A method for rooting of aseptic shoots of a jatropha

Abstract

The application discloses a sterile gemmation rooting method of Aegiceras corniculatum, and belongs to the technical field of plant tissue culture, and comprises the following steps: providing Aegiceras corniculatum hypocotyls, culturing the Aegiceras corniculatum hypocotyls in a bud induction medium after disinfection, and obtaining top buds of the Aegiceras corniculatum hypocotyls that germinate; and inoculating the top buds of the Aegiceras corniculatum hypocotyls that germinate obtained by culture for 12 months into a rooting culture medium to culture, and obtaining rooting tissue culture seedlings. The application solves the technical bottleneck of difficulty in rooting of bud induction of the Aegiceras corniculatum hypocotyls and low survival rate in existing Aegiceras corniculatum tissue culture by means of double technical innovation of suitable physiological age period screening and a special rooting culture medium formula, has high rooting efficiency, and has good growth of seedlings, fills the blank of rooting technology of bud age period regulation of the Aegiceras corniculatum hypocotyls at home and abroad, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a method for aseptic bud rooting of tung trees. Background Technology

[0002] Paulownia tree ( Aegiceras corniculatum Also known as candle fruit, it belongs to the Myrsinaceae family (… Myrsinaceae *Candlefruit* is a shrub or small tree naturally distributed along tropical coasts from Asia to Oceania. In my country, it is mainly distributed in Hainan, Guangxi Zhuang Autonomous Region, Guangdong, and Fujian. *Candlefruit* primarily grows in low to mid-tidal flats, is highly adaptable, and reproduces via vegetative propagation, easily forming monotypic (dominant) plant communities. It is a pioneer species in mangrove succession in low-salinity areas, and its distribution area in my country is second only to *Avicennia marina* (white mangrove). Avicennia marina *Tung tree* (Laminaria japonica) is one of my country's typical native mangrove species. This species exhibits remarkable ecological adaptability: its root system is well-developed, and it maintains salt balance by excreting salt through unique salt-secreting glands; its cryptovivic fruits drift with the water after ripening and can quickly take root and grow in mudflats. As a key species in the mangrove ecosystem, *Tung tree* forests function as windbreaks and wave absorbers, purify water, and fix and store carbon, while the understory fosters a rich benthic community. Economically, its flowers are a high-quality nectar source, its bark can be used to extract tannin, and its wood is suitable for firewood. Furthermore, *Tung tree* contains flavonoids, phenylpropanes, and other active substances, possessing anti-inflammatory, antioxidant, and potential anti-cancer value.

[0003] Currently, research on tung trees mainly focuses on their growth physiological characteristics and seedling cultivation techniques, with no reports of aseptic bud rooting and seedling production. Summary of the Invention

[0004] In view of this, this application provides a method for aseptic bud rooting of tung trees, aiming to solve the problem that there are currently no reports on the successful seedling production from aseptic bud rooting.

[0005] The embodiment of this application is implemented as follows: the method for aseptic bud rooting of tung tree includes the following steps: S01. Provide the hypocotyl of the tung tree, disinfect it, and then culture it in a bud induction medium to obtain the apical buds germinating from the hypocotyl of the tung tree; S02. The terminal buds that germinated from the hypocotyl of the 12-month-old tung tree were inoculated into a rooting medium to obtain rooted tissue culture seedlings.

[0006] Optionally, remove the outer shell of the tung tree hypocotyl, rinse the hypocotyl with running tap water for time t1, then soak it in a detergent solution diluted with pure water for time t2, rinse it under running water for time t3, then soak it in potassium permanganate solution for time t4, then disinfect the tung tree hypocotyl with alcohol in a clean bench for time t5, then soak it in HgCl2 for time t6, rinse it with sterile water W times, and finally blot the moisture off the surface of the hypocotyl with sterile filter paper.

[0007] Optionally, t1 is 8 min to 12 min; and / or t2 is 1 min to 5 min; and / or t3 is 2 min to 7 min; and / or t4 is 1 min to 5 min; and / or t5 is 8 s~20 s; and / or t6 is 1 min to 6 min; and / or W is 1 to 3 times; and / or The concentration of the potassium permanganate solution is 0.1%; and / or The alcohol concentration is 75%; and / or The concentration of HgCl2 is 0.1%.

[0008] Optionally, each liter of the bud induction medium includes: 0.5 g / L to 2.0 g / L activated carbon, 1.0 mg / L 6-BA, 0.3 mg / L NAA, and the remainder is MS medium with a pH of 5.8.

[0009] Optionally, after sterilization, the samples are placed in a bud induction medium for culture, with culture conditions including light intensity X1, photoperiod Y1, and temperature T1; and / or The terminal buds sprouting from the explants of the tung tree were inoculated into a rooting medium and cultured under the following conditions: light intensity X2, photoperiod Y2, and temperature T2.

[0010] Optionally, X1 is 20 μmol / m 2 •s ~30 μmol / m 2 •s; and / or Y1 is 8 h / d ~ 12 h / d; and / or T1 is 20℃~24℃; and / or X2 is 20 μmol / m 2 •s ~30 μmol / m 2 •s; and / or Y2 is 8 h / d ~ 12 h / d; and / or T2 is 20 ℃~24 ℃.

[0011] Optionally, each liter of the rooting medium includes: 1.0 mg / L to 2.5 mg / L KT, 0.1 mg / L to 0.3 mg / L NAA, 0 mg / L to 0.05 mg / L zinc oxide, and the remainder is MS medium.

[0012] Optionally, each liter of the rooting medium contains: 2.0 mg / L KT, 0.1 mg / L NAA, 0.05 mg / L zinc oxide, and the remainder is MS medium.

[0013] In this application: Compared with the prior art, the present invention has the following beneficial effects: Existing literature only reports on the budding of the hypocotyl of the tung tree, and there are no records of research on the independent rooting and seedling formation of aseptic buds. This invention successfully created a method for inducing buds to root in vitro from the hypocotyl, which is the first breakthrough in the tissue culture technology of tung trees and is the first of its kind at home and abroad.

[0014] This invention effectively solves the technical bottlenecks of difficult rooting and low survival rate of hypocotyl-induced buds in existing tung tree tissue culture by combining appropriate physiological age screening with a special rooting culture medium formula. It has high rooting efficiency and excellent seedling growth, filling the gap in the domestic and foreign technology for regulating the age of hypocotyl-induced buds for rooting of tung trees, and has broad application prospects.

[0015] The 3-month-old tender aseptic buds of this invention do not have the ability to root and are prone to death. The 10-month-old tender aseptic buds also do not have the ability to root. Therefore, the aseptic buds need to be subcultured for 12 months to reach physiological maturity. After that, they are in vitro inoculated into the special rooting culture medium optimized by this invention for rooting culture, thereby obtaining rooted tissue culture seedlings. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months in MS + KT 2.0 mg / L + NAA 0.1 mg / L formulation. Figure 2 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months in MS + 6-BA 0.8 mg / L + NAA 0.4 mg / L formulation. Figure 3 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months of culture in MS + 2,4-D 1.0 mg / L + NAA 0.4 mg / L formulation. Figure 4 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months of MS + 2.4-D 1.0 mg / L + 6-BA 0.1 mg / L. Figure 5 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months in MS + KT 2.0 mg / L + NAA 0.3 mg / L formulation. Figure 6 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 10 months in MS + TDZ 0.4 mg / L + NAA 0.2 mg / L formulation. Figure 7 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.1 mg / L formulation. Figure 8 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.1 mg / L + zinc oxide 0.05 mg / L formulation. Figure 9 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.1 mg / L + zinc oxide 0.1 mg / L formulation. Figure 10 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.3 mg / L formulation. Figure 11 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.3 mg / L + zinc oxide 0.05 mg / L formulation. Figure 12 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 12 months in MS + KT 2.0 mg / L + NAA 0.3 mg / L + zinc oxide 0.1 mg / L formulation. Figure 13 This is a figure showing the apical bud of the 12-month-old tung tree hypocotyl cultured for 4.0 months under the MS + KT 2.0 mg / L + NAA 0.1 mg / L + zinc oxide 0.05 mg / L formulation of this application; Figure 14 This is a figure showing the apical bud of the 12-month-old tung tree hypocotyl cultured for 4.0 months under MS + KT 2.0 mg / L + NAA 0.3 mg / L formulation. Figure 15 This is a figure showing the apical buds of the tung tree hypocotyl cultured for 2.5 months after 3 months in MS + KT 2.0 mg / L + NAA 0.1 mg / L + zinc oxide 0.05 mg / L formulation. Figure 16 This is a diagram of the terminal buds that germinated from the hypocotyl of a tung tree after 3 months of cultivation, as described in this application. Figure 17 This is a diagram of the terminal buds that germinated from the hypocotyl of a tung tree after 12 months of cultivation, as described in this application. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Furthermore, it should be understood that the specific embodiments described herein are only for illustration and explanation of this application and are not intended to limit this application.

[0019] In the description of this application, the term "comprising" means "including but not limited to".

[0020] In this application, "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0021] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one," "at least one of the following," or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b, or c," or "at least one of a, b, and c," can both mean: a, b, c, a~b (i.e., a and b), a~c, b~c, or a~b~c, where a, b, and c can be single or multiple.

[0022] Various embodiments of this application may exist in the form of a range; it should be understood that the description in the form of a range is merely for convenience and brevity and should not be construed as a hard limitation on the scope of this application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within the range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Furthermore, whenever a numerical range is referred to herein, it means including any referenced number (fraction or integer) within the referred range.

[0023] For rapid propagation of woody plants in vitro, the formation of adventitious roots is usually a key step in the transformation of detached buds into complete plants. At the same time, the survival rate of micropropagated woody plants is often affected during the hardening-off and transplanting stages due to the small number of roots, weak root function, or insufficient root adaptability.

[0024] Therefore, establishing a stable rooting technology for explants of Tung tree is not only a necessary step in improving the rapid propagation system of Tung tree tissue culture, but also a key technical foundation for realizing the in vitro preservation of superior Tung tree germplasm, large-scale production of seedlings with consistent genetic background, and standardized supply of seedlings for mangrove ecological restoration.

[0025] The technical solution of this application is as follows: This application provides a method for aseptic bud rooting of tung trees, including the following steps: S01. Provide tung tree explants, disinfect them, and culture them in bud induction medium to obtain the apical buds germinating from the tung tree explants; S02. The terminal buds sprouting from the explants of the tung tree are inoculated into the rooting medium and cultured to obtain rooted tissue culture seedlings.

[0026] In S01: In some embodiments, the tung tree explant includes a tung tree hypocotyl, and the apical bud germinating from the tung tree hypocotyl is obtained.

[0027] Furthermore, apical buds sprouting from the hypocotyls of tung trees that have been cultured for 3 to 12 months are obtained. For example, apical buds sprouting from hypocotyls of tung trees that have been cultured for 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, and 12 months are obtained.

[0028] Furthermore, the apical buds that germinated from the hypocotyl of a tung tree after 12 months of cultivation were obtained.

[0029] For example, the tung tree hypocotyl is a mature tung tree hypocotyl.

[0030] Further, a tung tree hypocotyl is provided, the outer shell of the tung tree hypocotyl is removed, the hypocotyl is rinsed with running tap water for time t1, then soaked in a detergent solution diluted with pure water for time t2, then rinsed under running water for time t3, then soaked in potassium permanganate solution for time t4, then disinfected with alcohol in a clean bench for time t5, then soaked in HgCl2 for time t6, rinsed with sterile water W times, and finally the surface moisture of the hypocotyl is dried with sterile filter paper.

[0031] Furthermore, t1 is 8 min to 12 min, for example, it can be 8 min, 9 min, 10 min, 11 min, 12 min, etc.; t2 can be 1 min to 5 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, etc.; t3 is 2 min to 7 min, for example, it can be 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, etc.; t4 is 1 min to 5 min, for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, etc.; t5 is 8 s to 20 s, for example, it can be 8 s, 10 s, 12 s, 15 s, 16 s, 18 s, 20 s, etc.; t6 can be 1 min to 6 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, etc.; W can be 1 to 3 times, for example, it can be 1 time, 2 times, 3 times, etc.; Furthermore, the concentration of the potassium permanganate solution is 0.1%.

[0032] Furthermore, the alcohol concentration is 75%.

[0033] Furthermore, the concentration of HgCl2 was 0.1%.

[0034] In some embodiments, the bud induction medium per liter includes: 0.5 g / L to 2.0 g / L activated carbon, 1.0 mg / L 6-BA, 0.3 mg / L NAA, and the remainder is MS medium with a pH of 5.8.

[0035] Furthermore, each liter of the bud induction medium comprises: 0.5 g / L to 2.0 g / L activated carbon, preferably 0.8 g / L to 1.8 g / L activated carbon, more preferably 1 g / L to 1.5 g / L activated carbon, even more preferably 1.2 g / L activated carbon; 1.0 mg / L 6-BA; 0.3 mg / L NAA; the remainder being MS medium; and the pH is 5.8.

[0036] For example, each liter of bud induction medium contains: 1 g / L activated carbon, 1.0 mg / L 6-BA, 0.3 mg / L NAA, and the remainder is MS medium with a pH of 5.8.

[0037] Furthermore, the MS medium includes: Macroelements: NH4NO3 1650 mg / L, KNO3 1900 mg / L, CaCl2·2H2O 440 mg / L, MgSO4·7H2O 370 mg / L, KH2PO4 170 mg / L; Trace elements: KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L; Iron salts: FeSO4·7H2O 27.8 mg / L, Na2-EDTA·2H2O 37.3 mg / L; Organic ingredients: IVA inositol 100 mg / L, IVB niacin 0.5 mg / L, pyridoxine hydrochloride (vitamin B6) 0.5 mg / L, thiamine hydrochloride (vitamin B1) 0.1 mg / L, glycine 2.0 mg / L.

[0038] To prepare 1 L of MS medium: accurately weigh the corresponding compounds mentioned above, dissolve them in an appropriate amount of distilled water, stir with a glass rod to promote dissolution, adjust the pH to 5.8 with NaOH, and finally bring the volume to 1 L.

[0039] Furthermore, gently agitate the product while soaking it in a dish soap solution diluted with pure water.

[0040] Furthermore, gently agitate the container while soaking it in potassium permanganate solution.

[0041] Furthermore, the mixture was constantly shaken during the soaking with HgCl2.

[0042] In some embodiments, after sterilization, the sample is placed in a bud induction medium for culture, and the culture conditions include a light intensity of X1, a photoperiod of Y1, and a temperature of T1.

[0043] Furthermore, X1 is 20 μmol / m 2 •s ~30 μmol / m 2 •s, for example, can be 20 μmol / m 2 •s, 22 μmol / m 2 •s, 25 μmol / m 2 •s, 26 μmol / m 2 •s, 28 μmol / m 2 •s, 30 μmol / m 2 •s etc.; Y1 can be 8 h / d to 12 h / d, for example, it can be 8 h / d, 8.5 h / d, 9 h / d, 10 h / d, 10.5 h / d, 11 h / d, 12 h / d, etc.; T1 is 20 ℃~24 ℃, for example, it can be 20 ℃, 20.5 ℃, 21 ℃, 21.5 ℃, 22 ℃, 23 ℃, 24 ℃, etc.

[0044] For example, the hypocotyl with its surface moisture dried is placed horizontally in a bottle containing bud induction medium.

[0045] In S02: In some embodiments, each liter of rooting medium comprises: 1.0 mg / L KT to 2.5 mg / L KT, 0.1 mg / L to 0.3 mg / L NAA, 0 mg / L to 0.05 mg / L zinc oxide, and the remainder is MS medium.

[0046] Further, each liter of the rooting medium comprises: 1.0 mg / L KT to 2.5 mg / L KT, preferably 1.2 mg / L KT to 2.2 mg / L KT, more preferably 1.5 mg / L KT to 2 mg / L KT, even more preferably 1.7 mg / L KT; 0.1 mg / L to 0.3 mg / L NAA, preferably 0.15 mg / L to 0.25 mg / L NAA, even more preferably 0.2 mg / L NAA; 0 mg / L to 0.05 mg / L zinc oxide, preferably 0.01 mg / L to 0.04 mg / L zinc oxide, even more preferably 0.02 mg / L to 0.03 mg / L zinc oxide, even more preferably 0.025 mg / L zinc oxide, with the remainder being MS medium.

[0047] For example, each liter of rooting medium contains: 2.0 mg / L KT, 0.1 mg / L NAA, and the remainder is MS medium.

[0048] For example, each liter of rooting medium contains: 2.0 mg / L KT, 0.1 mg / L NAA, 0.05 mg / L zinc oxide, and the remainder is MS medium.

[0049] For example, each liter of rooting medium contains: 2.0 mg / L KT, 0.3 mg / L NAA, and the remainder is MS medium.

[0050] In some embodiments, the terminal buds germinating from the explants of the tung tree are inoculated into a rooting medium and cultured under the following conditions: light intensity X2, photoperiod Y2, and temperature T2.

[0051] Furthermore, X2 is 20 μmol / m 2 •s ~30 μmol / m 2 •s, for example, can be 20 μmol / m 2 •s, 22 μmol / m 2 •s, 25 μmol / m 2 •s, 26 μmol / m 2 •s, 28 μmol / m 2 •s, 30 μmol / m 2 •s etc.; Y2 can be 8 h / d to 12 h / d, for example, it can be 8 h / d, 8.5 h / d, 9 h / d, 10 h / d, 10.5 h / d, 11 h / d, 12 h / d, etc.; T2 is 20 ℃~24 ℃, for example, it can be 20 ℃, 20.5 ℃, 21 ℃, 21.5 ℃, 22 ℃, 23 ℃, 24 ℃, etc.

[0052] Example 1

[0053] Select mature tung tree hypocotyls, remove the outer shell, rinse with running tap water for 10 minutes, then soak in a diluted detergent solution for 3 minutes (with gentle agitation), rinse under running water for 3 minutes, and finally soak in a 0.1% potassium permanganate solution for 3 minutes (with gentle agitation). After treatment, disinfect the tung tree hypocotyls in a clean bench with 75% alcohol for 15 seconds, then soak in 0.1% HgCl2 for 5 minutes (with continuous agitation), rinse three times with sterile water, and finally blot dry the surface of the explant with sterile filter paper. Place the sterilized tung tree hypocotyls in prepared bud induction medium (MS + activated carbon 1.0 g / L + 6-BA 1.0 mg / L + NAA 0.3 mg / L), one hypocotyl per bottle, replicated 10 times, with the hypocotyls placed horizontally. After inoculation, place on a light-controlled culture rack for cultivation under the following conditions: light intensity 20–30 μmol / m². 2 •s, photoperiod of 10 h / d, temperature of 20~24℃. Terminal buds of the hypocotyl of *Paulownia tomentosa* trees were obtained at 3 months of age. See... Figure 16 ; apical buds of the hypocotyl of the 10-month-old and 12-month-old tung tree (see Figure 17 ).

[0054] Example 2

[0055] Please see Figure 8Terminal buds of 12-month-old tung tree hypocotyls were inoculated into rooting medium. Ten bottles were used, with two plants per bottle. After inoculation, the plants were placed on a light-controlled culture rack under the following conditions: light intensity 20-30 μmol / m². 2 •s, photoperiod of 10h / d, temperature of 20~24℃. Rooting medium per liter includes: 2 mg / L KT, 0.1 mg / L NAA, 0.05 mg / L zinc oxide, with the remainder being MS medium.

[0056] After 2.5 months of cultivation, roots begin to form at the base of the stem of the tung tree terminal bud, with a rooting rate of over 90%.

[0057] The root system is over 1.5cm thick and robust.

[0058] Example 3

[0059] Please see Figure 7 The rooting medium per liter includes: MS + KT 2.0 mg / L + NAA 0.1 mg / L, and the rest is the same as in Example 2.

[0060] After 2.5 months of cultivation, roots begin to form at the base of the stem of the tung tree terminal bud, with a rooting rate of over 90%.

[0061] The root system is short and thick.

[0062] Example 4

[0063] Please see Figure 10 The rooting medium per liter includes: MS + KT 2.0 mg / L + NAA 0.3 mg / L, and the rest is the same as in Example 2.

[0064] After 2.5 months of cultivation, roots begin to form at the base of the stem of the tung tree terminal bud, with a rooting rate of over 25%.

[0065] The root system is relatively short.

[0066] Example 5

[0067] Please see Figure 9 The rooting medium per liter includes: MS + KT 2.0 mg / L + NAA 0.1 mg / L + zinc oxide 0.1 mg / L, and the rest is the same as in Example 2.

[0068] After 2.5 months of cultivation, roots begin to form at the base of the stem of the tung tree terminal bud, with a rooting rate of over 25%.

[0069] The root system is relatively short.

[0070] Example 6

[0071] The tissue culture seedlings with roots formed from the basal part of the terminal bud of the tung tree in Example 2 were further cultured. After 4 months, the growth of one bottle was as follows: Figure 13 As shown.

[0072] Example 7

[0073] The tissue culture seedlings with roots formed from the basal part of the terminal bud of the tung tree in Example 4 were further cultured. After 4 months, the growth of one bottle was as follows: Figure 14 As shown.

[0074] Comparative Example 1 Please see Figure 12 The rooting medium per liter includes: MS + KT 2.0 mg / L + NAA 0.3 mg / L + zinc oxide 0.1 mg / L, and the rest is the same as in Example 2.

[0075] After 2.5 months of cultivation, the terminal buds of the tung tree remained green but did not develop roots.

[0076] Comparative Example 2 Please see Figure 11 The rooting medium per liter includes: MS + KT 2.0 mg / L + NAA 0.3 mg / L + zinc oxide 0.05 mg / L, and the rest is the same as in Example 2.

[0077] After 2.5 months of cultivation, no roots were observed to form on the terminal buds of the tung tree.

[0078] Comparative Example 3 Please see Figure 15 Take the apical bud of the hypocotyl of a 3-month-old tung tree, and follow the same procedure as in Example 2.

[0079] After 2.5 months of cultivation, it was observed that no roots were grown at the base of the terminal bud stem, but the bud itself remained green.

[0080] Test case The apical buds and lower hypocotyls of *Paulownia tomentosa* trees cultured for 10 months were placed in culture media containing different hormone combinations, formulations 1–6. After 7 days of culture, browning of the lower hypocotyls was observed under different media treatments. With prolonged culture time, the lower hypocotyls gradually dried up and died, indicating that none of the hormone combinations were suitable for the growth of the lower hypocotyl. The apical buds remained green for the first month under different media treatments, but after 2.5 months of culture, the buds under formulations 2, 3, 4, and 6 began to yellow from the base until the entire bud turned yellow and died (see [link to relevant documentation]). Figure 2 , Figure 3 , Figure 4 and Figure 6 The terminal buds of tung trees cultured with formulas 1 and 5 can still remain green after 2.5 months (see Formula 1). Figure 1 and Figure 5Among the treatments, the buds under formulation 1 grew better, indicating that the hormone combination of KT and NAA is beneficial to the growth of the terminal buds of the tung tree. In addition, the growth was also related to hormone concentration; as the NAA concentration increased, the growth of the terminal buds of the tung tree deteriorated. None of the tung tree explants treated with any of the formulations rooted. This application is inspired by this research.

[0081] Formula 1: MS + KT 2.0 mg / L + NAA 0.1 mg / L Formula 2: MS + 6-BA 0.8 mg / L + NAA 0.4 mg / L Formula 3: MS + 2,4-D 1.0 mg / L + NAA 0.4 mg / L Formula 4: MS + 2,4-D 1.0 mg / L + 6-BA 0.1 mg / L Formula 5: MS + KT 2.0 mg / L + NAA 0.3 mg / L Formula 6: MS + TDZ 0.4 mg / L + NAA 0.2 mg / L.

[0082] This invention reveals that the aseptic bud rooting of the tung tree hypocotyl has strict physiological age requirements; 3-month-old tender aseptic buds, such as... Figure 16 They lack the ability to root and are prone to death; therefore, aseptic buds need to be subcultured for up to 12 months. Figure 17 After reaching physiological maturity, the seedlings are then inoculated into the specially formulated rooting medium optimized by this invention for rooting culture. This invention, through dual technological innovation of suitable physiological age screening and a dedicated rooting medium formula, effectively solves the technical bottlenecks of difficulty in rooting hypocotyl-induced buds and low survival rates in existing tung tree tissue culture. It achieves high rooting efficiency and superior seedling growth, filling a gap in domestic and international technology for regulating the age of hypocotyl-induced buds for rooting in tung trees, and has broad application prospects.

[0083] The embodiments described above merely illustrate specific implementation methods of this application, and while the descriptions are detailed and specific, they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the technical solution of this application, and these modifications and improvements all fall within the scope of protection of this application.

Claims

1. A method for aseptic bud rooting of tung trees, characterized by: Includes the following steps: The hypocotyl of the tung tree was provided, sterilized, and cultured in a bud induction medium to obtain the apical buds germinating from the hypocotyl. Each liter of the bud induction medium contained: 0.5 g / L to 2.0 g / L activated carbon, 1.0 mg / L 6-BA, 0.3 mg / L NAA, and the remainder was MS medium with a pH of 5.

8. The apical buds germinating from the hypocotyls of 12-month-old tung trees were inoculated into rooting medium to obtain rooted tissue culture seedlings. The rooting medium consisted of 1.0 mg / L to 2.5 mg / L KT, 0.1 mg / L to 0.3 mg / L NAA, 0 mg / L to 0.05 mg / L zinc oxide, and the remainder was MS medium.

2. The method for aseptic bud rooting of tung trees according to claim 1, characterized in that: Provide a tung tree hypocotyl, remove the outer shell of the tung tree hypocotyl, rinse the hypocotyl with running tap water for time t1, then soak it in a detergent solution diluted with pure water for time t2, rinse it under running water for time t3, then soak it in potassium permanganate solution for time t4, then disinfect the tung tree hypocotyl with alcohol in a clean bench for time t5, then soak it in HgCl2 for time t6, rinse it with sterile water W times, and finally blot the surface moisture of the hypocotyl with sterile filter paper.

3. The method for aseptic bud rooting of tung trees according to claim 2, characterized in that: t1 is 8 min to 12 min; and / or t2 is 1 min to 5 min; and / or t3 is 2 min to 7 min; and / or t4 is 1 min to 5 min; and / or t5 is 8 s~20 s; and / or t6 is 1 min to 6 min; and / or W is 1 to 3 times; and / or The concentration of the potassium permanganate solution is 0.1%; and / or The alcohol concentration is 75%; and / or The concentration of HgCl2 is 0.1%.

4. The method for aseptic bud rooting of tung trees according to claim 1, characterized in that: After sterilization, the buds were placed in a bud induction medium and cultured under the following conditions: light intensity X1, photoperiod Y1, and temperature T1; and / or The terminal buds sprouting from the explants of the tung tree were inoculated into a rooting medium and cultured under the following conditions: light intensity X2, photoperiod Y2, and temperature T2.

5. The method for aseptic bud rooting of tung trees according to claim 4, characterized in that: X1 is 20 μmol / m 2 •s ~30μmol / m 2 •s; and / or Y1 is 8 h / d ~ 12 h / d; and / or T1 is 20℃~24℃; and / or X2 is 20 μmol / m 2 •s ~30 μmol / m 2 •s; and / or Y2 is 8 h / d ~ 12 h / d; and / or T2 is 20 ℃~24 ℃.

6. The method for aseptic bud rooting of tung trees according to claim 1, characterized in that: The rooting medium per liter includes: 2.0 mg / L KT, 0.1 mg / L NAA, 0.05 mg / L zinc oxide, and the remainder is MS medium.

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