method for callus induction, differentiation and plant regeneration by taking rhododendron petals as explants and application thereof, and belongs to the technical field of tissue culture, and the specific steps comprise the following steps: S1, explant selection and disinfection; S2, callus induction; S3, callus subculture; S4, adventitious bud differentiation; and S5, adventitious root differentiation. In the above method, the defined conditions and culture medium components of each step are specifically limited, and the culture medium and the related condition control method provided by the application can effectively reduce the contamination rate of rhododendron petal tissue culture, improve the callus emergence rate and differentiation rate, and further improve the survival rate of regenerated plants.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology, and more specifically, to a method for inducing, differentiating, and regenerating callus tissue using rhododendron petals as explants, and its application. Background Technology

[0002] Rhododendrons are a collective term for plants in the genus *Rhododendron* (Ericaceae family), and are often referred to as the "Xi Shi of flowers" due to their high ornamental and ecological value. There are over 1200 species of rhododendrons worldwide, with my country boasting the largest population, approximately 600 species. Except for arid deserts in Ningxia and Xinjiang, rhododendrons are distributed throughout China, with the richest resources found in the Hengduan Mountains of Southwest China and the Qinghai-Tibet Plateau. Classic propagation methods for rhododendrons include sowing, cuttings, grafting, and layering. These methods are all constrained to varying degrees by factors such as species availability, seed germination, and the number of parent plants, resulting in a relatively slow overall development of the rhododendron industry. Plant tissue culture, on the other hand, offers high propagation efficiency and rapid reproduction, allowing for the production of large numbers of test-tube seedlings in a shorter time and with less space. Azalea tissue culture typically uses leaves and stem segments as explants. However, azalea leaves have pubescence on their surface, which carries many pathogens, making tissue culture sterilization difficult and resulting in a high contamination rate. In addition, due to the physiological characteristics 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 purpose of this invention is to provide a rhododendron tissue culture method with high survival rate, simplicity, and low contamination rate.

[0004] To achieve the above objectives, this invention provides a method for callus induction and rapid plant propagation using rhododendron petals as explants, specifically including the following steps:

[0005] S1: Explant selection and disinfection

[0006] After treating the rhododendrons in their early blooming stage at 2-4℃, take the rhododendron 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 moisture of the petals, and use the hand tearing method to remove the browned parts and the edges of the petals to create wounds.

[0007] S2: Callus induction

[0008] After the petals were treated in step S1, they were inoculated into callus induction medium and cultured at 25±2℃, 400~600Lx, and 16h / d light for 25~30d. The composition and ratio of the callus induction medium were as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0~1.2g / L, sucrose at 30g / L, agar at 7g / L, NAA at 0.1~0.2mg / L, and TDZ at 0.1~0.2mg / L.

[0009] S3: Callus Subgeneration

[0010] Callus was subcultured using a callus subculture medium, subcultured every 18–20 days for a total of 2 subcultures. The culture conditions were 25±2℃, 400–600 Lx, and 16 h / d light. The composition and ratio of the callus subculture medium were as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0–1.2 g / L, sucrose at a final concentration of 30 g / L, agar at a final concentration of 7 g / L, NAA at a final concentration of 0.1–0.2 mg / L, and TDZ at a final concentration of 0.1–0.2 mg / L.

[0011] S4: Adventitious bud induction

[0012] Callus was induced and cultured using an adventitious shoot induction medium at 25±2℃, 2000~2200Lx, and 16h / d light for 25~30d. The composition and ratio of the adventitious shoot induction medium were as follows: WPM medium supplemented with 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] Callus was induced and cultured using an adventitious root differentiation induction medium at 25±2℃, 2000~2200Lx, and 16h / d light for 25~30d. The composition and ratio of the adventitious root differentiation induction medium were as follows: WPM medium supplemented with calcium nitrate at a final concentration of 1.0~1.2g / L, sucrose at 30g / L, agar at 7g / L, activated carbon at 0.3~0.4g / L, NAA at 0.25~0.3mg / L, and IBA at 2.0~2.2mg / L.

[0015] Preferably, step S1 specifically includes the following steps: after treating the rhododendrons in their early blooming stage at 2-4℃, take the outer petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash them twice with sterile water, soak them in 6% sodium hypochlorite for 7-8 minutes, wash them three times with sterile water, absorb the moisture from the surface of the petals, and remove the browned parts and the edges of the petals by hand to create wounds.

[0016] Furthermore, a second aspect of the present invention provides a callus induction culture medium for rhododendron tissue culture, wherein the composition and ratio of the callus induction culture medium are as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0-1.2 g / L, sucrose at a final concentration of 30 g / L, agar at a final concentration of 7 g / L, NAA at a final concentration of 0.1-0.2 mg / L, and TDZ at a final concentration of 0.1-0.2 mg / L.

[0017] Furthermore, a third aspect of the present invention provides a callus subculture medium for rhododendron tissue culture, wherein the composition and ratio of the callus subculture medium are as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0-1.2 g / L, sucrose at a final concentration of 30 g / L, agar at a final concentration of 7 g / L, NAA at a final concentration of 0.1-0.2 mg / L, and TDZ at a final concentration of 0.1-0.2 mg / L.

[0018] Furthermore, a fourth aspect of the present invention provides an adventitious bud induction medium for rhododendron tissue culture, wherein the composition and ratio of the adventitious bud induction medium are as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0-1.2 g / L, sucrose at a final concentration of 30 g / L, agar at a final concentration of 7 g / L, NAA at a final concentration of 0.2-0.25 mg / L, and ZT at a final concentration of 1.0-1.2 mg / L.

[0019] Furthermore, the fifth aspect of the present invention also provides an adventitious root differentiation induction medium for rhododendron tissue culture, wherein the composition and ratio of the adventitious root differentiation induction medium are as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0-1.2 g / L, sucrose at a final concentration of 30 g / L, agar at a final concentration of 7 g / L, activated carbon at a final concentration of 0.3-0.4 g / L, NAA at a final concentration of 0.25-0.3 mg / L, and BA at a final concentration of 2.0-2.2 mg / L.

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

[0021] This invention establishes an azalea regeneration system using azalea petals as explants. The method for inducing callus tissue and rapidly propagating plants using azalea petals as explants provided by this invention can significantly improve the success rate and differentiation rate of azalea callus induction, thereby increasing the survival rate of regenerated azalea plants. Attached Figure Description

[0022] Figure 1 The images show the steps of callus induction, differentiation, and plant regeneration using rhododendron petals as explants, as well as the finished sterile seedlings, provided by this invention. Detailed Implementation

[0023] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, 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 methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.

[0024] It should be noted that the endpoints and any values ​​of the ranges disclosed herein 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 the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0025] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In some instances, terms having a conventional meaning are defined herein for clarification or ease of reference, and such definitions should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0026] As described in the background art, due to the physiological characteristics of azaleas, the survival rate of regenerated azalea plants is usually much lower than that of other varieties. Based on this, this 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 disinfection

[0028] After treating the rhododendrons in their early blooming stage at 2-4℃, take the rhododendron petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash them twice with sterile water, soak them in 6% sodium hypochlorite for 7-8 minutes, wash them three times with sterile water, dry the surface moisture of the petals, and remove the browned parts and petal edges by hand to create wounds.

[0029] S2: Callus induction

[0030] After the petals were treated in step S1, they were inoculated into callus induction medium and cultured at 25±2℃, 400~600Lx, and 16h / d light for 25~30d. The composition and ratio of the callus induction medium were as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0~1.2g / L, sucrose at 30g / L, agar at 7g / L, NAA at 0.1~0.2mg / L, and TDZ at 0.1~0.2mg / L.

[0031] S3: Callus Subgeneration

[0032] Callus was subcultured using a callus subculture medium, with subcultures every 18–20 days for a total of 2 subcultures. The culture conditions were 25±2℃, 400–600 Lx, and 16 h / d light. The callus subculture medium consisted of WPM medium supplemented with 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.

[0033] S4: Adventitious bud induction

[0034] Callus was induced and cultured using an adventitious shoot induction medium at 25±2℃, 2000~2200Lx, and 16h / d light for 25~30d. The composition and ratio of the adventitious shoot induction medium were as follows: WPM medium supplemented with 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] Callus was induced and cultured using an adventitious root differentiation induction medium at 25±2℃, 2000~2200Lx, and 16h / d light for 25~30d. The composition and ratio of the adventitious root differentiation induction medium were as follows: WPM medium supplemented with calcium nitrate at a final concentration of 1.0~1.2g / L, sucrose at 30g / L, agar at 7g / L, activated carbon at 0.3~0.4g / L, NAA at 0.25~0.3mg / L, and IBA at 2.0~2.2mg / L.

[0037] Figure 1 The images depict the steps of the rapid propagation method using azalea petals as explants, as described above. Figure 1 In this context, A represents the flower explant. Figure 1 In the image, B is a photograph of callus tissue obtained by petal induction. Figure 1 C in the image is a photograph of callus subculture. Figure 1 D in the image represents the differentiation of adventitious shoots from callus tissue. Figure 1 E in the image represents the differentiation of adventitious roots. Figure 1 F in the image represents the final sterile vaccine.

[0038] The specific implementation of this invention uses a novel culture medium, while strictly limiting the wound initiation method, callus induction, light intensity during subculture, and culture medium composition ratio, thereby improving the survival rate of rhododendron tissue culture.

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

[0040] Example 1

[0041] The effect of sodium hypochlorite disinfection time on explant contamination rate and browning rate

[0042] The test material was a healthy, disease-free 8-year-old rhododendron. Flowers in their initial blooming stage were collected at 8:00 AM and treated at 4℃ for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and immersed in 75% ethanol for 30 seconds in a clean bench. They were then washed twice with sterile water, and subsequently immersed in 6% sodium hypochlorite for 2, 4, 6, 8, and 10 minutes, followed by three washes with sterile water. The petals were then blotted dry with sterile paper towels, and browned parts and petal edges were cut off to induce wounds. The petals were then inoculated, back side down, onto WPM medium (without calcium nitrate) to induce callus formation. The medium was supplemented with 0.5 g / L calcium nitrate, 30 g / L sucrose, and 7 g / L agar. One petal was inoculated per bottle, with 10 bottles inoculated per treatment, and the treatment was repeated three times. The culture temperature was set at 25℃, the light intensity at 400 Lx, and the photoperiod at 16 h / d. The contamination rate and browning rate were calculated after 14 days of culture. The contamination rate was calculated as (number of contaminated explants / number of inoculated explants) × 100%; the browning rate was calculated as (number of browned explants / number of inoculated explants) × 100%. The relevant experimental results are shown in Table 1.

[0043] Table 1

[0044]

[0045]

[0046] As can be seen from the data in Table 1, the explant contamination rate was 0% when the sodium hypochlorite disinfection time was 8 and 10 min. However, the browning rate of explants was lower when the disinfection time was 8 min compared with 10 min. Therefore, the optimal disinfection time for sodium hypochlorite is 8 min.

[0047] Example 2

[0048] Effects of different hormone formulations on callus induction

[0049] The basic culture 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. Three hormone formulations were prepared as shown in Table 2.

[0050] Flowers in their initial blooming stage were collected at 8:00 AM and treated at 4℃ for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and immersed in 75% ethanol for 30 seconds in a clean bench. They were then washed twice with sterile water, soaked in 6% sodium hypochlorite for 8 minutes, and washed three times with sterile water. The petals were then blotted dry with sterile paper towels, and browned parts and petal edges were cut off to induce wounds. The petals were then inoculated, with the back side facing down, onto callus induction medium containing different hormone formulations. Ten bottles were inoculated per treatment, with one explant per bottle, and the inoculation was repeated three times. The culture temperature was set at 25℃, light intensity at 400 Lx, and photoperiod at 16 h / d. Callus rate and browning rate were calculated after 30 days of culture. The callus rate was calculated as (number of explants forming callus / number of inoculated explants) × 100%. The experimental results are shown in Table 2 and... Figure 1 As shown.

[0051] Table 2

[0052]

[0053] As shown in Table 2, after culturing explants with the three hormone formulations for 30 days, only the NT-numbered treatment group successfully induced callus tissue. The petals of this group paled in color, changing from rose-red to white, and the callus tissue was yellowish-green and relatively loose. Figure 1 As shown, the callus emergence rate was 73%. Explants in both the DT and DNZ treatment groups exhibited browning and death, making them unsuitable for petal callus induction. Therefore, the optimal hormone combination in the callus induction medium is 0.1 mg / L NAA + 0.1 mg / L TDZ.

[0054] Example 3

[0055] The Influence of Wound Induction Mode on Callus Induction

[0056] The basic culture 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] Flowers in their initial opening stage were collected at 8:00 AM and treated at 4℃ for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and immersed in 75% ethanol for 30 seconds in a clean bench, followed by two washes with sterile water, immersion in 6% sodium hypochlorite for 6 minutes, and three washes with sterile water. The petals were then blotted dry with sterile paper towels. Browned areas and petal edges were removed using tearing and cutting methods to induce wounds. The petals were then inoculated onto callus induction medium with the back facing down. Ten bottles were inoculated per treatment, with one explant per bottle, and the treatment was repeated three times. The culture temperature was set at 25℃, light intensity at 400 Lx, and photoperiod at 16 h / d. After 30 days of culture, the callus rate and browning rate were calculated. The experimental results are shown in Table 3.

[0058] Table 3

[0059] How wounds are caused Recovery rate (%) Browning rate (%) Tearing method 87 13 knife cutting method 73 27

[0060] As shown in Table 3, both the tearing method and the knife-cutting method can induce callus formation in azalea petals. However, compared with the knife-cutting method, the tearing method has a higher callus formation rate and a lower browning rate, indicating that the tearing method is suitable for inducing petal callus formation.

[0061] Example 4

[0062] Effects of flowering period on callus induction

[0063] The basic culture 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 at the bud stage, early bloom stage, and full bloom stage were collected at 8:00 AM. After being treated at 4℃ for 24 hours, the outermost petals were taken. The petal surface was rinsed with running water to remove dust. The petals were then soaked in 75% ethanol for 30 seconds in a clean bench, washed twice with sterile water, soaked in 6% sodium hypochlorite for 6 minutes, and washed three times with sterile water. The surface moisture of the petals was blotted dry with sterile paper towels. Browned parts and petal edges were removed by hand to induce wounds. The petals were then inoculated onto callus induction medium with the back side down. Ten bottles were inoculated per treatment, with one explant per bottle, and the treatment was repeated three times. The culture temperature was set at 25℃, the light intensity at 400 Lx, and the photoperiod at 16 h / d. After 30 days of culture, the callus induction rate and browning rate were calculated. The experimental results are shown in Table 4.

[0065] Table 4

[0066] Flowering period Recovery rate (%) Browning rate (%) Flower bud stage 60 40 initial stage 87 13 Peak bloom 37 63

[0067] As shown in Table 4, compared with petals in the initial opening stage, petals in the bud stage and full bloom stage had a lower callus induction rate and a higher browning rate. This may be because petals in the bud stage are too tender, and high concentrations of hormones can cause cell damage, while petals in the full bloom stage have lower cell totipotency and are less prone to differentiation. The results indicate that petals in the initial opening stage are suitable for callus induction.

[0068] Example 5

[0069] Effects of different temperature pretreatments on callus induction

[0070] The basic culture 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] Flowers in their initial blooming stage were collected at 8:00 AM and treated at different temperatures (4℃, 25℃, and 37℃) for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and soaked in 75% ethanol for 30 seconds in a clean bench. They were then washed twice with sterile water, soaked in 6% sodium hypochlorite for 6 minutes, and washed three times with sterile water. The petals were then blotted dry with sterile paper towels, and browned parts and petal edges were removed by hand to induce wounds. The petals were then inoculated onto callus induction medium with the back of the petals facing down. Ten bottles were inoculated per treatment, with one explant per bottle, and the treatment was repeated three times. The culture temperature was set at 25℃, light intensity at 400 Lx, and photoperiod at 16 h / d. After 30 days of culture, the callus induction rate and browning rate were calculated. The experimental results are shown in Table 5.

[0072] Table 5

[0073] Temperature (°C) Recovery rate (%) Browning rate (%) 4 87 13 25 53 47 37 40 60

[0074] As shown in Table 5, compared with the 4℃ treatment, the petal callus emergence rate was lower and the browning rate was higher after the 25℃ and 37℃ treatments. This may be because the low temperature treatment can improve the petal cell state and increase the endogenous hormone level, indicating that the 4℃ 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 culture 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 calcium nitrate concentrations were set at 0.1 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, and 2.0 g / L.

[0078] Flowers in their initial blooming stage were collected at 8:00 AM and treated at 4℃ for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and immersed in 75% ethanol for 30 seconds in a clean bench. They were then washed twice with sterile water, soaked in 6% sodium hypochlorite for 6 minutes, and washed three times with sterile water. The petals were then blotted dry with sterile paper towels, and browned parts and petal edges were removed by hand to induce wounds. The petals were then inoculated, with the back side facing down, onto callus induction medium containing different concentrations of calcium nitrate. Ten bottles were inoculated per treatment, with one explant per bottle, and the treatment was repeated three times. The culture temperature was set at 25℃, light intensity at 400 Lx, and photoperiod at 16 h / d. After 30 days of culture, the callus induction rate and browning rate were calculated. The experimental results are shown in Table 6.

[0079] Table 6

[0080]

[0081]

[0082] As shown in Table 6, both excessively low and excessively high calcium nitrate concentrations are not conducive to the induction of petal callus. Among them, 1.0 g / L calcium nitrate is the most suitable for the induction of petal callus, with a callus emergence rate of 100%.

[0083] Example 7

[0084] Effects of light on callus induction

[0085] The basic culture 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] Flowers in their initial blooming stage were collected at 8:00 AM and treated at 4℃ for 24 hours. The outermost petals were then removed, rinsed with running water to remove dust, and immersed in 75% ethanol for 30 seconds in a clean bench. They were then washed twice with sterile water, soaked in 6% sodium hypochlorite for 6 minutes, and washed three times with sterile water. The petals were then blotted dry with sterile paper towels, and browned parts and petal edges were removed by hand to induce wounds. The petals were then inoculated onto callus induction medium with the back side facing down. The culture temperature was set at 25℃, the light intensity at 400 Lx and 0 Lx, and the photoperiod at 16 h / d. Ten bottles were inoculated per treatment, with one explant per bottle, and the treatment was repeated three times. After 30 days of culture, the callus induction rate and browning rate were calculated. The experimental results are shown in Table 7.

[0087] Table 7

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

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

[0090] Example 8

[0091] Callus subculture

[0092] The basic culture 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] Callus tissue was excised using a scalpel and transferred to callus subculture medium. Six callus tissues were inoculated per bottle. The culture temperature was set at 25℃, the light intensity at 400 Lx, and the light duration at 16 h. Subculture was performed every 20 days, for a total of two subcultures. The browning rate of the callus tissue was then recorded. After two subcultures, the callus tissue was relatively dense, yellowish-green in color, and the browning rate was 0%.

[0094] Example 9

[0095] Induction of adventitious buds

[0096] The basic culture medium for adventitious shoot induction was WPM medium, supplemented with 1.0 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, NAA, and ZT. An orthogonal experimental design was used to study the effect of hormone combinations on adventitious shoot induction. Callus tissue was transferred to the adventitious shoot induction medium, with 6 callus pieces inoculated per bottle. The culture temperature was set at 25℃, the light intensity at 2000 Lx, and the photoperiod at 16 h / d. After 30 days, the browning rate and budding rate were recorded. 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] As shown in Table 8, the germination rate was relatively high when the hormone formula was 0.2-0.3 mg / L NAA + 1.0-2.0 mg / L ZT. Among them, the germination rate reached 83% when the hormone formula was 0.2 mg / L NAA + 1.0 mg / L ZT, which was most suitable for bud differentiation.

[0100] Example 10

[0101] Effects of hormone formulation on adventitious root differentiation

[0102] The basic culture medium for adventitious root differentiation was WPM medium, supplemented with 1.0 g / L calcium nitrate, 30 g / L sucrose, 7 g / L agar, activated carbon (AC), NAA, and IBA. An orthogonal experimental design was used to study the effects of hormone combinations on adventitious root differentiation. Adventitious shoots and lower callus tissue were excised and transferred to the adventitious root differentiation medium, with 6 adventitious shoots inoculated per bottle. The culture temperature was set at 25℃, the light intensity at 2000 Lx, and the photoperiod at 16 h / d. The browning rate and rooting rate were statistically analyzed after 30 days, and the 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] As shown in Table 9, the rooting rate was relatively high when the hormone and activated carbon formulation was 0.3–0.6 g / L activated carbon, 0.25–0.5 mg / L NAA, and 1.0–2.0 mg / L IBA. Among them, the rooting rate was the highest, reaching 80%, when the hormone formulation was 2.0 mg / L IBA + 0.25 mg / L NAA and 0.3 g / L AC, which was most suitable for the differentiation of adventitious roots.

[0106] As can be seen from the above embodiments, the culture medium and related condition control methods provided by the present invention can effectively reduce the contamination rate of rhododendron petal tissue culture, increase the callus emergence rate and differentiation rate, and thus improve the survival rate of regenerated plants obtained by using rhododendron petals as explants.

[0107] While the disclosure is as stated above, its scope of protection is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of this disclosure, and all such changes and modifications will fall within the protection scope of this invention.

Claims

1. A method for inducing callus, differentiating, and regenerating plants using rhododendron petals as explants, characterized in that, Includes the following steps: S1: Explant selection and disinfection After treating the rhododendrons in their early blooming stage at 2-4℃, take the outer petals, remove the dust with running water, soak the petals in 70-75% ethanol for 30 seconds, wash them twice with sterile water, soak them in 6% sodium hypochlorite for 7-8 minutes, wash them three times with sterile water, dry the surface moisture of the petals, and remove the browned parts and petal edges by hand to create wounds. The wounds can be created by cutting or tearing. S2: Callus induction After the petals were treated in step S1, they were inoculated into callus induction medium and cultured at 25±2℃, 400~600 Lx, and 16h / d light for 25~30 days. The composition and ratio of the callus induction medium were as follows: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0~1.2 g / L, sucrose at 30 g / L, agar at 7 g / L, NAA at 0.1~0.2 mg / L, and TDZ at 0.1~0.2 mg / L. S3: Callus Subgeneration Callus was subcultured using a callus subculture medium, subcultured every 18-20 days for a total of 2 subcultures. The culture conditions were 25±2℃, 400-600 Lx, and 16h / d light. The composition and ratio of the callus subculture medium were: WPM medium, supplemented with calcium nitrate at a final concentration of 1.0-1.2 g / L, sucrose at 30 g / L, agar at 7 g / L, NAA at 0.1-0.2 mg / L, and TDZ at 0.1-0.2 mg / L. S4: Adventitious bud differentiation Callus tissue was cultured using adventitious shoot differentiation medium at 25±2℃, 2000~2200 Lx, and 16h / d light for 25~30 days. The composition and ratio of the adventitious shoot differentiation medium were as follows: WPM medium supplemented with 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 Callus tissue was cultured using adventitious root differentiation medium at 25±2℃, 2000~2200 Lx, and 16h / d light for 25~30 days. The composition and ratio of the adventitious root differentiation medium were as follows: WPM medium supplemented with 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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