A tissue culture rapid propagation method of taiwania flousiana

Abstract

The application discloses a fast propagation method of Gkitia blakei tissue culture, and relates to the technical field of seedling cultivation, which comprises an explant preparation pretreatment stage, a light-regulated organogenesis culture stage and a bottle-out rhizobium inoculation and transplanting stage, wherein a piece of juvenile stem tip tissue is inserted between the scion and the stock in the explant preparation pretreatment stage by using a sandwich grafting method. The sandwich grafting method inserts the juvenile stem tip tissue between the scion and the stock, effectively breaks the mature effect of the explant of the adult superior tree through the physiological age resetting of the triple structure, greatly reduces the polyphenol oxidase activity of the explant, greatly improves the germination rate, and makes the new shoots keep the excellent genetic characteristics of the mother tree, thereby solving the contradiction between the regeneration capacity recession of the adult material and the genetic uncertainty of the young material. The phased application of the adjustable LED light module realizes the directional regulation of organogenesis, and the overall cultivation cycle is compressed. Stable production is realized through a standardized operation process, and the demand of large-scale afforestation for high-quality seedlings is met.

Description

Technical Field

[0001] This invention relates to the field of seedling cultivation technology, and in particular to a method for rapid propagation of Typha orientalis by tissue culture. Background Technology

[0002] As a national second-class protected plant and a Paleotropical relict species from the Tertiary period, *Taiwanese bald cypress* is an important and rare timber species in southern my country. Its wood is dense and highly resistant to decay, possessing extremely high economic value in construction and furniture manufacturing. Simultaneously, it plays an irreplaceable ecological role in maintaining forest ecosystem diversity and conserving water resources. However, *Taiwanese bald cypress* has a weak natural regeneration capacity, and its large-scale cultivation and resource restoration have always faced severe challenges due to its own biological characteristics and limitations in existing propagation techniques. Currently, the propagation techniques for *Taiwanese bald cypress* mainly rely on traditional methods, which have the following significant drawbacks:

[0003] The seeds of bald cypress have low yields and unstable germination rates. Seeds harvested in the same year typically have a germination rate of less than 30%, which drops sharply to below 10% after a year of cold storage. More importantly, bald cypress seedlings require 15-20 years to reach reproductive maturity, resulting in a lengthy breeding cycle that cannot meet the demand for rapid dissemination of superior germplasm. Furthermore, seed propagation leads to a high rate of genetic variation (over 70%), making it difficult to guarantee the uniformity of seedling traits and severely impacting the quality of timber.

[0004] Traditional cutting propagation techniques are limited by the age and location effects of the mother plant. The rooting rate of cuttings taken from mature, superior trees is generally less than 40%, and surviving seedlings are prone to crown imbalance, leading to bent trunks and significantly reducing the utilization value of the timber. In addition, cutting propagation depends on the number of branches of the mother plant, which is significantly limited by resources and cannot achieve large-scale seedling production.

[0005] Tissue culture of *Taxus chinensis* faces three major challenges: First, severe browning of explants, as the polyphenol oxidase activity in *Taxus chinensis* cells is significantly higher than that in other Taxodiaceae plants, resulting in a browning rate as high as 60%-80% in traditional tissue culture, greatly reducing the survival rate of explants; Second, difficulty in inducing rooting, as conventional auxin treatment has limited effect on promoting root differentiation in *Taxus chinensis*, with a rooting rate often below 50%, and poor root development leading to low transplant survival rates; Third, limited explant selection, as explants from mature superior trees exhibit a "maturation effect," resulting in a decline in morphogenesis ability, while young materials, although possessing strong regeneration ability, have not undergone genetic evaluation, easily leading to the loss of desirable traits.

[0006] Existing technologies suffer from problems such as long cycles, high costs, and poor stability. For example, traditional tissue culture takes up to 120 days, and the cost per seedling is 8-10 times that of conventionally sown seedlings. Furthermore, it is significantly affected by environmental factors, making commercial application difficult. Simultaneously, existing technologies lack a systematic design for regulating environmental factors such as light quality and temperature, failing to precisely control organ differentiation processes and further hindering the improvement of propagation efficiency. In summary, addressing the technical bottlenecks in the propagation of *Cephalotaxus fortunei*, developing a rapid tissue culture propagation technology that can overcome explant age limitations, efficiently control browning, precisely regulate organogenesis, and be applicable on a large scale is of great significance for the protection of *Cephalotaxus fortunei* germplasm resources, the promotion of superior varieties, and industrial breeding. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for rapid propagation of *Taxus chinensis* via tissue culture. The technical solution is as follows:

[0008] A rapid propagation method for bald cypress tissue culture includes an explant creation pretreatment stage, a light-regulated organogenesis culture stage, and an exogenous mycorrhizal transplantation stage. In the explant creation pretreatment stage, a juvenile shoot tip tissue is inserted between the scion and the rootstock using a sandwich grafting method.

[0009] In the photo-regulated organogenesis culture stage, an adjustable LED light module is used to assist in the cultivation of grafted seedlings: monochromatic red light is used to promote cell division during the seedling subculture stage, the light is converted to red and blue combination light during the seedling callus differentiation stage, and far-red light pretreatment is used to activate phytochromes before the seedlings take root.

[0010] The culture medium used in the ectorooting transplantation stage of the bottle-grown mycorrhizal fungi uses a dynamic anti-browning liquid layer with the synergistic effect of natural polyphenol oxidase inhibitors and thiamine to inhibit the browning rate.

[0011] Optionally, the explant creation pretreatment stage includes the following sub-steps: Step 11, cut semi-lignified branches of the current year from the bald cypress tree, with the cut at a 40°-50° angle;

[0012] Step 12, rootstock cultivation: Select 2-year-old bald cypress seedlings and plant them in nutrient pots. Apply slow-release fertilizer to the nutrient pots and cultivate for 6-10 days.

[0013] Step 13, Preparation of juvenile intermediate layer: Take the stem tip of a sterile tissue culture seedling, and under sterile conditions, longitudinally cut it into a juvenile intermediate layer slice with a thickness of 0.4cm-0.6cm, and place it in sterile water at 3℃-5℃ for later use;

[0014] Step 14: Make a transverse cut on the rootstock 4cm-6cm from the base, and make a T-shaped cut on one side of the cut surface to cut off part of the xylem and expose the cambium.

[0015] Step 15: Insert the juvenile shoot tip slice into the incision, aligning the juvenile intermediate layer cambium with the rootstock cambium;

[0016] Step 16: Cut the base of the scion into a wedge shape and insert it above the juvenile intermediate layer, so that the cambium layer of the scion and the cambium layer of the juvenile intermediate layer are in contact;

[0017] Step 17: Use an elastic grafting tape to spirally wrap from 2cm-4cm below the graft union to the top of the scion; Step 18: Spray a mixture of IBA and 6-BA onto the graft union and cover it with a transparent moisture-retaining cover.

[0018] Optionally, the explant preparation stage may also include post-grafting acclimatization management steps:

[0019] From day 9 to 15, place the container in a greenhouse with a shading rate of 75%-85% and a temperature of 24℃-26℃. Open the humidity cover for ventilation for 8-12 minutes every day at noon to maintain the humidity inside the cover at ≥90%.

[0020] Reduce the shading rate by 4.5%-5.5% daily from day 16 to 30. Remove the moisturizing cover from day 29 to 31 and spray with a 0.2% potassium dihydrogen phosphate solution.

[0021] Maintain a greenhouse temperature of 25℃-28℃ from day 31 to 60, and water at least once a week. Collect new shoots sprouting from the scion from day 59 to 61. If the polyphenol oxidase activity is less than or equal to 40 U / g・min, then proceed to the photoregulatory organogenesis culture stage.

[0022] Optionally, the photoregulated organogenesis culture stage includes the following sub-steps:

[0023] Step 21: After soaking the new stem segments in carbendazim, disinfecting them with alcohol and mercuric chloride, inoculate them into a double-layer anti-browning medium, first incubate them in the dark for 4-5 days, and then transfer them to a low-light culture medium of 400-600 lx.

[0024] Step 22: When the sprouts grow to 1.9cm-2.1cm and have 3-4 unfolded leaves, cut off the top of the sprouts under aseptic conditions and transfer them to the proliferation medium.

[0025] Step 23: Turn on the red light mode of the adjustable LED light module, cultivate at 24℃-26℃, and transfer once every 24-26 days. Calculate the proliferation coefficient. If the proliferation coefficient is greater than or equal to 5.0, select healthy seedlings with a height of 3cm-4cm and transfer them to the rooting medium.

[0026] Step 24: Select healthy seedlings that are 3cm-4cm tall, transfer them to the rooting medium, turn on the red and blue combination light of the adjustable LED light module, and culture them at 22℃-24℃ for 5-7 days.

[0027] Step 25: Switch the adjustable LED light module to far-red light mode, provide light for 7-9 hours a day to activate the conversion of photosensitive pigments Pr / Pfr, turn off the light source and carry out dark culture, and maintain the temperature at 22-24℃ for 3-5 days.

[0028] Step 26: Turn off the light source and incubate in the dark, maintaining a temperature of 22℃-24℃ for 5-7 days;

[0029] Step 27: Switch the adjustable LED light module to 1000lx low light and provide 11-13 hours of light per day to promote root elongation. By day 59-61, the root length should be greater than or equal to 2cm and the number of roots should be 3-5.

[0030] Optionally, the bottom layer of the double-layer anti-browning medium is a 0.9cm-1.1cm thick 1 / 2MS solid medium, and the top layer is a 0.15cm-0.25cm thick anti-browning liquid layer, which includes the following components: 15g / L purslane extract, 0.1% activated carbon and 5mg / L thiamine.

[0031] Optional, the red light mode uses an adjustable LED lighting module to control the light wavelength at 634nm and the light intensity at 80μmol / m². 2 •s; The red-blue combined light is controlled by an adjustable LED lighting module, with the ratio of red to blue light set to 3:1, and a total light intensity of 100 μmol / m². 2 •s; The far-red light mode uses an adjustable LED lighting module to control the light wavelength at 730nm and the light intensity at 15μmol / m². 2 ・s.

[0032] Optionally, the ectorooting mycorrhizalization transplantation stage includes the following sub-steps:

[0033] Step 31, prepare the rooting substrate;

[0034] Step 32: Fill the rooting substrate into the seedling trays, autoclave, and then cool to room temperature;

[0035] Step 33: Add colored puffball fungus agent. Evenly mix 4g-6g of fungus agent into the substrate of each well of the seed tray and adjust the substrate pH to 5.5-6.0.

[0036] Step 34: Remove the rooted seedlings from the culture bottle, immerse the roots in a mixture of IBA and carbendazim for 8-12 seconds, then remove and drain.

[0037] Step 35: Make a small hole, 1.3cm-1.8cm deep, in the center of the seedling tray, place the seedling in the hole, and compact the surrounding substrate.

[0038] Step 36: Immediately after transplanting, spray water to help the roots establish until the substrate is thoroughly soaked.

[0039] Optionally, the rooting substrate is prepared by mixing vermiculite and peat in a volume ratio of 3:1, adding 0.15wt%-0.25wt% chitosan and 0.04wt%-0.06wt% controlled-release fertilizer, with the nitrogen, phosphorus and potassium weight ratio of the controlled-release fertilizer being (15-17):(4-6):20.

[0040] Optionally, in step 33, colored puffball fungus agent is added at a ratio of 9%-11%, with mycelial activity greater than 80%.

[0041] Optional, a seedling hardening management stage may also be included:

[0042] Step 41: Place the seedling trays after transplanting in step 36 in a humidity chamber with a humidity of 85%-90%, a shading rate of 75%-85%, a daytime temperature of 24℃-26℃, and a nighttime temperature of 21℃-23℃. Ventilate 1-2 times a day for 7-10 days.

[0043] Step 42: Reduce the humidity to 75%-80%, adjust the shading rate to 45%-55%, and spray water 2-3 times a day for 5-7 days.

[0044] Step 43: Remove the humidity chamber, apply full light conditions, and allow the nighttime temperature to drop to 19℃-21℃. Spray with 0.2% compound fertilizer for 5-7 days.

[0045] Step 44: Move the plant to an outdoor rain shelter for natural light acclimatization. Water it 1-2 times a week. If the transplant survival rate is high on days 39-41, the cultivation is complete.

[0046] In summary, the present invention has at least one of the following beneficial technical effects:

[0047] This invention provides a rapid propagation method for *Cephalotaxus fortunei* through tissue culture. The sandwich grafting method involves inserting juvenile shoot tip tissue between the scion and rootstock. This triple-structure physiological age reset effectively overcomes the maturation effect of explants from mature, superior trees. It significantly reduces the activity of polyphenol oxidase in the explants, greatly increases the germination rate, and ensures that new shoots retain the superior genetic characteristics of the maternal parent, thus resolving the contradiction between the declining regenerative capacity of mature materials and the genetic uncertainty of young materials.

[0048] The phased application of adjustable LED lighting modules enables targeted regulation of organogenesis: monochromatic red light promotes cell division, stabilizing the proliferation coefficient at over 5.0; red-blue combined light optimizes callus differentiation quality; far-red light pretreatment activates the phytochrome system, significantly increasing the rooting rate to over 90%, greatly shortening the rooting time, and compressing the overall cultivation cycle.

[0049] The dynamic liquid layer design, featuring the synergistic effect of natural polyphenol oxidase inhibitors and thiamine, forms a dual barrier of physical isolation and chemical inhibition, significantly reducing explant browning rate and minimizing material loss due to browning. Simultaneously, it avoids the inhibitory effect of single antioxidants on cell division, ensuring proliferation efficiency. The synergistic application of colored bean puffball fungus inoculant and the substrate constructs a symbiotic nutrient network, increasing transplant survival rate to over 90% and mycorrhizal infection rate to over 60%, thus promoting improved seedling absorption efficiency of nutrients such as phosphorus and nitrogen.

[0050] Stable mass production is achieved through standardized operating procedures, reducing the cost per seedling by more than 50% compared to traditional tissue culture, and the annual propagation coefficient can reach more than 1,000 times, meeting the demand for high-quality seedlings for large-scale afforestation and providing a replicable technical paradigm for the protection and industrialization of bald cypress germplasm resources. Attached Figure Description

[0051] Figure 1 This is a flowchart illustrating a rapid propagation method for *Cephalotaxus fortunei* tissue culture according to the present invention.

[0052] Figure 2 This is a schematic diagram of the bald cypress seedlings located in the seedling tray during the ectorooting transplantation stage of the present invention. Detailed Implementation

[0053] The present invention will be further described in detail below with reference to the accompanying drawings.

[0054] This invention discloses a method for rapid propagation of *Cephalotaxus fortunei* via tissue culture.

[0055] Reference Figure 1 and Figure 2 Example 1, a method for rapid propagation of bald cypress tissue culture, includes an explant creation pretreatment stage, a light-regulated organogenesis culture stage, and an exogenous mycorrhizal transplantation stage. In the explant creation pretreatment stage, a juvenile shoot tip tissue is inserted between the scion and the rootstock using the sandwich grafting method.

[0056] In the photo-regulated organogenesis culture stage, an adjustable LED light module is used to assist in the cultivation of grafted seedlings: monochromatic red light is used to promote cell division during the seedling subculture stage, the light is converted to red and blue combination light during the seedling callus differentiation stage, and far-red light pretreatment is used to activate phytochromes before the seedlings take root.

[0057] The culture medium used in the ectorooting transplantation stage of the bottle-grown mycorrhizal fungi uses a dynamic anti-browning liquid layer with the synergistic effect of natural polyphenol oxidase inhibitors and thiamine to inhibit the browning rate.

[0058] By employing the aforementioned technical solution, the sandwich grafting method inserts juvenile shoot tip tissue between the scion and rootstock. Through the physiological age reset of this triple structure, it effectively overcomes the maturation effect of explants from mature superior trees. This significantly reduces the activity of polyphenol oxidase in the explants, greatly increases the germination rate, and ensures that the new shoots retain the excellent genetic characteristics of the maternal parent, thus resolving the contradiction between the declining regeneration capacity of mature materials and the genetic uncertainty of young materials.

[0059] The phased application of adjustable LED lighting modules enables targeted regulation of organogenesis: monochromatic red light promotes cell division, stabilizing the proliferation coefficient at over 5.0; red-blue combined light optimizes callus differentiation quality; far-red light pretreatment activates the phytochrome system, significantly increasing the rooting rate to over 90%, greatly shortening the rooting time, and reducing the overall cultivation cycle by over 40%.

[0060] The dynamic liquid layer design, which combines natural polyphenol oxidase inhibitors with thiamine, forms a dual barrier of physical isolation and chemical inhibition. The browning rate of explants is reduced from 60%-80% in traditional tissue culture to below 15%, significantly reducing material loss caused by browning. At the same time, it avoids the inhibitory effect of single antioxidants on cell division and ensures proliferation efficiency.

[0061] The synergistic application of colored puffball fungicide and substrate creates a symbiotic nutrient network, increasing the transplant survival rate to over 90% and the mycorrhizal infection rate to over 60%, thus improving the seedlings' absorption efficiency of nutrients such as phosphorus and nitrogen.

[0062] Stable mass production is achieved through standardized operating procedures (such as grafting parameters, photoperiod settings, and quantitative control of substrate ratio). The cost per seedling is reduced by more than 50% compared to traditional tissue culture, and the annual propagation coefficient can reach more than 1,000 times. This meets the demand for high-quality seedlings for large-scale afforestation and provides a replicable technical paradigm for the protection and industrialization of bald cypress germplasm resources.

[0063] Example 2, the explant creation pretreatment stage includes the following sub-steps: Step 11, cut semi-lignified branches of the current year from the bald cypress tree, with the cut at a 40°-50° angle;

[0064] Step 12, rootstock cultivation: Select 2-year-old bald cypress seedlings and plant them in nutrient pots. Apply slow-release fertilizer to the nutrient pots and cultivate for 6-10 days.

[0065] Step 13, Preparation of juvenile intermediate layer: Take the stem tip of a sterile tissue culture seedling, and under sterile conditions, longitudinally cut it into a juvenile intermediate layer slice with a thickness of 0.4cm-0.6cm, and place it in sterile water at 3℃-5℃ for later use;

[0066] Step 14: Make a transverse cut on the rootstock 4cm-6cm from the base, and make a T-shaped cut on one side of the cut surface to cut off part of the xylem and expose the cambium.

[0067] Step 15: Insert the juvenile shoot tip slice into the incision, aligning the juvenile intermediate layer cambium with the rootstock cambium;

[0068] Step 16: Cut the base of the scion into a wedge shape and insert it above the juvenile intermediate layer, so that the cambium layer of the scion and the cambium layer of the juvenile intermediate layer are in contact;

[0069] Step 17: Use an elastic grafting tape to spirally wrap from 2cm-4cm below the graft union to the top of the scion; Step 18: Spray a mixture of IBA and 6-BA onto the graft union and cover it with a transparent moisture-retaining cover.

[0070] Example 3, the explant wound pretreatment stage also includes post-grafting acclimatization management steps:

[0071] From day 9 to 15, place the container in a greenhouse with a shading rate of 75%-85% and a temperature of 24℃-26℃. Open the humidity cover for ventilation for 8-12 minutes every day at noon to maintain the humidity inside the cover at ≥90%.

[0072] Reduce the shading rate by 4.5%-5.5% daily from day 16 to 30. Remove the moisturizing cover from day 29 to 31 and spray with a 0.2% potassium dihydrogen phosphate solution.

[0073] Maintain a greenhouse temperature of 25℃-28℃ from day 31 to 60, and water at least once a week. Collect new shoots sprouting from the scion from day 59 to 61. If the polyphenol oxidase activity is less than or equal to 40 U / g・min, then proceed to the photoregulatory organogenesis culture stage.

[0074] By adopting the above technical solutions, the 40°-50° inclined design of the scion cut increases the contact area with the intermediate layer. Combined with the T-shaped cut to precisely expose the rootstock cambium, the alignment rate of the cambium layers in the rootstock-juvenile layer-scion triple structure is increased to over 90%, resulting in a grafting survival rate 35%-40% higher than traditional cleft grafting. The elastic grafting tape, spirally wrapped 2-4cm below the graft union, ensures a tight fit while avoiding tissue necrosis caused by excessive pressure, shortening the callus formation time of the scion to 10-12 days. Pretreatment with juvenile shoot tip slices, preserved in sterile water at 3℃-5℃, maintains a shoot tip tissue survival rate of over 95%, and the juvenile gene signals carried by these slices effectively activate the morphogenesis potential of the scion. Experimental data show that scions treated with this method have higher cytokinin content and lower lignin content in their new shoots compared to directly propagated scions, successfully resetting the physiological age of explants from mature superior trees.

[0075] Phased acclimatization management, through precise control of environmental parameters, simulates the natural microenvironment of new shoot growth in *Pterocarya stenoptera*, improving the uniformity of new shoot emergence from the scion and controlling the difference in branch length within ±0.5cm. Weekly watering combined with 0.2% potassium dihydrogen phosphate spraying promotes the accumulation of photosynthetic products in new shoots, increasing the soluble sugar content by 45% compared to extensive management, thus providing robust explants for subsequent tissue culture.

[0076] Using a polyphenol oxidase activity of ≤40 U / g・min as the threshold for proceeding to the next stage, explants with low browning risk were screened from the source. Testing showed that new shoots meeting this standard had a browning rate controlled below 15% in subsequent tissue culture, a 50%-60% reduction compared to unscreened materials. This significantly reduced culture failures due to explant quality issues and lowered production costs.

[0077] The 6-10 day rootstock pre-cultivation process keeps the seedlings in an active growth state, and combined with 18-20 days of acclimatization under a moisture-retaining cover, the entire pretreatment stage is shortened by 20-25 days compared to traditional grafting seedling cultivation. At the same time, the new shoot germination rate reaches over 85%, providing a stable source of explants for large-scale tissue culture.

[0078] Example 4, the photo-regulated organogenesis culture stage includes the following sub-steps:

[0079] Step 21: After soaking the new stem segments in carbendazim, disinfecting them with alcohol and mercuric chloride, inoculate them into a double-layer anti-browning medium, first incubate them in the dark for 4-5 days, and then transfer them to a low-light culture medium of 400-600 lx.

[0080] Step 22: When the sprouts grow to 1.9cm-2.1cm and have 3-4 unfolded leaves, cut off the top of the sprouts under aseptic conditions and transfer them to the proliferation medium.

[0081] Step 23: Turn on the red light mode of the adjustable LED light module, cultivate at 24℃-26℃, and transfer once every 24-26 days. Calculate the proliferation coefficient. If the proliferation coefficient is greater than or equal to 5.0, select healthy seedlings with a height of 3cm-4cm and transfer them to the rooting medium.

[0082] Step 24: Select healthy seedlings that are 3cm-4cm tall, transfer them to the rooting medium, turn on the red and blue combination light of the adjustable LED light module, and culture them at 22℃-24℃ for 5-7 days.

[0083] Step 25: Switch the adjustable LED light module to far-red light mode, provide light for 7-9 hours a day to activate the conversion of photosensitive pigments Pr / Pfr, turn off the light source and carry out dark culture, and maintain the temperature at 22-24℃ for 3-5 days.

[0084] Step 26: Turn off the light source and incubate in the dark, maintaining a temperature of 22℃-24℃ for 5-7 days;

[0085] Step 27: Switch the adjustable LED light module to 1000lx low light and provide 11-13 hours of light per day to promote root elongation. By day 59-61, the root length should be greater than or equal to 2cm and the number of roots should be 3-5.

[0086] Example 5: The bottom layer of the double-layer anti-browning culture medium is a 0.9cm-1.1cm thick 1 / 2MS solid culture medium, and the top layer is a 0.15cm-0.25cm thick anti-browning liquid layer, which includes the following components: 15g / L purslane extract, 0.1% activated carbon and 5mg / L thiamine.

[0087] Example 6: The red light mode is controlled by an adjustable LED lighting module, with a light wavelength of 634nm and a light intensity of 80μmol / m². 2 •s; The red-blue combined light is controlled by an adjustable LED lighting module, with the ratio of red to blue light set to 3:1, and a total light intensity of 100 μmol / m². 2 •s; The far-red light mode uses an adjustable LED lighting module to control the light wavelength at 730nm and the light intensity at 15μmol / m². 2 ・s.

[0088] By employing the above technical solution, a dynamic protective barrier is formed between the bottom 1 / 2 MS solid culture medium and the upper 0.15-0.25 cm thick anti-browning liquid layer, containing 15 g / L purslane extract, 0.1% activated carbon, and 5 mg / L thiamine. The natural polyphenol oxidase inhibitor in the purslane extract specifically inhibits enzyme activity, activated carbon adsorbs free phenolic substances, and thiamine enhances the cell's antioxidant capacity. The synergistic effect of these three components reduces the explant browning rate from 60%-80% in traditional single-layer culture media to below 15%, while avoiding the inhibition of cell division by a single antioxidant, ensuring a stable germination rate of over 85%.

[0089] Red light mode (634nm, 80μmol / m) 2 •s): Targeted promotion of cytokinin signal transduction, stabilizing the subgeneration proliferation coefficient at over 5.0, and increasing seedling vigor by over 40%.

[0090] Red-blue combination light (3:1, 100 μmol / m) 2 •s): Optimizes chloroplast development and photosynthetic efficiency, increases callus differentiation rate to 90%, an increase of 25% compared to monochromatic light treatment, and increases chlorophyll content of differentiated seedlings by more than 30%.

[0091] Far-red light pretreatment (730nm, 15μmol / m) 2•s): By activating the conversion of phytochrome Pr / Pfr, the expression of auxin-responsive genes is upregulated, increasing the root primordium induction rate to 92%, which is more than 50% higher than that without far-red light treatment.

[0092] Dark culture for 4-5 days combined with a weak light transition of 400-600 lx avoids stress-induced browning caused by strong light, thus shortening the initial culture cycle.

[0093] A subculture cycle of 24-26 days and constant temperature control of 24-26℃ standardize the proliferation process and reduce the batch-to-batch variation coefficient.

[0094] The alternating light and dark regulation during the rooting stage reduces the root elongation time from the traditional 45-60 days to 35 days, and the proportion of healthy roots with 3-5 roots of 2cm or more reaches 90%, which is significantly better than the proportion of deformed roots in conventional methods.

[0095] The step-by-step sterilization process of carbendazim soaking, alcohol disinfection, and mercuric chloride treatment, combined with strict aseptic operation, keeps the contamination rate of the culture process below 5%, greatly reducing material waste and batch loss caused by contamination.

[0096] Example 7, the ectorooting mycorrhizal transplantation stage includes the following sub-steps:

[0097] Step 31, prepare the rooting substrate;

[0098] Step 32: Fill the rooting substrate into the seedling trays, autoclave, and then cool to room temperature;

[0099] Step 33: Add colored puffball fungus agent. Evenly mix 4g-6g of fungus agent into the substrate of each well of the seed tray and adjust the substrate pH to 5.5-6.0.

[0100] Step 34: Remove the rooted seedlings from the culture bottle, immerse the roots in a mixture of IBA and carbendazim for 8-12 seconds, then remove and drain.

[0101] Step 35: Make a small hole, 1.3cm-1.8cm deep, in the center of the seedling tray, place the seedling in the hole, and compact the surrounding substrate.

[0102] Step 36: Immediately after transplanting, spray water to help the roots establish until the substrate is thoroughly soaked.

[0103] Example 8: The rooting substrate is prepared by mixing vermiculite and peat in a volume ratio of 3:1, adding 0.15wt%-0.25wt% chitosan and 0.04wt%-0.06wt% controlled-release fertilizer. The nitrogen, phosphorus and potassium weight ratio of the controlled-release fertilizer is (15-17):(4-6):20.

[0104] In Example 9, step 33 involves adding colored puffball fungus at a ratio of 9%-11%, resulting in mycelial activity greater than 80%.

[0105] By adopting the above technical solution, vermiculite retains water and is breathable. When mixed with peat moss at a volume ratio of 3:1 for slow-release nutrients, it forms a loose structure with a porosity of over 60%. Combined with 0.15%-0.25% chitosan, this avoids the compaction problem of traditional single-substrate methods and forms a protective film on the root surface, reducing water loss and pathogen invasion. The precise ratio of controlled-release fertilizer (N-P2O5-K2O=15-17:4-6:20) meets the phosphorus and potassium preferences of bald cypress seedlings, resulting in increased seedling growth compared to ordinary substrates. The fertilizer effect lasts for more than 3 months, reducing the frequency of topdressing.

[0106] The colored puffball fungus inoculant, added at a ratio of 9%-11%, exhibits mycelial activity exceeding 80%, forming a stable symbiotic relationship with the roots of the bald cypress, achieving a mycorrhizal infection rate of over 60%. The Haty network structure expands the nutrient absorption area, improving the seedlings' utilization of soil phosphorus and nitrogen, effectively addressing the problem of weak roots in tissue-cultured seedlings. Simultaneously, the secondary metabolites secreted by the mycorrhizae enhance the root system's antioxidant capacity; under drought stress, the relative water content of leaves is 20%-25% higher than that of seedlings without mycorrhizae, and the incidence of root rot decreases from 30% to below 5%.

[0107] Dip seedling roots in a mixture of IBA (500 mg / L) and carbendazim (0.3%) for 8-12 seconds to form double protection: IBA promotes adventitious root elongation and increases root fresh weight, while carbendazim forms a film on the root surface, reducing the risk of fungal infection after transplanting. Combined with a precise planting depth of 1.3-1.8 cm, avoiding root suffocation from planting too deep or dehydration from planting too shallow, the root recovery time is shortened from the traditional 15 days to 7 days.

[0108] Standardized control of the transplanting process is achieved by mixing 4-5g of microbial agent into each cell of the seedling tray, strictly controlling the substrate pH at 5.5-6.0, and spraying water to ensure thorough saturation at the bottom of the substrate. This ensures a stable batch survival rate of over 90%, with coefficients of variation for seedling height and diameter at ground level both less than 10%, providing uniform and high-quality seedlings for subsequent afforestation.

[0109] Example 10 also includes a seedling hardening management stage:

[0110] Step 41: Place the seedling trays after transplanting in step 36 in a humidity chamber with a humidity of 85%-90%, a shading rate of 75%-85%, a daytime temperature of 24℃-26℃, and a nighttime temperature of 21℃-23℃. Ventilate 1-2 times a day for 7-10 days.

[0111] Step 42: Reduce the humidity to 75%-80%, adjust the shading rate to 45%-55%, and spray water 2-3 times a day for 5-7 days.

[0112] Step 43: Remove the humidity chamber, apply full light conditions, and allow the nighttime temperature to drop to 19℃-21℃. Spray with 0.2% compound fertilizer for 5-7 days.

[0113] Step 44: Move the plant to an outdoor rain shelter for natural light acclimatization. Water it 1-2 times a week. If the transplant survival rate is high on days 39-41, the cultivation is complete.

[0114] By adopting the above technical solution, the high humidity (85%-90%) and high shading (75%-85%) conditions in step 41 simulate the microenvironment inside the tissue culture bottle, so that the transpiration water loss rate of the newly transplanted seedlings is controlled below 10%, thus avoiding wilting and death.

[0115] Steps 42-44 involve gradually decreasing humidity, shading, and temperature, with each stage's environmental parameter variation controlled within 10%-15%, allowing seedlings to gradually establish physiological mechanisms for drought and light stress tolerance. This reduces the stress-induced leaf drop rate from 25% in traditional methods to below 5%.

[0116] The gradual transition from low light to full light increases the chlorophyll a / b ratio in the leaves and increases the content of carotenoids, effectively avoiding damage to the photosynthetic apparatus caused by strong light.

[0117] Step 43, with full-light acclimatization and moderate nighttime cooling, promoted the improvement of seedling photosystem II repair efficiency, and the average daily photosynthetic accumulation increased by 15% compared with the constant temperature treatment.

[0118] Spraying with 0.2% compound fertilizer can specifically replenish the nutrients consumed during the hardening-off stage, thereby increasing the soluble sugar content and free amino acid content of the seedlings and providing a material basis for stress resistance.

[0119] Throughout the hardening process, the seedling root vigor (TTC reduction strength) was enhanced, and the new shoot growth reached more than 1.5cm, which is more than 60% higher than that of the unhardened seedlings, laying a growth foundation for subsequent field transplanting.

[0120] Standardized seedling hardening-off cycle keeps the difference in survival rate between batches within 5%, and ultimately stabilizes the transplant survival rate at over 90%.

[0121] The natural light acclimatization under the outdoor rain shelter significantly enhances the seedlings' adaptability to diurnal temperature differences and light intensity fluctuations. The recovery period after transplanting in the field is shortened from 15 days to 7 days, and the survival rate is higher than that of direct indoor transplanting.

[0122] The following specific embodiments illustrate the implementation principle of the present invention:

[0123] This embodiment was conducted at a bald cypress germplasm resource bank in a certain city. A 25-year-old superior tree with a diameter at breast height of 42cm and a height of 28m was selected as the parent tree. The implementation period was 180 days. The specific operation is as follows:

[0124] Innovative pretreatment of explants:

[0125] Scion: Take a semi-lignified branch from the current year (6cm long, with 2 axillary buds), and make a 45° angled cut;

[0126] Rootstock: 2-year-old seedlings (0.6cm in diameter at ground level), transplanted into 15cm seedling pots, and 5g of slow-release fertilizer with a N-P2O5-K2O ratio of 16:5:20 is applied;

[0127] Juvenile intermediate layer: Take the stem tip (0.8cm long) of a sterile tissue culture seedling, cut it longitudinally into a 0.5cm thick slice, and store it in sterile water at 4℃;

[0128] Post-grafting acclimatization: Maintain 80% shading and 90% humidity from day 9 to 15; spray with 0.2% potassium dihydrogen phosphate on day 30; collect new branches on day 60; polyphenol oxidase activity is 38 U / g・min.

[0129] Photoregulatory organogenesis culture:

[0130] Disinfection process: Soak in 0.1% carbendazim for 30 minutes, 75% alcohol for 30 seconds, 0.1% mercuric chloride for 8 minutes, and rinse with sterile water 5 times;

[0131] Double-layer culture medium: bottom layer 1cm thick 1 / 2 MS solid medium, top layer 0.2cm anti-browning solution, 15g / L purslane extract, 0.1% activated charcoal and 5mg / L thiamine.

[0132] Light control parameters:

[0133] Proliferation stage: 634nm red light (80μmol / m 2 •s), 25℃, subculture once every 25 days

[0134] Differentiation stage: Red-blue combination light (3:1, 100 μmol / m 2 •s), 23℃

[0135] Before rooting: 730nm far-red light (15μmol / m 2 •s), 8 hours of sunlight per day

[0136] Mycorrhizal transplantation via ectorooting:

[0137] Rooting substrate: vermiculite: peat moss volume ratio 3:1, with the addition of 0.2% chitosan and 0.05% controlled-release fertilizer, pH 5.8;

[0138] Inoculation with fungal agent: Mix 5g of colored puffball fungus agent (mycelial activity 85%) into each well of substrate;

[0139] Transplanting procedure: Dip seedling roots in a mixture of 500mg / LIBA + 0.3% carbendazim for 10 seconds, and plant at a depth of 1.5cm;

[0140] Seedling hardening management:

[0141] Phase 1 (8 days): Humidity 88%, shading rate 80%, daytime temperature 25℃ / nighttime temperature 22℃;

[0142] Phase 2 (6 days): Humidity 78%, shading rate 50%, spray water twice a day;

[0143] Phase 3 (6 days): Full sunlight, nighttime temperature 20℃, spray with 0.2% compound fertilizer;

[0144] Phase 4 (20 days): Outdoor rain shelter acclimatization, water once a week.

[0145] Complete cultivation process.

[0146] Table 1 shows a comparison of the effects with traditional methods:

[0147] Table 1

[0148] Comparison Projects Traditional tissue culture methods This technical solution Increase Explant pretreatment Grafting survival rate The survival rate of cleft grafting is 55%-60%. The survival rate of sandwich grafting is 92%. Increase by 32-37 percentage points Polyphenol oxidase activity 85-100 U / g·min of mature scions The new shoots after treatment had a concentration of 38 U / g·min. Reduced by 55.3%-62% Preprocessing cycle 90-100 days 60 days Shortened by 33.3%-40% Photoregulatory organ culture browning rate of explants 60%-80% 12% Reduced by 48-68 percentage points Proliferation coefficient 3.0-3.5 5.2 Increase by 50%-73.3% Rooting rate 45%-50% 91% An increase of 41-46 percentage points Rooting cycle 45-60 days 35 days Shortened by 22.2%-41.7% Transplanting and hardening off Mycorrhizal infection rate The natural infection rate is less than 10%. 65% Increase by more than 55 percentage points Transplant survival rate 50%-55% 92% Increase by 37-42 percentage points New shoot growth after hardening 0.8-1.0cm 1.6cm Increase by 60%-100% Overall benefits Annual reproductive coefficient 300-400 times 1050 times Increase by 162.5%-250% Single seedling production cost 2.97 yuan 1.45 yuan Reduced by 51.2% Total breeding cycle 180-210 days 110 days Shortened by 38.1%-47.6% Field transplant survival rate 60%-65% 90% Increase by 25-30 percentage points

[0149] Sandwich grafting resets the physiological age of explants from mature superior trees, reduces polyphenol oxidase activity by 62%, and shortens the pretreatment cycle by 40%, solving the problem of poor rooting caused by the maturation effect of traditional cuttings. Light-regulated culture: The dynamic anti-browning system and staged light quality regulation work synergistically to reduce the browning rate from 70% to 12%, increase the proliferation coefficient by 54%, and shorten the rooting cycle by 33%, breaking through the triple bottleneck of browning-proliferation-rooting in traditional tissue culture.

[0150] Transplanting and hardening-off: Combining mycorrhizal symbiosis with tiered hardening-off increased the transplant survival rate from 52% to 92%, doubled the growth of new shoots, and shortened the recovery period after field planting from 15 days to 7 days, significantly enhancing the environmental adaptability of seedlings.

[0151] Industrialization value: The annual propagation coefficient reaches 1050 times, and the cost per seedling is reduced by more than 50%, providing an economically feasible technical solution for the large-scale seedling cultivation of bald cypress, with a comprehensive benefit increase of 2.3 times compared with traditional methods.

[0152] The above are all preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of *Taxus chinensis* via tissue culture, characterized in that: The process includes the explant creation pretreatment stage, the light-regulated organogenesis culture stage, and the bottle exogenous mycorrhizal transplantation stage. In the explant creation pretreatment stage, a piece of juvenile shoot tip tissue is inserted between the scion and the rootstock using the sandwich grafting method. In the photo-regulated organogenesis culture stage, an adjustable LED light module is used to assist in the cultivation of grafted seedlings: monochromatic red light is used to promote cell division during the seedling subculture stage, the light is converted to red and blue combination light during the seedling callus differentiation stage, and far-red light pretreatment is used to activate phytochromes before the seedlings take root. The culture medium used in the photoregulated organogenesis stage employed a dynamic anti-browning liquid layer to synergistically inhibit browning rate with natural polyphenol oxidase inhibitors and thiamine. If the new shoots sprouting from the scion are collected and the polyphenol oxidase activity is less than or equal to 40 U / g·min, they will enter the photoregulated organogenesis culture stage. The photoregulatory organogenesis culture stage includes the following sub-steps: Step 21: After soaking the new stem segments in carbendazim, disinfecting them with alcohol and mercuric chloride, inoculate them into a double-layer anti-browning medium, first incubate them in the dark for 4-5 days, and then transfer them to a low-light culture medium of 400-600 lx. Step 22: When the sprouts grow to 1.9cm-2.1cm and have 3-4 unfolded leaves, cut off the top of the sprouts under aseptic conditions and transfer them to the proliferation medium. Step 23: Turn on the red light mode of the adjustable LED light module, cultivate at 24℃-26℃, and transfer once every 24-26 days. Calculate the proliferation coefficient. If the proliferation coefficient is greater than or equal to 5.0, select healthy seedlings with a height of 3cm-4cm and transfer them to the rooting medium. Step 24: Select healthy seedlings that are 3cm-4cm tall, transfer them to the rooting medium, turn on the red and blue combination light of the adjustable LED light module, and culture them at 22℃-24℃ for 5-7 days. Step 25: Switch the adjustable LED light module to far-red light mode, provide light for 7-9 hours a day to activate the conversion of photosensitive pigments Pr / Pfr, turn off the light source and carry out dark culture, and maintain the temperature at 22-24℃ for 3-5 days. Step 26: Turn off the light source and incubate in the dark, maintaining a temperature of 22℃-24℃ for 5-7 days; Step 27: Switch the adjustable LED light module to 1000lx low light and provide 11-13 hours of light per day to promote root elongation. By day 59-61, the root length should be greater than or equal to 2cm and the number of roots should be 3-5. The bottom layer of the double-layer anti-browning medium is a 0.9cm-1.1cm thick 1 / 2MS solid medium, and the top layer is a 0.15cm-0.25cm thick anti-browning liquid layer, which includes the following components: 15g / L purslane extract, 0.1% activated carbon and 5mg / L thiamine; The red light mode uses an adjustable LED lighting module to control the light wavelength at 634nm and the light intensity at 80μmol / m²·s; the red-blue combined light mode uses an adjustable LED lighting module to control the ratio of red light to blue light at 3:1 and the total light intensity at 100μmol / m²·s; and the far-red light mode uses an adjustable LED lighting module to control the light wavelength at 730nm and the light intensity at 15μmol / m²·s.

2. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 1, characterized in that: The pretreatment stage of explant creation includes the following sub-steps: Step 11, cut semi-lignified branches of the current year from the superior bald cypress tree, with the cut at a 40°-50° angle; Step 12, rootstock cultivation: Select 2-year-old bald cypress seedlings and plant them in nutrient pots. Apply slow-release fertilizer to the nutrient pots and cultivate for 6-10 days. Step 13, Preparation of juvenile intermediate layer: Take the stem tip of a sterile tissue culture seedling, and under sterile conditions, longitudinally cut it into a juvenile intermediate layer slice with a thickness of 0.4cm-0.6cm, and place it in sterile water at 3℃-5℃ for later use; Step 14: Make a transverse cut on the rootstock 4cm-6cm from the base, and make a T-shaped cut on one side of the cut surface to cut off part of the xylem and expose the cambium. Step 15: Insert the juvenile shoot tip slice into the incision, aligning the juvenile intermediate layer cambium with the rootstock cambium; Step 16: Cut the base of the scion into a wedge shape and insert it above the juvenile intermediate layer, so that the cambium layer of the scion and the cambium layer of the juvenile intermediate layer are in contact; Step 17: Use an elastic grafting tape to spirally wrap from 2cm-4cm below the graft union to the top of the scion; Step 18: Spray a mixture of IBA and 6-BA onto the interface and cover it with a transparent moisturizing cover.

3. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 2, characterized in that: The explant preparation stage also includes post-grafting acclimatization management steps: From day 9 to 15, place the container in a greenhouse with a shading rate of 75%-85% and a temperature of 24℃-26℃. Open the humidity cover for ventilation for 8-12 minutes every day at noon to maintain the humidity inside the cover at ≥90%. Reduce the shading rate by 4.5%-5.5% daily from day 16 to 30. Remove the moisturizing cover from day 29 to 31 and spray with a 0.2% potassium dihydrogen phosphate solution. Maintain a greenhouse temperature of 25℃-28℃ from day 31 to 60, and water at least once a week. Collect new shoots sprouting from the scion from day 59 to 61. If the polyphenol oxidase activity is less than or equal to 40 U / g・min, then proceed to the photoregulatory organogenesis culture stage.

4. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 3, characterized in that: The ectorooted mycorrhizal transplantation stage includes the following sub-steps: Step 31, prepare the rooting substrate; Step 32: Fill the rooting substrate into the seedling trays, autoclave, and then cool to room temperature; Step 33: Add colored puffball fungus agent. Evenly mix 4g-6g of fungus agent into the substrate of each well of the seed tray and adjust the substrate pH to 5.5-6.

0. Step 34: Remove the rooted seedlings from the culture bottle, immerse the roots in a mixture of IBA and carbendazim for 8-12 seconds, then remove and drain. Step 35: Make a small hole 1.3cm-1.8cm deep in the center of the seedling tray, put the seedling into the hole, and compact the surrounding substrate. Step 36: Immediately after transplanting, spray water to help the roots establish until the substrate is thoroughly soaked.

5. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 4, characterized in that: The method for preparing the rooting substrate is as follows: vermiculite and peat are mixed in a volume ratio of 3:1, and 0.15wt%-0.25wt% of chitosan and 0.04wt%-0.06wt% of controlled-release fertilizer are added. The weight ratio of nitrogen, phosphorus and potassium in the controlled-release fertilizer is (15-17):(4-6):

20.

6. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 5, characterized in that: Step 33: Add colored puffball fungus at a ratio of 9%-11%, with mycelial activity greater than 80%.

7. The method for rapid propagation of *Taxus chinensis* via tissue culture according to claim 6, characterized in that: It also includes the seedling hardening management stage: Step 41: Place the seedling trays after transplanting in step 36 in a humidity chamber with a humidity of 85%-90%, a shading rate of 75%-85%, a daytime temperature of 24℃-26℃, and a nighttime temperature of 21℃-23℃. Ventilate 1-2 times a day for 7-10 days. Step 42: Reduce the humidity to 75%-80%, adjust the shading rate to 45%-55%, and spray water 2-3 times a day for 5-7 days. Step 43: Remove the humidity chamber, apply full light conditions, and allow the nighttime temperature to drop to 19℃-21℃. Spray with 0.2% compound fertilizer for 5-7 days. Step 44: Move the seedlings to an outdoor rain shelter for natural light acclimatization. Water them 1-2 times a week. If the transplant survival rate meets the standard on days 39-41, the cultivation is complete.

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