Method for tissue culture and rapid propagation of hybrid rhododendron by taking rhododendron ovatum as female parent

Abstract

The invention discloses a tissue culture and rapid propagation method for hybrid rhododendron by taking flos lonicerae as a female parent, and belongs to the technical field of plant tissue culture.The tissue culture and rapid propagation method comprises the steps that semi-lignified tender stem segments of the hybrid rhododendron are selected as explants, and adventitious bud induction, multiplication culture, seedling strengthening and rooting, seedling hardening and transplanting are conducted. The transplanting survival rate of tissue culture seedlings reaches 85% or above, the rooting rate and the survival rate are high, a large number of hybrid rhododendron can be rapidly obtained, and the method has important significance in production and application.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically a method for rapid propagation of hybrid rhododendrons using Rhododendron simsii as the female parent through tissue culture. Background Technology

[0002] *Rhododendron ovatum*, belonging to the genus *Rhododendron* and subgenus *Rhododendron* of the family Ericaceae, is an evergreen ornamental flower with a corolla of 2-4 cm. The flowers are pale pink to purple or white and have a certain fragrance, making it highly valued for its horticultural appeal and worthy of development. *Rhododendron simsii* var. *putuoense*, a variant of *Rhododendron* distributed in coastal areas, has light purplish-red flowers. Compared to other rhododendrons in the *Rhododendron* subgenus, *Rhododendron ovatum* has a slower rate of reaching maturity and flowering, and requires more sophisticated cultivation techniques, hindering its widespread application; currently, there are virtually no products available on the market. *Rhododendron simsii* grows near the sea and has some salt tolerance and drought resistance than *Rhododendron ovatum*, but its propagation and cultivation are also limited, therefore, no seedlings are available on the market. Hybrids have a shorter time to maturity and flowering, and improved drought resistance, making them more suitable for horticultural applications; however, their propagation remains unresolved.

[0003] Currently, tissue culture technology is used for hybrid propagation in many plants, but due to species differences, each species has its own tissue culture system, as follows:

[0004] Rhododendron simsii was used as explants. The explants were soaked in laundry detergent for 10-20 minutes, rinsed under running tap water for 2 hours, soaked in 75% alcohol for 30 seconds, and rinsed twice with sterile water. Then, they were disinfected with 2% NaClO for 25-30 minutes, rinsed six times with sterile water, and finally blotted dry with sterile filter paper. Individual florets were removed from the explants using a scalpel and scissors. The induction medium was WPM + TDZ (0.5 mg / L) + IBA (1.0 mg / L), and the subculture medium was WPM + TDZ (0.2 mg / L) + IBA (0.1 mg / L) + GA3 (3 mg / L).

[0005] Rhododendron was induced using leaves as explants. The callus induction medium consisted of 1 / 4 Anderson + 1.0 mg / L 2,4-D + 0.5 mg / L 6-BA, 1 / 4 Anderson + 0.1 mg / L IBA + 1.0 mg / L TDZ for bud induction, 1 / 4 Anderson + 0.1 mg / L NAA + 1.0 mg / L ZT for seedling strengthening, and 1 / 4 Anderson + 2.0 mg / L IAA and 0.1 mg / L NAA for rooting.

[0006] Rhododendron simsii was grown using terminal buds as explants. The explants were disinfected with 75% alcohol for 20 seconds, followed by 0.1% mercuric chloride for 5 minutes, and then rinsed with sterile water. Induction medium consisted of WPM + ZT 3.0 mg / L (hereinafter the same) + NAA 0.1; subculture medium consisted of WPM + TDZ 1.0 + NAA 0.1; seedling strengthening medium consisted of WPM + ZT 0.5 + GA3 0.5; and rooting medium consisted of WPM + ZT 0.1 + NAA 0.5.

[0007] Rhododendron molle was propagated using young stems as explants. Treatment involved 70% ethanol for 10 seconds, soaking in 2% NaClO for 15 minutes, and rinsing 5-6 times with sterile water. The optimal culture medium for propagation was WPM + ZT 2.0 mg / L + NAA 0.5 mg / L + sucrose 30 g / L; the optimal medium for seedling strengthening was WPM + ZT 0.5 mg / L + GA3 2.0 mg / L + sucrose 30 g / L; and the optimal medium for rooting was WPM + IAA 0.1 mg / L + 1% sucrose.

[0008] Red horse honeysuckle was used as explants, which were disinfected in 0.1% HgCl2 solution for 5 min, sterilized in 2% NaClO solution for 5 min, and rinsed with sterile water. Induction medium consisted of WPM + 6-BA 1.8-2.2 mg / L + 2,4-D 2.8-3.2 mg / L; subculture medium consisted of WPM + ZT 0.8-1.2 mg / L + NAA 0.05 mg / L + AC 0.5 g / L; and rooting medium consisted of WPM + IBA 0.8-1.2 mg / L + NAA 0.4-0.6 mg / L + AC 1 g / L. The transplanting substrate was a mixture of sandy loam and humus in a 6:4 volume ratio.

[0009] There are currently no reports on tissue culture techniques for hybrids of Rhododendron simsii and Rhododendron putuoense. Summary of the Invention

[0010] To address the lack of suitable tissue culture techniques for hybrids of *Rhododendron simsii* and *Rhododendron putuoense* in existing technologies, this invention provides a rapid propagation method for hybrid rhododendrons using tissue culture with *Rhododendron simsii* as the female parent. The hybrid rhododendron selected in this invention is a hybrid rhododendron cultivated through artificial hybridization using superior *Rhododendron simsii* female parent and *Rhododendron putuoense* male parent single plants. This hybrid rhododendron can flower after more than 3 years of cultivation, with a significantly shortened flowering period, and combines the characteristic traits of both evergreen and deciduous rhododendrons, retaining the characteristics of both parent plants.

[0011] Technical solution: A rapid propagation method for hybrid rhododendrons using Rhododendron simsii as the female parent through tissue culture, comprising the following steps:

[0012] Step 1, Material Selection and Sterilization: Select semi-lignified tender stem segments of hybrid azaleas as explants and sterilize them; the hybrid azaleas refer to azaleas that are hybrids of Rhododendron simsii and Rhododendron putuoensis, with Rhododendron simsii as the female parent and Rhododendron putuoensis as the male parent.

[0013] Step 2, Adventitious bud induction stage: Cut the explants that have been sterilized in step 1 into stem segments of 1-1.5cm, inoculate them into adventitious bud induction medium for culture, and obtain aseptic cluster buds after adventitious bud differentiation;

[0014] Step 3, Proliferation Culture Stage: The explants that produced sterile shoot clusters in Step 2 were cut into small clusters with 2-3 buds and inoculated into the proliferation culture medium to produce shoot clusters.

[0015] Step 4, Seedling Rooting Stage: When the clustered buds in Step 3 grow to 1-2 cm, the explants are inoculated into the seedling rooting culture medium for seedling rooting culture to obtain tissue culture seedlings;

[0016] Step 5, Hardening-off stage: When the tissue culture seedlings from Step 4 grow to 4-6cm and the roots are 1.5-3cm long, open the culture bottle, add an amino acid aqueous solution, and place it under weak light for hardening-off treatment.

[0017] Step 6, Transplanting stage: Clean the culture medium from the roots of the tissue culture seedlings with warm water, soak them in a carbendazim solution, and then transplant them into the substrate for cultivation.

[0018] Furthermore, in step 1, the sterilization process is as follows: the semi-lignified tender stem segments of the hybrid azalea are cleaned with a soft brush dipped in dish soap and water, wrapped in gauze, rinsed under running water for 30 minutes, placed on a clean bench, disinfected with 75% alcohol for 30-40 seconds, rinsed 3 times with sterile water, surface disinfected with 5% sodium hypochlorite solution for 9-10 minutes, and finally rinsed 5 times with sterile water.

[0019] Furthermore, in step 2, the formulation of the adventitious bud induction medium is as follows:

[0020] WPM medium was used as the basal medium, with the addition of TDZ, NAA, sucrose and agar powder. The concentration of TDZ was 0.8-1.2 mg / mL, the concentration of NAA was 0.1-0.2 mg / mL, the amount of sucrose added was 28-32 g / L, and the amount of agar powder added was 6.0-6.8 g / L. The pH value of the medium before sterilization was 5.3-5.7.

[0021] The cultivation conditions in step 2 are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and time of 20-35 days.

[0022] Furthermore, in step 3, the formulation of the proliferation medium is as follows: WPM medium is used as the basal medium, with the addition of ZT and NAA, and the concentration of ZT is 1.8-2.2 mg / ml and the concentration of NAA is 0.2 mg / ml.

[0023] The cultivation conditions for step 3 are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and time of 15-25 days.

[0024] Furthermore, in step 4, the formula for the seedling rooting medium is as follows:

[0025] Using 1 / 2 WPM medium as the basal medium, NAA, IBA and activated carbon were added, with the concentration of NAA being 0.3 mg / ml, the concentration of IBA being 0.1 mg / ml, and the mass percentage of activated carbon being 0.5%.

[0026] In step 4, the cultivation conditions are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and the growth time of the clustered shoots in the seedling rooting medium is 25-30 days.

[0027] Furthermore, in step 5, the volume ratio of the added amino acid aqueous solution to the culture medium in the culture flask is 1:(4-3).

[0028] In step 5, the cultivation conditions are as follows: time is 5-7 days, humidity is 75-85%, light intensity is 800-1100 lx, and temperature is 24℃-28℃.

[0029] Furthermore, in step 6: the soaking time in the chlorothalonil solution is 5-6 minutes, and the mass percentage of chlorothalonil in the chlorothalonil solution is 0.2%.

[0030] The substrate consists of coconut coir and loess, with a mass ratio of coconut coir to loess of (5-4):1. Before use, the coconut coir is soaked and rinsed with water to ensure that its EC value is less than 0.3.

[0031] Furthermore, in step 6, the cultivation conditions in the substrate are as follows: when transplanting to an outdoor greenhouse, the light intensity is 1000-1300 lx, with some shading, and the temperature is maintained at 16℃-28℃; after growing in the greenhouse for half a year, it can be transplanted to the open field in February-April and October-November.

[0032] Beneficial effects:

[0033] 1) Through research on the tissue culture rapid propagation method of hybrid azalea with Rhododendron simsii as the female parent, the tissue culture rapid propagation method provided by this invention has a transplant survival rate of more than 85% for tissue culture seedlings, and a high rooting rate and survival rate. It can quickly and in large quantities obtain this hybrid azalea, which is of great significance in production application.

[0034] 2) Using hybrid rhododendron tender stems as explants to induce adventitious buds can realize its industrialized seedling production. The method of this invention is an efficient, safe and low-cost tissue culture method. Attached Figure Description

[0035] Figure 1 This is the state of explants cultured on adventitious bud culture medium No. 1 for 10 days during the adventitious bud induction stage of the example.

[0036] Figure 2 This is the state of the explants cultured in proliferation medium 1 for 30 days during the proliferation stage of the example.

[0037] Figure 3 As an example of seedling vigorous rooting, the state of explants cultured on seedling vigorous rooting medium No. 1 after 27 days;

[0038] Figure 4 This is a diagram showing the transplanting stage of tissue culture seedlings 30 days after transplanting them into the greenhouse. Detailed Implementation

[0039] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.

[0040] In this embodiment of the invention, the culture medium and related additives were purchased from Beijing Kulaibo Technology Co., Ltd.; chlorothalonil solution was purchased from Limin Chemical Co., Ltd.; amino acid fertilizer was purchased from Shandong Shikefeng Ecological and Chemical Engineering Co., Ltd.; coconut coir was imported from India; and loess was obtained from garden soil. The main components of WPM culture medium and MS culture medium are as follows:

[0041] Table 1. Major components of MS medium (mg / L)

[0042]

[0043] Table 2. Main components of WPM culture medium (mg / L)

[0044]

[0045] Example 1

[0046] Step 1, Material Selection and Sterilization: Select semi-lignified tender stem segments of hybrid rhododendron as explants. Clean them with a soft brush dipped in dish soap and water, wrap them in gauze, rinse them under running water for 30 minutes, place them on a clean bench, disinfect them with 75% alcohol for 40 seconds, rinse them three times with sterile water, then disinfect them with 5% sodium hypochlorite solution for 10 minutes, and finally rinse them five times with sterile water.

[0047] In this embodiment, the hybrid azalea refers to a hybrid of *Rhododendron simsii* and *Rhododendron putuoense*, with *Rhododendron simsii* (subgenus *Rhododendron*) as the female parent and *Rhododendron putuoense* (subgenus *Rhododendron*) as the male parent. *Rhododendron simsii* is an evergreen shrub with fragrant purple flowers; *Rhododendron putuoense* is a deciduous shrub with red flowers. This invention uses the tender stems of the hybrid azalea as explants to induce adventitious buds, enabling industrialized seedling production. It is an efficient, safe, and low-cost tissue culture method.

[0048] Step 2, Adventitious bud induction stage: Cut the sterilized explants from Step 1 into stem segments of 1-1.5 cm and inoculate them into different adventitious bud induction media for culture. Adventitious buds differentiate into aseptic clusters of buds. The culture conditions are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and culture time of 40 days.

[0049] The explants were cultured on adventitious bud culture medium No. 1 for 10 days. The differentiation status of the adventitious buds was as follows: Figure 1 As shown in Table 3, the formulations, differentiation time, and number of adventitious shoots induction media for different adventitious shoot induction media are as follows. In Table 3, the formulation for adventitious shoot induction medium No. 1 is as follows: TDZ and NAA are added to WPM medium, with TDZ concentration at 1.0 mg / mL and NAA concentration at 0.1 mg / mL; sucrose and agar powder are added, with sucrose added at 32 g / L and agar powder added at 6.8 g / L.

[0050] Adventitious bud induction media No. 1-4, with a pH of 5.7 before sterilization. Table 3 shows: WPM medium, a commonly used tissue culture medium for woody plants; TDZ (thiafenuron-methyl, chemical name: N-phenyl-N'-(1,2,3-thiadiazol-5-yl)urea); NAA (α-naphthaleneacetic acid); BA (6-benzyladenine).

[0051] Table 3. Effects of different adventitious shoot induction media on adventitious shoot induction

[0052]

[0053] Step 3, Proliferation Stage: The explants (containing sterile shoot clusters) obtained in Step 2 on Adventitious Bud Medium No. 1 are cut into small clusters with 2-3 buds each and inoculated into different proliferation media to induce shoot clustering. The culture conditions for Step 3 are: light intensity 1000-1300 lx, temperature 24℃-28℃, and culture time 25 days.

[0054] The formulations and proliferation quantities of different proliferation media are shown in Table 4. In Table 4, the formulation for proliferation medium 2 is: ZT and NAA are added to WPM medium, with a ZT concentration of 2.0 mg / ml and a NAA concentration of 0.2 mg / ml. In Table 2, ZT refers to zeatin.

[0055] Table 4. Effects of different proliferation media on proliferation

[0056]

[0057] Figure 2 The state of explants cultured in proliferation medium 1 for 30 days.

[0058] Step 4, Seedling Strengthening and Rooting Stage: When the explant shoots cultured in Proliferation Medium 1 in Step 3 grow to 1-2 cm, they are cut off and inoculated into different seedling strengthening and rooting media for seedling strengthening and rooting culture to obtain tissue culture seedlings. The culture conditions are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and the growth time of the shoots in the proliferation medium is 35 days.

[0059] Figure 3 The image shows the state of explant shoot clusters cultured on seedling rooting medium No. 1 after 27 days. The right image is a magnified schematic diagram of the explants in the left image. Table 5 shows the formulations, rooting times, and quantities of different seedling rooting media. In Table 5, seedling rooting medium No. 1 is: 1 / 2 WPM medium with added NAA, IBA, and activated charcoal, achieving a NAA concentration of 0.3 mg / mL, an IBA concentration of 0.1 mg / mL, and activated charcoal accounting for 0.5% of the medium's mass. IBA refers to indolebutyric acid; 1 / 2 WPM medium means the concentration of macroelements in the medium is half that of the standard WPM medium.

[0060] Table 5. Effects of different rooting media on rooting of seedlings

[0061]

[0062] Step 5, Hardening-off Stage: When the tissue culture seedlings cultured in the No. 1 rooting medium from Step 4 reach a length of 4-6 cm and the root length is 1.5-3 cm, open the culture bottle and add an amino acid aqueous solution. The volume ratio of the added amino acid aqueous solution to the culture medium in the culture bottle is 1:3. Culture conditions: Harden-off treatment is carried out under weak light (800-1100 lx) for 5 days, maintaining a humidity of 75-85% and a temperature of 24℃-28℃.

[0063] Amino acid aqueous solution refers to a solution prepared by diluting amino acid fertilizer with water 2000 times. Amino acid fertilizer is a nutrient solution with an amino acid content >10%, nitrogen, phosphorus, and potassium >10%, organic matter >6%, and trace elements >2%, purchased from Shandong Shikefeng Ecological Educational Engineering Co., Ltd. The ratio of the mass of amino acids to the total mass of nitrogen, phosphorus, and potassium in the amino acid fertilizer is 1:1.

[0064] Step 6, Transplanting stage: Clean the culture medium from the roots of the tissue culture seedlings with warm water, soak them in a 0.2% chlorothalonil solution for 5-6 minutes, and then transplant them into a substrate of coconut coir: yellow soil = 5:1 (volume ratio) for cultivation; soak and rinse the coconut coir before use to ensure that its EC value is less than 0.3.

[0065] Cultivation conditions in the substrate: Move to an outdoor greenhouse for planting, increase shading measures to achieve a light intensity of 1000-1300 LX, and maintain the temperature between 16℃ and 28℃. Figure 4 The image shows the transplanted seedlings 30 days after transplanting into the greenhouse. After six months of growth in the greenhouse, they were transplanted to the open field in March, where they received 1000-1500 LX of sunlight and were watered as needed. 180 days after transplanting, the survival rate of the tissue culture seedlings reached over 85%.

[0066] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.

Claims

1. A rapid propagation method for hybrid rhododendrons using *Rhododendron simsii* as the female parent through tissue culture, characterized in that... Includes the following steps: Step 1, Material Selection and Sterilization: Select semi-lignified tender stem segments of hybrid azaleas as explants and sterilize them; the hybrid azaleas refer to azaleas that are hybrids of Rhododendron simsii and Rhododendron putuoensis, with Rhododendron simsii as the female parent and Rhododendron putuoensis as the male parent. Step 2, Adventitious bud induction stage: Cut the explants that have been sterilized in step 1 into stem segments of 1-1.5cm, inoculate them into adventitious bud induction medium for culture, and the adventitious buds differentiate to form aseptic cluster buds; Step 3, Proliferation Culture Stage: The explants that produced sterile shoot clusters in Step 2 were cut into small clusters with 2-3 buds and inoculated into the proliferation culture medium to produce shoot clusters. Step 4, Seedling Rooting Stage: When the clustered shoots of the explants in Step 3 grow to 1-2 cm, the explants are inoculated into the seedling rooting medium for seedling rooting culture to obtain tissue culture seedlings; Step 5, Hardening-off stage: When the tissue culture seedlings from Step 4 grow to 4-6cm and the roots are 1.5-3cm long, open the culture bottle, add an amino acid aqueous solution, and place it under weak light for hardening-off treatment. Step 6, Transplanting stage: Clean the culture medium from the roots of the tissue culture seedlings with warm water, soak them in a carbendazim solution, and then transplant them into the substrate for cultivation.

2. The method according to claim 1, characterized in that, In step 1, the sterilization process is as follows: the semi-lignified tender stem segments of hybrid azalea are cleaned with a soft brush dipped in dish soap and water, wrapped in gauze, rinsed under running water for 30 minutes, placed on a clean bench, disinfected with 75% alcohol for 30-40 seconds, rinsed 3 times with sterile water, surface disinfected with 5% sodium hypochlorite solution for 9-10 minutes, and finally rinsed 5 times with sterile water.

3. The method according to claim 1, characterized in that, In step 2, the formulation of the adventitious bud induction medium is as follows: WPM medium was used as the basal medium, with the addition of TDZ, NAA, sucrose and agar powder, and the concentration of TDZ was 0.8-1.2 mg / mL, the concentration of NAA was 0.1-0.2 mg / mL, the amount of sucrose added was 28-32 g / L, and the amount of agar powder added was 6.0-6.8 g / L. The pH value of the medium before sterilization was 5.3-5.

7. The cultivation conditions are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and cultivation time of 20-35 days.

4. The method according to claim 1, characterized in that, In step 3, the formulation of the proliferation medium is as follows: WPM medium is used as the basal medium, with the addition of ZT and NAA, and the concentration of ZT is 1.8-2.2 mg / ml and the concentration of NAA is 0.2 mg / ml. The cultivation conditions are: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and cultivation time of 15-25 days.

5. The method according to claim 1, characterized in that, In step 4, the formula for the seedling rooting medium is as follows: Using 1 / 2 WPM medium as the basal medium, NAA, IBA, and activated charcoal were added, with the concentrations of NAA (0.3 mg / ml), IBA (0.1 mg / ml), and activated charcoal (0.5% by mass). The cultivation conditions were: light intensity of 1000-1300 lx, temperature of 24℃-28℃, and the growth time of the clustered shoots in the seedling rooting medium was 25-30 days.

6. The method according to claim 1, characterized in that, In step 5, the volume ratio of the added amino acid aqueous solution to the culture medium in the culture flask is 1:(4-3). The cultivation conditions are as follows: 5-7 days, humidity 75-85%, light intensity 800-1100 lx, and temperature 24℃-28℃.

7. The method according to claim 1, characterized in that, In step 6: Soak in a chlorothalonil solution for 5-6 minutes. The chlorothalonil solution contains 0.2% chlorothalonil by mass. The substrate consists of coconut coir and loess, with a mass ratio of coconut coir to loess of (5-4):

1. Before use, the coconut coir is soaked and rinsed with water to ensure that its EC value is less than 0.

3.

8. The method according to claim 1, characterized in that, In step 6, the cultivation conditions in the substrate are as follows: when transplanting to an outdoor greenhouse, the light intensity should be 1000-1300 LX, with some shading, and the temperature should be maintained at 16℃-28℃. After growing in the greenhouse for half a year, the plants can be transplanted to the open field in February-April and October-November.

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