Rapid propagation method for woody oil prinsepia utilis royle

Abstract

The invention belongs to the technical field of plant tissue culture, and discloses a woody oil prinsepia utilis royle rapid propagation method. The efficient tissue culture and rapid propagation method is established by taking a naturally growing prinsepia utilis royle plant, selecting healthy current-year semi-lignified branches without diseases and insect pests and taking a stem section with an axillary bud as an explant. The method comprises the steps of disinfection treatment of stem segments with axillary buds, induction of axillary buds of explants, multiplication culture, rooting induction and acclimatization and transplantation. The axillary bud induction rate reaches 83.3%, the proliferation multiple reaches 4.2, the rooting rate reaches 90% or above, and the transplanting survival rate reaches 95.0% or above. The method can effectively solve the problems of low germination rate, character separation, low propagation coefficient and the like in seed propagation and green branch cutting, can obtain a large number of excellent seedlings with consistent genetic information in a short time, and has important significance in propagation and popularization and utilization of improved prinsepia utilis royle seeds with excellent economic characters such as high yield and oil content.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant tissue culture, and particularly relates to a rapid propagation method of woody oil plant Prinsepia uniflora Batal. BACKGROUND

[0002] Prinsepia uniflora Batal. Prinsepia utilis Prinsepia uniflora Batal. (Royle) also known as total flower flat core wood, is a small shrub of Rosaceae subfamily of Prinsepia, which is evergreen or deciduous. Prinsepia uniflora Batal. is distributed in the mountain slopes, valleys, wastelands and jungles with an altitude of 1800-3200m in Yunnan, Sichuan, Tibet and Guizhou of China, and the main production area is in Lijiang, Dali and Diqing of northwest Yunnan, and Lijiang is the main production area in northwest Yunnan. Prinsepia uniflora Batal. has a history of several thousand years of medicinal and edible use in the minority areas of Lijiang and Shangri-La in northwest Yunnan. The inner stem, leaf, root and fruit of Prinsepia uniflora Batal. can be used as medicine, which has the effects of attacking toxin, promoting blood circulation, removing blood stasis, tonifying deficiency, anemia, setting bone and detumescence. The main fatty acid composition of Prinsepia uniflora Batal. oil is oleic acid, linoleic acid and saturated fatty acid, and the content ratio of the three fatty acids is 1:1:0.8, which is extremely close to the structure of human fatty acid 1:1:1, and very meets the strict requirements of the Food and Agriculture Organization of the United Nations on dietary nutrition. The linoleic acid is 4 times higher than the world's most famous olive oil, and is a high-grade health edible oil. At the same time, Prinsepia uniflora Batal. oil has good affinity with the skin, and is used as skin care oil with fast penetration and absorption, which can moisturize the skin and prevent sunburn. In addition, modern medical research shows that Prinsepia uniflora Batal. oil has good effects on preventing and treating high blood pressure, reducing blood lipids, preventing cardiovascular and cerebrovascular diseases, treating diabetes and the like. A large number of research results show that Prinsepia uniflora Batal. is a woody oil plant with great development and utilization value.

[0003] At present, the seedling of Prinsepia uniflora Batal is mainly obtained by traditional breeding method, and artificial seedling is carried out by seed sowing or cutting, but it is limited by season, climate and other factors, and cannot be produced throughout the year, and the propagation coefficient is low. There are other research reports on the in vitro rapid propagation technology of Prinsepia uniflora Batal, which mainly obtains Prinsepia uniflora Batal tissue culture seedlings through sterile seed sowing and then tissue culture of sterile seedlings (Zeng N et al., 2007) or seedling seed sowing and then tissue propagation culture of seedling seedlings (Yao Li-wen, 2018). However, the tissue culture seedlings obtained by seed breeding have the following disadvantages: (1) compared with the mother plant, there is a genetic trait separation phenomenon, and the genetic information of the offspring seedling cannot be completely retained, and the genetic information of the offspring seedling is inconsistent. (2) The obtained tissue culture seedlings have inconsistent properties, some of which lose the excellent economic properties required for production, such as high yield, disease resistance, seed oil content, etc., and lose the significance of propagating excellent seedlings. (3) The seed germination rate is low, and the Prinsepia uniflora Batal seed will also appear seed inactivation phenomenon with the extension of storage time, and the germination rate of the seed stored at room temperature after 6 months of seed harvesting is almost zero. In order to solve the above problems, the present application uses the naturally grown Prinsepia uniflora Batal stem segments for tissue culture, which can completely retain the genetic information of the mother plant, so as to obtain seedlings with stable excellent economic properties, and also has the advantages of high propagation coefficient, high survival rate, can be produced throughout the year and the like, which lays a foundation for rapid propagation of good seed in the future.

[0004] Therefore, the present application aims to use the naturally grown Prinsepia uniflora Batal stem segments with axillary buds as explants to provide detailed procedures and methods for establishing axillary bud induction, proliferation and differentiation, and in-vitro rooting regeneration system. SUMMARY

[0005] In order to achieve the above purpose, the present application provides a method for establishing a high-efficiency regeneration system by using the stem segments of woody oil Prinsepia uniflora Batal, which comprises the following steps: Step 1. Collection of explants: in 4-8 months, the semi-lignified Prinsepia uniflora Batal branches growing healthily and free of diseases and pests are cut, the leaves on the branches are carefully peeled off, part of the petiole is left to protect the axillary buds, and the branches are cut into 1cm-long stem segments with one axillary bud each by using scissors for standby.

[0006] Step 2. Sterilization treatment of explants: soak the explants in detergent for 30 minutes, shake the detergent well to foam, then rinse the explants with flowing tap water for 30 minutes, sterilize the explants with 75% alcohol for 30 seconds under sterile conditions, immerse the explants in sterile water for 3 times, then perform surface sterilization on the explants with 6% sodium hypochlorite solution for 12 minutes, immerse the explants in sterile water repeatedly for 5 times, and then absorb the water on the explants with sterile filter paper. The glass bottle should be shaken constantly during the sterilization and cleaning process, so that the explants can be fully contacted with the sterilizing solution.

[0007] Step 3. Axillary bud induction: the stems treated with detoxification were inoculated in the axillary bud induction medium, which was WPM+ZT 0.8mg / L, pH 5.8, with 30g / L sucrose and 6g / L agar. The inoculated tissue culture bottles were placed in the culture room for light culture, with the culture conditions being temperature 25℃, light intensity 2000-3000lx, light time 12h / d, and culture time 6-8 weeks.

[0008] Step 4. Proliferation culture: the aseptic seedlings cultured in Step 3 were cut into 1cm-long stem segments, which were inoculated in the proliferation medium, which was WPM+ZT 0.5mg / L, pH 5.8, with 30g / L sucrose and 6g / L agar. The culture conditions were the same as those in Step 3.

[0009] Step 5. Rooting culture: when the seedlings cultured in the proliferation culture grew to about 6cm, they were cut into 1cm-long stem segments, which were transplanted into the rooting medium of 1 / 2WPM+IBA 1mg / L, with 30g / L sucrose and 6g / L agar, to promote rooting and seedling growth. The culture conditions were the same as those in Step 3.

[0010] Step 6. Hardening and transplanting: when the roots of the tissue culture seedlings grew to about 2cm, the culture bottles were moved to a place with scattered light indoors, the bottle caps were slightly opened after 3d, and then fully opened after another 2d, during which water was sprayed to maintain a certain humidity. After 4d, the seedlings were hardened and transplanted. Before transplanting, the substrate of the plug tray was mixed at a ratio of 1:1:1 of coconut husk, grass charcoal and perlite, and the substrate was disinfected with carbendazim before use. The shade was controlled, the humidity and temperature were controlled at about 70% and about 30℃ respectively, and the temperature should not exceed 35℃. After 1 week of transplanting, 1 / 2WPM mother liquor was sprayed twice, once every 1 week, and then leaf fertilizer was sprayed after the growth recovered.

[0011] The collection time of the explants in Step 1 was from April to August every year. The semi-lignified branches that were healthy and free of pests and diseases were cut in dry and sunny weather, and the inoculation material was 1cm-long stem segments with axillary buds.

[0012] The disinfection treatment in Step 1 was first soaked in detergent for 30min, the detergent was fully shaken to foam, and then rinsed with running water for 30min. Under sterile conditions, the explants were disinfected with 75% alcohol for 30s, then immersed in sterile water for 3 times, and then disinfected with 6% sodium hypochlorite solution for 12min. The explants were repeatedly immersed in sterile water for 5 times, and the process was continuously shaken to ensure that the explants were fully contacted with the disinfectant.

[0013] The amount of macroelements, microelements and organic matter contained in the 1 / 2WPM rooting medium described in step 5 is halved, and the rest remains unchanged.

[0014] The pH of the medium in steps 3, 4 and 5 is 5.8, the culture conditions are a temperature of 25°C, a light intensity of 2000-3000lx, a light time of 12h / d, and a culture time of 4-8 weeks.

[0015] Compared with the prior art, the application has the following advantages and effects: breaking the tissue culture mode of using seeds as explants or obtaining explants after sowing seeds, using naturally grown Chinese hawthorn stem segments as explants, which can completely retain the genetic information and excellent economic traits of the mother plant, and obtain stable seedlings.

[0016] The application has the advantages of fast axillary bud germination, fast axillary bud proliferation, high proliferation coefficient, high rooting rate, and healthy plant growth, and the axillary bud induction rate is 83.3%, the proliferation multiple is 4.2, the rooting rate is more than 90%, and the transplanting survival rate is more than 95.0%.

[0017] The application effectively solves the problems of low propagation coefficient and limitation by climate conditions in the propagation of Chinese hawthorn by seed sowing or cutting, and the problems of obtaining Chinese hawthorn tissue culture seedlings by seed sowing, which cannot completely retain the genetic information of the mother plant and has the problem of genetic separation of tissue culture seedlings obtained by seed sowing, and provides an important way for obtaining a large number of Chinese hawthorn seedlings with excellent economic traits in a short period of time and realizing efficient use of resources. BRIEF DESCRIPTION OF DRAWINGS Figure 1 The application utilizes stem segments to induce axillary buds; Figure 2 The application utilizes stem segments to induce axillary buds; Figure 3 The application utilizes stem segments to induce axillary buds; DETAILED DESCRIPTION

[0018] The application will be further described in detail below in combination with examples, but the implementation of the application is not limited thereto. The ordinary experimenters in the technical field can realize the purpose of the application according to the above disclosed content of the application and the range of parameters.

[0019] Step 1. Explant collection: in 4-8 months when the weather is fine, cut the semi-lignified Chinese hawthorn branches that are healthy and free of pests and diseases, carefully peel off the leaves on the branches, leave part of the petiole to protect the axillary buds, cut the stems into 1cm-long segments with one axillary bud each, and reserve them for use.

[0020] Step 2. Sterilization treatment of explants: soak in detergent for 30 min, shake well to foam, then rinse with running tap water for 30 min, sterilize with 75% alcohol for 30 s under sterile conditions, wash with sterile water for 3 times, then sterilize the surface with 6% sodium hypochlorite solution for 12 min, repeatedly wash with sterile water for 5 times, and dry with sterile filter paper. Shake the glass bottle constantly during the sterilization and cleaning process to ensure that the explants are fully exposed to the sterilizing solution.

[0021] The sterilization effect of 6% sodium hypochlorite on stem segments in this example was obtained through screening experiments under different treatment times, and the results are as follows.

[0022] Table 1 Sterilization effect of 6% sodium hypochlorite under different treatment times.

[0023] Disinfection time (min) Inoculation number Pollution number Pollution rate (%) Survival number Survival rate (%) 5 60 30 50 25 41.7 12 60 10 16.7 49 81.7 15 60 6 10 42 70 18 60 5 8.3 28 46.7 20 60 2 3.3 12 20

[0024] As shown in Table 1, the sterilization effect of 6% sodium hypochlorite for 12 min on the stem segments of Qingci fruit is good and the survival rate is high.

[0025] Step 3. Axillary bud induction: the sterilized stem segments are inserted into the axillary bud induction medium, which is WPM + ZT 0.8 mg / L, pH 5.8, with the addition of 30 g / L sucrose and 6 g / L agar. The inoculated tissue culture bottles are placed in a culture room for light culture, with a temperature of 25°C, a light intensity of 2000-3000 lx, a light time of 12 h / d, and a culture time of 6-8 weeks.

[0026] Table 2 Effect of different ZT concentrations on axillary bud induction and differentiation culture.

[0027] Different ZT concentration medium Inoculation number Germination number Germination rate (%) Germination days (d) WPM+ZT 0.5 mg / L 30 10 33.3 25 WPM+ZT 0.6 mg / L 30 15 50 18 WPM+ZT 0.8 mg / L 30 25 83.3 12 WPM+ZT 1 mg / L 30 22 73.3 15 WPM+ZT 1.5 mg / L 30 20 66.7 15

[0028] Each medium is inoculated with 30 stem segments, and 3 repeated treatments are set. The induction culture results are shown in Table 2. After about 25 days of light culture, it is found that buds are differentiated on all media, among which small buds first appear in the WPM + ZT 0.8 mg / L medium, and bud emergence occurs in 12 d, with the highest germination rate of 83.3%, good bud quality, and faster growth rate than the other two media. Therefore, WPM + ZT 0.8 mg / L is the most suitable medium formula for axillary bud induction.

[0029] Step 4. Proliferation culture: the sterile seedlings cultured in step 3 are cut into 1 cm long stem segments and inoculated into the proliferation medium, which is WPM + ZT 0.5 mg / L, pH 5.8, with the addition of 30 g / L sucrose and 6 g / L agar. The culture conditions are the same as step 3.

[0030] Table 3. Effects of different hormone combinations on proliferation culture

[0031] Medium Hormone combination Inoculation number Induced axillary bud number Proliferation times WPM WPM+ZT 0.3 mg / L 30 45 1.5 WPM WPM+ZT 0.4 mg / L 30 50 1.7 WPM WPM+ZT 0.5 mg / L 30 126 4.2 MS MS+6-BA 2 mg / L+NAA 0.15 mg / L 30 98 3.3 MS MS+6-BA 1.5 mg / L+NAA 0.1 mg / L 30 46 1.5 MS MS+6-BA 2 mg / L+NAA 0.1 mg / L 30 38 1.3

[0032] The most suitable proliferation medium for Malus robusta stem segments was obtained by adding different concentrations of auxin (NAA) and cytokinin (ZT and 6-BA) to WPM and MS medium. The test results of three repeated treatments are shown in Table 3. The aseptic seedlings obtained by axillary bud induction culture were cut into stem segments with one axillary bud, then inoculated on proliferation medium containing different hormone combinations, and the test results were observed and counted. As shown in Table 3, the proliferation fold of ZT was generally higher than that of 6-BA; in the proliferation medium of WPM+ZT 0.5 mg / L, the axillary bud proliferation fold was the highest, 4.2 times, while in the proliferation medium of MS+6-BA 2 mg / L+NAA 0.15 mg / L, the proliferation fold was 3.3 times, which was lower than that of WPM+ZT 0.5 mg / L, and the proliferation seedlings grew slowly and were weak. In the six proliferation media, Malus robusta stem segments did not proliferate and differentiate into cluster seedlings, but induced different numbers of axillary buds.

[0033] Step 5. Rooting culture. When the proliferation culture seedlings grow to about 6 cm, they are cut into stem segments of about 1 cm, and the seedlings are transplanted into 1 / 2 WPM+IBA 1 mg / L rooting medium to promote rooting and seedling growth. After about 20 days of culture, 90% of the seedlings can grow 3-5 roots. It was found in the test that adding appropriate activated carbon and banana juice to the rooting medium is beneficial to seedling growth and root formation, and shortens the rooting culture period.

[0034] Step 6. Acclimation and transplantation. Since the test tube seedlings rooted in the bottle are not suitable for the external climate, direct transplantation is easy to die, so acclimation and domestication is also an essential part of plant tissue culture technology. When the roots of the tissue culture seedlings grow to about 2 cm, the culture bottle is moved to a place with scattered light at room temperature, the bottle cap is slightly opened after 3 days, and the bottle cap is completely opened after another 2 days. During this period, water should be sprayed to maintain a certain humidity. After 4 days, the seedlings are acclimated and transplanted. Before transplantation, the substrate of the plug tray is mixed at a ratio of 1:1:1 of coconut husk, grass charcoal, and perlite. The substrate is sterilized before use. The seedlings are shaded and the temperature and humidity are controlled. The relative humidity is controlled at about 70%, and the temperature is controlled at about 30°C, not exceeding 35°C. After 1 week of transplantation, the seedlings are sprayed with 1 / 2 WPM mother liquor twice every 1 week. After the seedlings recover, they are sprayed with foliar fertilizer. The survival rate can reach more than 95%.

Claims

1. A method for rapid propagation of the woody oilseed plant *Prickly pear*, characterized in that... Includes the following steps: (1) Explant collection: During the sunny months of April to August, cut healthy, disease-free, semi-lignified branches of *Rhizophora stylosa*. Carefully peel off the leaves from the branches, leaving some petioles to protect the axillary buds. Cut the branches into 1cm sections with one axillary bud each. (2) Sterilization treatment of explants: Soak the explants in detergent for 30 minutes, ensuring the detergent is thoroughly shaken to create foam. Rinse with running tap water for 30 minutes. Disinfect with 75% alcohol for 30 seconds under sterile conditions. Rinse three times with sterile water. Then, disinfect the surface with 6% sodium hypochlorite solution for 12 minutes. Rinse repeatedly with sterile water five times. Blot dry with sterile filter paper. During the disinfection and cleaning process, continuously shake the tissue culture bottle to ensure the explants are fully in contact with the disinfectant. (3) Axillary bud induction: The sterilized stem segments were inoculated into axillary bud induction medium, which was WPM+ZT 0.8mg / L, pH 5.8, with 30g / L sucrose and 6g / L agar. The inoculated tissue culture bottles were placed in a culture room for light culture. The culture conditions were 25℃, light intensity of 2000-3000lx, light duration of 12h / d, and culture time of 6-8 weeks. (4) Proliferation culture: The sterile seedlings cultured in step 3 were cut into stem segments of about 1cm and inoculated into proliferation medium, which was WPM+ZT 0.5mg / L, pH 5.8, with 30g / L sucrose and 6g / L agar. The culture conditions were the same as in step 3. (5) Rooting culture: When the seedlings from the propagation culture grow to about 6cm, cut them into stem segments of about 1cm and insert the stem segments upright into the rooting culture medium, which is 1 / 2 WPM + IBA 1mg / L, with 30g / L sucrose and 6g / L agar added to promote rooting and strong seedlings. The culture conditions are the same as in step 3. (6) Hardening and transplanting: When the roots of the tissue culture seedlings grow to about 2cm, move the culture bottles to a place with diffused light indoors. After 3 days, slightly open the bottle cap, and after another 2 days, fully open the bottle cap. During this period, spray water to maintain a certain humidity. After 4 days, harden and transplant the seedlings. Before transplanting, the substrate for the seedling trays is a mixture of coconut coir, peat moss, and perlite in a ratio of 1:1:

1. The substrate is disinfected with carbendazim before use. Shade and sun protection are provided, and the humidity and temperature of the greenhouse are well controlled. The relative humidity is controlled at about 70%, and the temperature is controlled at about 30℃, not exceeding 35℃. One week after transplanting, spray with 1 / 2 WPM stock solution twice, once every week. After the plant resumes growth, apply foliar fertilizer in conjunction with the spray.

2. The method for rapid propagation of the woody oilseed plant *Prickly pear* as described in claim 1, characterized in that: The explant collection time mentioned in step 1 is from April to August each year. Collect healthy, disease-free, semi-lignified branches in sunny and dry weather. The inoculation material is a stem segment with axillary buds of about 1 cm, and part of the petiole is retained to protect the axillary buds.

3. The method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: In step 1, the disinfection process involves first soaking the surface in detergent for 30 minutes, rinsing it under running tap water for 30 minutes, disinfecting it with 75% alcohol for 30 seconds under sterile conditions, rinsing it three times with sterile water, and then disinfecting the surface with 6% sodium hypochlorite solution for 12 minutes, followed by repeated rinsing with sterile water five times.

4. The method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: In step 3, the axillary bud induction medium is WPM+ZT 0.8 mg / L, pH 5.8, with 30 g / L sucrose and 6 g / L agar added.

5. A method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: In step 4, the proliferation medium is WPM+ZT 0.5 mg / L, pH 5.8, supplemented with 30 g / L sucrose and 6 g / L agar.

6. The method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: In step 5, the rooting medium is 1 / 2 WPM + IBA 1 mg / L, pH 5.8, with 30 g / L sucrose and 6 g / L agar added.

7. A method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: The amounts of macro-elements, micro-elements, and organic matter in the 1 / 2 WPM rooting medium described in step 5 are halved, while the rest remain unchanged.

8. A method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: In steps 3, 4, and 5, the pH of the culture medium is 5.8, the culture conditions are 25°C, the light intensity is 2000-3000 lx, the light duration is 12 h / d, and the culture time is 6-8 weeks.

9. A method for rapid propagation of the woody oilseed plant *Prickly pear* according to claim 1, characterized in that: For hardening off and transplanting, use a substrate made of coconut coir, peat moss, and perlite in a 1:1:1 ratio. Maintain the relative humidity in the greenhouse at around 70% and the temperature at around 30℃, not exceeding 35℃. One week after transplanting, spray twice with a 1 / 2 WPM stock solution, once a week. After the plant resumes growth, apply foliar fertilizer in conjunction with the spray.

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