Tissue culture breeding method for endangered plant tung paulownia

Abstract

The invention discloses a tissue culture breeding method of an endangered plant tung tree, which comprises the following steps: selecting mature and complete tung tree fruits without wormholes, cleaning, peeling off, taking seeds, sterilizing the seeds, inoculating the seeds into a germination induction culture medium to induce germination, shearing, carrying out multiplication culture to form clumpy buds, shearing terminal buds, and inducing rooting to obtain tissue culture bottle seedlings; and acclimatizing and transplanting to obtain tissue culture seedlings of the tung yunnanensis. According to the method, the seedling cultivation period can be obviously shortened, the multiplication coefficient, the rooting rate and the transplanting survival rate are greatly improved, the seedling cultivation cost is obviously reduced, and more Yunnan tung seedlings with consistent quality can be propagated at a higher speed; sufficient nursery stock guarantee is provided for further development of field regression, ex-situ conservation, development and utilization and the like of the Yunnan paulownia, and the method has important significance for effectively increasing the population number of the Yunnan paulownia and achieving scientific protection and even reasonable development and utilization of germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture breeding method for the endangered plant Thunbergia yunnanensis. Background Art

[0002] Yunnan tung ( Craigia yunnanensis ) is a species of the genus Thunbergia (Tliaceae) Craigia W.W. Smith & W.E. Evans, a deciduous tree, is a rare species endemic to southwest China and a Class II National Protected Plant. It is found in the southern subtropical dry-wet alternating hot climate of Yunnan, Guangxi, and Guizhou, as well as in mountain forests at altitudes of 500 to 1,700 meters. The tree can reach 20 to 30 meters in height. Its papery, elliptical leaves are short-pointed at the apex and rounded at the base. The basal veins are three and the margins are serrated. Its inflorescences are axillary, with two to five flowers. The winged capsule is elliptical, 3.5 cm long and 2.5 to 3 cm wide, with thin, membranous, and five-ribbed wings. The seeds are approximately 1 cm long. As the fruit matures, the papery pericarp changes from emerald green to a rough, dry, and brittle dark brown, while the seeds change from tender white and smooth to wrinkled dark brown. The wood of the Tung tree is hard, heavy, wear-resistant, and well-processed. Its tall, straight trunk makes it suitable for use as a pillar, making it a valuable and economically valuable tree species. Its strong adaptability to limestone karst habitats makes it suitable for afforestation in these habitats. Furthermore, the genus Tung is an oligospecies, and Tung, as the type species, holds significant scientific value in floristic and geographical studies, with its extant populations serving as a key group for systematic evolutionary studies.

[0003] However, Tung is a typical species with a very small population, with a narrow and sporadic distribution in the wild. Its population is extremely small, and due to environmental constraints, its natural reproductive capacity is weak, making natural regeneration very difficult, resulting in slow population growth. Furthermore, habitat encroachment, environmental destruction, and plant felling have severely threatened its survival. According to records, there are only 5 to 9 remaining wild distribution sites of Tung, with a wild population of only 11 to 100 plants. It has been listed as an endangered plant species requiring key protection and rescue in the China Red List (Volume I) and the IUCN.

[0004] In order to protect and develop the rare and endangered plant resources of Tung oil tree, scholars have carried out research on population conservation genetics, chloroplast genome, pollination and breeding systems, and seed characteristics of Tung oil tree, but there are few studies on seedling breeding. Expanding populations through conservation measures and artificial intervention is the most effective way to save endangered plants. Plant tissue culture technology has become an important means of breeding and preserving endangered plants. The technology for breeding Tung oil tree using tissue culture is only found in patent publication number CN 110663552 B. This patent proposes a method for breeding tissue culture seedlings using young Tung oil tree tissue as an explant, undergoing 30 days of induced differentiation culture, 60 days of subculture proliferation culture, and 30 days of seedling rooting culture. The induction differentiation rate is only 92%, the proliferation coefficient is 4.7, the rooting rate is 91% after 30 days, and the transplant survival rate after 30 days is 90%. However, this technology requires at least 150 days to grow seedlings, which has the problems of a long seedling cultivation cycle, low proliferation coefficient, rooting rate and survival rate, and high seedling cultivation costs, making it unfavorable to meet the demand for obtaining a large number of seedlings in a short period of time. It is urgent to save the rare and endangered plant Tung oil tree. It is necessary to establish a Tung oil tree tissue culture propagation technology with a shorter seedling cycle, higher proliferation coefficient, and higher rooting and transplanting survival rate, so as to achieve the propagation of more Tung oil tree seedlings of consistent quality at a faster rate, provide sufficient seedling guarantee for further research on the return of Tung oil tree to the wild, ex situ protection, and development and utilization, and have important significance for effectively expanding the population of Tung oil tree and realizing the scientific protection of germplasm resources and even the rational development and utilization. Summary of the Invention

[0005] The present invention overcomes the shortcomings of the prior art and provides a tissue culture propagation technology for Tung oilseed oil plant with a shorter seedling raising period, higher proliferation coefficient and rooting rate, and better seedling quality.

[0006] In order to achieve the above object, the technical solution of the present invention is as follows: A tissue culture propagation method for the endangered plant Tung oil tree, the main operating steps are as follows: (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 5 min to 10 min, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 10 min to 20 min, rinse with pure water 3 to 5 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 20 s to 30 s, rinse with sterile water 3 to 5 times, then put them into 2% sodium hypochlorite solution and stir and soak for 8 min to 10 min, rinse with sterile water 3 to 5 times, and place the seeds on sterilized filter paper to absorb the moisture; (2) Germination induction culture: Place the sterilized seeds flat on the germination induction medium for germination induction; (3) Subculture and proliferation: After the seeds germinate and grow into seedlings, cut the apical buds and the area below the hypocotyl and insert them into the subculture and proliferation medium. After 20 to 25 days of proliferation and proliferation, clusters of buds will form. When subculture and proliferation are repeated, the clusters of buds will be cut and transferred to the subculture and proliferation medium for further subculture and proliferation. (4) Rooting culture: insert the single apical bud with a height of ≥1 cm obtained in step (3) vertically into the rooting induction medium to obtain the tissue culture bottle seedling. When inserting the bud vertically into the culture medium, ensure that the lowest stem node is 0.1 cm to 0.5 cm above the culture medium; (5) Hardening and transplanting: The rooted tissue culture seedlings cultured in step (4) are moved to an outdoor environment with a shade shed for hardening and then transplanted into a transplanting medium. Conventional seedling management is performed to obtain the Yunnan Paulownia seedlings.

[0007] Preferably, the bud induction medium described in step (2) is modified WPM+GA3 0.2 mg / L~0.4 mg / L+NAA 0.1 mg / L~0.2 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.2.

[0008] Preferably, the subculture proliferation medium in step (3) is modified WPM + 2-ip 0.6 mg / L~0.8 mg / L + NAA 0.2 mg / L~0.3 mg / L + IAA 0.2 mg / L~0.3 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8~6.2.

[0009] Preferably, when cutting the terminal buds in step (3), care is taken to retain all leaves and petioles without trimming, retaining a height of 2 cm to 4 cm; when cutting below the hypocotyl, the fibrous roots are removed, retaining a height of 3 cm to 5 cm; and the method for dividing the cluster of buds during the second subculture is to cut the large cluster into small clusters with 2 to 3 buds retained.

[0010] Preferably, the rooting induction medium in step (4) is 1 / 2 modified WPM + NAA 0.1 mg / L~0.2 mg / L + IAA 1 mg / L~3 mg / L + IBA 1 mg / L~3 mg / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8~6.2.

[0011] Preferably, the culture temperature and light control conditions in step (2), step (3), and step (4) are: temperature 25±3°C, light intensity 2000 Lx to 8000 Lx, and light duration 12 h / d to 14 h / d; the composition of the modified WPM and 1 / 2 modified WPM culture medium is:

[0012] Preferably, the seedling hardening method in step (5) is to harden the seedlings for 1 to 2 days with the bottle cap closed in a greenhouse with a shading rate of 60% to 80%, and then harden the seedlings for 2 to 5 days with the bottle cap opened.

[0013] Preferably, the transplanting matrix in step (5) is red soil, sand, and vermiculite in a volume ratio of 5:3:2. Before transplanting and raising seedlings, the transplanting matrix is ​​first sterilized by pouring a potassium permanganate solution with a volume concentration of 0.1%, and then rinsed with clean water to remove the liquid.

[0014] Preferably, the conventional seedling management described in step (5) is as follows: the seedlings are placed in an outdoor nursery with a shade net, the shading rate is 50% to 60% within one week after transplanting, the light intensity is gradually increased after one week, the shading rate is maintained at 10% to 20% after one month, the water content of the substrate is maintained at 60% to 80%, and the seedlings are sprayed once or twice a month with a 0.3% to 0.5% mass concentration compound fertilizer (N:P:K=15:15:15) aqueous solution.

[0015] Compared with the prior art, the present invention has the following advantages and positive effects: 1. Application The present invention makes up for the deficiencies of the prior art by optimizing and screening the culture medium formula and culture scheme at each stage of the tissue culture propagation technology of Tung oil tree, significantly shortens the seedling cultivation cycle to 92 days to 105 days, greatly improves the technical effect of each stage of tissue culture, the germination rate reaches 95.6% to 96.3%, the proliferation coefficient reaches 6.7 to 8.6, the rooting rate reaches 95.4% to 96.7%, the survival rate is 94.3% to 95.8%, the seedlings are strong, the seedling cultivation cost is reduced, and large-scale mass production can be achieved, solving the difficult problem of scarce wild Tung oil tree seedlings, laying a seedling foundation for expanding the population size by returning the seedlings to the population and ex situ protection, and is of great significance for the preservation and utilization of this rare and endangered species.

[0016] 2. When sterilizing seeds, the present invention optimizes the existing technology of using mercuric chloride as a sterilizer and selects a combination of ethanol and sodium hypochlorite. After sterilization, the sterilizer can be directly discharged without treatment, which is non-toxic, pollution-free and easy to operate.

[0017] 3. The steps of the present invention are simple. The culture does not need to undergo callus tissue induction and redifferentiation before proliferation. After proliferation, there is no need to strengthen the seedlings and the single buds can be directly cut and rooted. After germination, a large number of effective buds can be obtained using only the proliferation culture medium, which avoids frequent replacement of the culture medium during the culture process, increases the risk of contamination, reduces workload and energy consumption, and reduces the cost of seedling cultivation.

[0018] 4. The present invention screened and optimized the usage ratio of a large number of elements in the basic culture medium based on the conditions of the seedlings of Tung tree at different growth stages during the cultivation process. On the basis of the WPM universal culture medium, the usage concentrations of NH4NO3, KNO3, and CaCl2·2H2O were increased. During the experiment, it was found that Tung tree was prone to yellow leaves and the leaf tips were hooked and curled before transplanting seedlings. Increasing the ratio of nitrogen and calcium elements can improve this situation. When the present invention increases the nitrogen ratio, the content of nitrate nitrogen and ammonium nitrogen is respectively increased. Nitrate nitrogen can promote the plant's absorption of other mineral elements, and ammonium nitrogen can improve the plant's resistance to stress. The two are selected in a suitable ratio to synergistically promote the plant's more robust growth.

[0019] 5. In the rooting stage, the present invention reduces the concentration of a large number of elements by half on the basis of the improved WPM, thereby reducing the concentration of a large number of elements N, P, and K, thereby reducing nutritional growth, and prompting plants to allocate more nutrients to root development. This is not only beneficial to rooting and making the root system stronger, but also can improve the plant's adaptability to the environment after transplanting, improve the transplant survival rate, and also save costs, making it more suitable for large-scale production.

[0020] 6. When optimizing the culture medium, the present invention flexibly changes the types and concentrations of different hormones according to the target direction. The combination of GA3 and NAA is used in the germination induction medium to promote rapid germination of seeds. In the subculture proliferation stage, the more effective 2-ip is selected to replace the conventional 6-BA, and NAA and IAA at appropriate concentrations are used to promote the expansion of the absorption surface to form adventitious buds, and the elongation of axillary buds is increased, significantly improving the proliferation coefficient. Adding an appropriate amount of NAA in the rooting stage can promote the rapid formation of root points after mild callus at the base, and work together with IAA and IBA to promote the formation of a strong root system.

[0021] 7. The present invention has obtained a substrate ratio suitable for the growth of Yunnan tung through a large number of experimental screenings during seedling hardening and transplanting. Red soil in the transplanting substrate provides the main nutrition, but its air permeability is relatively poor, which can easily lead to root hypoxia. Sand can increase the air permeability of the substrate, preventing seedlings from rotting due to root blockage, affecting the condition of the seedlings or even death. Vermiculite has a good water and fertilizer adsorption effect. The appropriate proportion of the three significantly improves the survival rate and growth performance of seedling transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the germination state of Tung oilseed seeds in Example 1.

[0023] Figure 2 This is a photo of Tung oilseed seeds germinating into seedlings in Example 1.

[0024] Figure 3 This is a photo of the initial stage of Tung oil tree proliferation culture in Example 1.

[0025] Figure 4 This is a photo of the Tung oil tree proliferation cultured for 20 days in Example 1.

[0026] Figure 5 This is a photo of the bottom of the bottle of the rooted bottle seedling of Tung oil tree in Example 1.

[0027] Figure 6 These are photos of transplanted surviving seedlings of Tung oil tree from Example 1.

[0028] Figure 7 These are photos of surviving Tung oil tree seedlings from Example 1.

[0029] Figure 8 This is a photo of the 30-day proliferation culture of Tung oil tree in comparative example 1.

[0030] Figure 9 This is a photo of the bottom of the bottle of the rooted bottle seedling of Tung oil tree in comparative example 1.

[0031] Figure 10 This is a photo of the comparative example 2, Tung oil tree proliferation culture for 30 days. Figure 11 This is a photo of the bottom of the bottle of the rooted bottle seedling of Tung oil tree in comparative example 2.

[0032] Figure 12 This is a photo of the bottom of the bottle of the rooted bottle seedling of Tung oil tree in comparative example 3. DETAILED DESCRIPTION

[0033] The following examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. However, it should be understood by those skilled in the art that various changes may be made thereto in form and detail without departing from the scope defined in the claims of the present invention.

[0034] In the following examples, the compositions of the MS, WPM, modified WPM, and 1 / 2 modified WPM media are as follows:

[0035] Example 1: A tissue culture propagation method for the endangered plant Tung oil tree, the main operating steps are as follows: (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel off the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 5 min to 10 min, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 10 min to 20 min, rinse with pure water 3 to 5 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 20 s to 30 s, rinse with sterile water 3 to 5 times, then put them into 2% sodium hypochlorite solution and stir and soak for 8 min to 10 min, rinse with sterile water 3 to 5 times, and place the seeds on sterilized filter paper to absorb the moisture. (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 8 minutes, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 15 minutes, rinse with pure water 5 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 30 seconds, rinse with sterile water 5 times, then put them into 2% sodium hypochlorite solution and stir and soak for 9 minutes, rinse with sterile water 5 times, and place the seeds on sterilized filter paper to absorb the moisture.

[0036] (2) Germination induction culture: The sterilized seeds were placed flat in a germination induction medium for germination induction. The germination induction medium was modified WPM + GA 30.3 mg / L + NAA 0.2 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8~6.2, and the culture conditions were temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light time 12 h / d~14 h / d.

[0037] (3) Subculture and proliferation culture: After the seeds germinate and grow into seedlings, cut the apical bud and the position below the hypocotyl and insert them into the subculture and proliferation culture medium. When cutting the apical bud, be careful to keep all leaves and petioles untrimmed, and keep the height at 3 cm. When cutting the position below the hypocotyl, cut off the fibrous roots, and keep the height at 4 cm. After 20 days of proliferation culture, clusters of buds are formed. When subculture and proliferation are carried out again, the clusters of buds are cut into small clusters with 2 to 3 buds, and then transferred to the subculture and proliferation culture medium for continued subculture and proliferation culture. The subculture and proliferation culture medium is modified WPM+2-ip 0.6 mg / L+NAA 0.25 mg / L+IAA 0.25 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.2, and the culture conditions are temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light time 12 h / d~14 h / d.

[0038] (4) Rooting culture: insert the single apical bud with a height of ≥1 cm obtained in step (3) vertically into the rooting induction medium to obtain the tissue culture bottle seedling. Note that when inserting the culture medium vertically, ensure that the lowest stem node is 0.3 cm above the culture medium. The rooting induction medium is 1 / 2 modified WPM+NAA 0.1 mg / L+IAA 1 mg / L+IBA 3 mg / L+agar 3.7 g / L+sugar 15 g / L, pH=5.8~6.2, and the culture conditions are temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light duration 12 h / d~14 h / d.

[0039] (5) Hardening and transplanting: The rooted tissue culture seedlings in step (4) were moved to an outdoor greenhouse with a shading rate of 60% and hardened for 2 days. The seedlings were then opened and hardened for 3 days. The seedlings were then transplanted into a matrix of red soil, sand, and vermiculite with a volume ratio of 5:3:2. Before transplanting, the transplanting matrix was first sterilized by drenching it with a potassium permanganate solution with a volume concentration of 0.1%, and then washed away with clean water. After transplanting, the seedlings were placed in an outdoor nursery with a shade net. Within one week after transplanting, the shading rate was 60%. After one week, the light was gradually increased. After one month, the shading rate was maintained at 20%. The water content of the matrix was maintained at 65%. The seedlings were sprayed with a 0.4% compound fertilizer (N:P:K=15:15:15) aqueous solution twice a month to obtain Yunnan tung seedlings.

[0040] Example 2: A tissue culture propagation method for the endangered plant Tung oil tree, the main operating steps are as follows: (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 5 minutes, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 10 minutes, rinse with pure water 3 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 20 seconds, rinse with sterile water 3 times, then put them into 2% sodium hypochlorite solution and stir and soak for 8 minutes, rinse with sterile water 3 times, and place the seeds on sterilized filter paper to absorb the moisture.

[0041] (2) Germination induction culture: The sterilized seeds were placed flat in a germination induction medium for germination induction. The germination induction medium was modified WPM + GA3 0.2 mg / L + NAA 0.1 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8~6.2, and the culture conditions were temperature 25±3 ℃, light intensity 2000 Lx~8000 Lx, and light time 12 h / d~14 h / d.

[0042] (3) Subculture and proliferation culture: After the seeds germinate and grow into seedlings, cut the apical bud and the position below the hypocotyl and insert them into the subculture and proliferation culture medium. When cutting the apical bud, be careful to keep all leaves and petioles untrimmed, and keep the height at 2 cm. When cutting the position below the hypocotyl, cut off the fibrous roots, and keep the height at 3 cm. After 25 days of proliferation culture, clusters of buds are formed. When subculture and proliferation are carried out again, the clusters of buds are cut into small clusters with 2 to 3 buds, and then transferred to the subculture and proliferation culture medium for continued subculture and proliferation culture. The subculture and proliferation culture medium is modified WPM + 2-ip 0.6 mg / L + NAA 0.2 mg / L + IAA 0.2 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8 ~ 6.2, and the culture conditions are temperature 25 ± 3 ℃, light intensity 2000 Lx ~ 8000 Lx, and light time 12 h / d ~ 14 h / d.

[0043] (4) Rooting culture: insert the single apical bud with a height of ≥1 cm obtained in step (3) vertically into the rooting induction medium to obtain tissue culture bottle seedlings. Note that when inserting the culture medium vertically, ensure that the lowest stem node is 0.5 cm above the culture medium. The rooting induction medium is 1 / 2 modified WPM+NAA 0.2 mg / L+IAA 3 mg / L+IBA 3 mg / L+agar 3.7 g / L+sugar 15 g / L, pH=5.8~6.2, and the culture conditions are temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light duration 12 h / d~14 h / d.

[0044] (5) Hardening and transplanting: The rooted tissue culture seedlings in step (4) were moved to an outdoor greenhouse with a shading rate of 60% and hardened for 1 day. The seedlings were then opened and hardened for 2 days. The seedlings were then transplanted into a matrix of red soil, sand, and vermiculite with a volume ratio of 5:3:2. Before transplanting, the transplanting matrix was first sterilized by drenching it with a potassium permanganate solution with a volume concentration of 0.1%, and then washed away with clean water. After transplanting, the seedlings were placed in an outdoor nursery with a shade net. Within one week after transplanting, the shading rate was 50%. After one week, the light was gradually increased. After one month, the shading rate was maintained at 10%. The water content of the matrix was maintained at 60%. The seedlings were sprayed with a 0.3% compound fertilizer (N:P:K=15:15:15) aqueous solution once a month to obtain Yunnan tung seedlings.

[0045] Example 3: A tissue culture propagation method for the endangered plant Tung oil tree, the main operating steps are as follows: (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 10 minutes, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 20 minutes, rinse with pure water 5 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 30 seconds, rinse with sterile water 5 times, then put them into 2% sodium hypochlorite solution and stir and soak for 10 minutes, rinse with sterile water 5 times, and place the seeds on sterilized filter paper to absorb the moisture.

[0046] (2) Germination induction culture: The sterilized seeds were placed flat in a germination induction medium for germination induction. The germination induction medium was modified WPM + GA3 0.4 mg / L + NAA 0.2 mg / L + agar 3.7 g / L + sugar 30 g / L, pH = 5.8~6.2, and the culture conditions were temperature 25±3 ℃, light intensity 2000 Lx~8000 Lx, and light time 12 h / d~14 h / d.

[0047] (3) Subculture and proliferation culture: After the seeds germinate and grow into seedlings, cut the apical bud and the position below the hypocotyl and insert them into the subculture and proliferation culture medium. When cutting the apical bud, be careful to keep all leaves and petioles untrimmed, and keep the height at 4 cm. When cutting the position below the hypocotyl, cut off the fibrous roots, and keep the height at 5 cm. After 23 days of proliferation culture, clusters of buds are formed. When subculture and proliferation are carried out again, the clusters of buds are cut into small clusters with 2 to 3 buds, and then transferred to the subculture and proliferation culture medium for continued subculture and proliferation culture; the subculture and proliferation culture medium is modified WPM+2-ip 0.8 mg / L+NAA 0.3 mg / L+IAA 0.3 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.2, and the culture conditions are temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light time 12 h / d~14 h / d.

[0048] (4) Rooting culture: insert the single apical bud with a height of ≥1 cm obtained in step (3) vertically into the rooting induction medium to obtain tissue culture bottle seedlings. Note that when vertically inserting into the culture medium, ensure that the lowest stem node is 0.1 cm above the culture medium. The rooting induction medium is 1 / 2 modified WPM + NAA 0.1 mg / L + IAA 1 mg / L + IBA 1 mg / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8~6.2, and the culture conditions are temperature 25±3℃, light intensity 2000 Lx~8000 Lx, and light duration 12 h / d~14 h / d.

[0049] (5) Hardening and transplanting: The rooted tissue culture seedlings in step (4) were moved to an outdoor greenhouse with a shading rate of 80% and hardened for 2 days. The seedlings were then opened and hardened for 5 days. The seedlings were then transplanted into a matrix of red soil, sand, and vermiculite with a volume ratio of 5:3:2. Before transplanting, the transplanting matrix was first sterilized by drenching it with a potassium permanganate solution with a volume concentration of 0.1%, and then washed away with clean water. After transplanting, the seedlings were placed in an outdoor nursery with a shade net. Within one week after transplanting, the shading rate was 60%. After one week, the light was gradually increased. After one month, the shading rate was maintained at 20%. The water content of the matrix was maintained at 80%. The seedlings were sprayed with a 0.5% compound fertilizer (N:P:K=15:15:15) aqueous solution once a month to obtain Yunnan tung seedlings.

[0050] Comparative Example 1: A tissue culture propagation method for Tung oil tree is provided, primarily comparing and illustrating the advantages of the present invention over the prior art. This method is essentially the same as Example 1, except that the optimal formulations for each tissue culture stage are disclosed in "CN 110663552 B: A tissue culture rapid propagation method for Tung oil tree," namely, the germination medium is formulated as MS + 1.0 mg / L 6-BA + 0.5 mg / L IAA, the secondary proliferation medium is formulated as MS + 0.8 mg / L 6-BA + 0.5 mg / L IAA, and the rooting induction medium is formulated as MS + 0.8 mg / L IAA + 0.8 mg / L IBA. The transplanting medium is a mixture of perlite, humus, and raw laterite in a volume ratio of 1:2:3.

[0051] Comparative Example 2: A tissue culture propagation method for Tung oil tree mainly compares and illustrates the advantageous effects of the present invention on the improvement of the basic culture medium. It is basically the same as Example 1, except that the basic culture medium in the formulas of the bud induction medium, the subculture proliferation medium and the root induction medium adopts WPM, that is, the bud induction medium is WPM+GA3 0.3 mg / L+NAA 0.2 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.2; the subculture proliferation medium is WPM+2-ip 0.6 mg / L+NAA 0.25 mg / L+IAA 0.25 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.2; the root induction medium is 1 / 2 WPM+NAA 0.1 mg / L+IAA 1 mg / L+IBA 3 mg / L+agar 3.7 g / L+sugar 15 g / L, pH=5.8~6.2.

[0052] Comparative Example 3: A tissue culture propagation method for Tung oil plants is provided, primarily comparing and illustrating the advantageous effects of halving the basal culture medium during the rooting stage. The method is substantially the same as Example 1, except that the basal culture medium during the rooting stage is modified WPM, without halving the basal culture medium. Specifically, the rooting induction medium comprises modified WPM, 0.1 mg / L NAA, 1 mg / L IAA, 3 mg / L IBA, 3.7 g / L agar, and 15 g / L sugar, with a pH of 5.8-6.2.

[0053] Tissue culture results of each case The treatment methods of Examples 1 to 3 of the present invention and Comparative Examples 1 to 3 were respectively used for treatment, and the statistical test results were as follows: contamination rate / %=(number of contaminated seeds / total number of inoculated seeds)×100%; germination rate / %=(number of germinated seeds / total number of inoculated seeds)×100%; proliferation coefficient=total number of effective buds grown for cutting / total number of single buds at inoculation; rooting rate / %=(number of rooted buds / total number of inoculated buds)×100%; and transplant survival rate / %=(number of surviving seedlings / total number of transplanted seedlings)×100%.

[0054] Table 1 Comparison of the effects of each stage of tissue culture

[0055] As shown in Table 1, there is no significant difference in the state of the buds of the three examples. They are growing vigorously, with stretched leaves, dark green, and strong root systems. However, there are relatively large differences in the seedling raising period and proliferation coefficient. The shortest germination time of Example 1 is 20 days, the shortest proliferation period is 20 days, and the shortest rooting time is 22 days, which are shorter than those of Example 2 and Example 3. At the same time, Example 1 has the lowest contamination rate, and the germination rate, proliferation coefficient, rooting rate, and transplant survival rate are all higher than those of Example 2 and Example 3. Therefore, compared with Example 2 and Example 3, Example 1 has the best effect in each stage of tissue culture and is the optimal example. The effect is as follows: Figures 1 to 7 shown.

[0056] Table 2 Comparison of the effects of tissue culture at each stage between Example 1 and Comparative Example 1

[0057] As shown in Table 2, the results of each stage of tissue culture in Example 1 are better than those in Comparative Example 1, which are most evident in germination rate, seedling period, proliferation coefficient and seedling state. The seedling proliferation and rooting state of Comparative Example 1 are shown in Table 2. Figures 8 and 9. In the initial germination stage, the germination rate of Comparative Example 1 was only 76.8%, which was significantly lower than 96.3% of Example 1. The germination rate was reduced by 19.5%, which was also lower than the effect of the disclosed technology. This may be because the induced differentiation co-culture medium of the disclosed technology is mainly suitable for young and tender tissues of apical buds or lateral buds, and is not suitable for seeds. The culture medium formula optimized for seed screening of the present invention can meet the mineral element nutrition and suitable hormone content required for seed germination, promote seed germination, and significantly improve the germination rate. In the secondary proliferation culture stage, the highest value of Comparative Example 1 was only 4.7, which was significantly lower than 8.6 of Example 1, and the proliferation coefficient was reduced by 3.9. The proliferation coefficient of the present invention was nearly 1 times higher than that of the prior art. The proliferation cycle of Comparative Example 1 was 60 days, which was 40 days longer than that of Example 1, and the proliferation cycle was shortened by 3 times, which is equivalent to Example 1 being able to proliferate 3 rounds, while Comparative Example 1 only completed 1 round of proliferation, indicating that the proliferation effect was increased by 4 times within the same time, and the seedling state was poor, the leaves were larger and yellower, and the state was worse than that of Example 1. During the rooting induction culture stage, the rooting rate of Comparative Example 1 was 90.8%, a 5.9% decrease from 96.7% in Example 1. The rooting time in Comparative Example 1 was 30 days, an 8-day increase from 22 days in Example 1. The roots were thinner and fewer in number. During the transplanting stage, the transplant survival rate of Comparative Example 1 was 89.5%, a 6.3% decrease from 95.8% in Example 1. The above analysis demonstrates that the present invention significantly outperforms existing technologies, demonstrating that the culture medium formulations optimized through extensive testing at each stage are suitable for the growth of Tung oil trees, allowing for the production of large quantities of robust, consistent seedlings in a short period of time.

[0058] Table 3 Comparison of the effects of each stage of tissue culture between Example 1 and Comparative Example 2

[0059] As shown in Table 3, in the initial germination stage, the germination rate of Comparative Example 2 was only 92.1%, which was significantly lower than 96.3% of Example 1, and the germination rate decreased by 4.2%. The shortest germination time of Comparative Example 2 was 26 days, which was 6 days longer than the 20 days of Example 1. In the secondary proliferation culture stage, the highest germination rate of Comparative Example 2 was only 5.9, which was 8.6 of Example 1, and the proliferation coefficient decreased by 2.7. The proliferation period of Comparative Example 2 was 30 days, which was 10 days longer than the 20 days of Example 1. In the rooting induction culture stage, the rooting rate of Comparative Example 2 was 92.3%, which was 4.4% lower than 96.7% of Example 1; the shortest rooting time of Comparative Example 2 was 30 days, which was 8 days longer than the 22 days of Example 1. The most prominent performance was in the state of the seedlings. The proliferation and rooting state of Comparative Example 2 were as shown in FIG. Figure 10-11In Comparative Example 2, the base callus was larger, the leaves were yellowish, the leaf tips appeared dead, and leaf drop was observed. The root system was short and small in number, mostly consisting of a single root. In summary, the results of tissue culture in Example 1 at each stage were significantly better than those in Comparative Example 2, indicating that the improved basic culture medium of the present invention is more in line with the growth requirements of Tung oil tree, achieving significant advantages.

[0060] Table 4 Comparison of rooting culture effects between Example 1 and Comparative Example 3

[0061] As shown in Table 4, the rooting rate of Comparative Example 3 is only 80.4%, which is 16.3% lower than the 96.7% of Example 1. The shortest rooting time of Comparative Example 3 is 32 days, which is 10 days longer than the 22 days of Example 1. Figure 12 , It was found that the roots of Comparative Example 3 were fewer in number and relatively short and thick, and the rooting culture effect of Example 1 was significantly better than that of Comparative Example 3, indicating that the method of the present invention is more conducive to rooting by reducing the basic culture medium by half after improvement.

[0062] Based on the above analysis, it can be shown that the culture medium formula and culture method at each stage obtained by the present invention through a large number of experimental screening are more suitable for the growth needs of Tung oil tree, greatly shorten the seedling raising period, and significantly improve the proliferation coefficient, rooting rate and transplant survival rate. It can achieve the propagation of more Tung oil tree seedlings with consistent quality at a faster speed, and can provide sufficient seedling guarantee for further research on the wild return, ex situ protection and development and utilization of Tung oil tree. It is of great significance for effectively expanding the population of Tung oil tree, realizing the scientific protection of germplasm resources and even the rational development and utilization.

Claims

1. A tissue culture propagation method for the endangered plant Tung oil palm, characterized by: The method comprises the steps of seed collection and processing, germination induction culture, subculture proliferation culture, rooting culture and seedling hardening and transplanting; selecting mature, complete and insect-free Tung oil fruits, washing them, peeling them and taking seeds; sterilizing the seeds, inoculating them in a germination induction medium for germination induction, shearing them and then inoculating them in a subculture proliferation medium for culture to form cluster buds; shearing the terminal buds and inserting them into a rooting induction medium for rooting induction to obtain tissue culture bottle seedlings; and hardening and transplanting them to obtain Tung oil seedlings. The main operation steps are as follows: (1) Seed collection and processing: Collect mature, intact, and insect-free Tung fruits, stir and wash them with laundry detergent containing Australian tea tree essential oil, peel the peel and take out the seeds, put them into a beaker, cover the beaker with gauze and rinse with running water for 5 min to 10 min, then pour 2% volume concentration of Sanisol disinfectant into the beaker and soak for 10 min to 20 min, rinse with pure water 3 to 5 times, put the seeds into 75% ethanol solution on a clean bench and stir and soak for 20 s to 30 s, rinse with sterile water 3 to 5 times, then put them into 2% sodium hypochlorite solution and stir and soak for 8 min to 10 min, rinse with sterile water 3 to 5 times, and place the seeds on sterilized filter paper to absorb the moisture; (2) Germination induction culture: Place the sterilized seeds flat on the germination induction medium for germination induction; (3) Subculture and proliferation: After the seeds germinate and grow into seedlings, cut the apical buds and the area below the hypocotyl and insert them into the subculture and proliferation medium. After 20 to 25 days of proliferation and proliferation, clusters of buds will form. When subculture and proliferation are repeated, the clusters of buds will be cut and transferred to the subculture and proliferation medium for further subculture and proliferation. (4) Rooting culture: insert the single apical bud with a height of ≥1 cm obtained in step (3) vertically into the rooting induction medium to obtain the tissue culture bottle seedling. When inserting the bud vertically into the culture medium, ensure that the lowest stem node is 0.1 cm to 0.5 cm above the culture medium; (5) Hardening and transplanting: The rooted tissue culture seedlings cultured in step (4) are moved to an outdoor environment with a shade shed for hardening and then transplanted into a transplanting medium. Conventional seedling management is performed to obtain the Yunnan Paulownia seedlings.

2. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: The bud induction medium described in step (2) is modified WPM+GA3 0.2 mg / L~0.4 mg / L+NAA 0.1 mg / L~0.2 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.

2.

3. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, wherein: The subculture proliferation medium described in step (3) is modified WPM+2-ip 0.6 mg / L~0.8 mg / L+NAA 0.2 mg / L~0.3 mg / L+IAA0.2 mg / L~0.3 mg / L+agar 3.7 g / L+sugar 30 g / L, pH=5.8~6.

2.

4. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: When cutting the terminal buds in step (3), be careful to keep all leaves and petioles untrimmed, keeping the height at 2 cm to 4 cm. When cutting below the hypocotyl, remove the fibrous roots, keeping the height at 3 cm to 5 cm. When subcultured again, the method of dividing the bud cluster is to cut the large cluster into small clusters with 2 to 3 buds.

5. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: The rooting induction medium described in step (4) is 1 / 2 modified WPM + NAA 0.1 mg / L~0.2 mg / L + IAA 1 mg / L~3 mg / L + IBA 1 mg / L~3 mg / L + agar 3.7 g / L + sugar 15 g / L, pH = 5.8~6.

2.

6. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: The culture temperature and light control conditions in step (2), step (3), and step (4) are: temperature 25±3°C, light intensity 2000 Lx to 8000 Lx, and light duration 12 h / d to 14 h / d; the composition of the modified WPM and 1 / 2 modified WPM culture medium is: 。 7. The method for tissue culture propagation of the endangered plant Tungia yunnanensis according to claim 1, characterized in that: The seedling hardening method described in step (5) is to harden the seedlings for 1 to 2 days with the bottle cap closed in a greenhouse with a shading rate of 60% to 80%, and then harden the seedlings for 2 to 5 days with the bottle cap opened.

8. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: The transplanting matrix described in step (5) is red soil, sand, and vermiculite in a volume ratio of 5:3:

2. Before transplanting and raising seedlings, the transplanting matrix is ​​first sterilized by pouring a potassium permanganate solution with a volume concentration of 0.1%, and then rinsed with clean water to remove the liquid.

9. The method for tissue culture propagation of the endangered plant Tung oil palm according to claim 1, characterized in that: The conventional seedling management described in step (5) is as follows: the seedlings are placed in an outdoor nursery with a shade net. Within one week after transplanting, the shading rate is 50% to 60%. After one week, the light intensity is gradually increased. After one month, the shading rate is maintained at 10% to 20%. The water content of the substrate is maintained at 60% to 80%. The seedlings are sprayed once or twice a month with a 0.3% to 0.5% mass concentration compound fertilizer (N:P:K=15:15:15) aqueous solution.

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