Method for inducing test tube tuberous roots under dahlia pinnata tissue culture condition

Abstract

The invention provides a method for inducing test tube tuberous roots under dahlia pinnata tissue culture conditions, which belongs to the technical field of plant tissue culture and comprises the following steps: step 1, obtaining sterile explants; step 2, primary culture: removing a color-changing part on the wound surface of the to-be-inoculated explant obtained in the step 1, and then inoculating the to-be-inoculated explant into a primary culture medium for culture to obtain a sterile test-tube plantlet; step 3, subculture multiplication culture: cutting the seedlings into cluster blocks, and inoculating the cluster blocks into a subculture multiplication culture medium to obtain multiplication test-tube seedlings; step 4, rooting culture; performing illumination culture in a rooting culture medium for 4 weeks to obtain rooted test-tube plantlets; 5, test tube root tuber induction: inoculating the rooted test tube plantlet into a root tuber induction culture medium for illumination culture for 4 weeks to obtain dahlia pinnata test tube sterile root tubers; through segmented induction and addition of different exogenous hormones, the breeding efficiency of dahlia pinnata can be effectively improved, and the breeding period is shortened.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for inducing test tube tube roots of dahlia under tissue culture conditions. Background Art

[0002] Dahlia (Dahlia pinnata Cav) is a perennial bulbous flower in the genus Dahlia in the Asteraceae family, comprising approximately 27 species. Also known as Dahlia, Dahlia, Passionflower, Western Peony, Dahlia, Sweet Potato Flower, and Red Potato Flower, it is native to Mexico, Colombia, and Guatemala, and later spread to Europe, Japan, and China. Dahlias are known for their graceful blooms, diverse shapes, long blooming period, and wide variety, earning them a reputation as the darling of flowers. Dahlias are the product of natural interspecific hybridization, with a relatively recent history of artificial breeding. They are widely cultivated worldwide, and are currently most widely cultivated in Northeast, Northwest, and North my country.

[0003] Dahlias have been artificially hybridized and selected to become allo-octoploids, with new varieties emerging in an endless stream. They have gorgeous colors, long flowering periods, strong adaptability, and are easy to cultivate. There are many varieties with rich and bright colors and beautiful flower shapes. There are not only a variety of single-color flowers, but also more and more gorgeous multi-color flowers with unique and diverse flower shapes. At present, China refers to international classification standards and divides dahlias into seven major color systems according to flower color, namely red, yellow, purple, white, black, pink and multi-color. According to flower shape, they are divided into twelve types, namely single-petal type, laurel type, decorative type, collar type, rosette type, ball type, hydrangea type, chrysanthemum type, peony type, cactus type, loose hair type and plate type, with extremely high ornamental value.

[0004] There are many ways to propagate dahlias, including seed propagation, root division, cuttings, and tissue culture. Root division and cuttings are commonly used in production. While these methods offer high survival rates, they have low propagation coefficients, far from meeting the needs of year-round propagation and cultivation. Tissue culture allows for rapid expansion, particularly by obtaining virus-free seedlings, which can preserve the variety's desirable traits, reduce the incidence of viral diseases, and effectively improve dahlia quality. The induction and efficient propagation of in vitro root tubers is a key technology for tissue culture seedling production, effectively improving the efficiency of large-scale tissue culture production and offering promising prospects for widespread application.

[0005] Previous patents on dahlia tissue culture and propagation have been partially reported. For example, the patent "A High-Efficiency Propagation Method for Virus-Free Dahlia Seedlings" (CN105028214A) discloses a method for efficiently propagating virus-free dahlia seedlings. The method includes pretreatment of virus-free dahlia seedlings, induction of adventitious buds, subculture of adventitious buds by cutting, seedling hardening, hydroponic rooting, hydroponic seedling strengthening, and transplanting. This method addresses the issues of low efficiency and recontamination during the propagation process, allowing for the rapid production of large quantities of high-quality virus-free dahlia seedlings in a short period of time. The patent "A Method for Preserving Dahlia Germplasm Propagated by Tissue Culture" (CN105123524A) discloses a method for preserving dahlia germplasm propagated by tissue culture, including explant selection and low-temperature pretreatment, inoculation, adventitious bud induction and transfer, slow-growth preservation of germplasm, rejuvenation, and rooting and transplanting. This method can preserve dahlia germplasm for up to four years, while also addressing the problems of large space requirements and high costs associated with germplasm preservation. It is simple to operate and offers stable and reliable technology. The patent "Dahlia Foot Bud Year-Round Rapid Propagation Technology" (CN200910205123.8) discloses a method that utilizes advanced solar greenhouses, cold storage, germination, and excellent cutting media to increase the number and frequency of dahlia foot bud propagation, significantly improving the survival rate of dahlia foot bud cuttings and creating conditions for the rapid propagation and application of new dahlia varieties. However, the above patents only cover the dahlia propagation process up to rooting culture and do not address the induction of tuber roots during dahlia propagation.

[0006] Previous research on dahlia tissue culture includes the following: Li David et al. (1986) used stem segments, flower buds, leaves, and axillary buds of dahlia as explants and induced callus tissue by adding an appropriate combination of hormones. Tian Songqing et al. (2020) optimized the optimal formula for primary culture, callus induction, subculture, and rooting using stem segments of the dahlia cultivar "Feluoga" as explants. Wei Sanli et al. (1996) used young leaves of adventitious buds from dahlia tubers as explants to induce bud differentiation and rooting. Ren Guihong et al. (2012) determined that dahlia apical buds and stem segments with axillary buds were the most suitable explants for in vitro rapid propagation. The hormone ratio suitable for the growth and differentiation of newly emerged axillary buds was 1 mg / L 6-BA + 0.2 mg / L NAA, while the hormone ratio suitable for adventitious bud differentiation from leaves was 3 mg / L 6-BA + 0.1 mg / L NAA. Zhao Yange et al. (2017) optimized tissue culture techniques using dahlia stem tips, stem segments with axillary buds, and leaves as explants, and determined that the optimal bud-inducing medium was MS supplemented with 0.5 mg / L 6-BA and 0.2 mg / L NAA, the optimal proliferation medium was MS supplemented with 2 mg / L 6-BA and 1 mg / L NAA, and the optimal rooting medium was 1 / 2 MS supplemented with 1 mg / L NAA, 4 mg / L agar, and 20 mg / L sucrose. However, the above studies were all about dahlia tissue culture, mostly focusing on adventitious bud induction, proliferation and rooted seedling transplanting, and there were few reports on in vitro tuber formation and transplanting. Summary of the Invention

[0007] Purpose of the invention: The purpose of the present invention is to address the deficiencies of the existing technology and provide a method for inducing tuber roots of dahlia under tissue culture conditions. By synergistically applying exogenous hormones through induction treatment at different stages, the reproductive efficiency of dahlia can be effectively improved and the breeding cycle can be shortened.

[0008] Technical solution: The method for inducing tuber roots of dahlia under tissue culture conditions of the present invention comprises the following steps:

[0009] Step 1: Obtaining sterile explants: Selecting current-year, pest-free, healthy, and well-developed dahlia branches, and obtaining explants by disinfection and cleaning; and culturing the explants in a primary culture medium to obtain explants to be inoculated;

[0010] Step 2, primary culture: removing the discolored part of the wound surface of the explant to be inoculated obtained in step 1, and then inoculating it into the primary culture medium for culture to obtain sterile test tube plantlets;

[0011] Step 3, subculture proliferation culture: selecting the sterile test tube seedlings obtained in step 2 with better and more consistent growth and cutting them into clusters containing 2-5 adventitious buds, and inoculating the clusters into subculture proliferation culture medium to obtain proliferated test tube seedlings;

[0012] Step 4, rooting culture; the proliferated test tube seedlings obtained from step 3 were inoculated into the rooting medium for light culture. The culture conditions were a temperature of 25±2°C, a light intensity of 1500-2500 lx, and a light exposure time of 6 h·d. -1 , humidity 60% to 70%, after culturing for 4 weeks, rooted test tube seedlings were obtained;

[0013] Step 5, in vitro root induction: the rooted test tube seedlings obtained in step 4 were inoculated into the root induction medium for light culture. The culture conditions were temperature 25±2℃, light intensity 1500~2500lx, and light duration 6h·d. -1 , humidity 60% to 70%, and after culturing for 4 weeks, sterile tube roots of dahlia were obtained.

[0014] Furthermore, step 1 is specifically as follows:

[0015] Step 1.1: Select a 2 cm stem segment with an axillary bud from a current-year, pest-free, healthy, and well-developed branch of the dahlia cultivar 'JOWEY LINDA' as an explant. Soak the explant in a 0.2% KMnO4 solution for 10 min, then rinse with water and place in a beaker.

[0016] Step 1.2: Soak the explants in detergent-containing water in a beaker for 5 minutes. Remove the explants, wrap them with gauze, and rinse them under running water for 30 minutes. Then, use filter paper to absorb the moisture on the explant surface.

[0017] Step 1.3: Place the explants, which have been dried, into a small beaker in a pre-sterilized pot and add 75% alcohol to sterilize for 30 seconds.

[0018] Step 1.4: Continue to add 0.1% HgCl2 and sterilize for 8 minutes. After sterilization, rinse the explants with sterile water several times to obtain explants to be inoculated.

[0019] Furthermore, step 2 is specifically as follows:

[0020] Step 2.1, placing the explant to be inoculated obtained in step 1 on sterile filter paper to absorb moisture, and cutting off the discolored part of the wound surface at the base of the stem segment;

[0021] Step 2.2: Inoculate the discolored explants into the primary culture medium. The culture conditions are: temperature 25±2℃, light intensity 1500~2500lx, and illumination time 16h·d. -1 , humidity 60-70%, and obtain sterile test tube seedlings.

[0022] Furthermore, step 3 is specifically as follows:

[0023] Step 3.1: Select the sterile test tube seedlings obtained in step 2, and cut them into clusters containing 2-5 adventitious buds, each cluster being 1 cm long;

[0024] Step 3.2: Inoculate the clumps into the subculture proliferation medium for light culture. The culture conditions are: temperature 25 ± 2°C, light intensity 1500-2500 lx, and light duration 16 h·d. -1 , humidity 60% to 70%, to obtain proliferated test tube seedlings;

[0025] Furthermore, the primary culture medium formula is MS+6-BA 2.0~3.0mg·L -1 +NAA 0.3~0.9mg·L -1 + sucrose 30g·L -1 + agar 6 g·L -1 , pH 5.6~6.0.

[0026] Furthermore, the proliferation culture medium formula is MS+6-BA 1.5~2.5mg·L -1 +NAA 0.005~0.015mg·L -1 +30g·L -1 + agar 6 g·L -1 , pH 5.6~6.0.

[0027] Furthermore, the rooting medium formula is MS+6-BA 0.05~0.15mg·L -1 +NAA 0.5~1.5mg·L -1 + sucrose 60g·L -1 + agar 6 g·L -1 , pH 5.6~6.0.

[0028] Furthermore, the tuber induction medium formula is WPM+6-BA 0.05~0.15mg·L -1 +NAA 0.5~1.5mg·L -1 + sucrose 40~60g·L -1 + agar 6 g·L -1 + activated carbon 2g·L -1 , pH 5.6~6.0.

[0029] Beneficial effects: Compared with the prior art, the advantages of the present invention are:

[0030] (1) In the prior art, dahlia tissue culture mostly involves adventitious bud induction, proliferation and rooted seedling transplantation, and rarely involves the induction of test tube tubers in the dahlia propagation process. The present invention adds the induction and cultivation of test tube tubers after the rooting culture of dahlia tissue culture is completed, and explores the induction and cultivation parameters suitable for in vitro tubers of dahlia, which is beneficial to improving the survival rate of dahlia tissue culture seedlings and shortening the breeding cycle.

[0031] (2) The present invention creatively adopts segmented induction, i.e., obtaining sterile explants, primary culture, subculture proliferation culture, rooting culture and in vitro tube root induction, and adds different exogenous hormones at different stages, which can effectively improve the reproduction efficiency of dahlia. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below with reference to the accompanying drawings, but the protection scope of the present invention is not limited to the embodiments.

[0034] Example 1

[0035] like Figure 1 The method for inducing tuber roots of dahlia under tissue culture conditions includes the following steps:

[0036] Step 1: Obtain sterile explants:

[0037] Step 1.1: Select a 2 cm stem segment with an axillary bud from a current-year, pest-free, healthy, and well-developed branch of the dahlia cultivar 'JOWEY LINDA' as an explant. Soak the explant in a 0.2% KMnO4 solution for 10 min, then rinse with water and place in a beaker.

[0038] Step 1.2: Soak the explants in detergent-containing water in a beaker for 5 minutes. Remove the explants, wrap them with gauze, and rinse them under running water for 30 minutes. Then, use filter paper to absorb the moisture on the explant surface.

[0039] Step 1.3: Place the explants, which have been dried, into a small beaker in a pre-sterilized pot and add 75% alcohol to sterilize for 30 seconds.

[0040] Step 1.4: Continue to add 0.1% HgCl2 and sterilize for 8 minutes. After sterilization, rinse the explants with sterile water several times to obtain explants to be inoculated.

[0041] Step 2: Primary Culture

[0042] Step 2 is as follows:

[0043] Step 2.1, placing the explant to be inoculated obtained in step 1 on sterile filter paper to absorb moisture, and cutting off the discolored part of the wound surface at the base of the stem segment;

[0044] Step 2.2: Inoculate the discolored explants into the primary culture medium. The primary culture medium is MS + 6-BA 2.5 mg·L -1 +NAA 0.5mg·L -1 + sucrose 30g·L -1 + agar 6 g·L -1 , pH 5.8; culture conditions were temperature 25±2℃, light intensity 1500~2500lx, and photoperiod 16h·d -1 , humidity 60-70%, and obtain sterile test tube seedlings.

[0045] Step 3: Subculture and proliferation:

[0046] Step 3.1: Select the sterile test tube seedlings obtained in step 2, and cut them into clusters containing 2-5 adventitious buds, each cluster being 1 cm long;

[0047] Step 3.2: Inoculate the clumps into the secondary proliferation medium for light culture. The proliferation medium formula is: MS + 6-BA 2.0 mg·L -1 +NAA 0.01mg·L -1 +30g·L -1 + agar 6 g·L -1 , pH 5.8; culture conditions are temperature 25±2℃, light intensity 1500~2500lx, and photoperiod 16h·d -1 , humidity 60% to 70%, and obtain proliferated test tube seedlings.

[0048] Step 4, rooting culture;

[0049] The proliferated test tube seedlings obtained in step 3 were inoculated into a rooting medium for light culture. The rooting medium formula was: MS + 6-BA 0.1 mg·L -1 +NAA 1.0mg·L -1 + sucrose 60g·L -1 + agar 6 g·L -1 , pH 5.8, culture conditions are temperature 25±2℃, light intensity 1500~2500lx, and photoperiod 6h·d -1 , humidity 60% to 70%, after culturing for 4 weeks, rooted test tube seedlings were obtained;

[0050] Step 5: Induction of tube root tubes:

[0051] The rooted test tube seedlings obtained in step 4 were inoculated into a tuber induction medium for light culture. The tuber induction medium was WPM + 6-BA 0.1 mg·L -1 +NAA 1.0mg·L -1 + sucrose 60g·L -1 + agar 6 g·L -1 + activated carbon 2g·L -1 , pH 5.8; culture conditions are temperature 25±2℃, light intensity 1500~2500lx, and light duration 6h·d -1 , humidity 60% to 70%, and after culturing for 4 weeks, sterile tube roots of dahlia were obtained.

[0052] The culture media prepared at each stage in Example 1 were used as culture media during dahlia tissue culture. Explants of dahlia at different stages were inoculated into the control culture media and the culture media at each stage. The comparative results of the various stages during tissue culture are shown in Tables 1-4.

[0053] Table 1 Effect of hormone addition on induction rate in induction culture

[0054]

[0055] Table 2 Effect of secondary proliferation medium on proliferation rate

[0056]

[0057] Table 3 Effect of hormone addition on rooting rate

[0058]

[0059] Table 4 Effects of basic culture medium for tuber induction on tuber formation of dahlia

[0060]

[0061] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A method for inducing tuber roots of dahlia under tissue culture conditions, characterized in that The steps include: Step 1: Obtaining sterile explants: Selecting current-year, pest-free, healthy, and well-developed dahlia branches, and obtaining explants by disinfection and cleaning; and culturing the explants in a primary culture medium to obtain explants to be inoculated; Step 2, primary culture: removing the discolored part of the wound surface of the explant to be inoculated obtained in step 1, inoculating it into the primary culture medium for culture, and obtaining sterile test tube plantlets; Step 3, subculture proliferation culture: selecting the sterile test tube seedlings obtained in step 2 with better and more consistent growth and cutting them into clusters containing 2-5 adventitious buds, and inoculating the clusters into subculture proliferation culture medium to obtain proliferated test tube seedlings; Step 4, rooting culture; the proliferated test tube seedlings obtained in step 3 were inoculated into the rooting medium for light culture. The culture conditions were a temperature of 25±2°C, a light intensity of 1500~2500lx, and a light exposure time of 6h·d. -1 , humidity 60% to 70%, after culturing for 4 weeks, rooted test tube seedlings were obtained; Step 5, in vitro root induction: the rooted test tube seedlings obtained in step 4 were inoculated into the root induction medium for light culture. The culture conditions were temperature 25±2℃, light intensity 1500~2500lx, and light duration 6h·d. -1 , humidity 60% to 70%, and after culturing for 4 weeks, sterile tube roots of dahlia were obtained.

2. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, wherein: Step 1 is as follows: Step 1.1: Select a 2 cm stem segment with an axillary bud from a healthy, pest-free, and full-budded branch of the dahlia cultivar 'JOWEY LINDA' as an explant. Soak the explant in a 0.2% KMnO4 solution for 10 min, then rinse with clean water and place in a beaker. Step 1.2: Soak the explants in detergent-containing water in a beaker for 5 minutes. Remove the explants, wrap them with gauze, and rinse them under running water for 30 minutes. Then, use filter paper to absorb the moisture on the explant surface. Step 1.3: Place the explants, which have been dried, into a pre-sterilized beaker and add 75% alcohol for 30 seconds. Step 1.4: After removing the alcohol solution in step 1.3, add 0.1% HgCl2 and sterilize for 8 minutes for secondary sterilization. After sterilization, rinse the explant several times with sterile water to obtain the explant to be inoculated.

3. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, wherein: Step 2 is as follows: Step 2.1, placing the explant to be inoculated obtained in step 1 on sterile filter paper to absorb moisture, and cutting off the discolored part of the wound surface at the base of the stem segment; Step 2.2: Inoculate the discolored explants into the primary culture medium. The culture conditions are: temperature 25±2℃, light intensity 1500~2500lx, and illumination time 16h·d. -1 , humidity 60-70%, and obtain sterile test tube seedlings.

4. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, wherein: Step 3 is as follows: Step 3.1: Select the sterile test tube seedlings obtained in step 2, and cut them into clusters containing 2-5 adventitious buds, each cluster being 1 cm long; Step 3.2: Inoculate the clumps into the subculture proliferation medium for light culture. The culture conditions are: temperature 25 ± 2°C, light intensity 1500-2500 lx, and light duration 16 h·d. -1 , humidity 60% to 70%, and obtain proliferated test tube seedlings.

5. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, wherein: The primary culture medium formula is: MS + 6-BA 2.0-3.0 mg·L -1 +NAA 0.3~0.9mg·L -1 + sucrose 30g·L -1 + agar 6 g·L -1 , pH 5.6~6.

0.

6. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, characterized in that: The proliferation culture medium formula is: MS+6-BA 1.5-2.5 mg·L -1 +NAA 0.005~0.015mg·L -1 +30g·L -1 + agar 6 g·L -1 , pH 5.6~6.

0.

7. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, characterized in that: The rooting medium formula is: MS+6-BA 0.05-0.15 mg·L -1 +NAA 0.5~1.5mg·L -1 + sucrose 60g·L -1 + agar 6 g·L -1 , pH 5.6~6.

0.

8. The method for inducing tube root growth of dahlia under tissue culture conditions according to claim 1, characterized in that: The root induction culture medium formula is: WPM + 6-BA 0.05-0.15 mg·L -1 +NAA 0.5~1.5mg·L -1 + sucrose 40~60g·L -1 + agar 6 g·L -1 + activated carbon 2g·L -1 , pH 5.6~6.0.

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