A cultivation method to promote the proliferation and rejuvenation of Albizia julibrissin subgeneration buds

Abstract

This invention discloses a method for promoting the proliferation and rejuvenation of Albizia julibrissin subculture buds. The method includes: dedifferentiation: cutting subculture buds after 22 generations into tissue blocks and inoculating them into callus induction medium, inducing dedifferentiation to form callus for 14-20 days under preset induction conditions; inducing clustered buds: cutting the callus and inoculating it into differentiation medium, placing it under preset differentiation conditions to induce clustered buds; rejuvenation culture: taking the stems or buds of the clustered buds and inoculating them into rejuvenation medium, placing them under preset culture conditions to obtain rejuvenated seedlings; subculture: cutting stem segments from the rejuvenated seedlings and inoculating them into subculture medium, culturing them under preset culture conditions. Compared with the prior art, this invention, through the induction of callus formation in subculture buds after 22 generations – induction of clustered buds – rejuvenation – subculture, restores the differentiation and regeneration reproductive capacity of Albizia julibrissin subculture buds that have shown signs of decline, thereby extending the tissue culture lifespan of Albizia julibrissin subculture buds.

Description

Technical Field

[0001] This invention relates to plant tissue culture, specifically to a culture method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds. Background Technology

[0002] *Albizia odoratissima* (Lf.) Benth, also known as hornwood or blackwood, is a tall deciduous tree belonging to the genus *Albizia* in the legume family. Currently, due to urban expansion and a lack of conservation awareness, wild *Albizia odoratissima* trees have been severely deforested, and natural regeneration is difficult to achieve. To preserve this species or quickly obtain high-quality seedlings, our research institute has conducted extensive research on plant tissue culture for *Albizia odoratissima*, for example:

[0003] CN202010880528.8 discloses a method for rapid propagation of Albizia julibrissin, a high-value timber tree species, through tissue culture. Specifically, it discloses steps such as disinfection of Albizia julibrissin explants, primary induction, subculture, rooting culture, hardening and transplanting, and seedling delivery to achieve an initial bud induction rate of over 97% and a cluster bud induction rate of over 98%, achieving a very good induction effect.

[0004] CN202010880492.3 discloses a method for effectively promoting the subculture and proliferation of Albizia julibrissin. Specifically, it discloses the process of selecting and treating Albizia julibrissin explants, primary induction, and subculture and proliferation to achieve an initial bud induction rate of over 98% and a cluster bud induction rate of over 99%, thus achieving a very good induction effect.

[0005] Plant tissue culture is an important way to preserve species genetic resources and rapidly propagate plant seedlings. However, through in-depth research on Albizia julibrissin tissue culture, our research team found that Albizia julibrissin buds show a decline trend after multiple subcultures. Specifically, Albizia julibrissin buds show decline after 22 subcultures. The decline of Albizia julibrissin buds is manifested by slow growth after inoculation, easy yellowing, and decreased differentiation, regeneration and reproductive capacity, resulting in poor tissue culture effect and low quality of tissue culture seedlings. However, re-using explants for tissue culture is a complicated and time-consuming process. Therefore, extending the tissue culture life of Albizia julibrissin buds is of great significance to the development of Albizia julibrissin tissue culture technology. Summary of the Invention

[0006] The purpose of this invention is to provide a cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin subculture buds, aiming to overcome the phenomena of slow growth, yellowing, browning of callus tissue, and decreased differentiation and regeneration reproductive capacity of Albizia julibrissin subculture buds after 22 generations, and to restore the differentiation and regeneration reproductive capacity of Albizia julibrissin subculture buds that have shown a declining trend, so as to extend the tissue culture life of Albizia julibrissin subculture buds.

[0007] To achieve the above objectives, the present invention provides a method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds, the method comprising:

[0008] S1, Dedifferentiation: Take the 22nd generation of successor shoots, cut them into tissue blocks, and inoculate them into callus induction medium. Induce dedifferentiation to form callus under preset induction conditions for 14-20 days. The callus induction medium consists of modified WPM medium, 0.3-0.9 mg / L methyl jasmonate, 0.8-1.6 mg / L LTDZ, 0.4-1.2 mg / L NAA, 1.0-2.0 mg / L IBA, 1.5-2.5 g / L sucrose, and 4.8-7.5 g / L agar.

[0009] S2, Inducing shoot clusters: The callus tissue is cut off, and the cut callus tissue is inoculated into a differentiation medium and placed under preset differentiation conditions to induce shoot clusters;

[0010] S3, Rejuvenation Culture: The stems or buds of the clustered buds are inoculated into a rejuvenation culture medium and cultured under preset conditions to obtain rejuvenated seedlings. The rejuvenation culture medium consists of WPM medium, 0.2-0.6 mg / L TDZ, 0.4-0.8 mg / L IBA, 0.3-0.8 mg / L methyl jasmonate, 0.5-1 mg / L chitosan, 0.1-0.5 mg / L riboflavin sodium phosphate, 3-6 g / L sucrose, and 4.8-7.5 g / L agar.

[0011] S4, Subculture: Cut stem segments from rejuvenated seedlings and inoculate them into subculture, where they are cultured under pre-set conditions.

[0012] Preferably, in the above technical solution, the modified WPM culture medium comprises 70-76 mg / L CaCl2, 470-475 mg / L Ca(NO3)2·4H2O, 170-175 mg / L KH2PO4, 340-360 mg / L K2SO4, 360-380 mg / L MgSO4, 180-210 mg / L KNO3, 0.2-0.3 mg / L CuSO4·5H2O, 40-50 mg / L FeNaEDTA, 2-4 mg / L H3BO3, 8-11 mg / L MnSO4·H2O, and 0.1-0.15 .... It is made from Na2MoO4·2H2O, 10-14 mg / L ZnSO4·7H2O, 0.5-0.8 mg / L nicotinic acid, 180-220 mg / L inositol, 0.4-0.6 mg / L vitamin B6 and 1-2 mg / L vitamin B1.

[0013] Preferably, in the above technical solution, the preset induction conditions include a temperature of 24-28℃, a light intensity of 300-600Lx, and a light exposure time of 12-14h / d.

[0014] Preferably, in the above technical solution, the specific steps in step S2, where the callus tissue is cut and the cut callus tissue is inoculated into the differentiation culture medium, are as follows:

[0015] Cut the callus tissue into 3-5 mm callus tissue blocks and insert the callus tissue blocks halfway into the differentiation culture medium, which is composed of WPM medium, 0.8-2.4 mg / L TDZ, 0.3-0.8 mg / L methyl jasmonate, 0.4-1.6 mg / L IBA, 0.5-1 mg / L brassinolide, 0.1-0.8 mg / L La(NO3)3, 3-6 g / L sucrose and 4.8-7.5 g / L agar.

[0016] Preferably, in the above technical solution, the method for preparing the differentiation culture medium is as follows:

[0017] (1) Prepare a mixed solution by taking WPM medium, TDZ, methyl jasmonate, IBA and La(NO3)3, and heat it to completely dissolve sucrose and agar in water. Then add the mixed solution to adjust the pH value to 6.5-6.8, make up the volume, dispense into tissue culture bottles, and sterilize the culture medium by high pressure steam sterilization.

[0018] (2) Prepare a 0.5-1 mg / L brassinolide solution by heating the 0.5-1 mg / L brassinolide solution in a water bath to 75-80°C and maintaining it for 25-35 min to obtain a sterile 0.5-1 mg / L brassinolide solution.

[0019] (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on a clean bench, and let it stand to solidify to obtain a differentiation culture medium.

[0020] Preferably, in the above technical solution, the preset differentiation conditions include a temperature of 24-28℃, a light intensity of 3000-4000Lx, and a light intensity of 16-18h / d.

[0021] Preferably, in the above technical solution, in step S4, the stem segment is a robust stem retaining 1-2 stem nodes.

[0022] According to the above technical solution, the principle behind specifying robust stems with 1-2 nodes as the inoculation target in subculture is that in previous experiments where the inoculation target was not limited to stems, it was found that the subcultured seedlings exhibited uneven growth, with a high proliferation coefficient but weak seedlings. Further observation and research suggested that the faster growth and greater proliferation of buds led to the weak seedlings. When a robust stem with 1 node is inoculated, the proliferation coefficient of the tissue culture seedlings obtained through subculture is 2-4, with larger leaves and stronger stems.

[0023] Preferably, in the above technical solution, the subculture medium is composed of WPM medium, 0.5 mg / L methyl jasmonate, 0.8 mg / L NAA and 0.6 mg / L IBA.

[0024] Preferably, in the above technical solution, the preset culture conditions include a temperature of 24-28℃, a light intensity of 6000-8000Lx, and a light intensity of 16h / d.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) Using the subcultured shoots of Albizia julibrissin after 22 generations as the research object of tissue culture, the problem of inability to subculture and germplasm preservation caused by subculture aging of Albizia julibrissin in the field of tissue culture technology is solved by inducing callus-inducing clustered shoots-rejuvenation-subculture, thereby extending the tissue culture life of Albizia julibrissin subcultured shoots.

[0027] (2) During the callus induction stage, by modifying the WPM medium and adjusting the concentrations of substances such as methyl jasmonate, TDZ, and sucrose, as well as by setting induction conditions, the tissue blocks were dedifferentiated to form callus tissue, while reducing the oxidation of tannins and other phenolic substances in the Albizia julibrissin tissue blocks into quinone structures and causing browning and necrosis.

[0028] (3) During the stage of inducing callus differentiation into shoot clusters, a differentiation medium was prepared by means of isolation and sterilization. TDZ, IBA, brassinolide and La(NO3)3 were set in the differentiation medium to promote the proliferation and differentiation of callus into shoot clusters.

[0029] (4) During the rejuvenation culture stage, the browning phenomenon of the inoculated tissue was reduced by adding methyl jasmonate to the culture medium, increasing the concentration of chitin and riboflavin sodium phosphate and reducing the concentration of TDZ and IBA, reducing the formation of callus tissue at the inoculation site and reducing the proliferation coefficient, so as to obtain stronger subculture seedlings with appropriate interstem length, avoid the excessive growth of tissue culture seedlings, and extend the tissue culture life of the obtained Albizia julibrissin subculture buds. Attached Figure Description

[0030] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0031] Figure 1 This is a figure showing the overall growth of the 22nd generation of tissue culture seedlings after inoculation by our research team.

[0032] Figure 2 Figure A shows the slow growth and yellowing of tissue culture seedlings after the 22nd generation of successor buds;

[0033] Figure 2 Figure B shows the low proliferation coefficient of tissue culture seedlings after 22 generations of successor buds;

[0034] Figure 3 Figure A in the diagram shows the poor callus induction effect in Example 1.

[0035] Figure 3 Figure B in the figure shows the effect of inducing callus tissue using the S1 dedifferentiation method in Example 3 of the present invention;

[0036] Figure 4 This is a diagram of obtaining tissue culture seedlings using the S2 induced shoot clustering method of the present invention;

[0037] Figure 5 This is a diagram showing the tissue culture seedlings obtained using the S3 rejuvenation culture method of the present invention;

[0038] Figure 6 This is a diagram of tissue culture seedlings obtained using the S4 subculture method of the present invention. Detailed Implementation

[0039] The buds of Albizia julibrissin exhibit a decline phenomenon, which can be seen from... Figure 1 and Figure 2 see, Figure 1 The tissue culture seedlings obtained after 22 generations of inoculation and subsequent bud cultivation showed low uniformity of growth. Some buds lost their differentiation, regeneration, and reproductive capacity, making it impossible to cultivate new plants from certain organs. Other buds exhibited reduced differentiation, regeneration, and reproductive capacity, resulting in a low proliferation coefficient (e.g., ...). Figure 2 (Figure B in the image) Some tissue culture seedlings that underwent bud differentiation and regeneration grew slowly and turned yellow (e.g., ...). Figure 2 As shown in Figure A, only some buds can obtain normal tissue culture seedlings, resulting in a low input-output ratio, poor tissue culture effect, and low uniformity of tissue culture seedlings.

[0040] Example 1

[0041] A culture method to promote the proliferation and rejuvenation of Albizia julibrissin sub-sprouts, and to screen culture media that induce dedifferentiation of Albizia julibrissin sub-sprouts.

[0042] Screening Experiment 1:

[0043] Twenty-two generations of non-yellowing subculture buds were taken, longitudinally cut along the stem, and the tissue blocks were inoculated into callus induction medium 1 and callus induction medium 2, respectively. They were placed under the conditions of 24℃, light intensity of 300Lx, and light intensity of 14h / d to induce dedifferentiation of subculture bud tissue for 14 days. Callus induction medium 1 was prepared by WPM medium + 1.6mg / L 6-BA + 0.8mg / L α-naphthaleneacetic acid (NAA) + 3g / L sucrose + 4.8g / L agar. Callus induction medium 2 was prepared by modified WPM medium + 1.6mg / L 6-BA + 0.8mg / L α-naphthaleneacetic acid + 3g / L sucrose + 4.8g / L agar.

[0044] The components of WPM medium and modified WPM medium are as follows:

[0045]

[0046] During the induction culture process, the induction rate using callus induction medium 1 was 38%, while the induction rate using callus induction medium 2 was 51%, indicating that the modified WPM medium can effectively promote the induction of Albizia julibrissin tissue blocks. The principle behind the improved WPM medium's promotion of induction may be twofold: firstly, reducing the K2SO4 content and increasing the MgSO4 content in the macronutrients; secondly, replacing NH4NO3 with KNO3 to convert nitrate nitrogen into ammonium nitrogen, allowing the tissue blocks to absorb NO3. - Ions alter the potential gradient, promoting the absorption of Mg by the tissue block. 2+ The absorption of Mg increases the dedifferentiation of Albizia julibrissin tissue blocks into callus; secondly, the increase of zinc and iron elements participates in activating, catalyzing, and regulating enzyme activity, promoting the dedifferentiation of tissue blocks into callus.

[0047] Screening Experiment 2:

[0048] Twenty-two generations of non-yellowing subspecies were taken, longitudinally cut along the stem, and divided into tissue blocks. Each tissue block was inoculated into callus induction medium and placed under conditions of 24℃, 300 Lx light intensity, and 14 h / d light to induce dedifferentiation of the subspecies tissue for 14 days. The medium consisted of modified WPM medium + 1.5 g / L sucrose + 4.8 g / L agar. A 5-factor, 4-level L24-wavelength ... 16 The orthogonal experiments are shown in Table 1. Callus induction rate (%) = number of inoculated cells that produced callus / total number of inoculated cells × 100%; Browning rate (%) = number of browned inoculated cells / total number of inoculated cells × 100%.

[0049] Table 1 Browning rate and induction rate of callus induction culture medium with different components

[0050]

[0051]

[0052] Table 1 shows that methyl jasmonic acid can reduce browning of Albizia julibrissin tissue blocks. The combination of methyl jasmonic acid and IBA resulted in less browning compared to the combination of methyl jasmonic acid and 6-BA. The mechanism by which methyl jasmonic acid and IBA reduce browning is that IBA inhibits the synthesis of phenolic substances, and methyl jasmonic acid inhibits polyphenol oxidase activity, thus reducing browning of Albizia julibrissin tissue blocks. The optimal callus induction medium for achieving the best induction effect consisted of modified WPM medium + 0.1 mg / L methyl jasmonic acid + 1.6 mg / L LTDZ + 0.4 mg / L NAA + 1.5 mg / L IBA, with an induction rate of 85%. The next best medium was modified WPM medium + 0.9 mg / L methyl jasmonic acid + 0.8 mg / L LTDZ + 1.2 mg / L NAA + 1 mg / L IBA, with an induction rate of 76%.

[0053] Example 2

[0054] A culture method to promote the proliferation and rejuvenation of Albizia julibrissin subgeneration shoots, and a screening differentiation culture medium.

[0055] Well-developed callus tissue was cut into smaller pieces from the stem segment and inoculated onto a differentiation induction medium to induce shoot differentiation and obtain clustered shoots. Each bottle contained 5-7 callus tissue pieces. The medium was based on WPM medium + 3 g / L sucrose + 4.8 g / L agar, and induced to differentiate into shoots for 35 days at 24℃, 4000 Lx light intensity, and 16 h / d. TDZ (0.4, 0.8, ...) was used as the induction medium. A 5-factor, 3-level L16 orthogonal experiment was designed with the following factors as factors: methyl jasmonate concentration (1.6, 2.4 mg / L), methyl jasmonate concentration (0.1, 0.3, 0.5, 0.8 mg / L), IBA concentration (0.4, 0.8, 1.2, 1.6 mg / L), brassinolide concentration (0.1, 0.5, 0.8, 1.2 mg / L), and La(NO3)3 concentration (0.1, 0.5, 0.8, 1.2 mg / L). The results are shown in Table 2.

[0056] Browning rate (%) = (Number of inoculated individuals that browned / Total number of inoculated individuals) × 100%.

[0057] Table 2. Proliferation coefficient and induction rate of different components of differentiation culture medium

[0058]

[0059] As shown in Table 2 above, when the concentrations of TDZ and IBA are the same or the concentration of IBA is greater than that of TDZ, the proliferation coefficient is significantly affected. When the concentration of brassinolide in the culture medium is greater than 1.0 mg / L, the proliferation coefficient changes little. Furthermore, when the concentration of La(NO3)3 is greater than 0.8 mg / L, proliferation is inhibited. Therefore, the differentiation medium consists of WPM medium + 0.4-2.4 mg / L TDZ + 0.3-0.8 mg / L methyl jasmonate + 0.4-1.6 mg / L IBA + 0.1-1 mg / L brassinolide + 0.1-0.8 mg / L La(NO3)3 + 3 g / L sucrose + 4.8 g / L agar.

[0060] Example 3

[0061] A method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds, the method comprising:

[0062] S1, Dedifferentiation: Take the 22nd generation of non-yellowing sub-sprouts, cut them longitudinally along the stem to form tissue blocks, and inoculate each tissue block into callus induction medium. Induce dedifferentiation of the sub-sprout tissue for 17 days under the conditions of temperature 24℃, light intensity 300Lx, and light intensity 14h / d. The callus induction medium is modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / L LTDZ + 0.4mg / L NAA + 1.5mg / L LIBA + 1.5g / L sucrose + 4.8g / L agar.

[0063] S2, Induction of Bud Clusters: Well-grown callus tissue was collected and cut into 3-5 mm pieces. Five to seven pieces were then inoculated into differentiation medium at a rate of 5-7 pieces per bottle. The medium was placed under conditions of 24°C, 4000 Lx light intensity, and 16 h / d light exposure to induce bud differentiation in the callus tissue pieces for 35 days, thus obtaining bud clusters. The differentiation medium was prepared as follows:

[0064] (1) Prepare a mixed solution consisting of WPM medium, 1.6 mg / L LTDZ, 0.5 mg / L methyl jasmonate, 0.8 mg / L IBA, and 0.5 mg / L La(NO3)3. Heat the solution to completely dissolve the sucrose and agar in water. Then add the mixed solution to adjust the pH to 6.5-6.8. Make up the volume, dispense into culture flasks, and sterilize the culture medium by autoclaving.

[0065] (2) Prepare a 0.8 mg / L brassinolide solution by heating the 0.8 mg / L brassinolide solution in a water bath to 75-80°C and maintaining it for 25 min to obtain a sterile 0.8 mg / L brassinolide solution.

[0066] (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on the ultra-clean workbench, let it stand to solidify, and obtain the differentiation culture medium.

[0067] S3, Rejuvenation Culture: Take the above-mentioned clustered bud divisions, retain one stem node and cut the inoculation port obliquely at 30-45°, inoculate them into the rejuvenation culture medium, and place them under the conditions of temperature 24℃, light intensity 6000Lx, and light intensity 16h / d to induce subculture bud rejuvenation for 45 days; the rejuvenation culture medium is composed of WPM medium + 0.4mg / L TDZ + 0.6mg / L IBA + 0.5mg / L methyl jasmonate + 0.8mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 3g / L sucrose + 4.8g / L agar.

[0068] S4, Subculture: Take the rejuvenated seedlings, cut off a healthy stem retaining one node, and inoculate the stem into the subculture medium. Place it under the conditions of 24℃, light intensity of 6000Lx, and light intensity of 16h / d for 45 days. The subculture medium consists of WPM medium, 0.5mg / L methyl jasmonate, 0.8mg / L NAA, and 0.5mg / L TDZ.

[0069] Example 4

[0070] A method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds, the method comprising:

[0071] S1, Dedifferentiation: Take the 22nd generation of non-yellowing sub-buds, cut them longitudinally along the stem to form tissue blocks, and inoculate each tissue block into callus induction medium. Induce dedifferentiation of the sub-bud tissue for 16 days under the conditions of temperature 26℃, light intensity 500Lx, and light intensity 12h / d. The callus induction medium is modified WPM medium + 0.3mg / L methyl jasmonate + 0.8mg / L LTDZ + 0.8mg / L NAA + 1mg / L IBA + 1g / L sucrose + 6.0g / L agar.

[0072] S2, Induction of Bud Clusters: Well-grown callus tissue was collected and cut into 3-5 mm pieces. Five to seven pieces were then inoculated into a differentiation medium at a rate of 26°C, 3000 Lx light intensity, and 18 h / d light exposure to induce bud differentiation in the callus tissue pieces for 40 days, resulting in bud clusters. The differentiation medium was prepared as follows:

[0073] (1) Prepare a mixed solution consisting of WPM medium, 0.8 mg / L LTDZ, 0.5 mg / L methyl jasmonate, 0.4 mg / L IBA, and 0.1 mg / L La(NO3)3. Heat the solution to completely dissolve the sucrose and agar in water. Then add the mixed solution to adjust the pH to 6.5-6.8. Make up the volume, dispense into culture flasks, and sterilize the culture medium by autoclaving.

[0074] (2) Prepare a 0.5 mg / L brassinolide solution by heating the 0.5 mg / L brassinolide solution in a water bath to 75-80°C and maintaining it for 30 min to obtain a sterile 0.5 mg / L brassinolide solution.

[0075] (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on the ultra-clean workbench, let it stand to solidify, and obtain the differentiation culture medium.

[0076] S3, Rejuvenation Culture: Take the above-mentioned clustered bud divisions, retain one stem node and cut the inoculation port obliquely at 30-45°, inoculate them into the rejuvenation culture medium, and place them under the conditions of temperature 26℃, light intensity 7000Lx, and light intensity 16h / d to induce subgeneration bud rejuvenation for 50 days; the rejuvenation culture medium is composed of WPM medium + 0.4mg / L TDZ + 0.6mg / L IBA + 0.5mg / L methyl jasmonate + 0.8mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 3g / L sucrose + 4.8g / L agar.

[0077] S4, Subculture: Take the rejuvenated seedlings, cut off a healthy stem retaining one node, and inoculate the stem into the subculture medium. Place it under the conditions of 26℃, light intensity of 7000Lx, and light intensity of 17h / d for 50 days. The subculture medium consists of WPM medium, 0.3mg / L methyl jasmonate, 0.6mg / L NAA, and 0.3mg / L TDZ.

[0078] Example 5

[0079] A method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds, the method comprising:

[0080] S1, Dedifferentiation: 22 generations of non-yellowing sub-sprouts were longitudinally cut along the stem to form tissue blocks. Each tissue block was inoculated into callus induction medium and placed under conditions of 28℃, 600 Lx light intensity, and 12 h / d light to induce dedifferentiation of the sub-sprout tissue for 16 days. The callus induction medium was a modified WPM medium + 0.9 mg / L methyl jasmonate.

[0081] +0.8mg / L LTDZ +1.2mg / L NAA +1mg / L LIBA +2g / L sucrose +7.5g / L agar;

[0082] S2, Induction of Bud Clusters: Well-grown callus tissue is taken and cut into 3-5 mm pieces. Five to seven pieces are placed in each bottle of differentiation medium and incubated for 40 days at 28°C, 4000 Lx light intensity, and 18 h / d light intensity to induce bud differentiation, thus obtaining bud clusters. The differentiation medium is prepared as follows:

[0083] (1) Prepare a mixed solution consisting of WPM medium, 2.4 mg / L LTDZ, 0.8 mg / L methyl jasmonate, 1.6 mg / L IBA, and 0.8 mg / L La(NO3)3. Heat the solution to completely dissolve the sucrose and agar in water. Then add the mixed solution to adjust the pH to 6.5-6.8. Make up the volume, dispense into culture flasks, and sterilize the culture medium by autoclaving.

[0084] (2) Prepare a 1 mg / L brassinolide solution by heating the 1 mg / L brassinolide solution in a water bath to 75-80°C and maintaining it for 35 min to obtain a sterile 1 mg / L brassinolide solution.

[0085] (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on the ultra-clean workbench, let it stand to solidify, and obtain the differentiation culture medium.

[0086] S3, Rejuvenation Culture: Take the above-mentioned clustered buds, retain 1-3 stem nodes and cut the inoculation port obliquely at 30-45°, inoculate them into the rejuvenation culture medium, and place them under the conditions of temperature 28℃, light intensity 8000Lx, and light intensity 18h / d to induce subgeneration bud rejuvenation for 40 days; the rejuvenation culture medium is composed of WPM medium + 0.8mg / L LTDZ + 0.8mg / L IBA + 0.8mg / L methyl jasmonate + 1mg / L chitin + 0.5mg / L riboflavin sodium phosphate + 6g / L sucrose + 7.5g / L agar.

[0087] S4, Subculture: Take the rejuvenated seedlings, cut off healthy stems retaining 2 nodes, and inoculate the stems into the subculture medium. Place them under the conditions of 28℃, light intensity of 8000Lx, and light intensity of 18h / d for 40 days. The subculture medium consists of WPM medium, 0.8mg / L methyl jasmonate, 1.2mg / L NAA, and 0.6mg / L TDZ.

[0088] Comparative Example 1

[0089] The dedifferentiation process is basically the same as in Example 3, except that the culture conditions are different.

[0090] Twenty-two generations of non-yellowing sub-sprouts were taken, longitudinally cut along the stem, and divided into tissue blocks. Each tissue block was inoculated into callus induction medium and placed under conditions of 24℃, 4000Lx light intensity, and 18h / d light intensity to induce dedifferentiation of the sub-sprout tissue for 14 days. The callus induction medium consisted of modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / L LTDZ + 0.4mg / L NAA + 1.5mg / L LIBA + 1.5g / L sucrose + 4.8g / L agar.

[0091] Comparative Example 2

[0092] The dedifferentiation process is basically the same as in Example 3, except that the culture conditions are different.

[0093] Twenty-two generations of non-yellowing sub-sprouts were taken, longitudinally cut along the stem, and divided into tissue blocks. Each tissue block was inoculated into callus induction medium and placed under conditions of 24℃, 4000Lx light intensity, and 18h / d light intensity to induce dedifferentiation of the sub-sprout tissue for 14 days. The callus induction medium consisted of modified WPM medium + 0.5mg / L methyl jasmonate + 1.6mg / L LTDZ + 0.4mg / L NAA + 1.5mg / L LIBA + 4.5g / L sucrose + 4.8g / L agar.

[0094] After dedifferentiation treatment, the samples from Example 3 and Comparative Examples 1-2 were cultured for 14 days, and their browning and callus induction were observed. The results are shown in Table 3 and... Figure 3 As shown in Figure A.

[0095] Table 3. Dedifferentiation browning rate and induction rate of Example 3 and Comparative Examples 1-2

[0096] Example 3 Comparative Example 1 Comparative Example 2 Browning rate (%) 5 6 11 Induction rate (%) 85 76 73

[0097] As can be seen from Table 3 above, compared with Example 3, increasing the light intensity and illumination time in Comparative Example 1 reduces the induction rate. Figure 3 As shown, Figure A is a diagram of the better callus obtained by inducing Albizia julibrissin tissue blocks in Comparative Example 1, and Figure B is a diagram of the better callus obtained by inducing Albizia julibrissin tissue blocks in Example 3. The comparison shows that the callus induced by each tissue block in Comparative Example 1 is smaller, which teaches that prolonged exposure of Albizia julibrissin tissue blocks to high-intensity light during the induction and dedifferentiation stage will lead to a lower induction rate and smaller callus blocks. Compared with Comparative Example 1, Comparative Example 2 shows that increasing the sucrose concentration will lead to a greater number of browning Albizia julibrissin tissue blocks and an increased browning rate.

[0098] Comparative Example 3

[0099] The induced shoot clusters are basically the same as those in Example 3, except that the culture conditions are different.

[0100] Well-grown callus tissue was cut into smaller callus tissue pieces from the whole stem segment. The callus tissue pieces were inoculated into the differentiation induction medium at a rate of 5-7 pieces per bottle and placed under the conditions of 24℃, 1000Lx light intensity, and 14h / d light intensity to induce the callus tissue pieces to differentiate into shoots for 20-30 days, thereby obtaining clustered shoots. The differentiation medium consisted of WPM medium + 1.6mg / L LTDZ + 0.5mg / L methyl jasmonate + 0.8mg / L LIBA + 0.8mg / L brassinolide + 0.5mg / L Lα(NO3)3 + 3g / L sucrose + 4.8g / L agar.

[0101] Comparative Example 4

[0102] The induced shoot clustering method is essentially the same as that in Example 3, except that the differentiation medium is prepared differently. The preparation method of the differentiation medium is as follows:

[0103] A mixed solution was prepared by taking WPM medium, 1.6 mg / L TDZ, 0.8 mg / L brassinolide solution, 0.5 mg / L methyl jasmonate, 0.8 mg / L IBA, and 0.5 mg / L La(NO3)3. Sucrose and agar were completely dissolved in water by heating. The pH of the mixed solution was then adjusted to 6.5-6.8. The solution was brought to a final volume and dispensed into tissue culture flasks. The culture medium was sterilized by autoclaving. After sterilization, the medium was allowed to solidify to obtain the differentiation medium.

[0104] The bud induction results of Example 3 were compared with those of Comparative Examples 3-4, and the results are shown in Table 4.

[0105] Table 4. Shoot induction results of Example 3 and Comparative Examples 3-4

[0106] Example 3 Comparative Example 3 Comparative Example 4 Proliferation coefficient (species) 5.7 4.6 4.4 Average plant height (cm) 12.8 5.3 8.9 Induction rate (%) 93.67 83.5 78.33 Browning rate (%) 3 2 3

[0107] As can be seen from Table 4, compared with Comparative Example 3, the proliferation coefficient and average plant height of Comparative Example 3 were significantly reduced, and the induction rate was also reduced. Compared with Comparative Example 4, Comparative Example 4 heated rapeseed lactone together with the culture medium, which led to structural changes of rapeseed lactone at high temperature and weakened its effect.

[0108] Comparative Example 5

[0109] The rejuvenation culture is basically the same as that in Example 3, except that the components of the rejuvenation culture medium are different.

[0110] The induced buds were inoculated into a rejuvenation medium and placed under conditions of 24°C, 6000 Lx light intensity, and 16 h / d light intensity to induce subculture bud rejuvenation for 20-60 days. The rejuvenation medium consisted of WPM medium + 1.6 mg / L LTDZ + 0.8 mg / L LIBA + 0.5 mg / L methyl jasmonate + 3 g / L sucrose + 4.8 g / L agar.

[0111] Comparative Example 6

[0112] The rejuvenation culture is basically the same as that in Example 3, except that the components of the rejuvenation culture medium are different.

[0113] The induced buds were inoculated into a rejuvenation medium and placed under conditions of 24°C, 6000 Lx light intensity, and 16 h / d light intensity to induce subculture bud rejuvenation for 20-60 days. The rejuvenation medium consisted of WPM medium + 0.4 mg / L LTDZ + 0.6 mg / L LIBA + 0.5 mg / L methyl jasmonate + 0.8 mg / L chitosan + 3 g / L sucrose + 4.8 g / L agar.

[0114] Comparative Example 7

[0115] The rejuvenation culture is basically the same as that in Example 3, except that the components of the rejuvenation culture medium are different.

[0116] The induced buds were inoculated into a rejuvenation medium and placed under conditions of 24°C, 6000 Lx light intensity, and 16 h / d light intensity to induce subculture bud rejuvenation for 20-60 days. The rejuvenation medium consisted of WPM medium + 0.4 mg / L LTDZ + 0.6 mg / L LIBA + 0.5 mg / L methyl jasmonate + 0.5 mg / L sodium riboflavin phosphate + 3 g / L sucrose + 4.8 g / L agar.

[0117] After culturing the rejuvenation culture of Example 3 and Comparative Examples 5-7 for 45 days, five bottles of rejuvenation tissue culture seedlings were randomly selected from each group to determine their stem diameter, callus condition at the inoculation site, and proliferation coefficient, as shown in Table 5.

[0118] Table 5. Results of rejuvenated tissue culture seedlings under different treatments.

[0119] Example 3 Comparative Example 5 Comparative Example 6 Comparative Example 7 Culture medium color pale yellow White White pale yellow Stem diameter(mm) 0.93 0.65 0.81 0.73 callus tissue Yes, very small Yes, relatively large Yes, smaller Yes, very small. Proliferation coefficient 3.1 4.6 3.3 3.6

[0120] As can be seen from Table 5, adding riboflavin sodium phosphate to the rejuvenation medium will make the medium color pale yellow. Adding chitosan or riboflavin will reduce the formation of callus at the inoculation site, and the proliferation coefficient will also change accordingly. This indicates that the combined use of chitosan and riboflavin will greatly reduce the formation of callus and reduce the proliferation coefficient.

[0121] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds, characterized in that, include: S1, Dedifferentiation: Secondary shoots from the 22nd generation are cut into tissue blocks and inoculated into callus induction medium. Dedifferentiation is induced to form callus under preset induction conditions for 14-20 days. The callus induction medium consists of modified WPM medium, 0.3-0.9 mg / L methyl jasmonate, 0.8-1.6 mg / L TDZ, 0.4-1.2 mg / L NAA, 1.0-2.0 mg / L IBA, 1.5-2.5 g / L sucrose, and 4.8-7.5 g / L agar. The modified WPM medium contains 70-76 mg / L CaCl2, 470-475 mg / L Ca(NO3)2·4H2O, 170-175 mg / L KH2PO4, 340-360 mg / L K2SO4, 360-380 mg / L MgSO4, and 180-210 mg / L... It is made from KNO3, 0.2-0.3 mg / L CuSO4·5H2O, 40-50 mg / L FeNaEDTA, 2-4 mg / L H3BO3, 8-11 mg / L MnSO4·H2O, 0.1-0.15 mg / L Na2MoO4·2H2O, 10-14 mg / L ZnSO4·7H2O, 0.5-0.8 mg / L nicotinic acid, 180-220 mg / L inositol, 0.4-0.6 mg / L vitamin B6 and 1-2 mg / L vitamin B1; S2, Inducing shoot clusters: The callus tissue is excised and inoculated into a differentiation medium, and placed under preset differentiation conditions to induce shoot clusters. The differentiation medium consists of WPM medium, 0.8-2.4 mg / L TDZ, 0.3-0.8 mg / L methyl jasmonate, 0.4-1.6 mg / L IBA, 0.5-1 mg / L brassinolide, 0.1-0.8 mg / L La(NO3)3, 3-6 g / L sucrose and 4.8-7.5 g / L agar. S3, Rejuvenation Culture: The stems or buds of the clustered buds are inoculated into a rejuvenation culture medium and cultured under preset conditions to obtain rejuvenated seedlings. The rejuvenation culture medium consists of WPM medium, 0.2-0.6 mg / L TDZ, 0.4-0.8 mg / L IBA, 0.3-0.8 mg / L methyl jasmonate, 0.5-1 mg / L chitosan, 0.1-0.5 mg / L riboflavin sodium phosphate, 3-6 g / L sucrose, and 4.8-7.5 g / L agar. S4, Subculture: Cut stem segments from rejuvenated seedlings and inoculate them into a subculture medium, which is then cultured under preset culture conditions. The subculture medium consists of WPM medium, 0.3-0.8 mg / L methyl jasmonate, 0.6-1.2 mg / L NAA, and 0.3-0.6 mg / L LTDZ.

2. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds according to claim 1, characterized in that, The preset induction conditions include a temperature of 24-28℃, a light intensity of 300-600 Lx, and a light exposure time of 12-14 h / d.

3. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin sub-sprouts according to claim 1, characterized in that, In step S2, the specific steps for cutting the callus tissue and inoculating the cut callus tissue into the differentiation culture medium are as follows: Cut the callus tissue into 3-5 mm callus tissue blocks and insert the callus tissue blocks halfway into the differentiation culture medium.

4. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin sub-sprouts according to claim 3, characterized in that, The method for preparing the differentiation culture medium is as follows: (1) Prepare a mixed solution by taking WPM medium, TDZ, methyl jasmonate, IBA and La(NO3)3, and heat it to completely dissolve sucrose and agar in water. Then add the mixed solution to adjust the pH value to 6.5-6.8, make up the volume, dispense into tissue culture bottles, and sterilize the culture medium by high pressure steam sterilization. (2) Prepare a 0.5-1 mg / L brassinolide solution by heating the 0.5-1 mg / L brassinolide solution in a water bath to 75-80℃ and maintaining it for 25-35 min to obtain a sterile 0.5-1 mg / L brassinolide solution. (3) Mix the brassinolide solution with the unsolidified culture medium next to the flame of an alcohol lamp on the ultra-clean workbench, let it stand to solidify, and obtain the differentiation culture medium.

5. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin sub-sprouts according to claim 1, characterized in that, The preset differentiation conditions include a temperature of 24-28℃, a light intensity of 3000-4000Lx, and a light exposure of 16-18h / d.

6. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds according to claim 1, characterized in that, In step S4, the stem segment is a robust stem that retains 1-2 nodes.

7. The cultivation method for promoting the proliferation and rejuvenation of Albizia julibrissin subgeneration buds according to claim 1, characterized in that, The preset culture conditions include a temperature of 24-28℃, a light intensity of 6000-8000 Lx, and a light exposure of 16-18 h / d.

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