A method for alternate grafting of rubber tree rootstocks

Abstract

The present application relates to the technical field of plant asexual cloning, and particularly relates to a root-stem alternating grafting method of rubber trees. The present application provides a rubber tree root-stem alternating grafting method. Compared with conventional plant grafting, the method grafts a rubber tree root segment as an intermediate scion between a rootstock and a terminal scion, and does not limit whether the types of the rootstock, the intermediate scion and the terminal scion are consistent, so that fusion of rubber tree plants of various types (different genotypes, different organs) can be realized. The present application aims to realize transplantation of rubber tree root segments and in-vivo preservation of excellent rootstock resources. In addition, after the fused plant is grafted to survive, aerial roots can be regenerated under the conditions of moisture and darkness through auxin induction, so that the proliferation and visualization analysis of the root segments can be realized. The present application provides a theoretical basis and technical support for the research and application of other perennial plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant asexual cloning, and particularly relates to a root-stem alternate grafting method of rubber trees. BACKGROUND

[0002] Rubber tree is a large arbor of Euphorbiaceae, which originates from the Amazon basin in South America. Natural rubber produced by rubber trees is the main raw material for making rubber. Natural rubber has unique elasticity, ductility and thermal conductivity, and is an important industrial raw material and an important strategic material.

[0003] In China, natural rubber trees are often cultivated by bud grafting propagation. Rubber tree bud grafting propagation is a common asexual propagation technology. By grafting bud pieces of excellent mother trees onto rootstocks (usually seedlings), variety improvement and efficient propagation are achieved. Among them, GT1 selected by a breeding institution in Malaysia is one of the traditional cultivation varieties with the strongest cold resistance in rubber trees. Since GT1 rootstock can significantly improve the cold resistance and drought resistance of scion varieties, it has strong resistance to rubber tree root rot and powdery mildew. The main root of GT1 seedling is developed and the lateral root is less. The bark of adult tree is thick (beneficial to tapping), and the milk ducts are arranged closely. Therefore, GT1 is widely used as rootstock or scion in rubber planting areas such as Yunnan and Hainan in China. However, the inventors found in the process of conceiving and implementing the present application that although GT1 is one of the excellent rootstocks, the rubber yield of GT1 as a scion (not a rootstock) is at a medium level, which is lower than that of modern high-yield varieties (such as RRIM600). Therefore, GT1 is only used as a rootstock. In addition, there is a PR107 strain of three-leaf rubber tree in Wufen Plant of Mengla Farm, Xishuangbanna Prefecture, with an annual dry rubber yield of 107.8 kg, which is 30 times that of ordinary rubber trees, and the single plant yield creates a record in the history of rubber planting, and is known as "rubber king tree". However, the high yield cannot be reproduced after grafting with its branches as scions. Scholars speculate that the high yield may be caused by excellent rootstock, but breeding experts have not been able to reproduce the rootstock of "rubber king tree" for a long time. The root segment of "rubber king tree" as an excellent rootstock has attracted attention from people in the field. In summary, the transplantation and preservation of rootstock is a problem to be solved by people in the field.

[0004] Therefore, a grafting method for root-stem alternation of rubber trees is needed to realize the transplantation and preservation of rootstock, so that excellent rootstock resources can be preserved in vivo.

[0005] The above content is only used to assist in understanding the technical solutions of the present application and does not mean that the above content is prior art. SUMMARY

[0006] The main purpose of the present application is to provide a root-stem alternate grafting method of rubber trees, which aims to solve the problem of how to realize the transplantation and preservation of rootstock through the root-stem alternate grafting method, so as to realize the fusion of roots and stems of different types of rubber trees.

[0007] To achieve the above object, the application provides a root-stem alternate grafting method of rubber trees, which comprises the following steps:

[0008] S1, preparing a stock: seeds of mature rubber trees are sowed to obtain rubber tree seedlings, and the rubber tree seedlings are transplanted and cultivated, and the seedlings are cultivated for 3-12 months to serve as the stock;

[0009] S2, preparing scions: root segments of rubber trees are cleaned and cut into 3-5 cm as intermediate scions, and stem segments of rubber trees are taken as terminal scions;

[0010] The root segment is a lateral root of a mature rubber tree or a main root of a rubber tree seedling, with a length of 3-5 cm and a diameter of 3-7 mm;

[0011] The stem segment is a stem segment of a rubber tree with apical buds, and all leaves are removed, with a length of 5-11 cm and a diameter of 3-7 mm;

[0012] The apical buds are in the dormant period or have just broken the dormant period;

[0013] S3, grafting the intermediate scion obtained in S2 to the morphological upper end of the stock in S1, and grafting the terminal scion to the morphological upper end of the intermediate scion, to form a to-be-fused plant after grafting, and the to-be-fused plant is cultivated under dark conditions for 20-30 days to obtain a root-stem alternate grafted fused plant.

[0014] Optionally, in S1, the growth cycle of the rubber seedling is 3 months, 6 months and 12 months, and the rubber seedling is used as the stock for grafting at the respective time.

[0015] Optionally, in S3, the grafting method is the wedge grafting method, the cut is cut in the shape of a "V", and the cut is wrapped and moisturized with sealing film and preservative film after wedge grafting.

[0016] Optionally, the cut is located at a position 40-60 cm away from the ground of the stock.

[0017] Optionally, the specific steps of cultivation in S3 are: the to-be-fused plant is cultured under dark conditions for 20-25 days, then the preservative film and the sealing film are removed, the to-be-fused plant is cultured under dark conditions for another 5 days, and then the to-be-fused plant is transferred to a normal growth environment, and water and fertilizer are continuously supplied; the dark conditions include a temperature of 20-30℃, a humidity of 60%-80%, and a light intensity of less than 100 μmol / m 2 / s; and the normal growth environment includes a temperature of 25-35℃, a humidity of 60%-90%, and a light intensity of more than 300 μmol / m 2 / s.

[0018] Optionally, the method further comprises the following steps:

[0019] S4, inducing aeration root to regenerate: the fusion plant obtained in S3 is cultivated by auxin induction under dark and moist conditions to regenerate aeration root.

[0020] Optionally, the specific step of inducing aeration root to regenerate in S4 is that 100 mg / L or 200 mg / L auxin is externally applied to the fusion plant under dark and moist conditions for continuous treatment for 2 months, and the treatment frequency is 3 times / day.

[0021] Optionally, the dark and moist conditions include temperature of 20-30 DEG C, humidity of 60%-80%, and light intensity less than 100 μmol / m 2 / s.

[0022] Optionally, the auxin is NAA or IBA.

[0023] Invention principle:

[0024] Grafting technology, that is, grafting a part of a plant to another plant body, so that the two parts grafted together grow into a complete new plant. The part grafted is called scion, and becomes the upper part or top of the plant body after grafting. The plant body to be grafted is called rootstock, and becomes the root system part of the plant body after grafting. Grafting is a kind of asexual reproduction. When grafting, the two wounded cambiums are close and tight together, and are healed into a whole by cell proliferation and connection of vascular tissues.

[0025] The rootstock used in industry includes root system and base stem section, but mainly uses the strong absorption capacity, stress resistance and disease resistance of the root system, and the existence of the stem section is only for the convenience of grafting operation; therefore, as long as the root section is preserved, the rootstock is preserved, and the lateral root of the root section is induced to grow, the root proliferation can be realized.

[0026] The present application mainly realizes the transplantation and preservation of root sections of various kinds of rubber trees by grafting the rubber tree root section as an intermediate scion between the rootstock and the terminal scion, and the root section is preserved, so the rootstock is preserved, and the transplantation and preservation of the rootstock are realized, so that the excellent rootstock resources can be preserved in vivo.

[0027] The technical scheme provided by the present application at least has the following beneficial effects:

[0028] 1. The application provides a rootstock alternate grafting method of rubber trees, which grafts the root section of the rubber tree as an intermediate scion between the rootstock and the terminal scion, so that the wound surface cell proliferation heals together to form a new fused plant, and the grafting does not limit the types of the rootstock, the intermediate scion and the terminal scion, which not only realizes the fusion of various types of rubber tree plants, but also realizes the transplantation of roots and the in vivo preservation of excellent rootstock resources.

[0029] 2. The application realizes the transformation from invisible analysis to visual analysis of the root section of the rubber tree by transplanting the root section of the underground part of the rubber tree to the middle part of the rubber tree as an intermediate scion.

[0030] 3. The application is carried out by taking rubber trees as materials, and the same operation can be extended to other perennial woody plants, which provides a theoretical basis and technical support for the research and application of perennial plants.

[0031] 4. After the fused plant is grafted to survive, the aerial roots can be regenerated by auxin induction under moist and dark conditions, which realizes the proliferation and visual analysis of the root section, and the successful induction of the aerial roots also realizes the proliferation and visual analysis of the rootstock.

[0032] 5. The application realizes the proliferation, transplantation and in vivo preservation of the excellent rootstock of the rubber tree through the rootstock alternate grafting method, which lays a foundation for the tissue culture of excellent rootstock.

[0033] 6. The application grafts the root section of the rubber tree as an intermediate scion between the rootstock and the terminal scion, and limits the plant size, growth condition and growth environment of the rootstock, the intermediate scion and the terminal scion, the whole culture process is simple in operation, simple in process, low in cost and independent of the genotype of the rubber tree.

[0034] 7. In the application, the number of intermediate scions and terminal scions is not limited to one, the terminal scion stem top after grafting to survive can graft the intermediate scion on the morphological top again, and the intermediate scion morphological top grafts a new terminal scion, which realizes the visual research and growth condition analysis of multiple intermediate scions (rootstock root sections). BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a plant grafted with root sections of different lengths and diameters as intermediate scions shown in the application;

[0036] Figure 2 is a terminal scion state at different development stages shown in the application;

[0037] Figure 3 is a schematic diagram of rubber rootstocks at different ages shown in the application;

[0038] Figure 4 is a schematic diagram of a plant normally grown after rhizome alternate grafting shown in the present application;

[0039] Figure 5 is a schematic diagram of a plant normally grown after rhizome alternate grafting shown in the present application; A-E, plants survived by rhizome alternate grafting in a greenhouse, F-I, plants survived by rhizome alternate grafting in an open environment;

[0040] Figure 6 is a schematic diagram of a plant normally grown after rhizome alternate grafting shown in the present application; A-E, plants survived by rhizome alternate grafting in a greenhouse, F-I, plants survived by rhizome alternate grafting in an open environment;

[0041] Figure 7 is a schematic diagram of a plant normally grown after rhizome alternate grafting shown in the present application; A-E, plants survived by rhizome alternate grafting in a greenhouse, F-I, plants survived by rhizome alternate grafting in an open environment;

[0042] Figure 8 is a schematic diagram of a plant normally grown after rhizome alternate grafting shown in the present application; A-E, plants survived by rhizome alternate grafting in a greenhouse, F-I, plants survived by rhizome alternate grafting in an open environment;

[0043] The implementation, functional characteristics and advantages of the present application will be further described with reference to the accompanying drawings. DETAILED DESCRIPTION

[0044] In order to better understand the above technical solutions, the exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to convey the scope of the present disclosure to those skilled in the art.

[0045] Invention concept: Genetic transformation of rubber trees is very difficult, and root transformation is relatively easy. The research team has established a genetic transformation system for rubber tree roots in the early stage and applied for a patent (Patent application number: 2024116714058), but the preservation, propagation and phenotype analysis of transgenic roots have not been effectively solved. The present application realizes plant organ transplantation by alternate grafting, grafts transgenic roots onto stems, facilitates the preservation, propagation and phenotype analysis of rubber tree tissues and organs, and provides convenience for inducing the regeneration of transgenic roots to break through the stable genetic transformation system of rubber trees. Based on the realization of stable genetic transformation of rubber tree roots, the present application develops a new method for phenotype identification of transgenic roots, which makes a great contribution to the basic research and breeding of rubber trees.

[0046] Plant materials: Rootstocks of rubber tree were obtained by germination of fresh rubber seeds, and cultivated in a greenhouse of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (North latitude 21°41'N / 101°25'E, altitude 570m); middle scions of rubber tree were collected from Xishuangbanna Tropical Botanical Garden and Mengla Farm, Xishuangbanna, Yunnan Province; terminal scions of rubber tree were collected from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.

[0047] Other materials: Double-sided blades, flowerpots and plastic wrap were purchased from supermarket, and flower soil was purchased from Chenggong Flower Market, Kunming City; auxin was purchased from Shanghai Bioengineering Co., Ltd.

[0048] Based on the above description, an embodiment of the rubber tree root and stem alternate grafting method is provided

[0049] First embodiment

[0050] The purpose of this embodiment experiment is to test the best conditions for the whole rubber tree root and stem alternate grafting

[0051] Preparation of rootstock:

[0052] Fresh rubber seeds were collected from Mengla Farm and Xishuangbanna Tropical Botanical Garden from August to October, and stored at low temperature and high humidity in a seed storage cabinet at 4°C. Before germination, full and undamaged seeds were selected from the refrigerator and planted in a sand pool. After covering the sand, carbendazim was sprayed, and water was poured twice a week. When the rubber seedlings grew to 10-20 cm, they were transplanted into 30 cm diameter flowerpots filled with flower soil. Regular fertilization and watering were carried out, and the rootstocks were used for grafting experiments at 3 months, 6 months and 12 months after transplantation.

[0053] Preparation of scion:

[0054] The surface lateral roots of adult "rubber king trees" were collected from Mengla Farm, with a diameter of 3-7 mm. After collection, they were placed in a self-sealing bag and sprayed with water to keep them moist. After being brought back to the laboratory, the soil was washed off and soaked or sprayed with water to keep them moist. Before grafting, the roots were cut into 3-5 cm long middle scions. The main roots of young rubber seedlings and the shallow lateral roots of adult plants were collected from Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, with a diameter of 3-7 mm. After collection, the soil was washed off and kept moist in the laboratory. They were cut into middle scions.

[0055] Grafting and moisture preservation:

[0056] A "V" shaped incision was made on the rootstock, and the middle scions (which could be multiple segments) were grafted onto the rootstock using the wedge grafting method. The middle scions were then grafted in sequence using the wedge grafting method. Finally, the stem segment with the terminal bud was grafted onto the middle scion as the terminal scion, also using the wedge grafting method. The wound was then wrapped tightly with sealing film, and all the scions were wrapped with plastic wrap to keep them moist. In general, the survival rate of multiple scions grafted at one time is greatly reduced, so the alternate grafting can be repeated again after the first grafting heals.

[0057] Experimental procedure:

[0058] 1) Comparison of survival rate of root-stem alternate grafting with different root length and diameter as middle scion

[0059] V-shaped cut was made at the position 50 cm away from the cotyledon of the rootstock, and rubber tree roots (middle scion) with different length and diameter were grafted onto the rootstock 6 months after transplanting. Then, the terminal scion was grafted onto the middle scion by means of wedge grafting. After grafting, the wound was wrapped with dry sealing film, and all the scions were wrapped with plastic wrap for moisture retention. The grafting was cultured in the dark for 20-25 days, and then the plastic wrap was removed and the grafting was cultured in the dark for another 5 days. After 1 month of culture under normal growth conditions, the survival rate was calculated.

[0060] 2) Comparison of survival rate of grafting in different seasons

[0061] Grafting experiments were carried out in different seasons of the year. In this study, the survival rate of root-stem alternate grafting in spring (February to April), summer (May to July), autumn (August to October), and winter (November to January) was compared. The grafting and statistical methods and procedures were the same as in process 1.

[0062] 3) Comparison of grafting survival rate of terminal scion with different development stages and lengths

[0063] One-year-old seedlings were used as rootstock, and V-shaped cut was made at the position 50 cm away from the cotyledon. Middle scions with root length of 5 cm and diameter of 7 mm were grafted onto the rootstock, and then terminal scions with length of 5 cm but in different stages of bud, leaf expansion, leaf formation, and dormancy were grafted onto the middle rootstock. After 2 months of grafting, the survival rate was calculated. In addition, stem segments with terminal buds in the bud stage and lengths of 5 cm, 7 cm, 9 cm, and 11 cm were grafted onto the middle scion, and the survival rate was calculated after 2 months of grafting.

[0064] 4) Comparison of grafting survival rate of different ages of rootstock

[0065] Three-month-old, six-month-old, and twelve-month-old rubber seedlings were used as rootstock, and middle scions with length of 5 cm and diameter of 7 mm were grafted onto the rootstock. Terminal scions with length of 5 cm and in dormancy were grafted onto the rootstock of different ages, and the survival rate was calculated after 2 months of grafting.

[0066] Experimental results:

[0067] 1) Comparison of survival rate of root-stem alternate grafting with different root length and diameter as middle scion, the experimental results are shown in Table 1.

[0068] Table 1 Comparison of survival rate of root-stem alternate grafting with different root length and diameter as middle scion

[0069]

[0070]

[0071] Note: the age of the rootstock is 6 months, and the grafting experiment is carried out in spring

[0072] In February, different lengths and diameters of rubber tree roots were used as intermediate scions to graft onto 6-month-old rootstocks by means of wedge grafting, and then 5 cm long stem segments with buds were grafted onto the upper part of the intermediate scions by means of wedge grafting. After 30 days of dark culture, the plants were cultured under light for another 30 days, and the survival rate was calculated. The results showed that the average survival rate was (36.12 ± 7.66) % when the root length was 3 cm, (41.57 ± 3.30) % when the root length was 4 cm, and (40.63 ± 6.69) % when the root length was 5 cm (Table 1). The results showed that the survival rate was lower when the root length was 3 cm, and the survival rate was higher when the root length was 4 cm and 5 cm, but there was no significant difference. Further comparison of the survival rate of different diameters of rubber tree roots as intermediate scions showed that the average survival rate was (34.79 ± 3.79) % when the root diameter was 3 mm, (37.40 ± 4.43) % when the root diameter was 5 mm, and (45.93 ± 1.78) % when the root diameter was 7 mm (Table 1). The survival rate increased with the increase of root diameter, and the survival rate was the highest when the root diameter was 7 mm.

[0073] 2) Comparison of the survival rate of root-stem alternate grafting in different seasons, the experimental results are shown in Table 1. Root-stem alternate grafting experiments were carried out in different seasons, and the age of the rubber seedlings was 1 year in this experiment. The main root of the seedling was used as the intermediate scion, and the survival rate was calculated after 2 months of grafting.

[0074] Table 2 Comparison of the survival rate of root-stem alternate grafting in different seasons

[0075]

[0076]

[0077] Note: the age of the rootstock is 12 months, the length of the intermediate scion is 5 cm, the diameter is 7 mm, and the terminal scion is in the dormant period, with a length of 5 cm

[0078] As shown in Table 2, the survival rate of grafting in spring and summer was higher than that in autumn and winter, and the survival rate of grafting in February to April was the highest, reaching 72.35%. The survival rate of grafting in winter was the lowest, only 21.61% (Table 2).

[0079] (3) Comparison of the survival rate of grafting of terminal scions at different developmental stages and lengths

[0080] The 1-year-old seedling was used as a stock, a V-shaped cut was made at a position 50 cm from the cotyledon, and a middle scion with a root length of 5 cm and a diameter of 7 mm was grafted onto the stock, and then end scions with a length of 5 cm and at different stages of germination, leaf expansion, leaf formation, and dormancy were grafted onto the middle stock. The survival rate was counted after 2 months of grafting. The results show that the survival rate of the end scion with a top bud at the initial germination and dormancy stages is the highest, reaching 56.05% and 69.60%, respectively; the survival rate of the end scion with a top bud at the leaf expansion and leaf formation stages is lower than 20%, reaching 9.70% and 18.94%, respectively (Table 3).

[0081] Table 3 Influence of end scions at different development stages on root-stem alternate grafting

[0082]

[0083] Note: Middle scion - root length 5 cm, diameter 7 mm; end scion length 5 cm, grafting experiment conducted in February

[0084] In addition, end scions with a length of 5 cm, 7 cm, 9 cm, and 11 cm at the germination stage were grafted onto the middle scion, and the grafting experiment was conducted in February. The survival rate was counted after 2 months of grafting. The results show that the survival rate decreases with the increase of the length of the end scion, and the longer the end scion, the lower the survival rate. The survival rate is the highest when the length of the end scion is 5 cm, reaching 50.83% (Table 4). Since root-stem alternate grafting produces at least two grafting interfaces at the same time, the initial water and nutrient transport is very weak, and too long an end scion will lead to insufficient water and nutrient supply, resulting in a low survival rate.

[0085] Table 4 Influence of end scions with different lengths on root-stem alternate grafting

[0086]

[0087] Note: Middle scion - root length 5 cm, diameter 7 mm; end scion at the germination stage, grafting experiment conducted in February

[0088] 4) Comparison of grafting survival rates of different seedling ages

[0089] The 3-month-old, 6-month-old, and 12-month-old rubber seedlings were used as stocks, a middle scion with a length of 5 cm and a diameter of 7 mm, and an end scion with a length of 5 cm and at the dormancy stage were grafted onto the stocks with different ages, respectively. The survival rate was counted after 2 months of grafting. The results show that the survival rate of root-stem alternate grafting with the 12-month-old rubber seedling as the stock is the highest, reaching 63.93%, and the survival rate of the 3-month-old rubber seedling as the stock is the lowest, only 14.17% (Table 5). Since the stock plays an important role in nutrient absorption and transport, the 3-month-old stock plant and root system have a weak growth, and therefore the survival rate is low.

[0090] Table 5 Comparison of grafting survival rate of different age stages of rootstock

[0091]

[0092] Note: the middle scion root length is 5 cm, the diameter is 7 mm, the terminal scion is 5 cm, and the grafting experiment is carried out in February

[0093] In summary, the best conditions for root-stem alternate grafting of rubber trees are: 12-month-old rubber seedlings as rootstock, 5 cm long and 7 mm thick roots as middle scion, 5 cm long dormant stem segments as terminal scion, and the best effect of grafting in spring.

[0094] Second embodiment

[0095] Based on the first embodiment, the purpose of this embodiment test is to show that through root-stem alternate grafting, the excellent rootstock of rubber trees can be preserved.

[0096] In the Wufen Plant of Mengla Farm, Xishuangbanna Prefecture, there is a PR107 strain of three-leaf rubber tree that produces 107.8 kg of dry rubber per year, which is 30 times that of ordinary rubber trees, and the single plant yield has set a record in the history of rubber planting, known as the "King of Rubber Tree". Many breeding experts and scholars have tried to replicate the "King of Rubber Tree"; however, grafting the branches of the King of Rubber Tree cannot achieve high yield. It is speculated that this may be due to the excellent rootstock and the optimal interaction between rootstock and scion. However, for a long time, breeding experts have not been able to replicate and propagate the "King of Rubber Tree" rootstock. On October 20, 2023, we took the surface lateral roots of the "King of Rubber Tree", cut them into 5 cm root segments as middle scions, and grafted them using the root-stem alternate grafting method. Due to the protection of the "King of Rubber Tree", we were unable to take a large sample, and only grafted 50 plants. Due to seasonal limitations, only 7 plants survived, with a survival rate of 7 / 50 = 14%. Currently, the plants are growing well, with a height of more than 2.5 m and a diameter of more than 2 cm. Figure 6 This experiment successfully transplanted the excellent rootstock of the "King of Rubber Tree" from the ground to the ground, away from soil microorganisms, and initially achieved detoxification and proliferation, laying the foundation for tissue culture of excellent rootstock.

[0097] Third embodiment

[0098] Based on the first embodiment, the purpose of this embodiment test is to show that through root-stem alternate grafting, the excellent rootstock of rubber trees can be preserved.

[0099] The previous inventors obtained transformed RUBY and GUS reporter gene rubber tree roots by Agrobacterium rhizogenes-mediated transformation method (patent application number: 2024116714058), but they could not achieve the preservation, propagation and phenotype identification of transgenic roots. Therefore, we made the transgenic roots into intermediate scions in vitro, and successfully preserved, transplanted and cultivated the transgenic roots by the root-stem alternate grafting method. Because the transgenic roots were weak and few in number, a total of 30 plants were grafted, and 5 plants survived, with a survival rate of 5 / 30 = 16.67% Figure 7 )

[0100] Fourth embodiment

[0101] Based on the first embodiment, the purpose of this embodiment is to show that auxin induction can make the rootstock root proliferate.

[0102] The root-stem alternate grafting method can achieve the transplantation and long-term preservation of roots, but cannot achieve root proliferation; in order to achieve the proliferation of excellent germplasm roots of rubber trees, we tried to induce the regeneration of lateral roots of grafted roots by exogenous NAA spraying. Therefore, the intermediate scion-root was wrapped with cotton, and 100 mg / L and 200 mg / L NAA were continuously sprayed every 3 days. The results showed that water treatment did not produce aerial roots, 17 / 30 = 56.67% of the plants produced aerial roots after 2 months of 100 mg / L NAA treatment; 22 / 30 = 73.33% of the plants produced aerial roots after 2 months of 200 mg / L NAA treatment Figure 8 ). In summary, this study achieved the transplantation, preservation and proliferation of excellent germplasm roots by alternate grafting and growth hormone treatment.

[0103] In summary, this study successfully established a root-stem alternate grafting method using rubber seedlings as rootstock. This method uses rubber tree root segments as intermediate scions grafted between the rootstock and the terminal scion, and does not limit the types of rootstock, intermediate scion and terminal scion, which can achieve the fusion of multiple types of rubber tree plants, the transplantation of roots and the in vivo preservation of excellent rootstock resources. Although the survival rates differ in different seasons and indoors and outdoors, grafted plants can be obtained, and the state of the rootstock, the length and diameter of the intermediate scion, and the development period and length of the terminal scion all have an impact on the survival rate of grafting. Using this method, the same or different types of rubber trees can be treated within 2 months to achieve the fusion of root segments and stem segments of the same or different types of rubber trees, and the grafted root segments can be induced to proliferate roots by continuous treatment with auxin. Later, combined with gene editing, RNAi and other technologies, the function of rubber tree genes can be simply and quickly studied, and technical support is provided for the replication of "rubber king trees".

[0104] It is to be understood that even though numerous characteristics and embodiments of the application have been set forth in the foregoing disclosure, the details can be varied without departing from the application, it is therefore desired that the appended claims be construed to cover all such modifications and changes as fall within the true spirit and scope of the application.

[0105] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A method for the stolonary grafting of rubber trees, characterized in that, The method comprises the following steps: S1, preparing a stock plant: seeds of mature rubber trees are sown to obtain rubber tree seedlings, and the rubber tree seedlings are transplanted and cultivated, and the seedlings are cultivated for 12 months to serve as the stock plant; S2, preparing a scion: a root section of a rubber tree is cleaned and cut into sections to serve as an intermediate scion, and a stem section of a rubber tree serves as a terminal scion; The root section is a lateral root of a mature rubber tree or a main root of a rubber tree seedling, has a length of 5 cm, and has a diameter of 7 mm; The stem section is a stem section of a rubber tree with a terminal bud, and all leaves are removed, has a length of 5 cm, and has a diameter of 7 mm; The terminal bud is in a dormant period or has just broken dormancy; S3, grafting the intermediate scion obtained in S2 to the morphological upper end of the stock plant in S1, and grafting the terminal scion to the morphological upper end of the intermediate scion, to form a to-be-fused plant after grafting, and the to-be-fused plant is cultivated under dark conditions for 20-30 days to obtain a rhizome-alternate-grafted fused plant; The grafting method is a wedge grafting method, the cut is a "V" cut, and the cut is wrapped and moisturized with sealing film and fresh-keeping film after wedge grafting, and the grafting is started in February-April; S4, inducing aeration roots to regenerate: the fused plant obtained in S3 is cultivated under dark and moisturizing conditions through auxin induction to regenerate aeration roots.

2. The method of claim 1, wherein, The cut is located at a position 40-60 cm away from the ground of the stock plant.

3. The method of claim 1, wherein, The specific steps of cultivation in S3 are as follows: the to-be-fused plant is cultured under dark conditions for 20-25 days, and then the fresh-keeping film and the sealing film are removed, the to-be-fused plant is cultured under dark conditions for another 5 days, and then the to-be-fused plant is transferred to a normal growth environment and continuously watered and fertilized; the dark conditions include a temperature of 20-30°C, a humidity of 60%-80%, and a light intensity of less than 100 μmol / m² / s; and the normal growth environment includes a temperature of 25-35°C, a humidity of 60%-90%, and a light intensity of greater than 300 μmol / m² / s.

4. The method of claim 1, wherein, The specific steps of inducing aeration roots to regenerate in S4 are as follows: 100 mg / L or 200 mg / L auxin is applied to the fused plant under dark and moisturizing conditions, and the treatment is continuously performed for 2 months, and the treatment frequency is 3 times per day.

5. The method of claim 1, wherein, The dark and moisturizing conditions include a temperature of 20-30°C, a humidity of 60%-80%, and a light intensity of less than 100 μmol / m² / s.

6. The method according to any one of claims 1 to 5, characterized in that, The auxin is NAA or IBA.

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