A method for regenerating seedlings from mature somatic embryos of rubber trees in vitro

Abstract

This invention provides a method for the germination and regeneration of mature rubber tree embryos into seedlings outside of test tubes, belonging to the field of seedling cultivation technology. The method uses somatic embryos derived from anther tissue culture as raw materials, and involves three stages of cultivation in a growth medium: dark culture, low-light culture, and strong-light culture. Then, the mature embryos that meet the requirements undergo closed-bottle / open-bottle acclimatization. After this acclimatization, they can be rooted and grown into seedlings in river sand outside the test tube. This method allows mature embryos to germinate and regenerate directly into seedlings outside the test tube, eliminating the need for a sterile culture room and specialized rooting medium, thus requiring less specialized knowledge from the operator. Furthermore, by combining the embryo germination and seedling regeneration with the in-test-tube and sand-bed acclimatization in one step, the cultivation process is simplified, the cultivation cycle is shortened by two months, and cultivation efficiency is improved while production costs are reduced.

Description

Technical Field

[0001] This invention belongs to the field of seedling cultivation technology, specifically relating to a method for the germination and regeneration of mature rubber tree embryos into seedlings outside a test tube. Background Technology

[0002] Natural rubber, as an important strategic material, plays an irreplaceable role in the production of high-end products such as aircraft tires, heavy-duty truck tires, mining machinery tires, and off-road tires.

[0003] The rubber tree, the only commercially available source of natural rubber ( Hevea brasiliensis Müll. is native to the tropical rainforests of the Amazon River basin in Brazil. It has strict requirements for growth conditions, and in my country, only some tropical areas in Yunnan, Hainan and Guangdong provinces have suitable conditions for rubber planting.

[0004] Currently, a core aspect of increasing domestic natural rubber production capacity lies in the coordinated development of superior varieties and high-quality seedlings. Superior varieties and seedlings directly determine the growth, resilience, and latex production efficiency of rubber trees throughout their entire life cycle, becoming the core guarantee for high and stable yields in rubber plantations. Somatic embryonic seedlings (also known as self-rooted clonal lines or somatic embryonic plants), with their significant advantages such as rapid growth and high yield, are hailed as a new generation of planting material. Under the premise of unchanged planting area and no higher-yielding varieties available, replacing traditional budded seedlings with somatic embryonic seedlings to upgrade old, low-yield, and low-quality rubber plantations is an effective measure to improve plantation production capacity. This approach can significantly increase yield per unit area, thereby achieving the dual goals of increasing rubber farmers' income and ensuring high and stable yields in rubber plantations.

[0005] The conventional process of propagating rubber tree seedlings from somatic embryos includes several steps, such as callus induction, somatic embryo induction and maturation culture, somatic embryo germination and regeneration into seedlings, and acclimatization and transplanting. Among these, somatic embryo germination and regeneration into seedlings and acclimatization and transplanting are key steps in achieving large-scale application of somatic embryo seedlings. According to existing reports, somatic embryo germination and regeneration into seedlings is usually carried out in sterile test tubes, where mature somatic embryos are inoculated into test tubes containing culture medium and cultured for 60-70 days to obtain seedlings. This process not only requires a sterile culture room with artificially controlled temperature and light, but the resulting seedlings are also tender and have weak resistance. They need to undergo acclimatization and hardening in test tubes (7-10 days), acclimatization and hardening in sand beds, and then a second cultivation in soil bags (≥4 months) before they can be planted in the field.

[0006] In summary, the process of germinating and regenerating seedlings from mature rubber tree embryos in test tubes must be carried out in a sterile and controlled environment, requiring production equipment such as air conditioners, dehumidifiers, and fluorescent lamps, which results in high production costs. Furthermore, the nutrients required for embryo germination and seedling regeneration are provided by a sterile, specialized rooting medium, the preparation and sterilization of which require skilled technicians. Moreover, the obtained seedlings must undergo acclimatization and hardening processes, including closed-tube and open-tube acclimatization, sand bed weaning, and hardening, before being transplanted into soil bags for secondary cultivation. Both the sand bed acclimatization and hardening and the secondary cultivation in soil bags result in seedling mortality at both stages, leading to a loss rate exceeding 40%, and both transplanting processes have a recovery period, resulting in a long overall cultivation cycle (≥8 months). Summary of the Invention

[0007] To simplify the cultivation process, shorten the cultivation cycle, and reduce costs while increasing efficiency, this invention provides a method for the germination and regeneration of mature rubber tree embryos into seedlings outside a test tube. This method reduces operational requirements, simplifies the cultivation process, improves cultivation efficiency, and lowers cultivation costs.

[0008] This invention provides a method for the in vitro germination of mature somatic embryos of rubber trees into seedlings, comprising the following steps:

[0009] (1) Normal somatic embryos obtained from rubber tree anther tissue culture were inoculated into growth medium and cultured in the dark, under weak light, and under strong light in sequence to obtain strong somatic embryos with well-developed radicles and plumules. The growth medium was based on MS medium and also included the following components at the following concentrations: 6-BA 1.2~1.5 mg / L, NAA 0.2~0.4 mg / L, ABA 0.3~0.6 mg / L, GA3 0.6~0.8 mg / L, KT 0.8~1.0 mg / L, agar 6 g / L, sucrose 70 g / L and activated carbon 1.2~1.5 g / L. The light intensity during the under weak light culture was 500~800 lx and the light intensity during the strong light culture was 1500~2000 lx.

[0010] (2) The strong embryos described in step (1) are transferred together with the culture medium to an outdoor greenhouse for acclimatization training by closing and opening bottles to obtain acclimatized embryos.

[0011] (3) After taking out the domesticated embryo described in step (2), rinse and disinfect it. Then, soak the radicle in 50-80 mg / L IBA solution for 2-6 seconds and transplant it into a seedling container for in vitro germination and regeneration into seedling culture. The seedling container is filled with sterilized clean river sand.

[0012] In a preferred embodiment of the present invention, the temperature of the dark culture in step (1) is 24.5~26.5 ℃, and the dark culture time is 20~25 days.

[0013] In a preferred embodiment of the present invention, in step (1), after the dark culture is completed, embryos with a length of 1.0 to 1.5 cm and a cotyledon width of 0.3 to 0.6 cm are selected for low-light culture. The temperature of the low-light culture is 26.5 to 28.5 ℃ and the time of the low-light culture is 7 to 10 days.

[0014] In a preferred embodiment of the present invention, the temperature of the strong light culture in step (1) is 26.5~28.5 ℃, and the strong light culture time is 6~8 days.

[0015] In a preferred embodiment of the present invention, the acclimatization training time in step (2) is 5 days, including 3 days of closed bottle and 2 days of open bottle.

[0016] In a preferred embodiment of the present invention, in step (3), a domesticated embryo with a radicle length of 0.5 to 1.2 cm and whose bud has begun to sprout is taken out.

[0017] In a preferred embodiment of the present invention, when cultivating seedlings by germination and regeneration outside the test tube in step (3), nutrient solution is sprayed into the seedling container and then sealed with a lid or gauze with ventilation holes, and a shade net is covered above the seedling container.

[0018] The culture medium is based on 3 / 4MS medium and also includes the following components at the following concentrations: IAA 0.6~0.8 mg / L, KT 0.5~1.0 mg / L, VB2 0.8~1.0 mg / L and GA3 1.5~2.0 mg / L.

[0019] In a preferred embodiment of the present invention, the culture medium is sprayed once every 7 days.

[0020] In a preferred embodiment of the present invention, the shading net has a shading rate of 80-90%. On the 25th to 28th day of cultivation, the shading net is replaced with a shading net with a shading rate of 50-60%. On the 50th day of cultivation, the shading net is removed.

[0021] The present invention also provides a method for cultivating rubber tree seedlings, which includes planting the seedlings obtained by the above method into soil bags for a second cultivation period of 2-3 months after removing the shade net one week later, to obtain rubber tree seedlings that can be planted in the field.

[0022] Beneficial Effects: This invention provides a method for the in vitro germination of mature somatic embryos of rubber trees into seedlings. Using somatic embryos obtained from anther tissue culture as raw materials, the method involves three stages of culture in a growth medium: dark culture, low-light culture, and strong-light culture. The maturation of the somatic embryo, the development of the radicle, and the plumule are completed on a single culture medium. Then, the qualified somatic embryos undergo closed-bottle / open-bottle acclimatization. After this acclimatization, they can be rooted and grown into seedlings in river sand outside the test tube. The method of this invention allows mature somatic embryos to germinate and regenerate into seedlings directly outside the test tube, eliminating the need for a sterile culture room and a special rooting medium, and requiring less specialized knowledge from the operator. Furthermore, by combining the germination and regeneration of somatic embryos with the in vitro and sand-bed acclimatization in one step, the cultivation process is simplified, shortening the cultivation cycle by 2 months, increasing cultivation efficiency by 2.5-4.0%, and reducing production costs by 2-3 yuan per plant. Attached Figure Description

[0023] Figure 1 These are field photos of the various stages of the method for regenerating seedlings from mature somatic embryos in vitro according to the present invention. In the figures, A represents a mature cotyledon-shaped somatic embryo; B represents a low-light somatic embryo cultured under low light conditions; C represents a high-light somatic embryo cultured under strong light conditions; D represents a somatic embryo undergoing acclimatization and training; E represents a robust acclimatized somatic embryo; F represents a somatic embryo germinating and regenerating into a seedling in vitro; G represents a somatic embryo resuming growth and beginning to sprout new buds; H represents a somatic embryo sprouting stems and growing new leaves; I represents a somatic embryo seedling regenerated from in vitro germination; J represents a somatic embryo seedling regenerated from in vitro germination after being implanted in a soil bag for a second cultivation period of 35 days; and K represents a somatic embryo seedling regenerated from in vitro germination that has reached the standards for field planting. Detailed Implementation

[0024] This invention provides a method for the in vitro germination of mature somatic embryos of rubber trees into seedlings, comprising the following steps:

[0025] (1) Normal somatic embryos obtained from rubber tree anther tissue culture were inoculated into growth medium and cultured in the dark, under weak light, and under strong light in sequence to obtain strong somatic embryos with well-developed radicles and plumules. The growth medium was based on MS medium and also included the following components at the following concentrations: 6-BA 1.2~1.5 mg / L, NAA 0.2~0.4 mg / L, ABA 0.3~0.6 mg / L, GA3 0.6~0.8 mg / L, KT 0.8~1.0 mg / L, agar 6 g / L, sucrose 70 g / L and activated carbon 1.2~1.5 g / L. The light intensity during the under weak light culture was 500~800 lx and the light intensity during the strong light culture was 1500~2000 lx.

[0026] (2) The strong embryos described in step (1) are transferred together with the culture medium to an outdoor greenhouse for acclimatization training by closing and opening bottles to obtain acclimatized embryos.

[0027] (3) After taking out the domesticated embryo described in step (2), rinse and disinfect it. Then, soak the radicle in 50-80 mg / L IBA solution for 2-6 seconds and transplant it into a seedling container for in vitro germination and regeneration into seedling culture. The seedling container is filled with sterilized clean river sand.

[0028] This invention uses normal rubber tree embryos as raw materials for seedling propagation. The embryos are cotyledon-shaped immature somatic embryos obtained from anther tissue culture. The embryos are inoculated onto a growth medium for dark culture. The growth medium is based on MS medium and includes 6-BA at a concentration of 1.2–1.5 mg / L, such as any concentration from 1.2 mg / L, 1.3 mg / L, 1.4 mg / L, and 1.5 mg / L, or any concentration within a range of two such concentrations. The synergistic effect of 6-BA and auxin NAA in this invention promotes normal development of somatic embryos.

[0029] The growth medium of this invention also includes NAA, wherein the concentration of NAA is 0.2~0.4 mg / L, such as any concentration selected from 0.2 mg / L, 0.3 mg / L, and 0.4 mg / L, or any concentration within a range of two such concentrations. The NAA of this invention, in synergistic effect with cytokinin 6-BA and KT, can promote the maturation of somatic embryos.

[0030] The growth medium of this invention also includes ABA, wherein the concentration of ABA is 0.3~0.6 mg / L, such as any concentration selected from 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, and 0.6 mg / L, or any concentration within a range of any two concentrations. The ABA of this invention is beneficial to the normal development of immature embryos.

[0031] The growth medium of this invention also includes GA3, wherein the concentration of GA3 is 0.6~0.8 mg / L, such as any concentration selected from 0.6 mg / L, 0.7 mg / L, and 0.8 mg / L, or any concentration within a range of any two concentrations. The GA3 of this invention promotes plumule germination and stem elongation. 。

[0032] The growth medium of this invention also includes KT, the concentration of which is 0.8~1.0 mg / L, such as any concentration selected from 0.8 mg / L, 0.9 mg / L, and 1.0 mg / L, or any concentration within a range of two such concentrations. The KT of this invention synergistically promotes somatic embryo maturation with auxin NAA.

[0033] The growth medium of this invention also includes activated carbon, the concentration of which is 1.2~1.5 g / L, such as any concentration from 1.2 g / L, 1.3 g / L, 1.4 g / L, and 1.5 g / L, or any concentration within a range of two such concentrations. The activated carbon of this invention adsorbs harmful substances (secondary metabolites such as phenols and quinones) released by the embryo during embryonic development, promoting embryonic maturation and root formation.

[0034] This invention adjusts light conditions to gradually enhance the resistance and autotrophic capacity of embryos during three stages: dark culture, low-light culture, and high-light culture. The total culture time of this invention is comparable to the conventional embryo maturation culture cycle, and the initial added hormones and nutrients are sufficient to meet the nutritional needs of the embryos throughout the maturation process, eliminating the need to supplement or replace the culture medium during cultivation. In this invention, the low-light and high-light culture stages aim to induce radicle germination in the somatic embryo and promote cotyledon thickening and greening, thus laying a solid foundation for subsequent in vitro germination and seedling regeneration. Therefore, by adjusting light conditions without changing the culture container or culture medium, the cultivation objective can be achieved, significantly reducing operating costs.

[0035] The dark culture temperature described in this invention is 24.5~26.5℃, which can be any temperature or a range of any two of the following: 24.5℃, 25℃, 25.5℃, 26℃, and 26.5℃. The dark culture time is 20~25 days, which can be any two of the following: 20 days, 21 days, 22 days, 23 days, 24 days, and 25 days. The dark culture described in this invention is beneficial for the maturation of the somatic embryo and the development of the radicle.

[0036] After the dark culture is completed, embryos with a length of 1.0-1.5 cm and a cotyledon width of 0.3-0.6 cm are selected for low-light culture. In one embodiment, the qualified normal embryos, along with the culture medium, are transferred to a low-light culture chamber for the low-light culture to obtain normal embryos with thickened cotyledons and a light green color. The low-light culture temperature is 26.5-28.5 ℃, which can be any temperature or a range of any two of 26.5 ℃, 27 ℃, 27.5 ℃, 28 ℃, and 28.5 ℃. The dark culture time is 7-10 days, which can be any time of 7 days, 8 days, 9 days, and 10 days, or a range of any two of 10 days.

[0037] After the initial low-light culture, the embryos are transferred to a high-light culture chamber for further high-light culture to obtain robust, normal embryos with well-developed radicles and plumules that are green in color. The high-light culture temperature is 26.5–28.5 °C, which can be any temperature from 26.5 °C, 27 °C, 27.5 °C, 28 °C, and 28.5 °C, or any temperature range between two such temperatures. The dark culture time is 6–8 days, which can be any time from 6 days, 7 days, and 8 days, or any time range between two such times.

[0038] After the strong light culture, the robust embryos are transferred along with the culture medium to a simple outdoor greenhouse for closed-bottle and open-bottle acclimatization training. The acclimatization training time is 5 days, including 3 days of closed-bottle training and 2 days of open-bottle training, to obtain acclimatized embryos.

[0039] In this invention, domesticated embryos with radicle lengths of 0.5-1.2 cm and whose plumules have begun to sprout are removed from the culture bottle. After washing the culture medium with tap water, they are disinfected by soaking in 400-500 times diluted carbendazim or chlorothalonil for 1-2 minutes. The radicles of the treated domesticated embryos are then soaked in 50-80 mg / L IBA solution for 2-6 seconds before being transplanted into a seedling container containing clean river sand with a particle size of 0.8-1.5 mm for in vitro germination and regeneration into seedlings. The clean river sand has been thoroughly disinfected before use, for example, by spraying with 500-700 times diluted carbendazim or chlorothalonil solution in the example.

[0040] After transplanting, the present invention sprays a nutrient solution (pH 5.8~6.2), which is based on 3 / 4MS medium and also includes the following components at the following concentrations: IAA 0.6~0.8 mg / L, KT 0.5~1.0 mg / L, VB2 0.8~1.0 mg / L and GA3 1.5~2.0 mg / L. Then, the container is sealed with a cap or gauze with ventilation holes, and finally, 80~90% shade netting is placed over the seedling container.

[0041] During this stage, the plants are cultivated in a simple greenhouse at 28-34℃ (daytime) and 25-30℃ (nighttime). Watering is done as needed to keep the river sand moist, and the relative humidity is maintained at 80-90%. The nutrient solution described above is sprayed every 7 days. After 10-15 days of cultivation, the embryos resume growth, new roots begin to emerge from the embryonic root tips, and the plumules elongate and shoot. On days 25-28, the plants are replaced with shade nets providing 50-60% shade. Cultivation continues for 40-50 days to obtain embryo seedlings (plant height ≥3cm, leaves ≥3). On day 50, the shade nets are removed, and after one week, the seedlings are transplanted into soil bags for a second cultivation, yielding embryo seedlings ready for field planting.

[0042] The present invention also provides a method for cultivating rubber tree seedlings, which includes planting the seedlings obtained by the above method into soil bags one week after removing the shade net, to obtain rubber tree seedlings suitable for field planting.

[0043] The cultivation method described in this invention, especially after planting in soil bags, adopts conventional management: when the seedlings begin to sprout new leaves, spray with 0.1% potassium dihydrogen phosphate. During this period, water on time as needed to keep the substrate moist. Spray with compound fertilizer (N:K:P=15:15:15) every 15-20 days. After 2-3 months, the standard for transplanting can be reached (plant height ≥40 cm, stem diameter ≥4.00 mm).

[0044] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a method for the germination and regeneration of mature rubber tree embryos into seedlings outside a test tube, should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] The method for germinating mature rubber tree embryos into seedlings outside of a test tube is as follows:

[0047] (1) The cotyledon-shaped embryos obtained from the anther tissue culture of the superior rubber tree variety Yunyan 73-477 were inoculated in MS medium supplemented with 1.2 mg / L 6-BA, 0.2 mg / L NAA, 0.3 mg / L ABA, 0.6 mg / L GA3, 0.8 mg / L KT, 6 g / L agar, 70 g / L sucrose and 1.2 g / L activated carbon and cultured in the dark for 25 days at a temperature of 24.5~26.5 ℃.

[0048] (2) Select well-developed, normal-shaped embryos with an embryo body length of 1.0–1.5 cm and a cotyledon width of 0.3–0.6 cm (e.g., Figure 1 (As shown in Figure A) Transferred along with the culture medium into a low-light culture chamber and incubated for 9 days (as shown in Figure A). Figure 1 As shown in Figure B), the cultivation temperature was 26.5~28.5 ℃, the light intensity was 600 lx, and normal embryos with thickened cotyledons and light green color were obtained.

[0049] (3) Then transfer it to a high-intensity light incubation chamber for 6 days of incubation (e.g. Figure 1 As shown in Figure C), the cultivation temperature was 26.5~28.5 ℃, and the light intensity was 1600 lx, to obtain strong, normal somatic embryos with well-developed radicles and plumules that were green in color (as shown in Figure C). Figure 1 (as shown in D).

[0050] (4) Transfer the strong light embryos along with the culture medium to a simple outdoor greenhouse for closed / open bottle acclimatization training for 3 / 2 days to obtain acclimatized embryos;

[0051] (5) Take out the domesticated embryo with a radicle length of 0.5~1.2 cm and the plumule has begun to sprout from the culture bottle, wash the culture medium with tap water, and then soak it in 400 times carbendazim or chlorothalonil for 2 min for disinfection.

[0052] (6) After soaking the radicles of the domesticated embryos treated in (5) in 80 mg / L IBA solution for 5-6 seconds, transplant them into a seedling container containing clean river sand with a particle size of 0.8-1.5 mm (thoroughly disinfected by spraying with 700 times diluted carbendazim or chlorothalonil solution) for in vitro germination and regeneration of seedlings. After transplanting, spray with 3 / 4MS supplemented with nutrient solution of IAA 0.8 mg / L, KT 0.5 mg / L, VB2 1.0 mg / L and GA3 1.5 mg / L (pH 5.8-6.2), then seal with a lid or gauze with ventilation holes, and finally place a shade net with a shading rate of 80-90% above the seedling container.

[0053] During this stage, the plants are cultivated in a simple greenhouse at 28~34 ℃ (daytime) / 25~30 ℃ (nighttime). During the cultivation period, water is applied according to the actual situation to keep the river sand moist, and the relative humidity of the air is maintained at 80~90%. Nutrient solution is sprayed once every 7 days.

[0054] (7) After 10-15 days of culture, the somatic embryo resumes growth, new roots begin to grow from the tip of the embryonic root, and the plumule elongates and forms a stem, such as Figure 1 As shown in G~H, on day 26 of cultivation, replace the shade net with one offering 50-60% shading, and continue cultivation for 40-50 days to obtain seedlings with somatic embryos (plant height ≥ 3 cm, leaves ≥ 3). Figure 1 As shown in Figure I. On day 50, remove the shade netting, and a week later, plant soil bags for secondary cultivation to obtain seedlings suitable for field planting. Figure 1 As shown in K.

[0055] Routine management: After planting the soil bags, spray with 0.1% potassium dihydrogen phosphate when the seedlings begin to sprout new leaves. During this period, water regularly as needed to keep the substrate moist. Spray with compound fertilizer (N:K:P=15:15:15) every 15-20 days. The plant can reach the standard for transplanting in 2-3 months (plant height ≥40 cm, stem diameter ≥4.00 mm).

[0056] Example 2

[0057] The method for germinating mature rubber tree embryos into seedlings outside of a test tube is as follows:

[0058] (1) The cotyledon-shaped embryos obtained from the anther tissue culture of the superior rubber tree variety 73397 were inoculated in MS medium supplemented with 1.4 mg / L 6-BA, 0.3 mg / L NAA, 0.5 mg / L ABA, 0.8 mg / L GA3, 1.0 mg / L KT, 6 g / L agar, 70 g / L sucrose and 1.3 g / L activated carbon and cultured in the dark for 25 days at a temperature of 24.5~26.5 ℃.

[0059] (2) Select well-developed normal embryos with cotyledon shape, embryo length of 1.2-1.5 cm and cotyledon width of 0.4-0.6 cm, and transfer them along with the culture medium into a low-light culture room for 9 days. The culture temperature is 26.5-28.5 ℃ and the light intensity is 600 lx to obtain normal embryos with thickened cotyledons and light green cotyledon shape.

[0060] (3) Then it is transferred to a high-light culture chamber for 6 days of cultivation at a temperature of 26.5~28.5 ℃ and a light intensity of 1600 lx to obtain a strong and normal cotyledon embryo with well-developed radicle and plumule.

[0061] (4) Transfer the strong light embryos along with the culture medium to a simple outdoor greenhouse for closed / open bottle acclimatization training for 3 / 2 days to obtain acclimatized embryos;

[0062] (5) Take out the domesticated embryo with a radicle length of 0.6~1.0 cm and the embryonic bud has begun to sprout from the culture bottle, wash the culture medium with tap water, and then soak it in 400 times carbendazim or chlorothalonil for 2 min for disinfection.

[0063] (6) After soaking the radicle of the domesticated embryo treated in (5) in 60 mg / L IBA solution for 5-6 seconds, transplant it into a seedling container containing clean river sand with a particle size of 0.8-1.5 mm (thoroughly disinfected by spraying with 700 times carbendazim or chlorothalonil solution). After transplanting, spray with 3 / 4MS nutrient solution supplemented with IAA 0.4 mg / L, KT 0.2 mg / L, VB2 1.0 mg / L and GA3 1.0 mg / L (pH 5.8-6.2), then seal with a lid or gauze with ventilation holes, and finally cover the seedling container with a shade net with a shading rate of 90%.

[0064] (7) Place the seedling containers with implanted embryos in a simple greenhouse at 28~34 ℃ (day) / 25~30 ℃ (night). During the cultivation period, water according to the actual situation to keep the river sand moist and maintain the relative humidity of the air at 80~90%. Spray nutrient solution once every 7 days.

[0065] (8) After 10-15 days of cultivation, new roots begin to grow from the root tip, and the plumule elongates and shoots out. On the 26th day of cultivation, replace the shade net with a shade net with a 50% shading rate and continue cultivation for 40-50 days to obtain strong embryonic seedlings. On the 50th day, remove the shade net, and after one week, plant the seedlings in soil bags for secondary cultivation to obtain embryonic seedlings suitable for field planting.

[0066] Routine management: After planting the soil bags, spray with 0.1% potassium dihydrogen phosphate when the seedlings begin to sprout new leaves. During this period, water regularly as needed to keep the substrate moist. Spray with compound fertilizer (N:K:P=15:15:15) every 15-20 days. The plant can reach the standard for transplanting in 2-3 months (plant height ≥40 cm, stem diameter ≥4.00 mm).

[0067] Comparative Example 1

[0068] (1) The cotyledon-shaped embryos obtained from the anther tissue culture of the superior rubber tree varieties Yunyan 73-477 and Reyan 73397 were inoculated in MS medium supplemented with 1.2 mg / L 6-BA, 0.3 mg / L NAA, 0.3 mg / L ABA, 0.7 mg / L GA3, 1.0 mg / L KT, 6 g / L agar, 70 g / L sucrose and 1.2 g / L activated carbon and cultured in the dark for 50 days at a temperature of 24.5~26.5℃.

[0069] (2) Select well-developed mature somatic embryos with cotyledon shape, embryo length of 1.0-1.5 cm and cotyledon width of 0.3-0.6 cm, and inoculate them in MS medium supplemented with IAA 0.8 mg / L, KT 0.5 mg / L, VB2 1.0 mg / L, GA3 1.5 mg / L, agar 6 g / L, sucrose 50 g / L and activated carbon 1.2 g / L for 40-50 days to obtain somatic embryo seedlings. The culture temperature is 26.5-28.5℃ and the light intensity is 1600 lx.

[0070] (3) Transfer somatic embryos with ≥3 leaves and ≥3 cm in height, along with their test tubes, from the sterile culture room to a sterile environment, and perform closed-tube (5-7 days) and open-tube (2-3 days) acclimatization in sequence. During this period, it is necessary to closely monitor the contamination of the culture medium to ensure that the somatic embryos are removed before complete contamination;

[0071] (4) Remove the somatic embryos that have undergone in vitro acclimatization from the test tubes, wash off the culture medium, and disinfect them by soaking in 400 times diluted carbendazim or chlorothalonil for 2 minutes. Then transplant them to a sand bed containing clean river sand with a particle size of 0.5~1.0 mm (thoroughly disinfected by spraying with 700 times diluted carbendazim or chlorothalonil solution) for acclimatization. After transplanting, spray with 3 / 4MS nutrient solution supplemented with IAA 0.4 mg / L, KT 0.2 mg / L, VB2 1.0 mg / L and GA3 1.0 mg / L (pH 5.8~6.2). Finally, build an arched shed above the sand bed, cover it with a film and shade net (shading rate 80~90%) to retain moisture and provide shade, and spray a sterilizing agent every two weeks to kill fungi and allow the somatic embryos to gradually adapt to the outdoor environment. During this stage, water needs to be sprayed regularly to maintain the temperature at 20~30 ℃ and the humidity at close to 90~100%. After 1.5 to 2 months of cultivation, the survival rate was investigated (number of surviving plants / number of transplanted plants * 100%).

[0072] (5) Plants that have survived transplanting from the sand bed are planted into soil bags for secondary cultivation, and seedlings that meet the standards for field planting can be obtained.

[0073] Routine management: After planting the soil bags, spray with 0.1% potassium dihydrogen phosphate when the seedlings begin to sprout new leaves. During this period, water as needed to keep the substrate moist. Spray with compound fertilizer (N:K:P=15:15:15) every 15-20 days. The plant can reach the standard for transplanting in 3-4 months (plant height ≥40 cm, stem diameter ≥4.00 mm).

[0074] Comparative Example 2

[0075] (1) The cotyledon-shaped embryos obtained from the anther tissue culture of the superior rubber tree varieties Yunyan 73-477 and Reyan 73397 were inoculated in MS medium supplemented with 1.2 mg / L 6-BA, 0.3 mg / L NAA, 0.3 mg / L ABA, 0.7 mg / L GA3, 1.0 mg / L KT, 6 g / L agar, 70 g / L sucrose and 1.2 g / L activated carbon and cultured in the dark for 50-60 days at a temperature of 24.5-26.5 ℃.

[0076] (2) Select well-developed mature embryos with cotyledon shape, 1.0-1.5 cm in length and 0.3-0.6 cm in width, and remove them from the culture bottle. After washing the culture medium with tap water, disinfect them by soaking in 400 times diluted carbendazim or chlorothalonil for 2 minutes.

[0077] (3) After soaking the mature embryos treated in (2) in 60 mg / L IBA solution for 5-6 seconds, transplant them into seedling containers containing clean river sand with a particle size of 0.8-1.5 mm (thoroughly disinfected by spraying with 700 times diluted carbendazim or chlorothalonil solution). After transplanting, spray with 3 / 4MS nutrient solution supplemented with IAA 0.4 mg / L, KT 0.2 mg / L, VB2 1.0 mg / L and GA3 1.0 mg / L (pH 5.8-6.2), then seal with a lid or gauze with ventilation holes, and finally cover the seedling containers with a shade net with a shading rate of 90%.

[0078] (4) Place the seedling containers with implanted embryos in a simple greenhouse at 28~34 ℃ (day) / 25~30 ℃ (night). During the cultivation period, water according to the actual situation to keep the river sand moist and maintain the relative humidity of the air at 80~90%. Spray nutrient solution once every 7 days.

[0079] (5) During the cultivation period, the germination and regeneration of mature embryos outside the test tube were observed every 5 days. According to the germination of embryos, the shading rate of the shade net was gradually reduced. After 40 to 50 days of cultivation, the regeneration rate of embryo plants was counted. The cultivation continued until the obtained embryo seedlings had more than 6 leaves and were taller than 8 cm. The shade net was removed, and after one week, the seedlings were planted in soil bags for secondary cultivation to obtain embryo seedlings that could be planted in the field.

[0080] Statistical analysis was performed on Examples 1, 2, Comparative Example 1, and Comparative Example 2. When compiling the statistical data, the cultivation efficiency was calculated as: embryo regeneration rate × sand bed survival rate × soil bag secondary cultivation survival rate.

[0081] Table 1. Statistical results of the cultivation effects of the examples and comparative examples.

[0082]

[0083] In Table 1, in Comparative Example 2, mature somatic embryos that had not undergone domestication were directly transplanted into test tubes to germinate and regenerate into seedlings. Their tissue structure was still immature, lacking effective resistance to external abiotic stresses (such as changes in moisture and temperature) and biotic stresses (such as microbial infection). Three to five days after transplantation, most of the somatic embryos exhibited contamination or wilting due to adaptation difficulties. Upon continued culture for 8 to 10 days, all somatic embryos died due to contamination or dehydration, ultimately resulting in no seedlings.

[0084] As shown in Table 1, this invention was applied to the cultivation of somatic embryos of two varieties, Yunyan 73-477 and Reyan 73397, respectively. It combines somatic embryo germination and regeneration with acclimatization, simplifying the cultivation process and achieving cost reduction and efficiency improvement. This method can shorten the cultivation cycle by 2 months, and increase cultivation efficiency by 3.75% and 12.08%, respectively.

[0085] A statistical analysis of the costs of cultivating Yunyan 73-477 embryos using the present invention and conventional methods is presented in Table 2. The results show that the production cost of the present invention is 2.15 yuan / plant lower than that of the conventional method.

[0086] Table 2. Production cost statistics of this invention and conventional cultivation methods (Yunnan Research 73-477)

[0087]

[0088] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for germinating mature somatic embryos of rubber trees into seedlings outside a test tube, characterized in that, Includes the following steps: (1) Normal somatic embryos obtained from anther tissue culture of rubber trees are inoculated into growth medium and cultured in the dark, low light and strong light in sequence to obtain strong somatic embryos with well-developed radicles and plumules; the somatic embryos are immature somatic embryos with cotyledon shape obtained from anther tissue culture; after the dark culture, somatic embryos with an embryo length of 1.0-1.5 cm and a cotyledon width of 0.3-0.6 cm are selected for low light culture for 7-10 days; The growth medium consists of MS medium, 6-BA 1.2~1.5 mg / L, NAA 0.2~0.4 mg / L, ABA 0.3~0.6 mg / L, GA3 0.6~0.8 mg / L, KT 0.8~1.0 mg / L, agar 6 g / L, sucrose 70 g / L, and activated carbon 1.2~1.5 g / L; the light intensity during the low-light culture is 500~800 lx, and the light intensity during the high-light culture is 1500~2000 lx. (2) The strong embryos described in step (1) are transferred together with the culture medium to an outdoor greenhouse for acclimatization training by closing and opening bottles to obtain acclimatized embryos. (3) Take out the domesticated embryos with a radicle length of 0.5~1.2 cm and the plumules starting to sprout in step (2), rinse and disinfect them, then soak the radicles in 50~80 mg / L IBA solution for 2~6 s and transplant them into a seedling container for in vitro germination and regeneration seedling culture; the seedling container is filled with sterilized clean river sand; when the in vitro germination and regeneration seedling culture is carried out, spray nutrient solution into the seedling container and seal it with a lid or gauze with ventilation holes, and cover the seedling container with a shade net; The nutrient solution consists of 3 / 4MS medium, IAA 0.6~0.8 mg / L, KT 0.5~1.0 mg / L, VB2 0.8~1.0 mg / L and GA3 1.5~2.0 mg / L.

2. The method according to claim 1, characterized in that, The temperature of the dark culture in step (1) is 24.5~26.5 ℃, and the dark culture time is 20~25 days.

3. The method according to claim 1 or 2, characterized in that, The temperature for the low-light culture in step (1) is 26.5~28.5 ℃.

4. The method according to claim 1, characterized in that, The temperature of the strong light culture in step (1) is 26.5~28.5℃, and the strong light culture time is 6~8 days.

5. The method according to claim 1, characterized in that, The acclimatization training in step (2) lasts for 5 days, including 3 days of closed-bottle training and 2 days of open-bottle training.

6. The method according to claim 1, characterized in that, The nutrient solution is sprayed once every 7 days.

7. The method according to claim 1, characterized in that, The shading net has a shading rate of 80-90%. On the 25th to 28th day of cultivation, the shading net is replaced with a shading net with a shading rate of 50-60%. On the 50th day of cultivation, the shading net is removed.

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