An efficient propagation method for Populus europaeus 107

Abstract

The present invention relates to the field of plant propagation technology, and in particular to an efficient propagation method for Populus occidentalis. The method uses one-year-old Populus occidentalis branches as cuttings to induce a large number of adventitious buds. These buds are then cut into stem segments and propagated in a micro-cutting device to induce rooted seedlings. After the rooted seedlings grow taller, they can also be cut into segments for further propagation. Compared with conventional hardwood cuttings, this method significantly increases the number of seedlings cultivated, which is three times the number of conventional hardwood cuttings. n times, has a high reproduction coefficient, saves land space used for reproduction, provides technical support for the efficient promotion and utilization of 107 poplar and the genetic transformation research based on field callus regeneration adventitious buds, and has important application value.

Description

Technical Field

[0001] The present invention relates to the technical field of plant breeding, and in particular to an efficient breeding method for Populus europaeus 107. Background Art

[0002] Populus × euramericana cv. 'Neva', a member of the Salicaceae family (Populus), is a superior clone selected from the "American Black Populus × European Black Populus" clone introduced from Italy. Populus × euramericana cv. 'Neva' is widely suitable for cultivation, being prevalent in the Huanghuai and Haihe River basins. Currently, it is widely cultivated in Northeast and Northwest China. It boasts diverse uses, boasts rapid growth and high yields, and is cold-resistant, boasts high density, a straight trunk, a graceful shape, small lateral branches, dense foliage, strong wind and pest resistance, and is easy to propagate with a high survival rate. Furthermore, Populus × euramericana has a strong budding ability, allowing for large-scale cultivation of seedlings through asexual propagation methods such as cuttings, grafting, layering, and tissue culture, providing a foundation for the development of an in vitro regeneration system for poplars. Compared to tissue culture-based adventitious bud regeneration, field callus regeneration offers greater adaptability. This approach eliminates the need for rooting, hardening, transplanting, and sterilization steps required for inducing adventitious buds through tissue culture. Furthermore, cuttings can be directly obtained for production, enabling a single-step approach. Furthermore, adventitious bud regeneration from cuttings is not only highly adaptable but also allows for clustering, significantly improving propagation efficiency. Furthermore, existing techniques for asexual propagation of Populus 107 remain limited in efficiency and require significant land space. Therefore, establishing an efficient propagation method for Populus 107 is crucial for its widespread utilization and for genetic transformation research based on field callus regeneration. Summary of the Invention

[0003] In response to the above technical problems, the present invention provides an efficient asexual propagation method using one-year-old branches of Populus europaeus 107 as cuttings in combination with a micro-cutting device. The method has a high propagation coefficient and occupies less land space, providing technical support for the efficient promotion and utilization of Populus 107 and the genetic transformation research based on field callus regeneration of adventitious buds.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides an efficient propagation method for Populus europaeus 107, comprising the following steps:

[0006] (1) Preparation of materials before breeding

[0007] One-year-old ungerminated Populus occidentalis 107 was collected as cuttings; the top of the cutting was soaked in a solution containing cytokinin and sealed with wax for later use;

[0008] (2) Cutting and cultivation

[0009] The cuttings treated in step (1) are inserted into a flower pot containing a culture medium; after dark cultivation for 8 to 12 days, they are placed under light and cultured for 26 to 30 days to obtain adventitious buds;

[0010] (3) Adventitious bud cuttings

[0011] When the adventitious buds obtained in step (2) grow to 2 to 3 cm, they are cut off from the cuttings and inserted into a container containing a culture medium. After culturing for 1 to 2 weeks, rooted adventitious buds are obtained;

[0012] (4) Repeated cuttings of adventitious buds

[0013] After the rooting, the adventitious buds are cultured until the plant height reaches 8-9 cm, and then cut into 2-3 cm stem segments and re-inserted into a container containing a culture medium. The culture method is the same as step (3). After 1-2 weeks of culture, rooted seedlings are obtained.

[0014] (5) Transplanting

[0015] Continue to cultivate the rooted seedlings until they are 6 to 8 cm tall and then transplant them to the field.

[0016] Preferably, the length of the cuttings in step (1) is 12 to 16 cm, and the thickness is 0.7 to 2.5 cm.

[0017] Preferably, the cytokinin-containing solution in step (1) contains 0.2-0.8 mg / L cytokinin and 0.05-0.15 mg / L NAA.

[0018] Preferably, the soaking time in step (1) is 12 to 17 minutes.

[0019] Preferably, the dark culture temperature in step (2) is 25° C. to 26° C., and the humidity is 45% to 65%.

[0020] Preferably, the temperature of the culture under light in step (2) is 25° C. to 26° C., and the humidity is 45% to 65%.

[0021] Preferably, the culture medium in step (2), step (3) and step (4) is sand, raw soil and vermiculite, with a mass ratio of 4-6:0.8-1.2:0.8-1.2.

[0022] Preferably, the culture temperature in step (3) is 25° C. to 26° C., and the humidity is 45% to 65%.

[0023] Preferably, the containers containing the culture medium in step (3) and step (4) are both micro-cutting containers.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses one-year-old European and American Poplar 107 branches as cuttings to induce a large number of adventitious buds. After that, the branches are cut into stem segments and inserted into a micro-cutting device to induce rooted seedlings. After the rooted seedlings grow taller, they can also be cut into segments and propagated by cuttings. Compared with conventional hardwood cuttings, this method can significantly increase the number of seedlings cultivated, which is 3 times that of conventional hardwood cuttings. n times (n is the number of repeated cuttings). The present invention provides a highly efficient asexual propagation method using one-year-old branches of Populus europaeus 107 as cuttings in combination with a micro-cutting device. This method has a high propagation coefficient, saves land space used for propagation, and provides technical support for the efficient promotion and utilization of Populus 107 and genetic transformation research based on field callus regeneration of adventitious buds, thus having important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0027] Figure 1 This is a diagram of the state of the cuttings of Populus europaeus 107 being planted in a flowerpot;

[0028] Figure 2 These are images of callus tissue growing from cut branches of Populus occidentalis 107. (A) shows the entire flowerpot, and (B) shows the callus tissue of a single branch.

[0029] Figure 3 These are images of adventitious buds growing from cut branches of Populus occidentalis 107. (A) shows the entire pot, (B) shows the adventitious buds on a single branch. (C) shows the adventitious buds when they are about 2 cm long. (D) shows the adventitious buds when they are about 3 cm long.

[0030] Figure 4 This is a diagram showing the state of adventitious buds being cultured on a micro-cutting device;

[0031] Figure 5 This is a diagram of the rooting state of adventitious buds. DETAILED DESCRIPTION

[0032] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified. The micro-insertion device used in the following examples is from ZL202321141212.2.

[0034] Example 1

[0035] An efficient propagation method for Populus europaeus 107, comprising the following steps:

[0036] (1) Collection and processing of Populus occidentalis 107 branches

[0037] On March 7, 2023, before the spring germination of Populus euphorbiae 107, one-year-old cuttings of Populus euphorbiae 107 seedlings were cut from their bases at the Yanshan Nursery in Cangzhou, Hebei Province. All cuttings were pre-germinated, and small branches were removed, leaving cuttings approximately 2 meters long. The cuttings were then cut into three sections and placed in a water-soaked chamber at the base of the artificial climate chamber at Hebei Agricultural University for two days.

[0038] (2) Preparation of culture medium and cuttings

[0039] Culture medium: sand: original soil: vermiculite (5:1:1);

[0040] Preparation of cuttings: Cut the soaked 107 poplar branches into cuttings with a length of 15 cm and a thickness of 1.5 cm, keeping the cross-section smooth and the cambium intact.

[0041] (3) Processing of cuttings

[0042] Immerse the top section of the cutting into a hormone solution containing cytokinin 6-BA 0.5 mg / L + NAA 0.1 mg / L for 15 minutes, and then seal with wax.

[0043] (4) Cutting and cultivation

[0044] The culture medium was thoroughly mixed and placed in a flower pot with an inner diameter of 15×15×10 mm. The amount of culture medium in each flower pot was ensured to be consistent. Eight treated cuttings were inserted into each flower pot at the same depth. The cuttings were kept in the dark for 10 days at a temperature of 25°C and a humidity of 50% (see Figure 1 Then, the cells were placed in an artificial climate chamber under light, cultured at 25°C and 50% humidity for 28 days, and callus tissue was induced (see Figure 2 ) and adventitious buds (see Figure 3 ), with an average of (43+81+74) / 3=66 buds per cutting.

[0045] (5) Adventitious bud cuttings

[0046] When the adventitious buds on the cuttings grow to 3 cm, cut them off from the cuttings and insert them into a micro-cutting container filled with culture medium. If there are too many leaves, trim them appropriately and keep 3 leaves. Then culture them in an artificial climate chamber (see Figure 4 ) , culture temperature is 25°C, humidity is 50%. Cover with plastic film initially to maintain humidity, remove after one week. Then, water normally. After two weeks of rooting, continue cultivation in the artificial climate chamber.

[0047] (6) Repeated cuttings of adventitious buds

[0048] After 60 days, when the plants in the micro-cutting device have taken root and grown to 9 cm in height, they are cut off and cut into 3 cm stem segments, which are then re-cut into the micro-cutting device and cultured for another 2 weeks. The culture method is the same as step (5). By mid-June, the cuttings can be repeated once, and finally rooted seedlings are obtained (see Figure 5 ).

[0049] (7) Transplanting rooted seedlings to the field

[0050] When the rooted seedlings grow to 7 cm, they can be transplanted to the field and carried out normal water and fertilizer management.

[0051] (8) Calculation of reproduction coefficient

[0052] The number of rooted 107 poplar seedlings that can be propagated from a one-year-old 107 poplar plant using this propagation method is expressed as the propagation coefficient, which is 200 / 15 × the average number of adventitious buds per cutting × (9 / 3). n , after calculation, the reproduction coefficient of this embodiment 1 = 200 / 15×66×(9 / 3) 1 =2640.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An efficient propagation method for Populus europaeus 107, characterized in that: The following steps are involved: (1) Preparation of materials before breeding One-year-old ungerminated Populus europaeus 107 were collected as cuttings; Soak the top of the cutting with a solution containing cytokinin and seal it with wax; (2) Cutting and cultivation The cuttings treated in step (1) are inserted into a flower pot containing a culture medium; after dark cultivation for 8 to 12 days, they are placed under light and cultured for 26 to 30 days to obtain adventitious buds; (3) Adventitious bud cuttings When the adventitious buds obtained in step (2) grow to 2 to 3 cm, they are cut off from the cuttings and inserted into a container containing a culture medium. After culturing for 1 to 2 weeks, rooted adventitious buds are obtained; (4) Repeated cuttings of adventitious buds After the rooting, the adventitious buds are cultured until the plant height reaches 8-9 cm, and then cut into 2-3 cm stem segments and re-inserted into a container containing a culture medium. The culture method is the same as step (3). After 1-2 weeks of culture, rooted seedlings are obtained. (5) Transplanting Continue to cultivate the rooted seedlings until they are 6-8 cm tall and then transplant them to the field. The cytokinin-containing solution in step (1) contains 0.2-0.8 mg / L cytokinin and 0.05-0.15 mg / L NAA; The soaking time in step (1) is 12 to 17 minutes; In step (2), the dark culture temperature is 25° C. to 26° C. and the humidity is 45% to 65%; The temperature of the culture under light in step (2) is 25°C to 26°C, and the humidity is 45% to 65%; The culture medium in step (2), step (3) and step (4) is sand, original soil and vermiculite, and the mass ratio is 4-6:0.8-1.2:0.8-1.

2.

2. The efficient breeding method according to claim 1, wherein The length of the cuttings in step (1) is 12 to 16 cm, and the thickness is 0.7 to 2.5 cm.

3. The efficient breeding method according to claim 1, wherein The culture temperature in step (3) is 25° C. to 26° C., and the humidity is 45% to 65%.

4. The efficient breeding method according to claim 1, characterized in that The containers containing the culture matrix in step (3) and step (4) are both micro-cutting containers.

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