Construction method of chimonanthus praecox stem tip culture system with high proliferation rate and capable of being used for genetic transformation

Abstract

The invention provides a method for constructing a chimonanthus red heart stem tip culture system which is high in proliferation rate and can be used for genetic transformation. According to the method disclosed by the invention, the batch rapid production of the chimonanthus praecox taking the stem tip as the explant is established and optimized from three links of sterile seedling cultivation, stem tip proliferation and rooting induction respectively. Compared with research reports of existing literatures, the method has the advantages that the aseptic seedling cultivation period and the rooting induction culture period are shortened, the multiplication coefficient, the rooting rate and the survival rate of the chimonanthus praecox are increased, and a transformed explant preparation and regeneration approach is provided for chimonanthus praecox molecular breeding.

Description

Technical Field

[0001] The present invention relates to the technical field of plant cultivation, and specifically, to a method for constructing a stem tip culture system of Chimonanthus praecox with a high proliferation rate and applicable to genetic transformation. Background Art

[0002] Chimonanthus praecox (L.) Link Chimonanthus praecox , a traditional famous flower in China, has a long cultivation history and rich cultural connotations. It has extremely high ornamental value in landscaping, with strong floral fragrance, and its roots, leaves and flowers can all be used as medicine, having the effects of regulating qi and relieving pain, dispelling cold and detoxifying. Constructing a high-proliferation coefficient culture system with stem tips as explants is of great significance for completing the closed-loop from virus-free conservation of endangered varieties, rescue of weak Chimonanthus praecox to industrialized batch rapid production of molecular breeding and sterile genetic transformation materials of Chimonanthus praecox, as well as rapid production and promotion of rare and excellent varieties.

[0003] Current research mostly uses one-year-old branch stem segments, uses a variety of culture medium formulations, and adds different types and concentrations of plant growth regulators to improve the formation rates of buds and roots. However, the composition of the culture medium and the hormone ratio need to be precisely adjusted according to the specific explant type and culture objective, and there is currently no universal optimal formula. In the relevant stages of stem tip proliferation, research on tissue culture of Chimonanthus praecox began in 1990. Researchers conducted a preliminary exploration of rapid propagation of Chimonanthus praecox using stem segments with axillary buds (Zhang Lin et al., 1990). Researchers tried test-tube propagation of Chimonanthus praecox and found that it was difficult for Chimonanthus praecox to redifferentiate, and there were serious browning problems and low differentiation rates (Zheng Junbao et al., 1995). Zeng Jing used the cotyledons of aseptic seedlings of Chimonanthus praecox seeds as explants, and established a regeneration system of Chimonanthus praecox through screening of different hormone combinations. The improved MS medium (MgSO4 and ZnSO4 doubled, KNO3 and NH4NO3 halved) + 6-BA 1.0 mg / L + NAA 0.1 mg / L was used to induce bud proliferation, and the proliferation coefficient was 2.7 (Zeng Jing, 2007). In the rooting stage, using 1 / 2MS + NAA 0.1 mg / L as the culture medium, selecting strong rootless seedlings with a height of 3 - 4 cm, cutting off the basal callus, and then inoculating them into the rooting medium, the rooting rate was 80%. Seven days after the aseptic seedlings were inoculated into the rooting medium, a small amount of callus began to form at the incision, white protrusions appeared at the callus after 12 days, and 2 - 5 thick roots about 1 cm long could be produced after 25 days. However, in current experimental repetitions and practices, the repeatability of the above research conclusions is relatively small, and there are phenomena such as leaf yellowing, withering, and stem tip browning, so the culture medium formula and culture conditions still need to be repeatedly verified and optimized (Zheng Junbao et al., 1995; Zeng Jing, 2007; Zhang Hui et al., 2008; Li Chengke and Lv Yingmin, 2012; Zhao Zhenli et al., 2017).

[0004] At present, the production technology of wintersweet by tissue culture is only in the initial stage of research, with a low proliferation coefficient. At the same time, there are problems such as browning, non-differentiation, low differentiation rate, and low experimental repeatability (Chen Longqing, 2012; Li Chengke and Lü Yingmin, 2012; Ji Kaiting, 2013). At the same time, there is still room for improvement in terms of production speed, propagation coefficient, and plant quality. Especially for the native excellent variety group of red-heart wintersweet. At the same time, the molecular breeding approach of wintersweet mediated by Agrobacterium has not been established, especially the selection of transformation materials and the establishment of cultivation methods after transformation. Summary of the Invention

[0005] The object of the present invention is to provide a method for constructing a stem tip culture system of red-heart wintersweet with a high proliferation rate and suitable for genetic transformation.

[0006] To achieve the object of the present invention, the present invention provides a method for constructing a stem tip culture system of red-heart wintersweet with a high proliferation rate and suitable for genetic transformation. Using the stem tip of red-heart wintersweet as an explant, inoculate it into the proliferation medium, and after culturing in the dark for 5 - 7 days, transfer it to the light-dark alternating condition of 12 h light / 12 h dark for culture, and change the medium every 25 - 30 days; select healthy young plants growing to 0.8 cm or above, inoculate them onto the rooting medium, culture in the dark for 5 - 7 days, and then transfer them to the light-dark alternating condition of 12 h light / 12 h dark for 20 - 30 days to obtain complete red-heart wintersweet regenerated plants.

[0007] Preferably, the formula of the proliferation medium is: SH medium + 1.3 mg / L 6 - BA + 0.11 mg / L NAA + 2 mg / L PVP + 20 g / L sucrose + 7 g / L agar, pH = 5.6.

[0008] Preferably, the formula of the rooting medium is: 1 / 2 SH medium + 1.0 mg / L IBA + 10 g / L sucrose + 7 g / L agar, pH = 5.8.

[0009] Furthermore, the light-dark alternating culture conditions are: light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%, 12 h light / 12 h dark.

[0010] Furthermore, the conditions for dark culture are: temperature 24 - 26 °C, humidity 60% - 80%.

[0011] Furthermore, the preparation method of the explant includes: Select the current-year mature fruit receptacles on healthy mother plants, pick plump and healthy Chimonanthus praecox seeds with red hearts. After washing with water, bruise the hard pericarp, soak it in deionized water for 12 - 18 h, and peel off the hard pericarp. Then, soak it in a 250 mg / L gibberellin solution for 4 - 6 h. After soaking, rinse it with running water for 1 h, place it on a super clean bench for disinfection treatment of the seeds, then peel off the membranous seed coat, place the hypocotyl with the extending direction downward, and plant it in an MS medium without hormones (MS + 30 g / L sucrose + 7 g / L agar, pH = 5.8 - 6.0). The seeds start to germinate in 4 - 8 d, and sterile seedlings with true leaves unfolded can be obtained after culturing for about 40 d. The shoot tips (preferably shoot tips about 5 mm) can be taken at any time as explants.

[0012] Preferably, the method for disinfecting the seeds includes: disinfecting with 75% alcohol for 1 min, rinsing with sterile water once, then disinfecting with 5% sodium hypochlorite solution for 15 - 20 min, and finally rinsing with sterile water 5 times.

[0013] Furthermore, the conditions for culturing the seeds in the MS medium without hormones are: light intensity 6620 - 9360 Lux, temperature 24 - 26 °C, humidity 60% - 80%.

[0014] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The present invention established and optimized the rapid propagation system of Chimonanthus praecox with shoot tips as explants from three aspects: the cultivation of sterile seedlings (preparation of explants), shoot tip proliferation, and rooting induction. Compared with the research reports in existing literature, the cultivation period of sterile seedlings and the rooting induction culture period are shortened: the germination time of sterile seedlings is shortened from 14 - 21 days to 4 - 8 days, and the rooting induction period is shortened from 30 - 50 days to 20 - 30 days. The proliferation coefficient and rooting rate of Chimonanthus praecox are improved. The proliferation coefficient is increased from 2.7 to 7.1, the browning rate of shoot tips is reduced from 80% to 40%, and the rooting rate and survival rate are increased from 80% to 100%, greatly improving the rapid propagation efficiency. On this basis, the establishment of the genetic transformation system of Chimonanthus praecox achieved a breakthrough in the production of transgenic Chimonanthus praecox plants mediated by Agrobacterium tumefaciens.

[0015] (2) Using shoot tips as explants in the present invention is beneficial for the detoxification and regeneration of Chimonanthus praecox plants infected by fungi and viruses, the detoxification and conservation of endangered varieties, and the rescue of Chimonanthus praecox with weak growth potential.

[0016] (3) The present invention can obtain a large number of Chimonanthus praecox seedlings with the same growth potential in a short time, which is beneficial for the molecular breeding of Chimonanthus praecox and the factory-scale rapid production of sterile genetic transformation materials, as well as the rapid production and promotion of rare and excellent varieties.

[0017] (4) By precisely controlling the culture medium formula and culture conditions, the present invention improves the repeatability and stability of the entire culture process and reduces the uncertainty in experiments.

[0018] (5) By improving the explants and the process of adventitious bud differentiation, the present invention has established an Agrobacterium-mediated genetic transformation system for Chimonanthus praecox, achieving a breakthrough in obtaining Agrobacterium-mediated transgenic Chimonanthus praecox plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It shows the state of the regenerated Chimonanthus praecox seedlings taken out of the bottle in the specific embodiment of the present invention.

[0020] Figure 2 It shows the browning situation of the callus in Example 3 of the present invention. Figure 3 and Figure 4 respectively show the rooting induction situation using 1 / 2MS medium and the curling and yellowing of leaves during rooting induction using 1 / 2WPM medium in Example 5 of the present invention. SPECIFIC EMBODIMENTS

[0021] In view of the problems in the existing Chimonanthus praecox production using tissue culture method, such as long seed germination time in the sterile seedling cultivation stage, low proliferation rate in the proliferation stage, serious browning problem and low differentiation rate, long culture period in the rooting stage, poor experimental repeatability, and phenomena such as leaf yellowing, withering, and stem tip browning during the culture process, the present invention provides a method for constructing a stem tip culture system of red-heart Chimonanthus praecox with high proliferation rate and suitable for genetic transformation, aiming to improve the differentiation rate, proliferation rate of the stem tip culture of red-heart Chimonanthus praecox, as well as the rooting rate and seedling formation rate of seedlings about 0.8 cm, and shorten the culture period.

[0022] The present invention adopts the following technical solutions: The present invention provides a method for constructing a stem tip culture system of red-heart Chimonanthus praecox with high proliferation rate and suitable for genetic transformation, and the plant material is the stem tip of a sterile red-heart Chimonanthus praecox seedling cultivated from the seeds of the current-year red-heart Chimonanthus praecox.

[0023] Step 1: Cultivation of sterile seedlings Select the current-year mature fruit receptacles on healthy mother plants, take plump and healthy red-heart Chimonanthus praecox seeds, wash them with water, and remove the seeds floating on the water surface. Bruise the hard pericarp, soak it in deionized water for 12 - 18 h, and peel off the hard pericarp. Soak it in a 250 mg / L gibberellin solution for 4 - 6 h. Rinse it with running water for 1 h and set aside.

[0024] On the ultra-clean workbench, disinfect with 75% alcohol for 1 min, rinse once with sterile water, disinfect with 5% sodium hypochlorite solution for 15 - 20 min, rinse 5 times with sterile water, peel off the membranous seed coat with forceps, place the hypocotyl with the stretching direction downward, and plant it in MS medium without hormones (MS + 30 g / L sucrose + 7 g / L agar, pH = 5.8 - 6.0). Culture conditions: light intensity 6620 - 9360 Lux, temperature 24 - 26 °C, humidity 60% - 80%. The seeds start to germinate after 4 - 8 days of culture. Shoot tips can be taken after about 40 days of culture.

[0025] Step 2: Shoot tip proliferation Take shoot tips about 5 mm long and inoculate them into the proliferation medium. The formula of the proliferation medium is: SH medium + 1.3 mg / L 6 - BA + 0.11 mg / L NAA + 2 mg / L PVP + 20 g / L sucrose + 7 g / L agar, pH = 5.6. After culturing in the dark for 5 - 7 days, transfer them to the light - dark alternating condition of 12 h light / 12 h dark for culture. Light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%. Replace the medium every 25 - 30 days. During the 45 - 50 - day culture cycle, the multiplication coefficient is 7.1.

[0026] Step 3: Root induction Select healthy shoot tips that have grown to more than 0.8 cm and inoculate them onto the rooting medium. The formula of the medium is: 1 / 2 SH medium + 1.0 mg / L IBA + 10 g / L sucrose + 7 g / L agar, pH = 5.8. After culturing in the dark for 5 - 7 days (culture temperature 24 - 26 °C, humidity 60% - 80%), transfer them to the light - dark alternating condition of 12 h light / 12 h dark for culture (culture conditions: light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%). After culturing under light for 20 - 30 days, 2 - 6 roots of 1 - 5 cm in length grow out, and both the rooting rate and the seedling formation rate can reach 100%. The state of the regenerated seedlings of Chimonanthus praecox when they are taken out of the bottle is shown in Figure 1 。

[0027] The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] The following examples take the soaking time of gibberellin, the disinfection time of sodium hypochlorite, the concentration of anti - browning agent (PVP), the hormone ratio in the proliferation stage, the type of basic medium, the height of the plants during root induction, and the type and concentration of hormones in the rooting stage as variables to optimize the entire production and cultivation process. Compared with the existing aseptic rapid propagation method of Chimonanthus praecox, the seedling formation time of red - heart Chimonanthus praecox is shortened, and the multiplication coefficient is increased.

[0029] The experimental reagents used in the following examples are as follows: deionized water, GA3 (gibberellin), 5% sodium hypochlorite solution, 75% alcohol, PVP (polyvinylpyrrolidone), NAA (naphthaleneacetic acid), 6-BA (6-benzyladenine), MS medium, SH medium, WPM medium, sucrose, agar, IBA (indolebutyric acid).

[0030] Example 1 Changing the soaking time of gibberellin during the rapid cultivation stage of Chimonanthus praecox sterile seedlings Step 1: Cultivation of sterile seedlings Select plump and healthy seeds of the current year's Chimonanthus praecox, wash them, and remove the seeds floating on the water surface. Bruise the hard pericarp, soak it in deionized water for 12 - 18 h, and peel off the hard pericarp. Soak it in 250 mg / L gibberellin solution for 0, 2, 4, 6, 8, 10 h respectively. Rinse it with running water for 1 h and set aside for use.

[0031] On the ultra-clean workbench, disinfect with 75% alcohol for 1 min, rinse with sterile water once, disinfect with 5% sodium hypochlorite solution for 20 min, rinse with sterile water 5 times, use forceps to peel off the membranous seed coat, and plant the hypocotyl with the extending direction downward in the MS medium without hormones (sucrose 30 g / L + agar 7 g / L, pH = 5.8 - 6.0). Culture conditions: light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%. During the culture process, remove the contaminated seeds, which are not included in the statistics, and record the initial germination date of the seeds, germination potential (number of germinated seeds on the 7th day / total number of seeds), germination peak date (the day with the most germinated seeds), germination duration, and germination rate (counted on the 30th d), as shown in Table 1.

[0032] Table 1 Effects of gibberellin treatment time on seed germination

[0033] Example 2 Changing the disinfection time of 5% sodium hypochlorite solution during the rapid cultivation stage of Chimonanthus praecox sterile seedlings Step 1: Cultivation of sterile seedlings Select plump and healthy seeds of the current year's Chimonanthus praecox, wash them, and remove the seeds floating on the water surface. Bruise the hard pericarp, soak it in deionized water for 12 - 18 h, and peel off the hard pericarp. Soak it in 250 mg / L gibberellin solution for 4 h. Rinse it with running water for 1 h and set aside for use.

[0034] On the ultra-clean workbench, disinfect with 75% alcohol for 1 min, rinse once with sterile water, disinfect with 5% sodium hypochlorite solution for 10, 15, 20, and 25 min respectively, rinse 5 times with sterile water, peel the membranous seed coat with forceps, and plant the hypocotyl with the extending direction facing down in the MS medium without hormones (sucrose 30 g / L + agar 7 g / L, pH = 5.8 - 6.0). Culture conditions: light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%. Record the initial germination date of the seeds, germination potential (number of germinated seeds on the 7th day / total number of seeds), germination rate (counted on the 30th d, and plants contaminated with bacteria are excluded from the statistics), contamination rate, and seedling formation rate (counted on the 40th d for plants that can be used in subsequent experiments), as shown in Table 2.

[0035] Table 2 Effects of disinfection time with 5% sodium hypochlorite solution on seed germination

[0036] Example 3 Adding different concentrations of anti-browning agent PVP during the proliferation culture process Step 2: Take stem tips about 5 mm in length and inoculate them into the proliferation medium. The formula of the proliferation medium is: SH medium + 1.3 mg / L 6 - BA + 0.11 mg / L NAA + 20 g / L sucrose + 7 g / L agar, pH = 5.6. Add PVP at concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, and 4 mg / L respectively. At a temperature of 24 - 26 °C and a humidity of 60% - 80%, culture in the dark for 5 - 7 days, then transfer to the light - dark alternating condition of 12 h light / 12 h dark for culture. The light intensity is 4680 - 6620 Lux, the temperature is 24 - 26 °C, and the humidity is 60% - 80%. Replace the medium every 25 - 30 d. During the 45 - 50 - day culture period, count the callus browning rate, browning index {the browning level is rated from 0 to 4 levels: levels 0, 1, 2, 3, and 4 represent no browning, mild browning (S < 1 / 3; where S represents the browning area), medium - low browning (1 / 3 < S < 1 / 2), medium - high browning (1 / 2 < S < 2 / 3), and severe browning (S > 2 / 3). Further calculate the browning index (BI): BI = ∑[(browning level) × (number of explants at this level)] / (4 × total number of explants)}, and proliferation coefficient (see Table 3). Some callus tissues in the differentiation stage with severe browning turn brown, stop differentiating and growing, and become hard within 20 - 30 d. The browning situation of the callus is shown in Figure 2 。

[0037] Table 3 Effects of PVP concentration on callus browning and stem tip proliferation

[0038] Example 4 Hormone ratio during the growth culture process Step 2: Shoot tip proliferation Take the stem tip of about 5 mm and inoculate it in the proliferation medium. The formula of the proliferation medium is: SH medium + 2mg / L PVP + 20g / L sucrose + 7g / L agar, pH = 5.6, 6-BA: NAA hormone ratios are 0.9:0.11, 1.1:0.11, 1.3:0.11, and 1.5:0.11, respectively. After 5-7 days of dark culture, transfer to 12h light / 12h dark alternating light and dark conditions for culture, light intensity 4680~6620Lux, temperature 24~26℃, humidity 60%~80%, replace the medium every 25~30 days, and within the 45-50 day culture period, the state description, callus induction rate and proliferation coefficient under each hormone ratio of the proliferation coefficient are statistically analyzed (Table 4).

[0039] Table 4 Effects of hormone ratio on callus induction and shoot tip proliferation

[0040] Example 5 Changing the type of basal culture medium during root induction culture Step 3: Rooting Culture Healthy stem tips that grew to more than 1.2 cm were selected and inoculated on rooting medium. The medium formula was: 1.0 mg / L IBA + 10 g / L sucrose + 7 g / L agar, pH = 5.8, and the basic culture medium was: 1 / 2MS medium, 1 / 2WPM medium, 1 / 2SH medium, temperature 24~26℃, humidity 60%~80%, and cultured in the dark for 5~7 days. Then, they were transferred to the light-dark alternating conditions of 12h light / 12h dark, with a light intensity of 4680~6620Lux, temperature 24~26℃, and humidity 60%~80%. After 30~40 days of culture under light, the rooting rate, number of roots, root length, growth status, and seedling rate were counted (Table 5).

[0041] Table 5 Effect of basal culture medium on root induction

[0042] See the photo of yellowing and wilting plants grown on 1 / 2MS medium. Figure 3 .

[0043] See the photo of the plants with curled and yellowed leaves grown on 1 / 2WPM medium. Figure 4 .

[0044] Example 6 Changing the height of explants during root induction culture Step 3: Rooting Induction Select healthy shoot tips with heights of <0.5 cm, 0.5 - 0.8 cm, 0.8 - 1.0 cm, 1.0 - 1.5 cm, >1.5 cm, and inoculate them onto the rooting medium. The medium formula is: 1 / 2 SH medium + 1.0 mg / L IBA + 10 g / L sucrose + 7 g / L agar, pH = 5.8. After culturing in the dark for 5 - 7 days at a temperature of 24 - 26°C and a humidity of 60% - 80%, transfer them to a light-dark alternating condition of 12 h light / 12 h dark for culturing, with a light intensity of 4680 - 6620 Lux, a temperature of 24 - 26°C, and a humidity of 60% - 80%. After culturing under light for 20 - 30 days, count the rooting rate (Table 6).

[0045] Table 6 Effect of explant height on root induction

[0046] Example 7 Changing the hormone concentration during the rooting induction culture process Select healthy shoot tips with a length of more than 0.8 cm and inoculate them onto the rooting medium. The medium formula is: 1 / 2 SH medium + 10 g / L sucrose + 7 g / L agar, pH = 5.8, and the added IBA concentrations are 0.1 mg / L, 0.3 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, and 2.0 mg / L respectively. After culturing in the dark for 5 - 7 days at a temperature of 24 - 26°C and a humidity of 60% - 80%, transfer them to a light-dark alternating condition of 12 h light / 12 h dark for culturing, with a light intensity of 4680 - 6620 Lux, a temperature of 24 - 26°C, and a humidity of 60% - 80%. After culturing under light for 20 - 30 days, count the rooting rate, the number of roots, the root length, the growth status, and the seedling formation rate (Table 7).

[0047] Grow 2 - 6 roots with a length of 1 - 5 cm, and both the rooting rate and the seedling formation rate can reach 100%.

[0048] Table 7 Effect of hormone concentration on root induction

[0049] Example 8 Genetic transformation process of Chimonanthus praecox var. intermedius mediated by Agrobacterium tumefaciens Take shoot tips of Chimonanthus praecox var. intermedius about 5 mm in length and inoculate them into the proliferation medium. The proliferation medium formula is: SH medium + 1.3 mg / L 6 - BA + 0.11 mg / L NAA + 2 mg / L PVP + 20 g / L sucrose + 7 g / L agar, pH = 5.6. After pre - culturing in the dark for 2 days, use the pre - prepared Agrobacterium tumefaciens pCAMBIA - super1300 - GFP (purchased from Beijing Tsingke Biotechnology Co., Ltd.) bacterial solution for infection. The infection conditions are: OD 600= 0.4, vacuum for 10 min. After the infection is completed, co-culture in the dark for 3 - 5 d. The culture conditions are: temperature 19°C, humidity 60% - 80%. After degerming with 500 mg / L cefotaxime for 30 min, transfer to the selection medium. The medium formula is: proliferation medium + 200 mg / L cef + 40 mg / L Kana, pH = 5.6. Transfer to light for cultivation, light intensity 4680 - 6620 Lux, temperature 21 - 24°C, humidity 60% - 80%. Replace the medium every 7 days. The transformation rate is 90%. Within the culture period of 90 - 120 days, the Agrobacterium-mediated transgenic positive seedlings of Chimonanthus praecox var. intermedius are obtained for the first time, laying a foundation for the subsequent research on transgenic technology.

[0050] The existing regeneration pathways of Chimonanthus praecox using stem segments, cotyledons, and hypocotyls as explants cannot achieve the differentiation of adventitious buds, resulting in the inability to obtain materials suitable for transgenic research. And in the existing technology, the induction rate of callus is low, and the differentiating ability of the induced callus is poor, unable to form healthy regenerated plants, directly affecting the efficiency and success rate of transgenic research. The present invention takes a different approach, selects the stem tip as the material to induce the differentiation and regeneration of adventitious buds, and successfully establishes a transformation system, providing a technical basis for the subsequent molecular breeding of Chimonanthus praecox and also providing new ideas for the establishment of transformation systems for woody plants.

[0051] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

[0052] References: 1. Chen Longqing. Research progress on plants of Calycanthaceae [J]. Chinese Landscape Architecture, 2012, 28(08): 49 - 53. 2. Li Chengke, Lü Yingmin. Research progress on cuttage and tissue culture of vegetative propagation of Chimonanthus praecox [C] / / Ornamental Horticulture Professional Committee of Chinese Society for Horticultural Science, National Engineering Research Center for Floriculture. Research Advances in Chinese Ornamental Horticulture 2012. School of Landscape Architecture, Beijing Forestry University & National Engineering Research Center for Floriculture;, 2012: 4. 3. Ji Kaiting, Lu Jianguo, Guo Congcong. Research progress on propagation techniques of plants of Calycanthaceae [J]. Journal of Beijing Forestry University, 2013, 35(S1): 120 - 123. DOI: 10.13332 / j.1000 - 1522.2013.s1.028. 4. Zhang Lin, Tian Xingfan, Zhang Jingfen. Tissue culture and plant regeneration of Chimonanthus praecox [J]. Plant Physiology Communications, 1990(03):44. DOI:CNKI:SUN:ZWSL.0.1990-03-015. 5. Zheng Junbao, Zhang Yuman, Wang Xuerui, et al. Tissue culture of Chimonanthus praecox [J]. Journal of Beijing Forestry University, 1995(S1):6. DOI:CNKI:SUN:BJLY.0.1995-S1-022. 6. Zeng Jing. In vitro culture of Chimonanthus praecox and optimization of its genetic transformation system [D]. Southwest University, 2007.

Claims

1. A method for constructing a stem tip culture system of Chimonanthus praecox with high proliferation rate and suitable for genetic transformation, characterized in that, Using the stem tip of Chimonanthus praecox 'Luteus' as the explant, inoculate it into the proliferation medium and culture it in the dark for 5 - 7 days. Then transfer it to a light - dark alternating condition of 12 h light / 12 h dark for culture, and change the medium every 25 - 30 days. Select healthy young plants that have grown to 0.8 cm or more, inoculate them onto the rooting medium, culture them in the dark for 5 - 7 days, and then transfer them to a light - dark alternating condition of 12 h light / 12 h dark for 20 - 30 days to obtain complete regenerated plants of Chimonanthus praecox 'Luteus'. The formula of the proliferation medium is: SH medium + 1.3 mg / L 6 - BA + 0.11 mg / L NAA + 2 mg / L PVP + 20 g / L sucrose + 7 g / L agar, pH = 5.6; The formula of the rooting medium is: 1 / 2 SH medium + 1.0 mg / L IBA + 10 g / L sucrose + 7 g / L agar, pH = 5.

8.

2. The method according to claim 1, wherein The light - dark alternating culture conditions are: light intensity 4680 - 6620 Lux, temperature 24 - 26 °C, humidity 60% - 80%, 12 h light / 12 h dark.

3. The method according to claim 1, characterized in that, The dark - culture conditions are: temperature 24 - 26 °C, humidity 60% - 80%.

4. The method according to claim 1, wherein The preparation method of the explant includes: Select the current - year mature fruit receptacle on a healthy mother plant, take plump and healthy seeds of Chimonanthus praecox 'Luteus', wash them with water, bruise the hard pericarp, soak them in deionized water for 12 - 18 h, and peel off the hard pericarp. Then soak them in a 250 mg / L gibberellin solution for 4 - 6 h, rinse them with running water for 1 h after soaking, place them on a super - clean workbench for disinfection of the seeds, then peel off the membranous seed coat, place the hypocotyl with the extension direction downward, and plant it in an MS medium without hormones. The seeds start to germinate in 4 - 8 days, and sterile seedlings with true leaves unfolded are obtained after culturing for 40 days. The stem tip can be taken at any time as the explant.

5. The method according to claim 4, characterized in that The method for disinfecting the seeds includes: disinfecting with 75% alcohol for 1 min, rinsing once with sterile water, then disinfecting with 5% sodium hypochlorite solution for 15 - 20 min, and finally rinsing 5 times with sterile water.

6. The method according to claim 4, characterized in that The culture conditions of the seeds in the MS medium without hormones are: light intensity 6620 - 9360 Lux, temperature 24 - 26 °C, humidity 60% - 80%; The formula of the MS medium is: MS + 30 g / L sucrose + 7 g / L agar, pH = 5.8 - 6.0.

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