A tissue culture rapid propagation medium and method for direct differentiation of eucalyptus tereticornis

Abstract

The application belongs to the technical field of plant tissue culture and specifically relates to a tissue culture and rapid propagation medium and method for direct differentiation of Eucalyptus robusta. The application provides a tissue culture and rapid propagation medium for direct differentiation of Eucalyptus robusta, which comprises an adventitious bud induction medium and a rooting medium. The application also provides a tissue culture and rapid propagation method for direct differentiation of Eucalyptus robusta, which comprises the following steps: subjecting young twigs of Eucalyptus robusta to aseptic treatment, inoculating the twigs into a rooting medium to culture and obtain aseptic tissue culture seedlings; dividing leaves of the aseptic tissue culture seedlings into small pieces, inoculating the small pieces into an adventitious bud induction medium to culture and obtain adventitious buds; dividing the adventitious buds into stem segments with one axillary bud, inoculating the stem segments into the rooting medium to form new complete plants. The tissue culture and rapid propagation method for Eucalyptus robusta provided by the application directly induces adventitious buds without passing through a callus stage, has the characteristics of rapid induction, high adventitious bud induction rate, high leaf proliferation coefficient, high rooting rate, and strong regenerated seedlings.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant tissue culture and specifically relates to a tissue culture and rapid propagation medium and a tissue culture and rapid propagation method for Populus qiongdaoensis direct differentiation. BACKGROUND

[0002] Populus qiongdaoensis is a deciduous large tree of the Populus in the Salicaceae family, is a characteristic distribution species of Populus in the tropical region, and has important genetic evolution research value. Meanwhile, Populus qiongdaoensis likes light, has strong fast-growing and sprouting ability, can be used as a pioneer tree species for ecological restoration, and the wood is suitable for making pulp, plywood, fiberboard and the like, and has high economic and ecological research value. The existing natural resources are few, and the natural population is small, and natural regeneration is difficult, and Populus qiongdaoensis has been in an endangered state. Therefore, it is of great significance to carry out research on the preservation and utilization of Populus qiongdaoensis germplasm resources. Existing research shows that Populus qiongdaoensis can be asexually propagated through high-branch layering and tender branch cutting technology, however, the survival rate of cutting is low and is affected by many exogenous factors such as time, substrate and hormone treatment, the culture time is long, the multiplication rate of cutting is low, and the preservation and utilization of Populus qiongdaoensis germplasm resources are difficult. SUMMARY

[0003] The application provides a tissue culture and rapid propagation medium for Populus qiongdaoensis direct differentiation, a culture method of aseptic tissue culture seedlings and a tissue culture and rapid propagation method, and has high adventitious bud induction rate, short culture time and high leaf proliferation coefficient.

[0004] To solve the above technical problems, the application provides the following technical solutions.

[0005] The application provides a tissue culture and rapid propagation medium for Populus qiongdaoensis direct differentiation, and the tissue culture and rapid propagation medium comprises an adventitious bud induction medium and a rooting medium.

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

[0007] The rooting medium takes 1 / 2MS as a basic medium and further comprises IBA 0.4-0.6 mg / L, 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, and the pH value of the rooting medium is 5.5-6.0.

[0009] The application provides a tissue culture and rapid propagation method for Populus qiongdaoensis direct differentiation, adopts the tissue culture and rapid propagation medium according to the above technical solutions, and comprises the following steps:

[0010] The stem segment of Populus yunnanensis is inoculated into rooting culture medium to obtain aseptic tissue culture seedling;

[0011] The leaf of the aseptic tissue culture seedling of Populus yunnanensis is inoculated into adventitious bud induction culture medium to obtain an adventitious bud; the leaf is the 3rd-6th leaf from the top to the root direction;

[0012] The adventitious bud is transferred into rooting culture medium to perform rooting culture to obtain a rooting seedling.

[0013] Preferably, the stem segment further comprises sequentially performing cleaning and disinfection before inoculation; the cleaning comprises flushing with tap water for 4-6 hours; the disinfection comprises soaking in alcohol with a volume concentration of 75% for 20-40 seconds, washing with sterile water for 3-5 times, soaking in NaClO solution with an effective chlorine content of 2% for 4-6 minutes, and washing with sterile water for 3-5 times.

[0014] Preferably, the leaf is inoculated after being cut into small pieces; the size of the small piece of leaf is 0.5-1.5 cm x 0.5-1.5 cm.

[0015] Preferably, the temperature of the induction culture and the rooting culture is 21-23 ℃ respectively.

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

[0017] The light time in the light culture is 15-17 hours per day respectively; the light intensity is 2400-2600 lx respectively.

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

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

[0020] Preferably, the length of the adventitious bud is 3.0-5.0 cm.

[0021] The present application provides a tissue culture and rapid propagation culture medium for direct differentiation of Populus yunnanensis; in the adventitious bud induction culture medium of the tissue culture and rapid propagation culture medium, the cell division element zeatin (ZT) and the 6-BA promote cell division; the auxin NAA cooperates with 6-BA and ZT to regulate the organ development direction; a high cell division element / auxin ratio is beneficial to inducing leaf redifferentiation of Populus yunnanensis to form an adventitious bud; in the rooting culture medium of the tissue culture and rapid propagation culture medium, the IBA can promote cell division and cell growth to induce the formation of an adventitious root.

[0022] The application also provides a tissue culture and rapid propagation method of Populus xiaojiangensis directly differentiated, the application takes Populus xiaojiangensis branch stem segments as explants, obtains sterile seedlings through disinfection, and then directly induces adventitious buds from leaf blades and petioles of the sterile seedlings without a callus stage, the adventitious buds directly differentiated regenerate to form new complete plants, and the method has the characteristics of rapid induction, high adventitious bud induction rate, high leaf blade proliferation coefficient, high rooting rate and strong regenerated seedlings. The application can not only realize rapid breeding of Populus xiaojiangensis seedlings, but also provides a basis for an efficient genetic transformation system and guarantees future breeding work. The application provides an efficient, stable and rapid Populus xiaojiangensis breeding technical system based on leaf blades directly differentiated to form regenerated plants. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 5 is a diagram of adventitious roots formed at the bottom of Populus xiaojiangensis stem segments on the 21st day after inoculation in rooting medium;

[0024] Figure 2 Figure 6 is a diagram of rooting seedlings formed from Populus xiaojiangensis stem segments on the 87th day after inoculation in rooting medium;

[0025] Figure 3 Figure 7 is a diagram of leaves of Populus xiaojiangensis sterile tissue culture seedlings initially inoculated in adventitious bud induction medium;

[0026] Figure 4 Figure 8 is a diagram of adventitious buds formed from leaves of Populus xiaojiangensis sterile tissue culture seedlings inoculated in adventitious bud induction medium for 45 days;

[0027] Figure 5 Figure 9 is a diagram of adventitious buds formed from leaves of Populus xiaojiangensis sterile tissue culture seedlings inoculated in adventitious bud induction medium for 60 days;

[0028] Figure 6 Figure 10 is a diagram of rooting seedlings obtained by inoculating Populus xiaojiangensis adventitious buds in rooting medium;

[0029] Figure 7 Figure 11 is a comparison diagram of adventitious buds formed from leaves of Populus xiaojiangensis sterile tissue culture seedlings inoculated in two different adventitious bud induction media for 50 days. DETAILED DESCRIPTION

[0030] The application provides a tissue culture and rapid propagation medium for cultivating Populus xiaojiangensis directly differentiated, and the tissue culture and rapid propagation medium comprises an adventitious bud induction medium and a rooting medium.

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

[0032] As an optional embodiment, the concentration of 6-BA in the adventitious bud induction medium provided by the application is 0.4-0.6 mg / L, and more preferably 0.5 mg / L; in the specific embodiments of the application, the concentration of 6-BA in the adventitious bud induction medium can 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 application is 4-6 mg / L, and more preferably 5 mg / L; the role of ZT is to promote the differentiation of leaves of the sterile tissue culture seedling of Populus×japonica to form buds. The concentration of NAA in the adventitious bud induction medium provided by the application is 0.08-0.12 mg / L, and more preferably 0.1 mg / L; the auxin NAA cooperates with the cytokinin 6-BA and ZT to regulate the direction of organ development, and a high cytokinin / auxin ratio is conducive to inducing the leaves of the sterile tissue culture seedling of Populus×japonica to redifferentiate to form adventitious buds. The concentration of sucrose in the adventitious bud induction medium provided by the application is 25-35 g / L, and can also be 28-32 g / L, and more preferably 30 g / L. In the specific embodiments of the application, the concentration of sucrose in the adventitious bud induction medium can be 25, 27, 28, 29, 30, 31 or 35 g / L. The concentration of agar in the adventitious bud induction medium provided by the application is 6-8 g / L. In the specific embodiments of the application, the concentration of agar in the adventitious bud induction medium can be 6, 7 or 8 g / L. In the application, the pH value of the adventitious bud induction medium can be 5.5-6.0, and can also be 5.8.

[0033] The rooting medium 1 / 2MS provided in the application is a basic medium, and further comprises IBA 0.4-0.6 mg / L, 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 application 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×japonica, and promote rooting of the adventitious buds of Populus×japonica. The concentration of sucrose in the rooting medium provided by the application is 12-18 g / L, and can also be 13-16 g / L. In the specific embodiments of the application, 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 application is 6-8 g / L. In the specific embodiments of the application, the concentration of agar in the rooting medium can be 6, 7 or 8 g / L. In the application, 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 application both contain sucrose, which can provide carbon source required by plant growth, promote the generation and proliferation of buds, the growth of buds and rooting.

[0036] The tissue culture and rapid propagation medium provided by the application has good application effect, and the leaf blades and petioles of the aseptic seedlings can directly induce Populus heterophylla adventitious buds without passing through a callus stage, the directly differentiated adventitious buds regenerate to form new complete Populus heterophylla plants, the somatic clonal variation of the system is small, the genetic stability is high, the culture cycle is short, the operation is simple, the adventitious bud induction rate and the leaf blade proliferation coefficient can be improved.

[0037] The application does not have special limitations on the sources of the components in the adventitious bud induction medium and the rooting medium, and conventional commercially available products can be used.

[0038] The application provides a tissue culture and rapid propagation method of Populus heterophylla direct differentiation, and the method comprises the following steps:

[0039] The stem segment of Populus heterophylla is inoculated into the rooting medium for culture to obtain aseptic tissue culture seedlings.

[0040] The leaves of the aseptic tissue culture seedlings of Populus heterophylla are inoculated into the adventitious bud induction medium for induction culture to obtain adventitious buds, and the leaves are the 3rd to 6th leaves from the top to the root direction of the aseptic tissue culture seedlings.

[0041] The adventitious buds are transferred into the rooting medium for rooting culture to obtain rooted seedlings.

[0042] The stem segment of Populus heterophylla is inoculated into the rooting medium for culture to obtain aseptic tissue culture seedlings.

[0043] As an optional implementation manner, the preparation method of the stem segment of the application comprises the following steps: cutting off leaves of the branch of Paulownia elongata, and cutting the branch into stem segments with bud points; the length of the stem segment of the Paulownia elongata is 7-9 cm, and more preferably 8 cm. The branch of the Paulownia elongata in the application is a semi-lignified branch of one year, and as long as it is a semi-lignified branch of one year newly grown on the plant, there is no special limitation to the source of the semi-lignified branch. The stem segment in the application comprises at least one bud point; the method further comprises the following steps before inoculation: sequentially cleaning and disinfecting the stem segment with the bud; the cleaning method is not specially limited in the application, and a conventional method can be used. In the specific embodiment of the application, the stem segment is washed with tap water for 5-6 hours.

[0044] As an optional implementation manner, after the disinfection of the stem segment, the stem segment of the Paulownia elongata is inoculated into a rooting culture medium to culture, so as to obtain sterile tissue culture seedlings. The inoculation method is not specially limited in the application, and a conventional method can be used. As an optional implementation manner, the culture temperature is 21-23℃, and more preferably 22℃; the culture time is 80-95 days. As an optional implementation manner, the culture is light culture. As an optional implementation manner, the light culture time is 15-17 hours per day, and more preferably 16 hours per day; the light culture intensity can be 2400-2600 lx, and more preferably 2500 lx.

[0045] As an optional implementation manner, when the height of the sterile tissue culture seedling is 8-12 cm, the leaves of the sterile tissue culture seedling are cut off and inoculated into an adventitious bud induction culture medium to culture. The leaves in the application are the 3rd-6th leaves from the top to the root direction of the sterile tissue culture seedling, and the 3rd-6th leaves are selected because they are tender leaves and are easy to differentiate.

[0046] As an optional embodiment, before inoculating in the adventitious bud inducing medium, the leaves are cut into small pieces, and the size of the cut leaves is not specially limited in the present application, and the conventional size can be adopted. In the specific embodiment of the present application, the size of the cut leaves is 0.5-1.5 cm x 0.5-1.5 cm. The temperature of the induction culture is 21-23℃, and can also be 22℃. The time of the induction culture is 55-65 d, and can also be 58-60 d. The induction culture is light culture, the light time of the light culture is 15-17 h / d, and can also be 14-16 h / d; the intensity of the light culture is 2400-2600 lx, and more preferably 2500 lx. In the induction culture process, the edges of the leaves and petioles of Populus yunnanensis are induced to differentiate to form the adventitious buds of Populus yunnanensis; and the induction culture is preferably stopped until the height of the cluster buds is 3-5 cm.

[0047] As an optional embodiment, when the adventitious buds grow to 3-5 cm, the adventitious buds are separated from the edges of the leaves or petioles, inoculated in the rooting medium, and subjected to rooting culture. The rooting culture can root in about 15-20 d, and after rooting, the culture is continued under the original culture condition for a period of time to obtain the rooted seedlings.

[0048] As an optional embodiment, the temperature of the rooting culture is 21-23℃, and can also be 22℃; the time of the rooting culture is 58-65 d, and can also be 60-62 d. The rooting culture is light culture, the light time of the light culture is 15-17 h / d, and preferably 16 h / d; the intensity of the light culture is 2400-2600 lx, and can also be 2500 lx. The rooting culture is stopped until the height of the tissue culture seedlings reaches about 10 cm. The rooting culture of the present application makes the aseptic seedlings grow into strong roots, and the rooting rate of the adventitious buds approaches 100%.

[0049] The present application takes the annual branch stem segments of Populus yunnanensis as the explants, obtains the aseptic seedlings through disinfection and culture, and then directly induces the adventitious buds from the leaves and petioles of the aseptic seedlings without the callus stage. The directly differentiated adventitious buds are cut into stem segments with one axillary bud, inoculated in the rooting medium for rooting culture, and regenerated to form new complete plants. The Populus yunnanensis tissue culture rapid propagation method provided by the present application directly induces the adventitious buds without the callus stage, and has the characteristics of rapid induction, high induction rate, large multiplication rate, high rooting rate, and strong regenerated seedlings.

[0050] The prior art uses stem segments of tissue culture seedlings for propagation, but one bud point on the stem segment can only propagate one plant using rooting medium, and the propagation coefficient is low. The present application uses leaves of tissue culture seedlings for tissue culture, and since the number of leaves of one plant is large and each leaf can propagate more plants, the leaf direct differentiation method of the present application is used for propagation of Populus junnanensis, and the leaf proliferation coefficient is high. The present application not only realizes rapid breeding of Populus junnanensis seedlings, but also provides a basis for an efficient genetic transformation system and provides a guarantee for future breeding work.

[0051] The technical scheme of the present application adopts a direct differentiation regeneration system. This method is short in experimental period and simple in operation because the process of inducing callus is omitted. Since the plants directly differentiate buds without dedifferentiation, the somatic clonal variation of the system is small, the genetic stability of the recipient plants is maintained, and the genetic stability of the transformed exogenous genes is also maintained. The genetic transformation culture of the direct differentiation approach is stable and has a very low variation rate.

[0052] The Populus junnanensis semi-lignified branches used in the following examples are of the same batch.

[0053] Example 1

[0054] 1. Disinfection. Take semi-lignified branches of Populus junnanensis grown from leaf axils, which are one year old, remove the leaves and cut the branches into small branches with a length of about 8 cm. Then immerse the small branches in a glass beaker with a detergent solution for 10 min, and then rinse them with flowing tap water for about 4 h.

[0055] Place the preliminarily washed Populus junnanensis small branches in a sterile beaker in an ultra-clean bench, immerse them in 75% alcohol for 30 s, then wash them with sterile water for 3 times, immerse them in a NaClO solution with an effective chlorine content of 2% for 5 min while stirring constantly, and finally wash them with sterile water for 4 times.

[0056] 2. Rooting culture. Cut the disinfected Populus junnanensis small branches into stem segments with a length of about 3 cm, and each stem segment contains at least one bud point. Inoculate the stem segments into a rooting medium. The rooting medium is composed of 1 / 2MS as a basic medium, and also contains IBA 0.5 mg / L, agar 6 g / L and sucrose 15 g / L, and the pH value of the rooting medium is 5.8. Then culture under the conditions of a temperature of 22±1℃, a light time of 16 h / d and a light intensity of 2500 lx. The stem segment forms adventitious roots at the bottom on the 21st day after inoculation into the rooting medium (as shown in Figure 1 ), and obtains healthy rooting seedlings with a height of 10 cm on the 87th day (as shown in Figure 2 ).

[0057] 3. Adventitious bud induction and rooting culture. The 3rd to 6th leaves (including petiole) from the top to the root of the rooted seedling of Populus adenopoda × P. nigra (i.e. the aseptic tissue culture seedling) were taken off, cut into small pieces of 0.5-1.5 cm x 0.5-1.5 cm, and inoculated into the adventitious bud induction medium YD1 (the state after inoculation is shown in Fig. 1). Figure 3 The composition of the adventitious bud induction medium YD1 is: 1 / 2MS as the basic medium, further containing 6-BA 0.5 mg / L, NAA 0.1 mg / L, ZT 5 mg / L, agar 6 g / L and sucrose 30 g / L, and the pH value of the medium is 5.8. The adventitious bud induction culture was carried out at a culture temperature of 22±1°C, a light time of 16 h / d and a light intensity of 2500 lx. On the 45th day of the adventitious bud induction culture, the edges of the leaves and petioles induced differentiation to form the adventitious buds of Populus adenopoda x P. nigra as shown in Fig. 2. Figure 4

[0058] The adventitious buds were separated from the edges of the leaves or petioles when they grew to 3-5 cm as shown in Fig. 3, inoculated into the rooting medium respectively, and cultured at a culture temperature of 22±1°C, a light time of 16 h / d and a light intensity of 2500 lx for 60 d to obtain the rooted seedlings as shown in Fig. 4. The rooted seedlings grew vigorously, all the adventitious buds inoculated into the rooting medium rooted, and the rooting rate of the adventitious buds was 100%. From the inoculation of the leaves into the adventitious bud induction medium to the obtaining of the seedlings of Populus adenopoda x P. nigra by rooting, it took a total of 120 d. Figure 5 Figure 6 Comparative Example 1

[0059] 1. Sterilization. The same as in Example 1.

[0060] 2. Rooting culture. The same as in Example 1.

[0061] 3. Adventitious bud induction and rooting culture. The same as in Example 1, except that the composition of the adventitious bud induction medium YD2 is: 1 / 2MS as the basic medium, further containing 6-BA 0.5 mg / L, NAA 0.1 mg / L, agar 6 g / L and sucrose 30 g / L, and the pH value of the medium is 5.8. When the adventitious buds grew to 3-5 cm, the induction rate of the adventitious buds was counted. The comparison chart of the adventitious buds formed by inoculating the leaves of the aseptic tissue culture seedlings of Populus adenopoda x P. nigra in Example 1 and Comparative Example 1 into two different adventitious bud induction media for 50 d is shown in Fig. 5, in which the left side represents Example 1 and the right side represents Comparative Example 1.

[0062] The induction rates of the adventitious buds in Example 1 and Comparative Example 1 were counted. Figure 7 Figure 7 The induction rates of the adventitious buds in Example 1 and Comparative Example 1 were counted.

[0063] The induction rates of the adventitious buds in Example 1 and Comparative Example 1 were counted.

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

[0065] In Example 1, the leaf blades (including petioles) of Populus deltoids Bartr. induced adventitious buds in the adventitious bud induction medium at an induction rate of about 57.1%. In Comparative Example 1, the leaf blades (including petioles) of Populus deltoids Bartr. induced adventitious buds in the adventitious bud induction medium at an induction rate of about 16%.

[0066] The multiplication of the leaf blades in Example 1 and Comparative Example 1 was calculated as follows:

[0067] The multiplication of the leaf blades of Populus deltoids Bartr. was calculated according to the following formula:

[0068] Y = m x n x p,

[0069] wherein m represents the number of leaf blades available for differentiation per plant, i.e. the number of leaf blades (including petioles) from the 3rd to 6th leaf blades from the top to the root of the rooted seedlings (i.e. aseptic tissue culture seedlings) of Populus deltoids Bartr., ≥ 4;

[0070] n represents the number of leaf pieces obtained by dividing each leaf blade, ≥ 9;

[0071] p represents the adventitious bud induction rate.

[0072] In the calculation of the present application, m is 4, n is 9, and both m and n are calculated by taking the minimum value, and the results are as follows:

[0073] In Example 1, when p = 57.1%, the number of complete plants that can be proliferated from one aseptic seedling of Populus deltoids Bartr. in about 120 days is: Y = 4 x 9 x 57.1% ≈ 21.

[0074] In Comparative Example 1, when p = 16%, the number of complete plants that can be proliferated from one aseptic seedling of Populus deltoids Bartr. in about 120 days is: Y = 4 x 9 x 16% ≈ 6.

[0075] In summary, the semi-lignified branches newly grown from the leaf axils of Populus deltoids Bartr. are subjected to explant disinfection. The disinfected explants are cultured in a rooting medium to obtain aseptic seedlings. When the aseptic seedlings grow to about 10 cm, the leaf blades and petioles are divided into small pieces, inoculated into an adventitious bud induction medium and cultured under light to directly induce adventitious buds. When the adventitious buds grow to about 3-5 cm, they are inoculated into a rooting medium to culture and obtain rooted seedlings.

[0076] The present application utilizes the adventitious bud induction medium to directly induce the adventitious buds from the leaf of Populus deltoids Bartr, effectively shortens the tissue culture time and simplifies the operation steps; the method provided by the present application can proliferate a piece of leaf into multiple seedlings, breaks through the limitation that one seed can only breed one seedling, and one Populus deltoids Bartr sterile seedling can proliferate at least 21 complete plants through the leaf differentiation in about 120 days (from the leaf inoculation to the adventitious bud induction medium to the rooting to obtain the Populus deltoids Bartr seedling).

[0077] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A tissue culture medium for rapid propagation of Populus tomentosa in Hainan Island, characterized in that, The tissue culture rapid propagation medium includes an adventitious shoot induction medium and a rooting medium; The adventitious bud induction medium consists of 1 / 2 MS medium, 6-BA 0.4~0.6 mg / L, NAA 0.08~0.12 mg / L, ZT 4~6 mg / L, sucrose 25~35 g / L and agar 6~8 g / L; The rooting medium consists of 1 / 2 MS medium, IBA 0.4~0.6 mg / L, sucrose 12~18 g / L and agar 6~8 g / L; The explants cultured in the adventitious bud induction medium were small leaf pieces cut from aseptic tissue culture seedlings of Populus tomentosa in Hainan Island, with the leaf pieces measuring 0.5~1.5cm × 0.5~1.5cm.

2. The tissue culture rapid propagation culture 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 rapid tissue culture propagation method for direct differentiation of Populus tomentosa from Hainan Island, characterized in that, The tissue culture rapid propagation medium according to claim 1 or 2 includes the following steps: Stem segments of Populus yunnanensis were inoculated into rooting medium and cultured to obtain sterile tissue culture seedlings; Leaves of aseptic tissue culture seedlings of Populus tomentosa were inoculated into an adventitious bud induction medium for induction culture to obtain adventitious buds; the leaves were the 3rd to 6th leaves counting from the top towards the root; the leaves were cut into small pieces and inoculated; the size of the small leaf pieces was 0.5~1.5cm × 0.5~1.5cm. The adventitious buds were transferred to a rooting medium for rooting culture to obtain rooted seedlings.

4. The tissue culture rapid propagation method according to claim 3, characterized in that, Before inoculation, the stem segments are further subjected to cleaning and disinfection in sequence. The cleaning includes rinsing with tap water for 4-6 hours, and the disinfection includes soaking in 75% alcohol for 20-40 seconds, rinsing with sterile water 3-5 times, soaking again in NaClO solution with 2% available chlorine content for 4-6 minutes, and rinsing with sterile water 3-5 times.

5. The rapid tissue culture propagation method according to claim 3, characterized in that, The induction culture and rooting culture were conducted at temperatures of 21~23℃.

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

7. The rapid tissue culture propagation method according to claim 3, 5, or 6, characterized in that, The induction culture time is 55-65 days; the rooting culture time is 58-65 days.

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

9. The tissue culture rapid propagation method according to claim 3, characterized in that, The length of the adventitious bud is 3.0~5.0cm.

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