A tissue culture propagation method of cupressus gigantea

Abstract

The application belongs to the technical field of plant tissue culture and particularly relates to a tissue culture propagation method of Cupressus gigantea. In order to solve the technical problems of difficult asexual reproduction, low seed germination and rooting rates of Cupressus gigantea, the application provides the tissue culture propagation method of Cupressus gigantea, which comprises the following steps: taking Cupressus gigantea seeds or branches as explants, sterilizing the explants, and then sequentially performing initiation culture and rooting culture; when the Cupressus gigantea seeds are taken as the explants, the initiation culture further comprises proliferation culture. The application obtains the optimal tissue culture condition of Cupressus gigantea by screening the sterilization mode, the initiation culture medium, the proliferation culture medium and the rooting culture medium, and the survival rate of transplanting is more than 90% after seedling transplanting and local transplanting. The application provides technical support for the mass propagation of Cupressus gigantea and has important significance for the field return and population recovery of Cupressus gigantea.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture propagation method for giant cypress. Background Technology

[0002] Plant tissue culture is a new asexual reproduction technique developed in recent decades based on the theory of totipotency of plant cells. Woody plant tissue culture seedling production involves culturing the tissues or organs of a living woody plant under sterile conditions that provide the necessary nutrients and environment for its growth and development, thus transforming it into a new, independent plant. The tissues or organs can be taken from any part of the woody plant, such as roots, stems, leaves, flowers, fruits, seeds, embryos, etc., including cells, single cells, and even protoplasts within cells.

[0003] Giant cypress (Cupressus gigantean) belongs to the genus Cupressus in the family Cupressaceae. It is endemic to Tibet and the middle and lower reaches of the Yarlung Tsangpo River. Listed as a Class I protected wild plant in China, it is an ancient relict species. With the implementation of engineering projects, the land occupied by these projects poses a threat to wild resources, necessitating urgent research into artificial propagation techniques and related areas to provide technical support for its conservation.

[0004] However, tissue culture rooting is a complex physiological and biochemical process, and rooting is generally difficult in cypress family plants. Although there are reports of tissue culture rooting in cypress family plants such as Platycladus orientalis and Juniperus chinensis, these explants are all from young trees, and there are no reports of ancient cypress family trees over 100 years old; moreover, the rooting situation is not ideal, and the rooting rate is extremely low. For example, Zhao Yuxin (Zhao Yuxin. Study on tissue culture regeneration of ancient Platycladus orientalis trees and cloning of telomere-related genes [D]. Chinese Academy of Forestry, 2015) collected healthy current-year branches from ancient Platycladus orientalis trees at the Yellow Emperor's Mausoleum in Shaanxi Province for rapid tissue culture propagation. He screened out suitable sterilization methods, primary culture media, proliferation culture media and rooting culture media for ancient Platycladus orientalis trees, and initially established a tissue culture system for Platycladus orientalis. 8% of the materials rooted in WPM rooting medium, with an average of 1-2 roots per tree and an average root length of 1 cm, but the rooting situation was not ideal and the rooting rate was extremely low.

[0005] The giant cypress community has a rich family and genera composition, but the tree layer has a relatively simple species composition, and the population structure is mostly declining. Seed germination is difficult, and the seed setting rate is low, far lower than that of red pine and Chinese fir, resulting in a low seed vigor and germination rate. Therefore, developing a suitable propagation technique for giant cypress is not easy, and successfully obtaining a large number of regenerated giant cypress plants through tissue culture propagation is of great significance for the protection of giant cypress. Summary of the Invention

[0006] To address the technical problems of difficult asexual propagation of *Juniperus chinensis* and low seed germination and rooting rates, this invention provides a tissue culture propagation method for *Juniperus chinensis*. This invention uses *Juniperus chinensis* seeds and branches as explant sources for tissue culture, achieving regeneration of tissue-cultured seedlings in both cases. Furthermore, after hardening off and transplanting, and localization transplanting, the survival rate after transplanting exceeds 90%.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] This invention provides a tissue culture propagation method for giant cypress, which includes the following steps: using giant cypress seeds or branches as explants, sterilizing the explants, and then sequentially performing initiation culture and rooting culture; when using giant cypress seeds as explants, the initiation culture also includes proliferation culture.

[0009] When using giant cypress seeds as explants,

[0010] The sterilization method is 75% alcohol for 60 seconds + 30% sodium hypochlorite for 10 minutes.

[0011] The starter medium was MS medium.

[0012] Both the proliferation medium and the rooting medium were MS + 0.3 mg / L NAA + 0.05 mg / L IAA.

[0013] Furthermore, the propagation culture involves selecting sterile cypress seedlings with a height of over 8cm after the culture has been initiated, cutting them into 2cm stem segments with buds, and inoculating them into the propagation culture medium.

[0014] Furthermore, the rooting culture involves cutting healthy seedlings after proliferation culture into stem segments of more than 3 cm and inoculating them into a rooting culture medium.

[0015] The giant cypress branches were collected from tender branches of ancient giant cypress trees with a diameter at breast height of 0.5 to 85 cm.

[0016] When using giant cypress branches as explants,

[0017] The sterilization method is 75% alcohol for 30 seconds + 30% sodium hypochlorite for 5 minutes.

[0018] The starting medium was 1 / 2NN69 + 0.1 mg / L IBA.

[0019] The rooting medium was 1 / 2 MS + 0.3 mg / L NAA + 0.05 mg / L IAA.

[0020] The rooting culture involves selecting robust seedlings that are over 3cm tall after the initial culture has begun, and then inoculating them into a rooting culture medium.

[0021] During tissue culture, the culture conditions were as follows: light / dark ratio of 14 / 10h, light intensity of 2000 Lux, temperature of 24℃±2℃, and humidity of 60%±10%.

[0022] The initiation culture time is 42-56 days, the proliferation culture time is 40-42 days, and the rooting culture time is 30-35 days.

[0023] The tissue culture propagation method also includes hardening off and transplanting the rooted tissue culture seedlings after rooting culture.

[0024] Furthermore, the seedling hardening and transplanting process involves first hardening the tissue-cultured rooted seedlings in closed bottles for 5-7 days, then hardening them in open bottles for another 5-7 days, washing off the culture medium from the roots with clean water, and then hardening them in a substrate with a peat moss:perlite volume ratio of 2-3:1.

[0025] Furthermore, during the seedling hardening and transplanting process, it is necessary to spray and moisturize the seedlings, spraying for 30-45 seconds each in the morning, noon and evening, and controlling the indoor temperature at 22℃-30℃ and the humidity at 80%±2℃.

[0026] Beneficial Effects: This invention uses *Juniperus chinensis* seeds or branches as explants. After sterilization, the explants undergo sequential initiation and rooting cultures. When using *Juniperus chinensis* seeds as explants, the initiation culture is followed by a proliferation culture. This invention achieves optimal culture conditions through screening sterilization methods, initiation media, proliferation media, and rooting media. Experimental results show that when using *Juniperus chinensis* seeds as explants, with optimal sterilization methods, initiation media, proliferation media, and rooting media components and ratios, the rooting rate is approximately 60%, and the survival rate after hardening-off transplanting and localization transplanting both reach over 90%. When using *Juniperus chinensis* branches as explants, the rooting rate can also reach 50% through optimization of sterilization methods, initiation media, and rooting media. Moreover, the survival rate after localization transplanting of tissue culture seedlings is over 90%. This provides technical support for the large-scale propagation of *Juniperus chinensis* and is of great significance for its reintroduction into the wild and population recovery. Attached Figure Description

[0027] Figure 1 This is a germination trend chart of tissue culture seeds from *Juniperus chinensis* in Example 1.2;

[0028] Figure 2 The image shows the seed germination of *Juniperus chinensis* in Example 1.2 (from left to right: early stage, middle stage, and late stage).

[0029] Figure 3 This is a comparison diagram of the initial screening of the proliferation culture medium for *Juniperus chinensis* in Example 1.3;

[0030] Figure 4 This is a rooting diagram of *Juniperus chinensis* tissue culture seedlings in Example 1.5 using 2E medium;

[0031] Figure 5 This is a diagram showing the survival rate of tissue culture seedlings of *Juniperus chinensis* in Example 1.6.

[0032] Figure 6 This is a comparison diagram of the initial culture of *Juniperus chinensis* explants in Example 2.2;

[0033] Figure 7 This is a comparison chart of screening basic culture media for *Juniperus chinensis* in Example 2.3;

[0034] Figure 8 This is a diagram of rooted cypress seedlings from Example 2.4;

[0035] Figure 9 This is a diagram illustrating the local adaptation of tissue-cultured *Juniperus chinensis* seedlings in Example 3. Detailed Implementation

[0036] To make the technical problems, solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with the embodiments. Unless otherwise defined, all technical terms used herein have the same meaning as understood by one of ordinary skill in the art.

[0037] This invention uses both seeds and branches of *Juniperus chinensis* as explants to study *Juniperus chinensis* tissue culture, and analyzes the effects of different sterilization methods and culture medium formulations on the rapid propagation system of *Juniperus chinensis* tissue culture.

[0038] In one embodiment of this invention, *Juniperus chinensis* seeds were used as explants for tissue culture. Screening of sterilization methods, different proliferation media, and rooting media revealed that sterilization of *Juniperus chinensis* seeds using 75% ethanol (C₂H₆O) for 60 seconds followed by 30% sodium hypochlorite (NaClO) for 10 minutes, followed by inoculation onto MS medium, yielded sterile tissue-cultured seedlings. After 6 weeks, the initial induction rate was 51%. The optimal proliferation medium was MS + 0.3 mg / L NAA + 0.05 mg / L IAA. Seedlings proliferated on this medium showed significant elongation growth, with green, robust new shoots and rooting. The induction rate was 100%, and the proliferation coefficient was 4.33. Furthermore, this medium simultaneously promoted rooting, with a rooting rate of approximately 60%. After hardening and transplanting, the survival rate after transplanting was over 90%.

[0039] In one embodiment of the present invention, using cypress branches as explants, screening different mother plant culture environments, different primary culture media, basic culture media, and rooting culture media revealed that placing the mother plant in a greenhouse and sterilizing the collected explants with a combination of 75% ethanol for 30 seconds and 30% sodium hypochlorite for 5 minutes, then inoculating them onto 1 / 2 NN69 + 0.1 mg / L IBA primary culture medium for primary induction, and then directly inoculating them onto 1 / 2 MS + 0.3 mg / L NAA + 0.05 mg / L IAA culture medium for rooting culture, can achieve direct rooting, with a green retention rate of 70%, a rooting rate of 50%, and an average of 3.25 roots.

[0040] The following specific embodiments will be provided to explain the solution of the present invention. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0041] All experimental data were analyzed using SPSS 25. Contamination rate, germination rate, proliferation coefficient, and rooting rate were all analyzed using one-way ANOVA. The culture medium components and ratios described in this invention are commercially available conventional culture media, using 75 v / v ethanol and 30 v / v sodium hypochlorite.

[0042] The hormones used in the following examples include chlorpyrifos (CPPU), naphthaleneacetic acid (NAA), kinetin (KT), zeatin (ZT), isopentenyl adenine (2-IP), gibberellin (GA3), rooting powder No. 1 (ABT1), 3-indolebutyric acid (IBA), indoleacetic acid (IAA), and 6-benzylaminopurine (6-BA).

[0043] Example 1: Tissue culture derived from giant cypress seed explants

[0044] The giant cypress seeds of Example 1 of this invention were collected from a village in Tibet.

[0045] 1.1 Effects of different sterilization methods on obtaining sterile seedlings of Giant Cypress

[0046] The collected giant cypress seeds were sterilized with 75% alcohol and then with 30% sodium hypochlorite before being inoculated onto MS medium. Contamination and germination status were observed and statistically analyzed. The optimal sterilization treatment method was screened.

[0047] Contamination rate = (Number of contaminated seeds / Total number of inoculated seeds) × 100%;

[0048] Germination rate = (Number of germinated seeds / Total number of inoculated seeds) × 100%.

[0049] Sterilization experiments were conducted on *Crassula ovata* seeds using a combination of 75% ethanol and 30% sodium hypochlorite. The results are shown in Table 1. Different sterilization times did not significantly affect the germination rate of *Crassula ovata* seeds. The highest average germination rate, 55%, was achieved with the combination of 75% ethanol (C₂H₆O) for 60 seconds and 30% sodium hypochlorite (NaClO) for 10 minutes.

[0050] Different sterilization times significantly affected the contamination rate of *Platycladus orientalis* seeds in tissue culture. The sterilization combination of 75% ethanol (C₂H₆O) for 30 seconds + 30% sodium hypochlorite (NaClO) for 5 minutes + 5 seconds resulted in a contamination rate of 12.5%, significantly higher than the other two treatment combinations. There was no significant difference in contamination rates between the treatments of 75% ethanol (C₂H₆O) for 60 seconds + 30% sodium hypochlorite (NaClO) for 10 minutes and 75% ethanol (C₂H₆O) for 90 seconds + 30% sodium hypochlorite (NaClO) for 15 minutes, with contamination rates of 6.67% and 3.33%, respectively. In summary, the sterilization method of 75% ethanol (C₂H₆O) for 60 seconds + 30% sodium hypochlorite (NaClO) for 10 minutes yielded a higher germination rate and a relatively lower contamination rate, while also saving treatment time, making it a better sterilization method for *Platycladus orientalis* seeds in tissue culture.

[0051] Table 1. Effects of different sterilization treatments on the acquisition of sterile seedlings of *Platycladus orientalis*.

[0052]

[0053]

[0054] 1.2 Giant Cypress Seed Initiation Culture

[0055] Sterilized seeds (75% ethanol (C₂H₆O) for 60 seconds + 30% sodium hypochlorite (NaClO) for 10 minutes) were inoculated onto MS medium for initiation culture. The culture environment was 14 / 10 hours light / dark, with a light intensity of 2000 Lux, a temperature of 24℃±2℃, and a humidity of 60%±10%. Germination rate was recorded every 2 weeks, seed germination status was observed, and germination time and initial induction rate were recorded.

[0056] Primary induction rate = number of germinating seeds / total number of inoculated seeds × 100%.

[0057] After sterilization, *Juniperus chinensis* seeds were inoculated onto MS medium. Germination began after 10 days of culture. Germination rate was recorded every two weeks. The germination rate increased with increasing culture time, reaching a maximum of 51% after 6 weeks. Further culture showed no change in germination rate. The germination trend of *Juniperus chinensis* tissue culture seeds is shown below. Figure 1 After germination, the tissue culture seedlings of giant cypress were observed Figure 2 .

[0058] 1.3 Preliminary screening of giant cypress proliferation culture medium

[0059] Aseptic seedlings of *Platycladus orientalis* with a height of 8cm or more from the initial culture were selected and cut into 2cm stem segments with buds. These segments were then inoculated into 1 / 2 MS and MS media with different hormone ratios. After inoculation, the seedlings were placed in a culture room under the same environmental conditions as the initial culture. After 40 days, the proliferation rate and proliferation coefficient were calculated to screen for the optimal proliferation medium.

[0060] Proliferation rate = Number of elongated growths or clustered shoots / Initial inoculation number × 100%;

[0061] Proliferation coefficient = Number of stem segments or single buds from subculture after 40 days of culture / Initial inoculation number × 100%.

[0062] Different culture media were used to subculture and proliferate *Platycladus orientalis*. The results are shown in Table 2 and... Figure 3 As shown in Table 2, the culture medium formulations refer to the addition of different concentrations (mg / L) of hormones to MS medium. In terms of proliferation, treatments 3, 5, 6, and 10 showed better results, with the proliferating seedlings exhibiting elongation or producing new shoots, thus achieving proliferation. The induction rate for treatment 3 was 70%, and for treatment 10 it was 74%, both significantly higher than the other treatments. Treatment 5 had a relatively high proliferation coefficient, and treatment 6 produced longer new shoots. Additionally, treatment 2 showed rooting.

[0063] Table 2. Preliminary Screening Statistics of Proliferation Culture Medium for Giant Cypress Tissue Culture Seedlings

[0064]

[0065]

[0066] 1.4 Secondary screening of giant cypress proliferation medium

[0067] To further screen suitable culture media for the propagation of *Juglans regia*, in-depth screening experiments were conducted using different basal media on the initial screening media (media 2, 5, 6, and 10), i.e., repeated experiments 1.3 were performed using these media. The media treatments are shown in Table 3. The results showed that treatment 2A did not produce elongation growth, and the propagated seedlings turned yellow; treatment 2B showed rooting, while non-rooted seedlings turned yellow and did not show elongation growth; treatment 2C showed significant elongation growth, with green seedlings and thicker new shoots; treatment 2D showed no elongation growth; and treatment 2E showed significant elongation growth, with green and relatively robust new shoots, and a greater growth height than treatment 2C. The induction rates of treatments 2C and 2E were both 100%, significantly higher than other treatments, and the propagation coefficient of treatment 2E was significantly higher than that of treatment 2C. Observing the overall growth status, the order of treatments was 2E > 2C > 2D > 2B > 2A. In summary, treatment 2E (MS + 0.3 mg / L NAA + 0.05 mg / L IAA) had the highest induction rate and propagation coefficient, making it a better culture medium for the propagation of *Juglans regia*.

[0068] Table 3. Statistical table of secondary screening of tissue culture medium for *Juglans regia* seedlings.

[0069]

[0070] 1.5 Screening of rooting culture medium for giant cypress

[0071] Rooting culture was conducted using 2B and 2E media, which showed rooting phenomena during propagation culture. Healthy seedlings from the propagation culture were selected, cut into stem segments longer than 3 cm, and inoculated onto 1 / 2 MS media with different hormone ratios. The culture environment was the same as the initial culture. Rooting rate was calculated after 30 days.

[0072] Rooting rate = (Number of rooted stem segments / Number of inoculated stem segments) × 100%.

[0073] The results are as follows Figure 4 As shown, the 2E medium resulted in a higher rooting rate, and the rooted seedlings were bright green and exhibited better growth. Using 2E as the rooting medium, the rooting rate was approximately 60%.

[0074] 1.6 Hardening and Transplanting of Tissue Culture Seedlings of Giant Juniper

[0075] Giant cypress tissue culture seedlings cultured in 1.5-part 2E medium were placed in a greenhouse. They were first hardened off in closed bottles for 7 days, then hardened off again in open bottles for 5 days. The culture medium was washed off the roots with clean water, and the seedlings were then hardened off and transplanted into a substrate of peat moss:perlite (volume ratio 2:1). Moisture was maintained by spraying water for 30 seconds each in the morning, noon, and evening. The indoor temperature was controlled between 22℃ and 30℃, and the humidity was controlled at around 80%. Results showed that the survival rate of the hardened seedlings was over 90%. Figure 5 This image shows the survival rate of tissue culture seedlings of *Juniperus chinensis* after transplanting.

[0076] Example 1 uses *Juniperus chinensis* seeds as explants for tissue culture. Through sterilization, initiation culture, proliferation culture, rooting culture, and hardening-off transplanting of the seeds, regenerated *Juniperus chinensis* plants were successfully obtained. By screening different sterilization methods, proliferation media, rooting media, and hardening-off transplanting techniques, a tissue culture propagation system based on *Juniperus chinensis* seed explants was established.

[0077] When *Juniperus chinensis* seeds were sterilized using a sterilization method of 75% ethanol (C₂H₆O) for 60 seconds followed by 30% sodium hypochlorite (NaClO) for 10 minutes, and then inoculated onto MS medium, sterile tissue-cultured seedlings of *Juniperus chinensis* were obtained. After 6 weeks, the primary induction rate was 51%. Simultaneously, the obtained primary cultured sterile seedlings were proliferated using MS medium supplemented with 0.3 mg / L NAA and 0.05 mg / L NAA. The proliferated seedlings showed significant elongation growth, with green and relatively robust new shoots, and rooting. The induction rate was 100%, and the proliferation coefficient was 4.33. Furthermore, this medium facilitated simultaneous rooting, with a rooting rate of approximately 60%. After hardening off and transplanting, the survival rate after transplanting was over 90%.

[0078] Through parameter screening in Example 1 above, a complete tissue culture propagation technology system for *Juniperus chinensis* was established. This system enables the regeneration of *Juniperus chinensis* tissue culture seedlings, establishing a rapid propagation technology system unaffected by the source of materials.

[0079] Example 2: Tissue culture derived from explants of giant cypress branches

[0080] The giant cypress branches of Embodiment 2 of this invention were collected from a village in Tibet. The collected branches were healthy, disease-free, current-year branches. The collected branches were cut into 2-3cm stem segments and the lower leaves were removed.

[0081] 2.1 Effects of different maternal culture methods on the acquisition of tissue culture aseptic seedlings

[0082] Giant cypress explants were sterilized with 75% alcohol for 30 seconds and then with 30% sodium hypochlorite for 5 minutes before being inoculated onto MS medium. Contamination and germination status were observed and statistically analyzed. The optimal sterilization treatment method was screened.

[0083] Contamination rate = (Number of contaminated explants / Total number of inoculated explants) × 100%;

[0084] Induction rate = Number of germinating explants / Total number of inoculated explants × 100%.

[0085] Explants from *Juniperus chinensis* were placed in greenhouses and outdoor environments year-round. The greenhouse temperature was maintained at 10-20℃ in winter and 26℃ in summer, with a humidity of 80%. Explants were sterilized using a combination of 75% alcohol for 30 seconds and 30% sodium hypochlorite for 5 minutes. The results are shown in Table 4: the explants placed in the greenhouse had a lower contamination rate of 6.67%, which was beneficial for obtaining sterile seedlings. The contamination rate of explants placed outdoors was significantly higher than that in the greenhouse, reaching 36.67%.

[0086] Table 4. Effects of different culture methods on the acquisition of sterile seedlings of *Juglans regia*.

[0087] serial number sterilization method Training methods Pollution rate (%) 1 75% alcohol for 30 seconds + 30% sodium hypochlorite for 5 minutes greenhouse 6.67 2 75% alcohol for 30 seconds + 30% sodium hypochlorite for 5 minutes Outdoor open-air environment 36.67

[0088] 2.2 Initial screening of primary culture medium for *Juniperus chinensis* branch explants (initial culture)

[0089] Sterilized explants were inoculated onto different culture media for initiation culture. The culture environment was 14 / 10 hours light / dark, with a light intensity of 2000 Lux, a temperature of 24℃±2℃, and a humidity of 60%±10%. Germination rate was recorded every two weeks to observe seed germination status, and germination time and initial induction rate were calculated.

[0090] Primary induction rate = Number of germinating explants / Total number of inoculated explants × 100%.

[0091] Explants from tender shoots of *Cypressa gigantea* with diameters at breast height (DBH) of 0.5 and 85 cm were sterilized (75% ethanol for 30 seconds + 30% sodium hypochlorite for 5 minutes) and then inoculated onto different culture media (see Table 5). The explants with DBH of 85 cm showed a relatively high induction rate (77.5%) on P1 medium. Both DBH explants exhibited yellowing with increasing culture time on all six culture media, requiring further optimization of the culture media. Figure 6 ).

[0092] Table 5. Effects of different culture media on the acquisition of sterile seedlings of *Giant Cypress*.

[0093]

[0094] 2.3 Rescreening of basic culture medium for cypress branch explant tissue culture (start-up culture)

[0095] Different basal culture media were used to initiate the culture of sterilized (75% alcohol for 30 s + 30% sodium hypochlorite for 5 min) *Juniperus chinensis* branch explants (collected from young branches with a diameter at breast height of 0.5 cm). The culture environment was as described in section 2.2. The results are shown in Table 6 and... Figure 7As shown, with increasing cultivation time, some branches failed to sprout or root, gradually turning yellow and dying, while others remained bright green. Based on the green retention rate of the culture media, treatments 2, 3, and 5 showed relatively better green retention. From the perspective of branch condition, media 2 and 5 were the most effective. Media 5 had half the macronutrients, resulting in lower cost; therefore, in this experiment, media 5 (1 / 2NN69 + 0.1 mg / LIBA) was determined to be the optimal primary culture medium.

[0096] Table 6 Screening of basic culture media for tissue culture of *Juniperus chinensis* branch explants

[0097] Processing Number Culture medium formulation (mg / L) Status Description Green coverage rate (%) 1 NN69+0.1IBA yellowing <![CDATA[0 a ]]> 2 DKW+0.1IBA shoot tip growth <![CDATA[83.33 b ]]> 3 WPM+0.1IBA shoot tip growth <![CDATA[75 b ]]> 4 NT+0.1IBA yellowing <![CDATA[25 a ]]> 5 1 / 2NN69+0.1IBA shoot tip growth <![CDATA[80 b ]]> 6 1 / 2DKW+0.1IBA yellowing <![CDATA[0 a ]]> 7 1 / 2 WPM + 0.1 IBA yellowing <![CDATA[10 a ]]> 8 1 / 2NT + 0.1IBA yellowing <![CDATA[10 a ]]>

[0098] 2.4 Rooting Culture

[0099] Select robust seedlings (over 3 cm) from the initial culture and inoculate them onto 1 / 2 MS medium with different hormone ratios. The culture environment is the same as the initial culture. Rooting rate is calculated after 30 days.

[0100] Rooting rate = (Number of rooted stem segments / Number of inoculated stem segments) × 100%.

[0101] To screen suitable rooting media for *Platycladus orientalis*, different hormone-rich media were prepared. The results are shown in Table 7. Figure 8 As shown, the green retention rates of L and G3 media were 70%, significantly higher than other media; the rooting rates of G2 and G3 media were 50%, significantly higher than other media; there was no significant difference in the number of roots among the media, with G1, G2, and G3 media showing relatively higher rates. Considering the actual growth status of the rooted seedlings, the seedlings grown on G3 medium (1 / 2 MS + 0.3 mg / L NAA + 0.05 mg / L IAA) were bright green with robust roots, and rooted earliest during the observation period, making it a better rooting medium.

[0102] Table 7 Screening of rooting culture medium for explants from *Juniperus chinensis* branches

[0103]

[0104]

[0105] Example 2 used tender shoots collected from ancient giant cypress trees with diameters at breast height (DBH) of 0.5 and 86 cm as explants. In a greenhouse, the shoots were sterilized using a combination of 75% ethanol for 30 seconds and 30% sodium hypochlorite for 5 minutes. The explants were then inoculated onto 1 / 2 NN69 medium supplemented with 0.1 mg / L IBA for primary induction, followed by direct inoculation onto 1 / 2 MS medium supplemented with 0.3 mg / L NAA and 0.05 mg / L IAA for rooting culture. The greenness retention rate was 70%, the rooting rate was 50%, and the average number of roots was 3.25. This method can achieve direct rooting.

[0106] Example 3: Localization Adaptability of Giant Juniper Tissue Culture Seedlings

[0107] Example 3 involved the hardening-off and domestication transplantation of tissue-cultured *Juniperus chinensis* seedlings at a base in Tibet to study their local adaptability. The transplanting site was leveled, removing construction debris, large stones, rocks, and coarse rocks from the original location. Since the original site contained a significant amount of construction debris, the soil was replaced with farmland soil from the adjacent area and the site was leveled.

[0108] The rooted cypress seedlings from Examples 1 and 2 were transported in 9.6m freight trucks, covered with tarpaulins for insulation. Cypress is highly drought-resistant, so watering was not necessary during transport. Transportation took place in February to avoid excessively high temperatures and the peak growth period. After unloading, the potted seedlings were placed in the local open environment for 3-5 days for acclimatization. The seedlings were observed for growth; those without obvious disease or death were ready for transplanting.

[0109] Dig transplanting holes at 30cm intervals, with a depth of at least 30cm. Transplant the tissue-cultured cypress seedlings, covering them with soil after transplanting. Cover the soil with a thin film to retain moisture, and water the soil after transplanting with rooting powder. Water after transplanting, depending on local climate conditions, when the soil is dry, generally once every 10-15 days. Remove any rootstock suckers and monitor for pests and diseases. Fertilize once per quarter.

[0110] One year after transplanting, the growth status of the cypress tissue culture seedlings was observed. The survival rate after transplanting was over 90%. The tissue culture seedlings showed good growth after transplanting to the ground, with lateral branching and new shoot growth. The branches and leaves were healthy, and the plant shape was good. Figure 9 As shown.

Claims

1. A tissue culture propagation method for giant cypress, characterized by: Includes the following steps: Using giant cypress seeds or branches as explants, after sterilizing the explants, proceed with initiation culture and rooting culture in sequence; When using giant cypress seeds as explants, the sterilization method is 75. v / v % alcohol 60 s + 30 v / v Sodium hypochlorite for 10 min; the starting medium is MS medium; after the starting culture, there is also a proliferation culture; the proliferation medium and the rooting medium are both MS + 0.3 mg / L NAA + 0.05 mg / L IAA; When using giant cypress branches as explants, the sterilization method is 75. v / v % alcohol 30 s + 30 v / v Sodium hypochlorite for 5 min; the starting medium was 1 / 2 NN69 + 0.1 mg / L IBA; the rooting medium was 1 / 2 MS + 0.3 mg / L NAA + 0.05 mg / L IAA.

2. The tissue culture propagation method for giant cypress according to claim 1, characterized in that: When using giant cypress seeds as explants, the proliferation culture involves selecting sterile giant cypress seedlings with a height of 8 cm or more after the culture has been initiated, cutting them into 2 cm stem segments with buds, and inoculating them into the proliferation culture medium; the rooting culture involves cutting robust seedlings after the proliferation culture into stem segments of 3 cm or more, and inoculating them into the rooting culture medium.

3. The tissue culture propagation method for giant cypress according to claim 1, characterized in that: The giant cypress branches were collected from tender branches of ancient giant cypress trees with a diameter at breast height of 0.5–85 cm.

4. The tissue culture propagation method for giant cypress according to claim 1, characterized in that: During tissue culture, the culture conditions were as follows: light / dark ratio of 14 / 10 h, light intensity of 2000 Lux, temperature of 24℃±2℃, and humidity of 60%±10%.

5. The tissue culture propagation method for giant cypress according to claim 1, characterized in that: The initiation culture time is 42–56 days, the proliferation culture time is 40–42 days, and the rooting culture time is 30–35 days.

6. The method for tissue culture propagation of giant cypress according to claim 1, characterized in that: It also includes hardening off and transplanting tissue culture seedlings after rooting culture.

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