Cultivation method for efficiently obtaining normal somatic embryo of rubber tree clone variety

Abstract

The invention relates to the technical field of rubber tree seedling cultivation, and particularly discloses a cultivation method for efficiently obtaining a normal somatic embryo of a rubber tree clone variety, and the cultivation method comprises the following steps: S1, taking an anther newborn somatic cell immature embryo of the rubber tree clone variety, and picking out a cotyledon small embryo; s2, placing the cotyledon small embryos in an improved MS culture medium for maturation culture to obtain mature normal somatic embryos; s3, transferring the mature normal somatic embryo into a somatic embryo germination culture medium for somatic embryo plant induction culture. The method for cultivating the normal somatic embryo of the rubber tree clone variety has better maturation culture efficiency and higher normal somatic embryo and somatic embryo plant obtaining rate, and large-scale propagation can be realized. The maturing rate of cotyledon small embryos reaches up to 82.50% or above, the obtaining rate of normal somatic embryos reaches up to 75.0% or above, and the obtaining rate of somatic embryo plants reaches up to 63.0% or above.

Description

Technical Field

[0001] The invention relates to the technical field of rubber tree seedling cultivation, and in particular to a cultivation method for efficiently obtaining normal somatic embryos of rubber tree clone varieties. Background Art

[0002] Compared with old bud-grafted seedlings of the same asexual varieties, self-rooted juvenile asexual lines of rubber trees (referred to as somatic embryo plants) have the advantages of rapid growth, strong stress resistance, uniform forest appearance and high rubber yield. They are known as superior planting materials and have broad application prospects, especially in the renewal and transformation of old, low-yield and low-quality rubber plantations.

[0003] The tissue culture technology of rubber trees is the only way to breed somatic embryo plants, among which anther tissue culture is one of the main technical approaches and one of the world's recognized problems. At present, only a few asexual varieties such as 'Reyan 73397' and 'Reyan 72059' can be mass-produced with anther somatic embryo plants, and the promotion and utilization effect is good. More than 30,000 acres of the three major natural rubber producing areas of Hainan, Yunnan and Guangdong have been promoted and planted. However, the anther tissue culture technology of excellent asexual varieties such as 'Yunyan 80-1983', 'Yunyan 77-4', ​​'Yunyan 77-2' and 'Yunyan 73-46' has not been conquered, and an efficient and stable somatic embryo plant regeneration technology system has not been established or achieved a zero breakthrough. In addition, the anthers of some excellent clone varieties such as 'Yunyan 73-477', 'Yunyan 614' and 'RRIM600' are very easy to dedifferentiate and produce callus under suitable culture medium and culture conditions. The callus induction rate is above 90.0%, and the obtained callus has strong embryogenicity and can differentiate into somatic embryos. The somatic embryo occurrence rate is 69.66-84.27%, but the normal somatic embryo acquisition rate is only 15.0-30%, which seriously restricts the large-scale propagation and promotion of somatic embryo plants of these excellent clone varieties. Previous studies have shown that the complete process of rubber tree somatic embryo development can be divided into four stages: globular embryo→heart-shaped embryo→torpedo embryo→cotyledon embryo, and somatic embryo development is not synchronous. In the somatic embryo differentiation culture medium, the same piece of embryonic callus often contains two or more somatic embryos of different development stages, including globular embryo, heart-shaped embryo, torpedo embryo and cotyledon embryo. In the past, culture medium with the same composition as differentiation medium was usually used for maturation culture, resulting in a high rate of somatic embryo deformity. The deformed embryos could not further develop and germinate to form complete plants, resulting in a low rate of obtaining somatic embryo plants. This technical bottleneck seriously restricted the large-scale mass production and promotion and application of rubber tree somatic embryo plants.

[0004] In summary, there is an urgent need for a method for cultivating normal somatic embryos of asexual rubber tree varieties with better maturity culture efficiency, higher rate of obtaining normal somatic embryos and somatic embryo plants, and the ability to achieve large-scale propagation. This can significantly improve the rate of obtaining somatic embryo plants, thereby promoting the large-scale mass production and popularization and application of somatic embryo plants, which is beneficial to the stable and healthy development of the natural rubber industry. Summary of the invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for cultivating normal somatic embryos of a rubber tree clone variety, so as to at least achieve better mature culture efficiency and higher normal somatic embryo and somatic embryo plant acquisition rate.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] A method for cultivating normal somatic embryos of a rubber tree clone variety comprises the following steps:

[0008] S1: Take the primary somatic embryos of the anthers of the rubber tree clones and select the cotyledon embryos;

[0009] S2: placing the cotyledon embryos in a maturation medium for culture to obtain mature normal somatic embryos;

[0010] S3: transferring the mature normal somatic embryos into a somatic embryo germination medium for somatic embryo plant induction culture.

[0011] Furthermore, in step S2, the maturation medium is cultured, and the preparation method of the cotyledon embryo maturation medium is: modified MS supplemented with 0.50 mg / L 6-BA, 0.15 mg / L NAA, 0.25 mg / L KT, 70 g / L sucrose, 5.8 g / L agar powder and 50 mL / L coconut juice.

[0012] Furthermore, in step S2, the modified MS medium has 4 / 5 of the macroelements of MS, 1.2 times of the trace elements of MS, and the iron salts and organic components are the same as those of MS.

[0013] The macroelements include: NH4NO3, KNO3, CaCl2·2H2O, MgSO4·7H2O, KH2PO4; the trace elements include KI, H3BO3, MnSO4·4H2O, ZnSO4·7H2O, Na2MoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O; the iron salts include: FeSO4·7H2O, Na2-EDTA·2H2O; the organic components include: inositol, niacin, pyridoxine hydrochloride, thiamine hydrochloride and glycine.

[0014] Furthermore, the pH of the mature culture medium needs to be adjusted to 5.8-6.2 before use, and then sterilized at high temperature and high pressure.

[0015] Furthermore, in step S2, the temperature of the maturation culture is 24±0.5°C.

[0016] Furthermore, in step S2, the maturation culture is dark culture.

[0017] Furthermore, in step S3, the somatic embryo germination medium is 4 / 5MS+GA31.0-2.0mg / L+IAA0.25-0.50mg / L+sucrose 50g / L+agar 5.8g / L+activated carbon 1g / L.

[0018] Furthermore, in step S3, the culture temperature for the somatic embryo plant induction culture is 26±0.5°C.

[0019] Furthermore, in step S3, the somatic embryo plant induction culture is light culture, the light intensity is 1000-1500lx, and the light time is 10h / d.

[0020] Furthermore, the rubber tree clone varieties are diploid clone varieties, including 'Yunyan 73-477', 'Reken 525' and RRIM600'.

[0021] It is worth noting that the complete somatic embryo development process is spherical embryo → heart-shaped embryo → torpedo embryo → cotyledon embryo. In previous studies, the applicant found that there were large differences in the maturation rate of somatic embryos at different developmental stages after maturation culture, the rate of obtaining normal somatic embryos and somatic embryo plants. By comparing the maturation culture efficiency of somatic embryos at different developmental stages, the maturation culture effect of anther somatic embryos increased with the increase in the degree of development of the embryos, and the maturation culture effect of cotyledonary embryos was the best.

[0022] The beneficial effects of the present invention are:

[0023] The method for cultivating normal somatic embryos of asexual rubber tree varieties of the present invention has good maturation culture efficiency, high normal somatic embryo and somatic embryo plant acquisition rate, and can achieve large-scale propagation. The cotyledon embryo maturation rate is as high as 82.50% or more, the normal somatic embryo acquisition rate is as high as 75.0% or more, and the somatic embryo plant acquisition rate is as high as 63.0% or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are pictures of somatic embryos at different developmental stages of rubber trees, including A: Callus cultured in differentiation medium for 30-35 days, with somatic embryos at different developmental stages in the same callus; B: heart-shaped embryo; C: torpedo embryo; D: cotyledon embryo;

[0025] Figure 2The chart below is a comparison of the maturation culture effects of three types of somatic embryos of the asexual variety 'Yunyan 73-477', where E~F are the newly inoculated torpedo embryos, cotyledonary embryos and conventionally inoculated mixed embryos; H~J are the maturation culture effects of fish-shaped embryos (A1), cotyledonary embryos (A2) and conventionally mixed inoculated embryos (A3) at 45 days of culture. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings, but the protection scope of the present invention is not limited to the following.

[0027] Example 1

[0028] 1.1 Experimental Materials

[0029] Improved MS medium: macroelements are 4 / 5 of MS, trace elements are 1.2 times of MS, iron salts and organic components are the same as MS, and the mature medium is supplemented with sucrose 70g / L, agar powder 5.8g / L, and coconut juice 50mL / L. The pH value is adjusted to 5.8-6.2 before high temperature and high pressure sterilization.

[0030] Somatic embryo germination medium: 4 / 5MS+GA31.0~2.0mg / L+IAA0.25~0.50mg / L+sucrose 50g / L+agar 5.8g / L+activated carbon 1g / L

[0031] Experimental tools such as scalpels and forceps were thoroughly sterilized using an inoculation heater.

[0032] 1.2 Experimental methods

[0033] (1) The primary torpedo embryos of the anthers of the superior clone of rubber tree cultivar 'Yunyan 73-477' were collected. Figure 1 -C), cotyledon embryo ( Figure 1 -D) and mixed immature embryos were inoculated into conventional maturation medium (A3) for maturation culture (A3 medium is the optimal maturation medium for mixed immature embryos). After 45 days of culture, the maturation culture of somatic embryos was investigated, and the somatic embryo maturation rate and the normal somatic embryo acquisition rate were calculated. A3 maturation medium: modified MS supplemented with 6-BA1.0mg / L, NAA0.20mg / L, KT 0.20mg / L, GA30.5mg / L and ABA0.20mg / L; mature normal somatic embryos were continuously cultured for 50 days and then transferred into somatic embryo germination medium for somatic embryo plant induction culture. After 60 days of culture, the somatic embryo plant induction was investigated, and the somatic embryo plant acquisition rate was calculated.

[0034] (2) Select the anther torpedo embryos of the excellent asexual line of rubber tree cultivar 'Yunyan 73-477' ( Figure 1 -C) and cotyledon embryo ( Figure 1-D) for maturation culture, torpedo embryo maturation medium (A1): modified MS with 6-BA2.0mg / L, NAA0.15mg / L, GA30.5mg / L and ABA0.40mg / L; cotyledon embryo maturation medium (A2): modified MS with 6-BA0.50mg / L, NAA0.15mg / L and KT 0.25mg / L (A1 and A2 culture media are the optimal maturation medium for fish embryos and cotyledon embryos, respectively). With conventional maturation culture as the control, globular embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos were mixed and inoculated into the same maturation medium (A3) for maturation culture. When cultured to 45 days, the maturation culture of somatic embryos was investigated, and the somatic embryo maturation rate and normal somatic embryo acquisition rate were calculated. The mature normal somatic embryos were continued to be cultured to 50 days and transferred to the somatic embryo germination medium for somatic embryo plant induction culture. After 60 days of culture, the somatic embryo plant induction was investigated and the somatic embryo plant acquisition rate was calculated.

[0035] In the above experiments, each material was inoculated with 5 dishes, with 12 to 15 young embryos in each dish; somatic embryo maturation rate (%) = total number of mature somatic embryos / total number of inoculated somatic embryos × 100; normal somatic embryo acquisition rate (%) = total number of mature normal somatic embryos / total number of inoculated somatic embryos × 100; somatic embryo plant acquisition rate (%) = total number of somatic embryo plants / total number of inoculated somatic embryos × 100.

[0036] In the above-mentioned experiment, during the maturation culture stage of somatic embryos, culture dishes with a breathable membrane (with a diameter of 8 cm) were used, and 30 ml of culture medium was filled into each dish, which was placed in a dark culture room at a temperature of 24±0.5°C. During the somatic embryo germination stage, test tubes with a breathable membrane were used, and 26-27 ml of culture medium was filled into each tube, which was placed in a light culture room at a temperature of 26±0.5°C (light intensity of 1000-1500 lx, light duration of 10 h / d).

[0037] 1.3 Experimental Results

[0038] The primary somatic embryos at the torpedo embryo and cotyledon embryo stages were inoculated into the maturation medium ( Figure 2 -F, Figure 2 -G) After 20 days of dark culture, most of the embryos grew significantly but were not mature. As the culture time continued, the embryos continued to mature. At 45 days, most of the embryos had matured ( Figure 2 -H, Figure 2 -I, Figure 2 -J). After 50 days of continuous culture, a small number of somatic embryos have grown new roots. At this time, the mature normal somatic embryos are transferred to the somatic embryo germination medium and cultured for 60 days to obtain complete somatic embryo plants. Tables 1 and 2 show the efficiency of maturation culture of the three somatic cell embryos.

[0039] Table 1 Maturation culture efficiency of different somatic embryos of 'Yunyan 73-477' in A3 medium Unit: %

[0040]

[0041] Table 1 shows that in the same maturation culture medium, there are differences in the maturation culture effects of different somatic embryos of the excellent asexual line variety of rubber tree 'Yunyan 73-477'. The maturation culture effect of cotyledon embryos is the best, with a somatic embryo maturation rate of 66.56±2.14%, which is 8.78% and 10.45% higher than that of torpedo embryos and mixed somatic embryos, respectively; the normal somatic embryo acquisition rate is 52.78±2.55%, which is 3.21 times and 2.03 times that of torpedo embryos and mixed somatic embryos, respectively; the somatic embryo plant acquisition rate is 42.22±1.92%, which is 3.52 times and 2.67 times that of torpedo embryos and mixed somatic embryos, respectively.

[0042] Table 2. Efficiency of maturation culture of different somatic embryos of 'Yunyan 73-477' Unit: %

[0043]

[0044]

[0045] Table 2 shows that in the somatic embryo maturation culture of the superior asexual line variety of rubber tree 'Yunyan 73-477', the torpedo embryos and cotyledon embryolets were selected and inoculated in A1 and A2 for maturation culture respectively. The maturation effects of both were better than those of conventional mixed culture. The somatic embryo maturation rates were 78.19±4.26% and 82.50±4.33%, respectively, which were 23.02% and 26.79% higher than those of conventional mixed maturation culture respectively. The normal somatic embryo acquisition rates were 49.86±5.53% and 79.17±8.78%, respectively, which were 2.15 times and 3.42 times of those of conventional mixed maturation culture respectively. Moreover, most of the mature somatic embryos were normal somatic embryos with a bipolar structure, which could germinate and develop into complete somatic embryo plants in subsequent culture. The somatic embryo plant acquisition rates were 41.25±2.17% and 65.0±8.66%, respectively, which were 2.55 times and 4.01 times of those of conventional mixed maturation culture.

[0046] It can be seen from Table 1 and Table 2 that the primary cotyledonary embryos of the anther of the excellent asexual line of rubber tree 'Yunyan 73-477' were inoculated into A2 and A3 maturation culture media for maturation culture, and the maturation culture effect of A2 was better than that of A3, with a somatic embryo maturation rate of 82.50±4.33%, 15.94% higher than that of A3 (66.56±2.14%), a normal somatic embryo acquisition rate of 79.17±8.78%, and a somatic embryo plant acquisition rate of 65.00±8.66%, which were 1.50 times and 1.54 times that of A3, respectively; for the primary somatic embryos of the anther of 'Yunyan 73-477' at different developmental stages, the suitable maturation culture media were significantly different, and the maturation culture effect of the cotyledonary embryos was the best, and the somatic embryo maturation rate, normal somatic embryos and somatic embryo plant acquisition rates were higher than those of the other two types of embryos, followed by torpedo embryos, and the worst of mixed somatic embryos.

[0047] In conclusion, the primary cotyledonary embryos in the anthers of the superior clone cultivar 'Yunyan 73-477' of rubber tree are the best somatic embryos for maturation culture, and A2 is a suitable culture medium for maturation culture of cotyledonary embryos. The somatic embryo maturation rate is as high as 82.50±4.33%, and the acquisition rates of normal somatic embryos and somatic embryo plants are 79.17±8.78% and 65.00±8.66%, respectively.

[0048] Example 2

[0049] 2.1 Experimental Materials

[0050] Improved MS medium: macroelements are 4 / 5 of MS, trace elements are 1.2 times of MS, iron salts and organic components are the same as MS, and the mature medium is supplemented with sucrose 70g / L, agar powder 5.8g / L, and coconut juice 50mL / L. The pH value is adjusted to 5.8-6.2 before high temperature and high pressure sterilization.

[0051] Somatic embryo germination medium: 4 / 5MS+GA31.0~2.0+IAA0.25~0.50+sucrose 50g / L+agar 5.8g / L+activated carbon 1g / L

[0052] Experimental tools such as scalpels and forceps were thoroughly sterilized using an inoculation heater.

[0053] 2.2 Experimental methods

[0054] (1) The primary torpedo embryos ( Figure 1 -C), cotyledon embryo ( Figure 1-D) and mixed immature embryos were inoculated into conventional maturation medium (A3) for maturation culture. After 45 days of culture, the maturation culture of somatic embryos was investigated, and the somatic embryo maturation rate and the normal somatic embryo acquisition rate were calculated. A3 maturation medium: modified MS supplemented with 6-BA1.0mg / L, NAA0.20mg / L, KT 0.20mg / L, GA30.5mg / L and ABA0.20mg / L; the mature normal somatic embryos were continuously cultured until 50 days and then transferred into somatic embryo germination medium for somatic embryo plant induction culture. After 60 days of culture, the somatic embryo plant induction was investigated, and the somatic embryo plant acquisition rate was calculated.

[0055] (2) Select the anther torpedo embryos of the superior rubber tree variety 'RRIM600' ( Figure 1 -C) and cotyledon embryo ( Figure 1 -D) for maturation culture, torpedo embryo maturation medium (A1): modified MS with 6-BA2.0mg / L, NAA0.15mg / L, GA30.5mg / L and ABA0.40mg / L; cotyledon embryo maturation medium (A2): modified MS with 6-BA0.50mg / L, NAA0.15mg / L and KT 0.25mg / L. Conventional maturation culture was used as a control, that is, spherical embryos, heart-shaped embryos, torpedo embryos and cotyledon embryos were mixed and inoculated into the same maturation medium (A3) for maturation culture. When cultured to 45 days, the maturation culture of somatic embryos was investigated, and the somatic embryo maturation rate and normal somatic embryo acquisition rate were counted. Mature normal somatic embryos were continuously cultured to 50 days and transferred to somatic embryo germination medium for somatic embryo plant induction culture. After 60 days of culture, the somatic embryo plant induction was investigated and the somatic embryo plant acquisition rate was counted.

[0056] In the above experiments, each material was inoculated with 5 dishes, with 12 to 15 young embryos in each dish; somatic embryo maturation rate (%) = total number of mature somatic embryos / total number of inoculated somatic embryos × 100; normal somatic embryo acquisition rate (%) = total number of mature normal somatic embryos / total number of inoculated somatic embryos × 100; somatic embryo plant acquisition rate (%) = total number of somatic embryo plants / total number of inoculated somatic embryos × 100.

[0057] In the above-mentioned experiment, during the somatic embryo maturation culture stage, culture dishes with a breathable membrane (with a diameter of 8 cm) were used, 30 ml of culture medium was filled into each dish, and the culture dishes were placed in a dark culture room at a temperature of 24±0.5°C; in the somatic embryo germination / somatic embryo plant induction culture stage, test tubes with a breathable membrane were used, 26-27 ml of culture medium was filled into each tube, and the culture dishes were placed in a light culture room at a temperature of 26±0.5°C (light intensity of 1000-1500lx, light duration of 10h / d).

[0058] 2.3 Test results:

[0059] The primary somatic embryos of the rubber tree elite variety 'RRIM600' at the torpedo embryo and cotyledon embryo stage were inoculated into the maturation medium. After 22 days of dark culture, most of the embryos grew up significantly, but did not mature. As the culture time continued, the embryos continued to mature. At 46 days, most of the embryos had matured. After 50 days of continuous culture, a small number of somatic embryos had grown new roots. At this time, the mature normal somatic embryos were transferred to the somatic embryo germination medium and cultured for 60 days to obtain complete somatic embryo plants. Tables 3 and 4 show the maturation culture efficiency of the three somatic embryos.

[0060] Table 3 Maturation culture efficiency of different somatic embryos of 'RRIM600' in A3 medium Unit: %

[0061]

[0062]

[0063] Table 3 shows that in the same maturation culture medium, there are differences in the maturation culture effects of different somatic embryos of the superior rubber tree variety 'RRIM600'. The maturation culture effect of cotyledon embryolets is the best, with a somatic embryo maturation rate of 73.70±1.97%, which is 21.95% and 15.42% higher than that of torpedo embryos and mixed somatic embryos, respectively; the normal somatic embryo acquisition rate is 51.06±1.29%, which is 2.62 times and 1.79 times that of torpedo embryos and mixed somatic embryos, respectively; the somatic embryo plant acquisition rate is 37.74±0.88%, which is 2.41 times and 1.79 times that of torpedo embryos and mixed somatic embryos, respectively.

[0064] Table 4. Maturation culture efficiency of different somatic embryos of 'RRIM600' Unit: %

[0065]

[0066] Table 4 shows that in the maturation culture of primary somatic embryos of anthers of the superior rubber tree variety 'RRIM600', torpedo embryos and cotyledon embryolets were selected and inoculated in two maturation culture media A1 and A2 for maturation culture, and the maturation effects of both were better than those of conventional mixed culture. The somatic embryo maturation rates were 72.86±5.42% and 86.63±0.89%, respectively, which were 12.51% and 26.28% higher than those of conventional mixed maturation culture, respectively. The normal somatic embryo acquisition rates were 55.51±7.83% and 75.08±9.72%, respectively, which were 2.13 times and 2.89 times of those of conventional mixed maturation culture, respectively. Moreover, most of the mature somatic embryos were normal somatic embryos with a bipolar structure, and could germinate and develop into complete somatic embryo plants in subsequent culture. The somatic embryo plant acquisition rates were 37.61±7.40% and 63.65±5.46%, respectively, which were 1.86 times and 3.15 times of those of conventional mixed maturation culture.

[0067] Combining Table 3 and Table 4, it can be seen that the primary cotyledon embryos of the anthers of the rubber tree elite variety 'RRIM600' were inoculated into A2 and A3 maturation culture media for maturation culture, and the maturation culture effect of A2 was better than that of A3, with a somatic embryo maturation rate of 86.63±0.89%, 12.93% higher than that of A3 (73.70±1.97%), and normal somatic embryos and somatic embryo plant acquisition rates of 75.08±9.72% and 63.65±5.46%, 1.47 times and 1.69 times that of A3, respectively; for the somatic immature embryos of the rubber tree 'RRIM600' at different developmental stages, the applicable maturation culture media had significant differences, and the maturation culture effect of the cotyledon embryos was the best, and the somatic embryo maturation rate, normal somatic embryos and somatic embryo plant acquisition rates were all higher than those of the other two immature embryos, followed by torpedo embryos, and the worst of mixed somatic embryos.

[0068] In conclusion, the primary cotyledonary embryos of the rubber tree elite variety 'RRIM600' are the best somatic embryos for maturation culture, and A2 is a suitable culture medium for maturation culture of cotyledonary embryos. The somatic embryo maturation rate is as high as 86.63±0.89%, and the acquisition rates of normal somatic embryos and somatic embryo plants are 75.08±9.72% and 63.65±5.46%, respectively.

[0069] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.

Claims

1. A method for cultivating normal somatic embryos of a rubber tree clone variety, characterized in that, The following steps are involved: S1: Take the primary somatic embryos of the anthers of the rubber tree clones and select the cotyledon embryos; S2: placing the cotyledon embryos in a maturation medium for culture to obtain mature normal somatic embryos; S3: transferring the mature normal somatic embryos into a somatic embryo germination medium for somatic embryo plant induction culture.

2. The cultivation method according to claim 1, characterized in that: In step S2, the preparation method of the maturation culture medium is: modified MS is supplemented with 0.50 mg / L 6-BA, 0.15 mg / L NAA, 0.25 mg / L KT, 70 g / L sucrose, 5.8 g / L agar powder and 50 mL / L coconut juice.

3. The cultivation method according to claim 2, characterized in that: In step S2, the modified MS medium has 4 / 5 of the macroelements of MS, 1.2 times of the trace elements of MS, and the iron salts and organic components are the same as those of MS.

4. The cultivation method according to claim 3, characterized in that: The pH of the mature culture medium also needs to be adjusted to 5.8-6.2 before use, and then sterilized at high temperature and high pressure.

5. The cultivation method according to claim 1, characterized in that: In step S2, the temperature of the maturation culture is 24±0.5°C.

6. The cultivation method according to claim 1, characterized in that: In step S2, the maturation culture is dark culture.

7. The cultivation method according to claim 1, characterized in that: In step S3, the somatic embryo germination medium is 4 / 5MS+GA31.0-2.0mg / L+IAA0.25-0.50mg / L+sucrose 50g / L+agar 5.8g / L+activated carbon 1g / L.

8. The cultivation method according to claim 1, characterized in that: In step S3, the culture temperature for the somatic embryo plant induction culture is 26±0.5°C.

9. The cultivation method according to claim 1, characterized in that: In step S3, the somatic embryo plant induction culture is light culture, the light intensity is 1000-1500lx, and the light time is 10h / d.

10. The cultivation method according to claim 1, characterized in that: The rubber tree asexual varieties are diploid asexual varieties, including Yunyan 73-477, Reken 525 and RRIM600.

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