A method for establishing an in vitro regeneration system of aseptic leaves of Rhododendron dahurica

Abstract

The present invention belongs to the technical field of plant tissue culture, and specifically relates to a method for establishing an in vitro regeneration system of aseptic leaves of Rhododendron decorum Franch., comprising the following steps: culturing aseptic leaves of Rhododendron decorum Franch.; inducing in vitro regeneration of aseptic leaves of Rhododendron decorum Franch.; proliferating culture of adventitious bud seedlings, and the proliferation medium used for the proliferation culture is: WPM + 0.5 - 0.7 mg / L TDZ + 0.4 - 0.6 mg / L NAA + 0.5 - 1 mg / L GA3 + 300 mg / L mancozeb + 35 - 45 mg / L streptomycin sulfate; rooting culture. By using the method for establishing the in vitro regeneration system of aseptic leaves of Rhododendron decorum Franch. of the present invention, an in vitro regeneration system of leaves of Rhododendron decorum Franch. can be established, providing technical support for solving the standardized production of improved varieties of Rhododendron decorum Franch. and establishing a genetic transformation system.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for establishing an in vitro regeneration system of aseptic leaves of Rhododendron dahurica. Background Art

[0002] Greater White Rhododendron Rhododendron decorum Franch.) is a wild germplasm resource with great development potential in the genus Rhododendron. It is endemic to my country and is mainly distributed in Sichuan, Yunnan, Guizhou and Tibet. Rhododendron grandiflorum has great ornamental and economic value. Its flowers are large, numerous, fragrant and have a long flowering period. The flowers are white, pink or rose-colored. The leaves are bright and beautiful, and the new leaves are tender red. Its corolla is edible. Rhododendron grandiflorum has been widely cultivated and applied in the southwest region, and sequence varieties have been cultivated. The breeding and rapid propagation of Rhododendron grandiflorum must rely on efficient and stable plant regeneration and genetic transformation systems. Using leaves as explants has the advantage of easy material availability. Rhododendron in vitro leaves can differentiate adventitious buds. The research on the regeneration of Rhododendron grandiflorum plants mainly focuses on seed in vitro germination, stem segment, stem tip and flower bud proliferation. The regeneration ability and regeneration mode of Rhododendron plants vary greatly among different species and different explants. It is necessary to explore the leaf regeneration bud pathway and establish an efficient plant regeneration system for specific species.

[0003] In the process of plant tissue culture, the disinfection of explants is the key to the success of tissue culture. Generally, only conventional disinfection of the surface of plant tissue is required to be used directly for tissue culture. However, for woody plants, there are multiple microorganisms inside their branches and trunks. When the explants are sterilized, it is difficult to kill these internal microorganisms, resulting in a very high infection rate, which greatly reduces the success rate of tissue culture. In the tissue culture process of Rhododendron davidii, the sterile materials that have been surface sterilized still have endophytic bacteria breeding, and the endophytic bacteria pollution is the most serious in the proliferation culture stage. The pollutants will gradually spread, causing the death of tissue culture materials, which becomes a great obstacle in the process of establishing the in vitro regeneration system of Rhododendron davidii. Therefore, it is necessary to select an antibacterial agent that can inhibit endophytic bacteria pollution and has no adverse effects on plant tissues for application in the tissue culture of Rhododendron davidii.

[0004] The invention adopts Rhododendron grandiflorum detached leaves to induce adventitious buds, establishes an efficient Rhododendron grandiflorum detached leaves bud regeneration system, and provides a reference basis for the establishment of its improved variety rapid propagation and genetic transformation system. Summary of the invention

[0005] One object of the present invention is to provide a method for establishing an in vitro regeneration system of aseptic leaves of Rhododendron dahurica, so as to provide a reference for the establishment of its improved variety rapid propagation and genetic transformation system. Specifically, the present invention provides the following technical solutions:

[0006] A method for establishing an in vitro regeneration system of aseptic leaves of Rhododendron dasyphylla, comprising the following steps:

[0007] (1) Sterile leaf culture of Rhododendron dahurica

[0008] The explant stem segments of Rhododendron dahurica are cleaned and disinfected, and then placed in a stem segment culture medium for bud induction culture to obtain Rhododendron dahurica sterile leaves;

[0009] (2) In vitro regeneration induction culture of sterile leaves of Rhododendron dahurica

[0010] The sterile leaves of Rhododendron dahurica explants were inoculated on the regeneration induction medium to induce adventitious shoots.

[0011] (3) Proliferation and culture of adventitious sprouts

[0012] The proliferation medium used for proliferation culture is: WPM+0.5-0.7mg / L TDZ+0.4-0.6mg / L NAA+0.5-1mg / L GA3+300mg / L mancozeb+35-45mg / L streptomycin sulfate;

[0013] (4) Rooting culture

[0014] The healthy seedlings after proliferation are transferred to rooting medium for rooting cultivation.

[0015] Furthermore, in step (1), the method for disinfecting the stem segments is sequentially soaking in 0.1% HgCl2 solution for 11-12 min + 1% KMnO4 for 9-10 min + 10% H2O2 for 3-4 min + 1200-1500 mg / L citric acid solution for 10 min.

[0016] Furthermore, in step (1), the stem segment culture medium is: WPM + 2.5-3 mg / L TDZ + 7.5 g / L agar + 30 g / L sucrose + 3 g / L activated carbon, pH 5.4.

[0017] Furthermore, in step (2), the regeneration induction medium is: WPM+0.8-0.15 mg / L TDZ+0.01 mg / L NAA+7.5 g / L agar+30 g / L sucrose.

[0018] Furthermore, in step (2), the regeneration induction medium is: WPM+0.1 mg / L TDZ+0.01 mg / L NAA+7.5 g / L agar+30 g / L sucrose.

[0019] Furthermore, in step (3), the culture medium used for proliferation culture is: WPM+0.6mg / LTDZ+0.05mg / LNAA+0.5mg / L GA3+300mg / L mancozeb+40mg / L streptomycin sulfate.

[0020] Furthermore, in step (4), the rooting culture is: 1 / 2WPM+0.7-0.9mg / L NAA+7.5g / L agar+30g / L sucrose+3g / L activated carbon.

[0021] Furthermore, in any of the methods described in the present invention, the culture environment is cultured in a light incubator with alternating lighting conditions of 15°C for 12 h and 21°C for 12 h, the light intensity at 21°C is 2000 lx, and there is no light at 15°C.

[0022] Another object of the present invention is to provide an application of a fungicide combination in a process of in vitro tissue culture of azalea, wherein the fungicide combination is 300 mg / L mancozeb and 35-45 mg / L streptomycin sulfate, and the application is to reduce the contamination rate in the process of azalea proliferation culture.

[0023] The technical effects achieved by the present invention are:

[0024] The method for establishing the in vitro regeneration system of the aseptic leaves of the large white azalea of ​​the present invention can establish the in vitro regeneration system of the large white azalea leaves, provide technical support for solving the standardized production of the improved varieties of the large white azalea and establishing the genetic transformation system, and through the follow-up observation of the tissue culture seedlings produced by the technical solution, the properties of the finished plants are highly consistent. The problem of severe separation of ornamental properties such as flower shape, flower color, leaf shape, etc. of the finished plants caused by seedlings obtained by sowing (including aseptic sowing) and high variation rate (up to 30-40%) is avoided.

[0025] By adding the fungicide combination provided by the present invention during the proliferation culture process, the endophyte contamination rate was reduced to 12.50%, and the survival rate was increased to 72.50%, which greatly reduced the production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 1. The state of obtaining sterile leaves by inducing buds from stem segments; Note: A: Growth of axillary buds from stem segments 14 days after inoculation (3 mg / L TDZ); B: Growth of axillary buds from stem segments 60 days after inoculation (3 mg / L TDZ);

[0027] Figure 2 Effects of different TDZ concentrations on leaf bud induction; Note: a: 0.02 mg / L TDZ treatment; b: 0.1 mg / L TDZ treatment; c: 0.3 mg / L TDZ treatment;

[0028] Figure 3Effects of different TDZ and NAA concentrations on the proliferation and growth of Rhododendron serrata; Note: A, B, and C are all 0.2 mg / L TDZ, and the NAA concentrations are 0.01 mg / L, 0.05 mg / L, and 0.1 mg / L, respectively; D, E, and F are all 0.6 mg / L TDZ, and the NAA concentrations are 0.01 mg / L, 0.05 mg / L, and 0.1 mg / L, respectively; G, H, and I are all 1 mg / L TDZ, and the NAA concentrations are 0.01 mg / L, 0.05 mg / L, and 0.1 mg / L, respectively;

[0029] Figure 4 Figure 1. Endophyte contamination of bottle seedlings;

[0030] Figure 5 Effects of different NAA concentrations on the rooting of Rhododendron dahurica tissue culture seedlings; Note: A: 0 mg / L NAA treatment; B: 0.1 mg / L NAA treatment; C: 0.5 mg / L NAA treatment; D: 0.8 mg / L NAA treatment; E: 1.5 mg / L NAA treatment; The scale bars in the figures are 2 mm. DETAILED DESCRIPTION

[0031] The following will clearly and completely describe the concept of the present application and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present application.

[0032] The purchased goods in the test method, if no specific conditions are specified, are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, can be conventional products purchased from the market. Unless otherwise defined herein, scientific and technical terms used in conjunction with the present disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice and testing of the present disclosure.

[0033] Example 1

[0034] 1. Obtaining Sterile Materials

[0035] (1) Cut the stem explants of Rhododendron dahliae into 2-3 cm long stem segments with 2-3 buds. Remove the leaves and place them in a glass container with detergent and water and clean them in an ultrasonic cleaner for 30 min. Then take out the material and gently scrub the stem segments and buds with a brush to remove debris and dust. Rinse with slow running water for 30 min and place in an ultra-clean bench for surface sterilization.

[0036] (2) After sterilization under ultraviolet light on a clean bench for 30 minutes, disinfect with 75% alcohol for 30-60 seconds, wash twice with sterile water, and then soak in 0.1% HgCl2 solution (add 1-2 drops of Tween) for 11-12 minutes + 1% KMnO4 for 9-10 minutes + 10% H2O2 for 3-4 minutes. After soaking in the above agents, wash three times with sterile water. After that, cut off the browned parts at both ends of the stem segment with sterilized scissors, soak in a citric acid solution with a concentration of 1200-1500 mg / L for 10 minutes, and then inoculate into the culture medium.

[0037] (3) Culture medium formula

[0038] WPM+2.5-3mg / L TDZ+7.5g / L agar+30g / L sucrose+3g / L activated carbon, pH 5.4. After inoculation, culture in a light incubator with alternating lighting conditions of 15℃ (12h) and 21℃ (12h), with a light intensity of 2000lx at 21℃ and no light at 15℃.

[0039] Table 1 Effects of different disinfectant combinations on stem segment sterilization

[0040]

[0041] Note: The data in the table are the mean ± standard error of 3 replicates of the same treatment; different capital letters in the same column indicate extremely significant differences ( P＜0.01 ). Each disinfectant treatment was treated with 0.1% HgCl2.

[0042] The explant stem segments of Rhododendron dahurica could not reduce the browning and contamination of the stem segments under the treatment of 0.1% HgCl2, so 5% NaClO, 1% KMnO4, and 10% H2O2 were sterilized for different times. As shown in Table 1, the contamination rates of treatment 1, treatment 2, and treatment 5 were all below 10%, significantly lower than those of other groups, and the contamination rate of treatment 2 was 0. The browning rates of treatments 3, 4, and 5 were low, and the survival rates of treatments 4 and 5 were also higher than those of other groups. By comparison, it was found that under the same sterilization time of 10% H2O2, the browning rates of 1% KMnO4 treatments for 10 minutes and 15 minutes were lower than those of 5% NaClO. The contamination rates of 10 minutes of 1% KMnO4 and 5% NaClO and 4 minutes of 10% H2O2 treatments were very low, and accompanied by a higher survival rate. All treatments could induce new shoots, and the growth of new shoots of Rhododendron dahurica under 1% KMnO4 treatments was better than that under other treatments.

[0043] The best disinfection method: 1% KMnO4 10min + 10% H2O2 4min + 1500mg / L citric acid, at this time the contamination rate is 6.67%; the survival rate is 46.67%, and the browning rate is the lowest, which is 0.

[0044] The germination time and induction rate are shown in Table 2. As the TDZ concentration increases, the induction rate is higher when TDZ is 3 mg / L than other treatments. The germination time is also shortened with the increase of TDZ concentration. Figure 1 (A) It can be seen that the buds can expand in 10-15 days, sprout after 15 days, and healthy sprouts can be obtained after 2 months of cultivation (see Figure 1 B). The sprout induction rate after 60 days was 66.67%.

[0045] Table 2 Effects of different TDZ concentrations on the induction of axillary buds of Rhododendron dasyphylla

[0046] Treatment (Group) TDZ concentration (mg / L) Induction rate (%) Germination time (days) A 1 50.00±11.55 ab The buds begin to swell in 15-20 days and sprout after 20 days. B 2 40.00±5.77 ab Buds begin to swell in 12-17 days and sprout after 17 days C 3 66.67±8.82 b The buds begin to swell in 10-15 days and sprout after 15 days. D 4 36.67±6.67 a The buds begin to swell in 10-15 days and sprout after 15 days.

[0047] Note: The data in the table are the mean ± standard error of 3 replicates of the same treatment; different lowercase letters in the table indicate significant differences ( P＜0.05 ).

[0048] 2. In vitro regeneration induction of sterile leaves

[0049] Take the upper tender leaves of the sterile seedlings grown for 50-60 days under sterile conditions, cut the leaves into two pieces with a scalpel, and then inoculate them into WPM medium. Culture conditions: culture in a light incubator with alternating lighting conditions of 15℃ (12h) and 21℃ (12h), with a light intensity of 2000lx at 21℃ and no light at 15℃.

[0050] The best medium formula is: WPM+0.1mg / L TDZ+0.01mg / L NAA+7.5g / L agar+30g / L sucrose, which has a lower browning rate and the highest green leaf rate, reaching a very significant difference level with other formulas. After 60 days, the adventitious bud induction rate is 66.67%. Each explant forms 9-16 adventitious buds. (Table 3)

[0051] Table 3 Effects of different formulas on adventitious bud induction from explant leaves

[0052] TDZ concentration NAA concentration Browning rate (%) Green leaf rate (%) Germination induction rate (%) 0.02 0.01 38.89±5.56 a 61.11±5.56 a 27.78±5.56 a 0.1 0.01 22.22±14.70 a 77.78±14.70 a 66.67±9.62 b 0.3 0.01 55.56±11.11 a 44.44±11.11 a 5.56±5.56 a

[0053] Note: The data in the table are the mean ± standard error of 3 replicates of the same treatment; different lowercase letters in the table indicate extremely significant differences ( P＜0.01 ).

[0054] 3. Proliferation Culture

[0055] The above-mentioned 1-2cm high sprouts were transferred to the proliferation medium. The medium was based on WPM medium and TDZ, NAA and GA3 were added. Agar 7.5g / L and sucrose 30g / L were added. To prevent endophytic contamination, two fungicides were added to the medium: 300mg / L mancozeb + 40mg / L streptomycin sulfate. The streptomycin sulfate was filtered through a filter in advance. After the medium was sterilized, it was cooled to about 45°C and the filtered and sterilized streptomycin sulfate was added to the clean bench. The other components were added before the medium was sterilized. The culture conditions were: cultured in a light incubator with alternating light intensity of 15°C (12h) and 21°C (12h). The light intensity was 2000lx at 21°C and no light at 15°C.

[0056] The higher the TDZ concentration, the slower the growth. The bud proliferation times increased with the increase of TDZ concentration, and the buds grew more densely. When TDZ was 0.6 mg / L, the proliferation times were excellent, the leaves were emerald green, and the growth was good. When TDZ was 1 mg / L, it was difficult to count the clustered buds of tissue culture seedlings. Therefore, 0.6 mg / L TDZ was temporarily determined as the appropriate dosage for bud proliferation of Rhododendron davidii. The NAA concentration did not significantly increase the growth of buds and their proliferation times. Figure 3 It can be seen that when the NAA concentration is 0.05 mg / L, the growth height of the bottle seedlings is basically the same, and the leaf color is greener than when the NAA concentration is 0.01 mg / L and 0.1 mg / L. Therefore, 0.05 mg / L NAA is the appropriate concentration for use.

[0057] The best proliferation medium formula was: WPM+0.6mg / L TDZ+0.05mg / L NAA+0.5mg / L GA3+300mg / L mancozeb+40mg / L streptomycin sulfate. The highest proliferation multiple after 60 days was 5.47.

[0058] Table 4 Effects of different concentrations of TDZ and NAA on the proliferation of sterile seedlings of Rhododendron dasyphylla

[0059] Treatment (Group) TDZ concentration (mg / L) NAA concentration (mg / L) Multiplication times Growth status 1 0.2 0.01 3.23±0.23 Aa Good growth, emerald green leaves 2 0.6 0.05 5.47±0.29 Bb Normal growth, emerald green leaves 3 1 0.1 Unable to count Slow growth, yellow-green leaves 4 0.2 0.05 3.10±0.30 Aa Good growth, emerald green leaves 5 0.6 0.1 4.9±0.17 Bb Slow growing, emerald green leaves 6 1 0.01 Unable to count Slow growth, yellow-green leaves 7 0.2 0.1 3.30±0.21 Aa Good growth, emerald green leaves 8 0.6 0.01 5.20±0.21 Bb Normal growth, emerald green leaves 9 1 0.05 Unable to count Slow growth, yellow-green leaves

[0060] Note: The data in the table are the mean ± standard error of 3 replicates of the same treatment; different letters in the same column indicate significant differences ( P<0.05 ), lowercase letters in the table represent the differences among different TDZ concentrations under the same NAA concentration treatment, and uppercase letters in the table represent the differences among different NAA concentrations under the same TDZ concentration treatment.

[0061] Rhododendron grandiflorum will be contaminated by endophytic bacteria during tissue culture, especially after multiple subcultures, the contamination becomes more serious, affecting the rapid propagation process of tissue culture. Therefore, bactericides are added to the culture medium to reduce endophytic bacteria contamination. The present invention combines mancozeb and streptomycin sulfate, and the results are shown in Table 5: compared with the case where no antibacterial agent (CK, the same below) is added, the contamination rate of each treatment is reduced, and the synergistic effect of the two antibacterial agents is better than the single addition of one antibacterial agent. However, under the treatment of a higher concentration of mancozeb, the browning rate is also higher, and the browning seedlings eventually die. From the perspective of the contamination rate and survival rate of the tissue culture seedlings, the antibacterial effect of 100mg / L mancozeb+60mg / L streptomycin sulfate treatment is the best. It has the lowest contamination rate and the highest survival rate.

[0062] Table 5 Effects of mancozeb and streptomycin sulfate combination on tissue culture seedlings of Rhododendron dasyphylla

[0063] deal with Mancozeb(mg / L) Streptomycin sulfate (mg / L) Pollution rate (%) Browning rate (%) Survival rate (%) ck 0 0 100.00±0.00 0.00±0.00 0.00±0.00 D1 500 0 72.50±10.00 90.00±13.6 0.00±0.00 L1 0 100 37.50±9.00 0.00 ±0.00 62.5±6.85 DL1 500 20 27.50±6.00 100.00±0.00 0.00±0.00 DL2 300 40 15.00±6.00 22.50±5.59 62.5±5.60 DL3 100 60 12.50±0.00 15.00±5.50 72.50±5.85

[0064] Note: The data in the table are the mean ± standard error of 5 replicates of the same treatment.

[0065] 4. Rooting Culture

[0066] After proliferation, the healthy sprouts (1-2 cm in height) were cut off individually and transferred to the rooting medium. The medium is based on 1 / 2 WPM, supplemented with NAA and conventional ingredients, and has a pH of 5.4.

[0067] The best rooting medium formula is: 1 / 2WPM+0.8mg / L NAA+7.5g / L agar+30g / L sucrose+3g / L activated carbon. After 60 days, the rooting rate is 40%, and the average number of roots is 5.17.

[0068] Table 6 Effects of different concentrations of NAA on rooting of Rhododendron dahurica

[0069] deal with NAA concentration (mg / L) Rooting rate (%) Average number of roots Average root length (mm) 1 0 0.00±0.00 a 0.00±0.00 0.00±0.00 2 0.1 6.67±3.33 a 1.67±0.88 2.07±1.06 3 0.5 26.67±3.33 b 3.27±0.89 6.22±2.12 4 0.8 40.00±5.77 bc 5.17±0.33 8.03±2.12 5 1.5 46.67±6.67 c 2.11±0.11 2.89±0.36

[0070] Note: The data in the table are the mean ± standard error of 3 replicates of the same treatment; different lowercase letters in the table indicate significant differences ( P＜0.05 ).

[0071] In order to explore the suitable NAA concentration for rooting of Rhododendron davidii, different NAA concentration treatments were set. The results are shown in Table 6: Sterile seedlings without NAA addition could not grow roots, and the rooting effect was poor at a lower concentration of NAA. The rooting rate was the highest when the NAA concentration was 1.5 mg / L, and there was no significant difference in rooting effect compared with the concentration of 0.8 mg / L. Figure 5It can be seen that the average number of roots and average root length reached the best level among all treatments when the dosage of NAA was 0.8 mg / L, and both decreased when it was increased to 1.5 mg / L. Therefore, 0.8 mg / L NAA was selected as the dosage.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for establishing an in vitro regeneration system of sterile leaves of Rhododendron dasyphylla, characterized in that: The following steps are involved: (1) Sterile leaf culture of Rhododendron dahurica The explant stem segments of Rhododendron dahurica are cleaned and disinfected, and then placed in a stem segment culture medium for bud induction culture to obtain Rhododendron dahurica sterile leaves; In step (1), the method for disinfecting the stem segments is to sequentially soak them in 0.1% HgCl2 solution for 11-12 min + 1% KMnO4 for 9-10 min + 10% H2O2 for 3-4 min + 1200-1500 mg / L citric acid solution for 10 min; In step (1), the stem segment culture medium is: WPM + 2.5-3 mg / LTDZ + 7.5 g / L agar + 30 g / L sucrose + 3 g / L activated carbon, pH 5.4; (2) In vitro regeneration induction culture of sterile leaves of Rhododendron dahurica The sterile leaves of Rhododendron dahurica explants were inoculated on the regeneration induction medium to induce adventitious shoots. In step (2), the regeneration induction medium is: WPM + 0.1 mg / LTDZ + 0.01 mg / L NAA + 7.5 g / L agar + 30 g / L sucrose; (3) Proliferation and culture of adventitious sprouts The proliferation medium used for proliferation culture was: WPM + 0.6 mg / L TDZ + 0.05 mg / L NAA + 0.5 mg / L GA3 + 300 mg / L mancozeb + 40 mg / L streptomycin sulfate; (4) Rooting culture The robust sprouts after proliferation are transferred to the rooting medium for rooting cultivation; In step (4), the rooting medium is: 1 / 2WPM + 0.7-0.9mg / L NAA + 7.5g / L agar + 30g / L sucrose + 3g / L activated carbon; The culture environment for all steps was cultured in an incubator with alternating conditions of 15°C for 12 h and 21°C for 12 h. The light intensity was 2000 lx at 21°C, and there was no light at 15°C.

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