Tissue culture and rapid propagation culture medium and tissue culture and rapid propagation method for direct differentiation of poplar macrocephalum

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a tissue culture and rapid propagation culture medium and a tissue culture and rapid propagation method for direct differentiation of poplar macrocephala. The invention provides a tissue culture and rapid propagation culture medium for direct differentiation of poplar macrocephala. The tissue culture and rapid propagation culture medium comprises an adventitious bud induction culture medium and a rooting culture medium. The invention also provides a tissue culture and rapid propagation method for direct differentiation of the poplar macrocephalum, which comprises the following steps: carrying out sterile treatment on young and tender branchlets of the poplar macrocephalum, and inoculating the young and tender branchlets into a rooting culture medium for culture to obtain sterile tissue culture seedlings; cutting leaves of the sterile tissue culture seedlings of the poplar macrocephalum into small pieces, and inoculating the small pieces into an adventitious bud induction culture medium for culture to obtain adventitious buds; and cutting the adventitious bud into a stem section with an axillary bud, and inoculating the stem section on a rooting culture medium to form a new complete plant. According to the tissue culture and rapid propagation method for the poplar macrocephala, adventitious buds are directly formed without a callus stage through induction, and the tissue culture and rapid propagation method has the advantages of being rapid in induction, high in adventitious bud induction rate, high in leaf multiplication coefficient, high in rooting rate, robust in regenerated seedlings and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a tissue culture rapid propagation medium and a tissue culture rapid propagation method for direct differentiation of Populus chinensis. Background Art

[0002] Qiongdao poplar (Populus qiongdaoensis) is a large deciduous tree of the genus Populus in the Salicaceae family. It is a characteristic distribution species of poplar in tropical areas and has important genetic evolution research value. At the same time, Qiongdao poplar likes light, has fast growth and strong germination ability, can be used as a pioneer tree species for ecological restoration, and its wood is suitable for making pulp, plywood, fiberboard, etc., with high economic and ecological research value. The existing natural resources are few, and the natural population is small, natural regeneration is difficult, and it is already in an endangered state. Therefore, it is of great significance to carry out research on the preservation and utilization of Qiongdao poplar germplasm resources. Existing studies have shown that Qiongdao poplar can be asexually propagated through high-branch layering propagation technology and softwood cutting technology. However, the survival rate of cuttings is low and is affected by multiple exogenous factors such as time, substrate, hormone treatment, etc. The cultivation time is long and the proliferation multiple of cuttings is low, which causes certain difficulties for the preservation and utilization of Qiongdao poplar germplasm resources. Summary of the invention

[0003] The invention provides a tissue culture rapid propagation medium for direct differentiation of Populus chinensis, a culture method for sterile tissue culture seedlings and a tissue culture rapid propagation method, which have high adventitious bud induction rate, short culture time and high leaf proliferation coefficient.

[0004] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0005] The present invention provides a tissue culture rapid propagation medium for direct differentiation of Populus chinensis, wherein the tissue culture rapid propagation medium comprises an adventitious bud induction medium and a rooting medium;

[0006] The adventitious bud induction medium uses 1 / 2MS as a basic medium, and also includes 0.4-0.6 mg / L 6-BA, 0.08-0.12 mg / L NAA, 4-6 mg / L ZT, 25-35 g / L sucrose and 6-8 g / L agar;

[0007] The rooting culture medium uses 1 / 2MS as the basic culture medium and further comprises 0.4-0.6 mg / L IBA, 12-18 g / L sucrose and 6-8 g / L agar.

[0008] Preferably, the pH value of the adventitious bud induction medium is 5.5-6.0; the pH value of the rooting medium is 5.5-6.0.

[0009] The present invention provides a tissue culture and rapid propagation method for direct differentiation of Populus chinensis, which adopts the tissue culture and rapid propagation medium described in the above technical solution and comprises the following steps:

[0010] The stem segments of Populus chinensis are inoculated into a rooting medium for cultivation to obtain sterile tissue culture seedlings;

[0011] Inoculating the leaves of the sterile tissue culture seedlings of Populus chinensis into an adventitious bud induction medium for induction culture to obtain adventitious buds; the leaves are the third to sixth leaves from the top to the root;

[0012] The adventitious buds are transferred to a rooting medium for rooting culture to obtain rooted plantlets.

[0013] Preferably, the stem segments are sequentially cleaned and disinfected before inoculation, wherein the cleaning comprises rinsing with tap water for 4 to 6 hours, and the disinfection comprises soaking in 75% alcohol by volume for 20 to 40 seconds, washing with sterile water for 3 to 5 times, and then soaking in a NaClO solution with an effective chlorine content of 2% for 4 to 6 minutes, and washing with sterile water for 3 to 5 times.

[0014] Preferably, the leaves are cut into small pieces before inoculation; the size of the small pieces of leaves is 0.5-1.5 cm×0.5-1.5 cm.

[0015] Preferably, the temperatures of the induction culture and the rooting culture are 21-23°C, respectively.

[0016] Preferably, the induction culture and rooting culture are light culture;

[0017] The illumination time in the illumination culture is 15 to 17 h / d; the illumination intensity is 2400 to 2600 lx.

[0018] Preferably, the induction culture time is 55 to 65 days; the rooting culture time is 58 to 65 days.

[0019] Preferably, the length of the stem segment is 7 to 9 cm.

[0020] Preferably, the length of the adventitious buds is 3.0 to 5.0 cm.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a tissue culture rapid propagation medium for direct differentiation of Populus australis, in which the cytokinin zeatin (ZT) and 6-benzylaminopurine (6-BA) in the adventitious bud induction medium of the tissue culture rapid propagation medium promote cell division, the auxin NAA coordinates with 6-BA and ZT to regulate and determine the direction of organ development, and a high cytokinin / auxin ratio is beneficial to inducing the redifferentiation of Populus australis leaves to form adventitious buds; in the rooting medium of the tissue culture rapid propagation medium, indolebutyric acid (IBA) can promote cell division and cell growth to induce the formation of adventitious roots.

[0022] The present invention also provides a tissue culture rapid propagation method for direct differentiation of Populus chinensis. The present invention uses Populus chinensis branches and stems as explants, obtains sterile seedlings through disinfection, and then directly induces adventitious buds from the leaves and petioles of the sterile seedlings without going through the callus stage. The directly differentiated adventitious buds regenerate to form new complete plants, which has the characteristics of rapid induction, high adventitious bud induction rate, high leaf proliferation coefficient, high rooting rate, and strong regenerated seedlings. The present invention can not only realize the rapid propagation of Populus chinensis seedlings, but also provide a basis for an efficient genetic transformation system, providing a guarantee for future breeding work. The present invention provides a set of efficient, stable, and rapid Populus chinensis breeding technology systems based on direct differentiation of leaves to form regenerated plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a picture of the adventitious roots formed at the bottom of the stem segment of Populus chinensis on the 21st day after being inoculated into the rooting medium;

[0024] Figure 2 This is a photo of rooted seedlings formed by stem segments of Populus chinensis on the 87th day after inoculation into rooting medium;

[0025] Figure 3 This is a diagram showing the initial state of leaves of the aseptic tissue culture seedlings of Populus chinensis inoculated into adventitious bud induction medium;

[0026] Figure 4 This is a picture of adventitious buds formed by inoculating the leaves of the sterile tissue culture seedlings of Populus chinensis into adventitious bud induction medium and culturing for 45 days;

[0027] Figure 5 This is a picture of adventitious buds formed by inoculating the leaves of the sterile tissue culture seedlings of Populus chinensis into adventitious bud induction medium and culturing for 60 days;

[0028] Figure 6 This is a diagram showing the state of rooted seedlings obtained by inoculating adventitious buds of Populus chinensis into rooting medium;

[0029] Figure 7 This is a comparison of the adventitious buds formed after the leaves of the sterile tissue culture seedlings of Populus Qiongdao were inoculated into two different adventitious bud induction media and cultured for 50 days. DETAILED DESCRIPTION

[0030] The invention provides a tissue culture rapid propagation medium for culturing direct differentiation of Populus australis. The tissue culture rapid propagation medium comprises an adventitious bud induction medium and a rooting medium.

[0031] The adventitious bud induction medium of the present invention uses 1 / 2MS as the basic medium and further comprises 0.4-0.6 mg / L 6-BA, 0.08-0.12 mg / L NAA, 4-6 mg / L ZT, 25-35 g / L sucrose and 6-8 g / L agar.

[0032] As an optional embodiment, the concentration of 6-BA in the adventitious bud induction medium provided by the present invention is 0.4-0.6 mg / L, more preferably 0.5 mg / L; in a specific embodiment of the present invention, the concentration of 6-BA in the adventitious bud induction medium may be 0.4, 0.5 or 0.6 mg / L. 6-BA promotes cell division and proliferation, and improves the induction rate of buds. The concentration of ZT in the adventitious bud induction medium provided by the present invention is 4-6 mg / L, more preferably 5 mg / L; the function of ZT is to promote the differentiation of leaves of the sterile tissue culture seedlings of Populus chinensis to form buds. The concentration of NAA in the adventitious bud induction medium provided by the present invention is 0.08-0.12 mg / L, more preferably 0.1 mg / L; the auxin NAA synergistically regulates with the cytokinin 6-BA and ZT to determine the direction of organ development, and a high cytokinin / auxin ratio is conducive to inducing the redifferentiation of leaves of the sterile tissue culture seedlings of Populus chinensis to form adventitious buds. The concentration of sucrose in the adventitious bud induction medium provided by the present invention is 25-35 g / L, or 28-32 g / L, more preferably 30 g / L. In a specific embodiment of the present invention, the concentration of sucrose in the adventitious bud induction medium may be 25, 27, 28, 29, 30, 31 or 35 g / L. The concentration of agar in the adventitious bud induction medium provided by the present invention is 6-8 g / L. In a specific embodiment of the present invention, the concentration of agar in the adventitious bud induction medium may be 6, 7 or 8 g / L. In the present invention, the pH value of the adventitious bud induction medium may be 5.5-6.0, or 5.8.

[0033] The rooting medium 1 / 2MS of the present invention is a basic medium, and further comprises 0.4-0.6 mg / L IBA, 12-18 g / L sucrose and 6-8 g / L agar.

[0034] As an optional embodiment, the concentration of IBA in the rooting medium provided by the present invention is 0.4-0.6 mg / L, and can also be 0.45-0.55 mg / L, and more preferably 0.5 mg / L. IBA can promote cell division and cell growth of Populus australis, and promote rooting of adventitious buds of Populus australis. The concentration of sucrose in the rooting medium provided by the present invention is 12-18 g / L, and can also be 13-16 g / L. In a specific embodiment of the present invention, the concentration of sucrose in the rooting medium can be 12, 13, 15, 16, 17 or 18 g / L. The concentration of agar in the rooting medium provided by the present invention is 6-8 g / L. In a specific embodiment of the present invention, the concentration of agar in the rooting medium can be 6, 7 or 8 g / L. In the present invention, the pH value of the rooting medium can be 5.5-6.0, and more preferably 5.8.

[0035] The adventitious bud induction medium and the rooting medium provided by the invention both contain sucrose, which can provide a carbon source required for plant growth and promote the generation and proliferation of buds, and the growth and rooting of buds.

[0036] The tissue culture rapid propagation culture medium provided by the invention has good application effect. Leaves and petioles of sterile seedlings can be directly induced to form adventitious buds of Populus villosa without going through the callus tissue stage. The directly differentiated adventitious buds can be regenerated to form new complete Populus villosa plants. The somatic cell asexual system of the system has small variation and high genetic stability. In addition, the culture cycle is short and the operation is simple, and the adventitious bud induction rate and the proliferation coefficient of leaves can be improved.

[0037] The present invention has no special limitation on the sources of the components in the adventitious bud induction medium and the rooting medium, and conventional commercial products can be used.

[0038] The present invention provides a tissue culture and rapid propagation method for direct differentiation of Populus chinensis, which adopts the tissue culture and rapid propagation medium described in the above technical solution and comprises the following steps:

[0039] The stem segments of Populus chinensis are inoculated into a rooting medium for cultivation to obtain sterile tissue culture seedlings;

[0040] Inoculating the leaves of the sterile tissue culture seedlings of Populus chinensis into an adventitious bud induction medium for induction culture to obtain adventitious buds; the leaves are the third to sixth leaves from the top to the root of the sterile tissue culture seedlings;

[0041] The adventitious buds are transferred to a rooting medium for rooting culture to obtain rooted plantlets.

[0042] The invention inoculates the stem segments of Populus chinensis into a rooting medium for cultivation to obtain sterile tissue culture seedlings.

[0043] As an optional embodiment, the preparation method of the stem segment of the present invention comprises: cutting off the leaves of the poplar branches, and then cutting them into stem segments with buds; the length of the stem segment of the poplar is 7 to 9 cm, more preferably 8 cm. The poplar branches of the present invention are annual semi-lignified branches, as long as they are annual semi-lignified branches newly grown on the plant, and there is no special limitation on the source of the semi-lignified branches. The stem segment of the present invention includes at least one bud point; the present invention also includes cleaning and disinfecting the stem segment with buds in sequence before inoculation; the present invention does not specifically limit the cleaning method, and conventional methods can be used. In a specific embodiment of the present invention, tap water is used for rinsing, and the rinsing time is 5 to 6 hours. As an optional embodiment, the disinfection of the present invention is first soaked in 75% ethanol by volume for 20 to 40 seconds, or 30 seconds; then rinsed with sterile water for 3 to 4 times; then soaked in sodium hypochlorite solution with an effective chlorine content of 2% for 4 to 6 minutes, preferably soaked for 5 minutes; then rinsed with sterile water for 3 to 5 times. The disinfection method of the present invention is simple, and sterile seedlings can be successfully obtained by using 75% alcohol and NaClO solution. HgCl2 will pollute the environment, and improper use will have adverse effects on the human body. Compared with HgCl2 solution, the disinfection technical solution of the present invention is non-toxic, harmless and low in cost.

[0044] As an optional embodiment, after the stem segments of the present invention are disinfected, the stem segments of Populus chinensis are inoculated into a rooting medium for culture to obtain sterile tissue culture seedlings. The present invention has no special limitation on the inoculation method, and a conventional method can be used. As an optional embodiment, the culture temperature is 21 to 23°C, more preferably 22°C; the culture time is 80 to 95 days. As an optional embodiment, the culture is light culture. As an optional embodiment, the illumination time of the light culture is 15 to 17h / d, preferably 16h / d; the intensity of the light culture can be 2400 to 2600lx, preferably 2500lx.

[0045] As an optional embodiment, when the height of the sterile tissue culture seedling is 8 to 12 cm, the leaves of the sterile tissue culture seedling are cut and inoculated into an adventitious bud induction medium for induction culture. The leaves of the present invention are the 3rd to 6th leaves from the top to the root of the sterile tissue culture seedling. The 3rd to 6th leaves are selected because they are relatively tender leaves and are easy to differentiate.

[0046] As an optional embodiment, before inoculating in the adventitious bud induction medium, the present invention cuts the leaves into small pieces. The present invention does not specifically limit the size of the cut leaves, and a conventional size can be used. In a specific embodiment of the present invention, the size of the cut leaves is 0.5-1.5 cm × 0.5-1.5 cm. The temperature of the induction culture of the present invention is 21-23°C, or 22°C; the time of the induction culture is 55-65 days, or 58-60 days. The induction culture is light culture, and the illumination time of the light culture is 15-17h / d, or 14-16h / d; the intensity of the light culture is 2400-2600lx, more preferably 2500lx. During the induction culture process of the present invention, the edges of the leaves and petioles of Populus chinensis are induced to differentiate into adventitious buds of Populus chinensis; the present invention preferably induces the culture until the height of the clustered buds is 3-5 cm.

[0047] As an optional embodiment, the present invention separates the adventitious buds from the edges of leaves or petioles when the adventitious buds grow to 3 to 5 cm, and inoculates them into a rooting medium for rooting culture. The rooting culture of the present invention can take root on the 15th to 20th day, and after taking root, continue to culture under the original culture conditions for a period of time to obtain rooted seedlings.

[0048] As an optional embodiment, the temperature of the rooting culture of the present invention is 21-23°C, or 22°C; the time of the rooting culture is 58-65d, or 60-62d. The rooting culture is light culture, and the illumination time of the light culture is 15-17h / d, preferably 16h / d; the intensity of the light culture is 2400-2600lx, or 2500lx. The rooting culture is carried out until the height of the tissue culture seedling reaches about 10 cm. The rooting culture of the present invention enables the sterile seedlings to produce strong roots, and the rooting rate of adventitious buds is close to 100%.

[0049] The present invention uses the annual branch stem segments of Populus chinensis as explants, obtains sterile seedlings through disinfection and cultivation, and then directly induces adventitious buds to form from the leaves and petioles of the sterile seedlings without going through the callus stage, directly differentiates the adventitious buds, and then divides the adventitious buds into stem segments with an axillary bud, inoculates them on a rooting medium for rooting culture, and regenerates new complete plants. The Populus chinensis tissue culture rapid propagation method provided by the present invention directly induces the formation of adventitious buds without going through the callus stage, and has the characteristics of rapid induction, high induction rate, large proliferation multiples, high rooting rate, and strong regenerated seedlings.

[0050] In the prior art, stem segments of tissue culture seedlings are used for propagation, but one bud point on the stem segment can only be propagated to obtain one plant by culture with rooting medium, and the propagation coefficient is low. The present invention uses leaves of tissue culture seedlings for tissue culture. Since a plant has a large number of leaves and each leaf can be propagated to obtain more plants, the direct differentiation of leaves of the present invention is used to propagate the poplar, and the proliferation multiple of the leaves is high. The present invention can not only realize the rapid propagation of poplar seedlings, but also provide a basis for an efficient genetic transformation system, and provide a guarantee for future breeding work.

[0051] The technical solution of the present invention adopts a direct differentiation and regeneration system. Since this method omits the process of inducing callus tissue, the experimental cycle is short and the operation is simple. Since the plants that have not undergone dedifferentiation directly differentiate and sprout, the somatic cell asexual system of this system has small variation, and while maintaining the genetic stability of the recipient plant, the genetic stability of the transformed exogenous gene can be better maintained; the genetic transformation culture of the direct differentiation pathway is relatively stable and the variation rate is very low.

[0052] The one-year-old semi-lignified branches of Populus chinensis used in the following examples are from the same batch.

[0053] Example 1

[0054] 1. Disinfection. Take the semi-lignified branches of Populus chinensis growing from the leaf axils. The semi-lignified branches are one-year-old. Remove the leaves from the branches and cut them into twigs. The length of the twigs is about 8 cm. Then, place the twigs in a glass beaker and soak them in detergent solution for 10 minutes, and then rinse them in running tap water for about 4 hours.

[0055] In a clean bench, place the initially cleaned Populus chinensis twigs in a sterile beaker and soak them in 75% alcohol for 30 seconds. Then, wash them three times with sterile water, soak them in 2% NaClO solution for 5 minutes with constant stirring, and finally wash them four times with sterile water.

[0056] 2. Rooting culture. Cut the disinfected Populus australis twigs into stem segments of about 3 cm, each containing at least one bud, and inoculate them in rooting medium. The composition of the rooting medium is: 1 / 2MS as the basic medium, and also contains IBA0.5mg / L, agar 6g / L and sucrose 15g / L. The pH value of the rooting medium is 5.8. Then culture at a temperature of 22±1℃, a light duration of 16h / d, and a light intensity of 2500lx. On the 21st day after inoculation of the rooting medium, adventitious roots (such as Figure 1 On the 87th day, a robust rooted seedling with a height of 10 cm was obtained (as shown in Figure 2 shown).

[0057] 3. Adventitious bud induction and adventitious bud rooting culture. Take the 3rd to 6th leaves (including petioles) from the top to the root of the rooted seedlings (i.e., sterile tissue culture seedlings) of the above-mentioned Populus chinensis, cut them into small pieces with a length of 0.5-1.5 cm × 0.5-1.5 cm, and inoculate them into adventitious bud induction medium YD1 (the state after inoculation is shown in Figure 3 The composition of adventitious bud induction medium YD1 is: 1 / 2MS as the basic medium, and also contains 6-BA 0.5mg / L, NAA 0.1mg / L, ZT 5mg / L, agar 6g / L and sucrose 30g / L, and the pH value of the medium is 5.8. Then, adventitious bud induction culture was carried out under the conditions of culture temperature 22±1℃, light time 16h / d, and light intensity 2500lx. On the 45th day of adventitious bud induction culture, the edges of leaves and petioles were induced to differentiate into adventitious buds of Populus chinensis. Figure 4 shown.

[0058] Continue to culture in the adventitious bud induction medium until the 60th day. When the adventitious buds grow to 3-5 cm, Figure 5 As shown in FIG. 1 , the adventitious buds were separated from the edge of the leaves or petioles and inoculated on the rooting medium, respectively, and cultured for 60 days under the conditions of a culture temperature of 22±1°C, a light duration of 16 h / d, and a light intensity of 2500 lx to obtain rooted seedlings. Figure 6 As shown, the rooted seedlings grew strong, all the adventitious buds inoculated on the rooting medium took root, and the rooting rate of the adventitious buds was 100%. It took a total of 120 days from the inoculation of leaves on the adventitious bud induction medium to the rooting of the poplar seedlings.

[0059] Comparative Example 1

[0060] 1. Disinfection: Same as Example 1.

[0061] 2. Rooting culture: Same as Example 1.

[0062] 3. Adventitious bud induction and adventitious bud rooting culture. Same as Example 1, the only difference is that the composition of the adventitious bud induction medium YD2 is: 1 / 2MS as the basic medium, and also contains 6-BA0.5mg / L, NAA 0.1mg / L, agar 6g / L and sucrose 30g / L, and the pH value of the medium is 5.8. When the adventitious buds grow to 3-5cm, the induction rate of the adventitious buds is counted. The comparison of the adventitious buds formed by the leaves of the aseptic tissue culture seedlings of Populus chinensis of Example 1 and Comparative Example 1 inoculated in two different adventitious bud induction mediums and cultured for 50d is shown in Figure 1. Figure 7 , Figure 7 The left side represents Example 1, and the right side represents Comparative Example 1.

[0063] The adventitious bud induction rates of Example 1 and Comparative Example 1 were statistically analyzed.

[0064] The calculation formula for the adventitious bud induction rate is: adventitious bud induction rate (%) = number of explants producing adventitious buds / total number of inoculated explants × 100%.

[0065] In Example 1, the adventitious bud induction rate of the leaves (including petioles) of Populus chinensis in the adventitious bud induction medium was about 57.1%. In Comparative Example 1, the adventitious bud induction rate of the leaves (including petioles) of Populus chinensis in the adventitious bud induction medium was about 16%.

[0066] The multiplication multiples of the leaves of Example 1 and Comparative Example 1 were calculated as follows:

[0067] The proliferation multiples of the leaves of Populus chinensis were calculated according to the following formula:

[0068] Y = m × n × p,

[0069] Wherein, m represents the number of leaves available for differentiation in each plant, i.e., the number of leaves from the 3rd to 6th leaves (including petioles) from the top to the root of the rooted seedlings (i.e., sterile tissue culture seedlings) of Populus chinensis, ≥4;

[0070] n represents the number of leaflets obtained by segmenting each leaf, ≥9;

[0071] p represents the adventitious bud induction rate.

[0072] When calculating in the present invention, m is taken as 4, n is taken as 9, and both m and n are taken as minimum values ​​for calculation, and the results are as follows:

[0073] In Example 1, when p=57.1%, the number of complete plants that can be obtained by multiplying a sterile seedling of Populus chinensis in about 120 days is: Y=4×9×57.1%≈21 plants.

[0074] In comparative example 1, when p=16%, the number of complete plants that can be obtained by multiplying a sterile seedling of Populus chinensis after about 120 days is: Y=4×9×16%≈6 plants.

[0075] In summary, the present invention sterilizes explants by taking newly grown semi-lignified branches from the leaf axils of Populus chinensis. The sterilized explants are cultured in a rooting medium to obtain sterile seedlings. When the sterile seedlings grow to about 10 cm, the leaves and petioles are cut into small pieces, inoculated on an adventitious bud induction medium and cultured under light to directly induce adventitious buds. When the adventitious buds grow to about 3 to 5 cm, they are inoculated on a rooting medium for rooting culture to obtain rooted seedlings.

[0076] The present invention utilizes an adventitious bud induction culture medium to directly induce the formation of adventitious buds from leaves of Populus chinensis, thereby effectively shortening the tissue culture time and simplifying the operation steps; the method provided by the present invention can enable a leaf to proliferate to form multiple seedlings, breaking through the limitation that one seed can only breed one seedling; a sterile seedling of Populus chinensis can proliferate to obtain at least 21 complete plants through leaf differentiation in about 120 days (from the time when the leaf is inoculated into the adventitious bud induction culture medium to the time when the Populus chinensis seedling is rooted and obtained), and the leaf proliferation coefficient is high.

[0077] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A tissue culture rapid propagation medium for direct differentiation of Populus chinensis, characterized in that: The tissue culture rapid propagation medium includes an adventitious bud induction medium and a rooting medium; The adventitious bud induction medium uses 1 / 2MS as a basic medium, and also includes 0.4-0.6 mg / L 6-BA, 0.08-0.12 mg / L NAA, 4-6 mg / L ZT, 25-35 g / L sucrose and 6-8 g / L agar; The rooting culture medium uses 1 / 2MS as the basic culture medium and further comprises 0.4-0.6 mg / L IBA, 12-18 g / L sucrose and 6-8 g / L agar.

2. The tissue culture rapid propagation medium according to claim 1, characterized in that The pH value of the adventitious bud induction medium is 5.5-6.0; the pH value of the rooting medium is 5.5-6.

0.

3. A tissue culture and rapid propagation method for direct differentiation of Populus chinensis, characterized in that: The method of using the tissue culture rapid propagation medium according to claim 1 or 2 comprises the following steps: The stem segments of Populus chinensis are inoculated into a rooting medium for cultivation to obtain sterile tissue culture seedlings; Inoculate the leaves of the sterile tissue culture seedlings of Populus chinensis into an adventitious bud induction medium for induction culture to obtain adventitious buds; the leaves are the third to sixth leaves from the top to the root; The adventitious buds are transferred to a rooting medium for rooting culture to obtain rooted plantlets.

4. The tissue culture rapid propagation method according to claim 3, characterized in that: The stem segments are also cleaned and disinfected in sequence before inoculation, wherein the cleaning comprises rinsing with tap water for 4 to 6 hours, and the disinfection comprises soaking in 75% alcohol by volume for 20 to 40 seconds, washing with sterile water for 3 to 5 times, and then soaking in a NaClO solution with an effective chlorine content of 2% for 4 to 6 minutes, and washing with sterile water for 3 to 5 times.

5. The tissue culture and rapid propagation method according to claim 3, characterized in that: The leaves were cut into small pieces for inoculation; the size of the small pieces of leaves was 0.5-1.5 cm × 0.5-1.5 cm.

6. The tissue culture and rapid propagation method according to claim 3, characterized in that: The temperatures of the induction culture and the rooting culture are 21-23°C respectively.

7. The tissue culture and rapid propagation method according to claim 3, characterized in that: The induction culture and rooting culture are light culture; The illumination time in the illumination culture is 15 to 17 h / d; the illumination intensity is 2400 to 2600 lx.

8. The tissue culture rapid propagation method according to claim 3, 6 or 7, characterized in that: The induction culture time is 55 to 65 days; the rooting culture time is 58 to 65 days.

9. The tissue culture rapid propagation method according to claim 3, characterized in that: The length of the stem segment is 7 to 9 cm.

10. The tissue culture and rapid propagation method according to claim 3, characterized in that: The length of the adventitious buds is 3.0 to 5.0 cm.

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