A method for in-vitro rapid propagation and brown prevention of Lindera glauca based on zygotic embryos

Abstract

The application discloses a submerged Cinnamomum micranthum in-vitro rapid propagation and brown-preventing seedling raising method based on zygote embryos, directly induces adventitious buds of the submerged Cinnamomum micranthum for the first time, significantly improves a propagation coefficient, and overcomes problems such as vitrification and browning of the adventitious buds of the submerged Cinnamomum micranthum. The application establishes an efficient and stable in-vitro regeneration system of the submerged Cinnamomum micranthum, and provides a basis for large-scale seedling raising of the submerged Cinnamomum micranthum.

Description

Technical Field

[0001] This invention belongs to the field of submerged camphor tree propagation, specifically involving a method for rapid in vitro propagation of submerged camphor trees based on zygotic embryos and large-scale seedling cultivation with browning prevention. Background Technology

[0002] *Cinnamomum camphora*, a large evergreen broad-leaved tree belonging to the genus *Cinnamomum* of the Lauraceae family, is prized for its excellent timber quality and high economic and ecological value. However, as a self-pollinating plant with significant inconsistencies in the maturation of male and female flowers, a unique phenological overlap exists in natural forests: the expansion period of the current year's young fruit coincides with the ripening period of the previous year's fruit. To avoid competition and conflict over nutrient supply within the tree, the young fruit enters a prolonged physiological dormancy state to ensure the growth of the previous year's fruit and the plant's reproduction. However, this prolonged dormancy and growing season makes the seeds highly susceptible to environmental damage, disease, and predation by birds and animals during maturation, leading to numerous hollow, deformed, and shriveled seeds, as well as severe fruit drop. Natural forests of *Cinnamomum camphora* have extremely poor natural regeneration capacity, resulting in a sharp decline in population size and a current serious threat of endangerment. To address the propagation challenges of *Cinnamomum camphora*, current production relies primarily on traditional cutting propagation, but this method suffers from low rooting rates, poor seedling genetic uniformity, and a tendency to cause severe varietal degeneration, such as declining tree vigor. In recent years, tissue culture technology has begun to be applied to the conservation and propagation of submerged camphor trees, but it generally faces many technical bottlenecks in practical applications.

[0003] Chinese invention patent CN103651145B discloses a method for tissue culture of *Cinnamomum camphora*, which uses newly extracted semi-lignified stem segments with axillary buds as explants. These stem segments are rich in phenolic substances and are prone to browning after cutting or damage, leading to a decrease in initial survival rate and proliferation efficiency. The patent describes an induction phase using MS + 0.2–3.0 mg / L 6-BA + 0.01–0.5 mg / L NAA, requiring 40–50 days to obtain explants. In the crucial subculture proliferation stage, this method relies on adding 50–300 mg / L of the strong reducing inorganic salt sodium thiosulfate to prevent browning and leaf drop, ultimately achieving a proliferation coefficient of only 2.5 at most. This technology uses somatic cells from adult mother trees as explants, severely inhibiting totipotency expression, and the stem segment explants have extremely high endophytic bacterial contamination rates, making sterilization difficult. The large-scale use of inorganic salts to prevent browning easily causes osmotic pressure and salt stress in plant cells, significantly limiting the efficiency of industrialized propagation. Chen Bihua et al. used terminal buds or semi-lignified branches of *Camphora sinensis* seedlings as explants and induced callus tissue with a very high concentration of cytokinin combination (MS + BA 5.0 mg / L + IBA 0.1 mg / L + Vc 5 mg / L), with an induction rate of only 35% (Chen Bihua, Zhang Juan, Fan Huihua. Research on tissue culture technology of *Camphora sinensis* [J]. Seeds, 2016, 35(02):121-123.). Chinese invention patent CN104186324B discloses a method for inducing somatic embryogenesis of mature zygote embryos of *Pinus sylvestris*. Although it involves the in vitro culture of mature zygote embryos of plants, it is aimed at the gymnosperm coniferous species *Pinus sylvestris*. The technical path is to induce embryogenic callus tissue and then carry out somatic embryogenesis, using DCR basic culture medium and a single concentration of 2 mg / L 2,4-D hormone system. This differs from the biological mechanisms by which broad-leaved angiosperms like the submerged camphor tree directly induce adventitious buds through zygotic embryo organs, as well as their nutrient requirements in the culture medium and their hormonal regulatory responses, at the species level.

[0004] In summary, existing tissue culture techniques for *Cinnamomum camphora* are largely limited by the selection of explant type (stem segments), resulting in low explant induction rates, low propagation multiples, and high mortality rates in the later stages of tissue culture seedling production. Therefore, there is an urgent need to develop a highly efficient in vitro rapid propagation system for *Cinnamomum camphora* that overcomes the challenges of vitrification and browning, in order to meet the practical needs of modern forestry for high-standard, exponentially increasing afforestation seedling production. Summary of the Invention

[0005] In order to overcome the problems of easy contamination of materials, low germination induction rate, limited multiplication rate of subculture, and easy occurrence of lethal vitrification and browning in existing submerged camphor tissue culture, the purpose of this invention is to provide a method for rapid in vitro propagation and browning prevention of submerged camphor seedlings based on zygotic embryos.

[0006] The objective of this invention is achieved by at least one of the following technical solutions.

[0007] A method for rapid in vitro propagation and browning prevention of submerged camphor trees based on zygotic embryos includes the following steps: (1) Explant collection: Submerged camphor seeds were collected, dewaxed and degreased, and the seed coat was broken under sterile conditions. The endosperm was removed and the zygotic embryo was taken out as the explant. (2) Adventitious shoot initiation induction: The explants obtained in step (1) were inoculated into the initiation induction medium and cultured to obtain adventitious shoots; the initiation induction medium was based on MS medium, with the addition of 1.0-2.0 mg / L of 6-benzylaminopurine (6-BA), 0.05-0.2 mg / L of gibberellin (GA3) and 0.05-0.2 mg / L of 2,4-dichlorophenoxyacetic acid (2,4-D). (3) Anti-vitrification and anti-browning culture of adventitious buds: The epicotyl part of the adventitious buds that have sprouted in step (2) with the cotyledons removed is cut off and inoculated into the proliferation medium for subculture to obtain adventitious bud clusters; the proliferation medium is based on MS medium, with the addition of 1.0-3.0 mg / L of 6-BA, 0.02-0.08 mg / L of diethylaminoethanol hexanoate (DA-6), 0.02-0.1 mg / L of naphthaleneacetic acid (NAA), and 1.0-5.0 mg / L of glutathione (GSH) and 20-50 ml / L of coconut juice; (4) Rooting induction culture: Single buds are cut from the adventitious bud clusters in step (3) and inoculated into the rooting medium for induction; the rooting medium is based on 1 / 2 MS medium, with the addition of naphthaleneacetic acid (NAA) at a mass concentration of 0.05-0.2 mg / L, indolebutyric acid (IBA) at a mass concentration of 0.5-1.5 mg / L and activated carbon (AC) at a mass concentration of 0.1-0.5 g / L. (5) Transplanting of strong seedlings: Transplant the tissue culture seedlings after the root system at the base of the seedlings from step (4) has grown to 0.5-1cm.

[0008] Furthermore, the submerged camphor seeds are those collected from August to November.

[0009] Furthermore, the submerged camphor seeds were collected in mid-September. In mid-September, the zygotic embryo cells reach their physiological peak regeneration and differentiation capacity during the pre-maturation stage, and with the combined action of 6-BA, GA3, and 2,4-D, dormancy can be broken.

[0010] Furthermore, the dewaxing and degreasing treatment described in step (1) involves soaking the submerged camphor seeds in warm water at 40-50°C containing detergent for 12-24 hours. After the waxy outer skin softens, it is rubbed off. Then, it is repeatedly rinsed and filtered with clean water until the seed coat is free of wax, lignin, and tannins, thus obtaining submerged camphor seeds with the seed coat removed.

[0011] Furthermore, the dewaxing and degreasing treatment also includes disinfecting the surface of the submerged camphor seeds after the seed coat has been removed.

[0012] More preferably, the surface disinfection involves first immersing the surface in 75% alcohol for 20-40 seconds, then immersing it in 0.1% mercuric chloride solution for 5-20 minutes, and finally rinsing it with sterile water at least three times.

[0013] Furthermore, the initiation culture medium formulation described in step (2) comprises MS + 1.0 mg / L 6-BA + 0.1 mg / L GA3 + 0.1 mg / L 2,4-D.

[0014] Furthermore, in step (2), the initiation induction medium is used for 5-10 days.

[0015] Furthermore, the proliferation medium formulation in step (3) comprises MS + 2.0 mg / L 6-BA + 0.05 mg / L LDA-6 + 0.05 mg / L NAA + 2 mg / L glutathione + 40 ml / L coconut juice. Under the synergistic biochemical effect of this concentration of DA-6 and GSH, vitrification (hydration and transparency) of proliferating cells can be avoided, toxic reactive oxygen species generated by the cut surface can be rapidly eliminated, the polyphenol oxidation pathway can be blocked at the molecular level, and the yellowing and browning rates can be reduced.

[0016] Furthermore, the culture time using the proliferation medium in step (3) is 15-45 days.

[0017] Furthermore, in step (3), the culture time using the proliferation medium is 30 days.

[0018] Furthermore, the rooting medium formulation in step (4) comprises 1 / 2MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / L AC.

[0019] Furthermore, in step (4), the rooting medium is used for culturing for 15-45 days.

[0020] Furthermore, in step (4), the rooting medium is used for 30 days.

[0021] Furthermore, the initiation induction medium in step (2), the proliferation medium in step (3), and the rooting medium in step (4) are all supplemented with 30 g / L of sucrose and 6.5 g / L of carrageenan.

[0022] Furthermore, the pH of the initiation induction medium in step (2), the proliferation medium in step (3), and the rooting medium in step (4) are all 5.8-6.0.

[0023] Furthermore, the environmental conditions for steps (2) to (4) are 25±2℃, the light conditions are 2700-3000 lux, and the light duration is 14-16 h / d.

[0024] Furthermore, the seedling substrate for transplanting in step (5) includes red soil, perlite, coconut coir, rice husk, and wood ash, with a volume ratio of 6:1:1:1:1. This lightweight substrate possesses excellent root aeration, moisture retention, and buffering capacity against weakly acidic trace elements.

[0025] Furthermore, the method for rapid in vitro propagation and browning prevention of submerged camphor trees based on zygotic embryos is used for the preservation of submerged camphor germplasm resources, rapid asexual propagation, and large-scale seedling production.

[0026] A culture medium for the rapid in vitro propagation and browning prevention of submerged camphor trees based on zygotic embryos, comprising an initiation and induction medium, a proliferation medium, and a rooting medium; the initiation and induction medium formula comprises MS + 1.0 mg / L 6-BA + 0.1 mg / L GA3 + 0.1 mg / L 2,4-D; the proliferation medium formula comprises MS + 2.0 mg / L 6-BA + 0.05 mg / L DA-6 + 0.05 mg / L NAA + 2 mg / L GSH + 40 ml / L coconut juice; the rooting medium formula comprises 1 / 2 MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / L AC.

[0027] The above-described in vitro rapid propagation and anti-browning seedling culture medium based on zygotic embryos for the propagation of Cinnamomum camphora is applied to the propagation of Cinnamomum camphora.

[0028] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) This invention analyzes the physiological dormancy characteristics of submerged camphor seeds and realizes for the first time the direct induction of adventitious buds by submerged camphor zygote embryos. The induction time of zygote embryos is short and the germination induction rate is as high as 78.67%, which solves the problem of the difficulty in initiating submerged camphor zygote embryos as explants. As an explant, submerged camphor zygote embryos have strong regeneration ability and are suitable for establishing an efficient in vitro propagation system, thus improving the genetic stability of submerged camphor propagation.

[0029] (2) This invention introduces DA-6 into submerged camphor zygote embryos for the first time for propagation, and, in combination with 6-BA and NAA, maximizes the proliferation potential of meristematic tissues, resulting in a single-cycle proliferation multiple of over 3.36, while avoiding cell vitrification caused by hormonal imbalance. At the same time, glutathione endogenously scavenge free radicals, and, in combination with coconut juice nutrition, reduces the rate of yellowing and browning in subsequent generations to 16.20%, cultivating robust seedlings with significantly broadened and greener leaves.

[0030] (3) This invention, through the ingenious ratio of NAA, IBA and activated carbon, successfully skips the cumbersome callus transition period, and induces healthy root primordia directly from the stem cortex within 7 days. The average number of roots per plant reaches more than 4.8, the roots are white, plump and without deformities, and the rooting rate is as high as 91.67%, ensuring the survival rate of transplanting and laying a technical foundation for saving the endangered species of Camphor tree and meeting its commercial afforestation needs.

[0031] (4) This invention establishes a complete technical system from obtaining sterile seedlings from zygotes to rapid propagation and rooting and then transplanting to seedlings, which is suitable for the preservation of submerged camphor germplasm, large-scale seedling cultivation and resource protection and utilization. Attached Figure Description

[0032] Figure 1 Figure 1 shows the effect of different sampling times on the embryo germination of submerged camphor zygotes; among them, Figure 1 Figure A in the figure shows the results of different sampling times on the embryo germination rate of submerged camphor zygotes; Figure 1 Figure B in the graph shows the results of different sampling times on the contamination rate of submerged camphor zygote embryos; Figure 1 In the figure, C represents the browning rate of submerged camphor zygote embryos at different sampling times.

[0033] Figure 2 Images of submerged camphor trees from zygotic embryo germination to rooting and transplanting, using suitable culture media at each stage; among them, Figure 2 In Example 2, A represents the initial bud image of the zygote embryo treated with A5 in September; Figure 2 In Example 4, B represents the first subculture seedling image processed by the combination of B8 and C6. Figure 2 C in the image represents the second generation seedling processed with a combination of B8 and C6 in Example 4. Figure 2 In this image, D represents the third generation seedling processed using a combination of B8 and C6 in Example 4. Figure 2 E in the image represents the rooted seedling processed by D5 in Example 5; Figure 2 F in the image represents the rooted seedlings of Example 5 after transplanting 15 days after treatment with D5.

[0034] Figure 3 The images shown are from Example 5 and illustrate the effects of different growth regulators on the rooting of submerged camphor tissue culture seedlings after transplanting. Figure 3 The AI ​​in the examples correspond to processes D1-D9 in Example 5.

[0035] Figure 4 Browning images of B8 adventitious buds processed for Example 3. Detailed Implementation

[0036] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0037] In the following examples, the MS medium consisted of the following components: potassium nitrate 1900 mg / L, ammonium nitrate 1650 mg / L, potassium dihydrogen phosphate 170 mg / L, magnesium sulfate 370 mg / L, calcium chloride 440 mg / L, potassium iodide 0.83 mg / L, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L, cobalt chloride 0.025 mg / L, nicotinic acid 0.5 mg / L, disodium EDTA 37.3 mg / L, ferrous sulfate 27.8 mg / L, inositol 100 mg / L, glycine 2 mg / L, thiamine hydrochloride 0.1 mg / L, and pyridoxine hydrochloride 0.5 mg / L, purchased from Beijing Solarbio Technology Co., Ltd., catalog number M8521.

[0038] In the following examples, the test materials were obtained from the submerged camphor tree resource conservation nursery of Jiangxi Academy of Forestry Sciences (1895 and 1835).

[0039] In the following examples, all culture media were supplemented with 30 g / L sucrose and 6.5 g / L carrageenan, with a pH of 5.8–6.0, a culture temperature of 25 ± 2 °C, a light intensity of 2800 lux, and a photoperiod of 14 h / d.

[0040] Example 1: Effect of different sampling times on embryo germination of submerged camphor zygotes Seeds from 20-year-old submerged camphor trees were collected between mid-August and mid-November. The seeds were placed in a container with a small amount of laundry detergent, and then soaked in warm water (40℃~50℃) for 12~24 hours. Once the waxy outer skin was no longer smooth and soft, the seeds were placed in a mesh bag and rubbed to detach the outer skin from the seed. The seeds were then poured into clean water and sieved to remove any floating seed coat. The seeds were then rinsed repeatedly with clean water and filtered until the seed coat surface was smooth and free of lignin and tannins. The seeds were then disinfected in a clean bench with 75% alcohol (v / v) and 0.1% mercuric chloride (w / w) for 30 seconds and 10 minutes respectively. Finally, the surface disinfectant was washed off with sterile water.

[0041] Shelling: Using pointed forceps, hold the seed and puncture the seed coat with a scalpel to remove the intact white zygotic embryo. Inoculate the embryo into MS medium containing 1.0 mg / L 6-BA, 0.1 mg / L GA3, and 0.1 mg / L 2,4-D. Collect materials in the middle of each month and conduct experiments in 4 batches. Each batch contains 10 bottles, with 2 embryos inoculated per bottle. After 15 days, the contamination rate and germination rate are recorded.

[0042] The regeneration potential of submerged camphor zygote embryos is closely related to the time of explant zygote collection, directly affecting the success or failure of system construction. Figure 1 As shown in Figure A, there are significant differences in cell regeneration capacity among zygotes of different maturity levels. Observation of germination status revealed that zygotes collected in September were the explant materials with the strongest germination and regeneration capacity, with a germination rate of up to 71.70%. Zygotes collected in August were not mature enough; the immature embryonic tissue was tender, and the cells easily differentiated in a disordered direction, producing multi-lobed malformed buds and loose callus, making it difficult to obtain sterile and robust buds; therefore, the bud germination rate was only 45.00%. Endosperm fully matured in October and November; after peeling off the seed coat, a thin, brownish-thick seed coat remained tightly adhered to the zygote, making it difficult to peel off, and turned completely black after being placed in the culture medium for one week. Figure 1 As can be seen from BC, the browning rate and contamination rate gradually increase with the increase of seed bearing time.

[0043] The closer the submerged camphor seeds are to maturity or full maturity, the lower their germination rate becomes. This may be due to the excessively long growth and development period, the vulnerability of the embryo to insect and fly damage during the mature stage, resulting in incomplete embryonic development and the gradual loss of cell division ability in most seeds. Alternatively, it may be due to the stabilization of physiological and biochemical characteristics in mature seeds, the peak accumulation of nutrients, and the decrease in enzyme activity. Based on experimental data and actual induction conditions, the optimal time for collecting submerged camphor zygotic embryos is September, when they reach maturity.

[0044] Example 2: Effects of different treatments on the induction of adventitious shoots from submerged camphor zygote embryos After the seeds are shelled, the zygotic embryos are placed on the surface of the following culture medium to conduct adventitious bud induction experiments: (1) MS, (2) MS + 1.0 mg / L 6-BA, (3) MS + 1.0 mg / L 6-BA + 0.1 mg / L 2,4-D, (4) MS + 1.0 mg / L 6-BA + 0.1 mg / L ZT + 0.1 mg / L 2,4-D, (5) MS + 1.0 mg / L 6-BA +0.1 mg / L GA3+ 0.1 mg / L 2,4-D, (6) MS + 1.0 mg / L 6-BA + 0.1 mg / L DA-6 + 0.1mg / L 2,4-D.

[0045] Ten bottles were randomly inoculated for each treatment, with 4-6 zygote embryos inoculated into each bottle. The bud germination induction was recorded after 20 days.

[0046] Germination rate = (Number of germinated buds / Total number of inoculated buds) × 100%.

[0047] After the seeds were shelled, they were placed in different culture media. The white endosperm gradually turned into green cotyledons. The hypocotyl inserted into the culture medium to form white roots, the epicotyl extended to develop into a stem, and the plumule developed into a green stem and leaves, eventually forming a complete seedling. As shown in Table 1, different growth regulators had significant differences in their induction effects on adventitious buds. In treatments 1-3, buds began to germinate after nearly 10 days, with a low germination rate. Furthermore, the zygotic embryo could only germinate a single bud, and adventitious buds could not form at the node between the epicotyl and cotyledon, directly affecting subsequent proliferation culture. In treatments A4 and A6, the addition of ZT or DA-6 to the culture medium improved the bud germination rate to some extent, possibly because cytokinins promoted cell division. However, light brown callus tissue appeared at the endosperm and embryo junction, and the buds were small. The initial bud image of treatment A5 is shown below. Figure 2 As shown in Figure A, treatment A5, using GA3, 6-BA, and 2,4-D, synergistically promoted seed germination, reducing the germination initiation time to only 5 days and achieving a germination rate of 78.67%. The combined use of these three agents activated the amylase synthesis in the corymb meristem and seed endosperm, breaking dormancy, stimulating cell division and elongation, alleviating apical dominance, promoting plant height, and directly inducing corymb differentiation into clustered shoots, thus shortening the developmental cycle. Analysis of the experimental results regarding zygotic embryo germination time, shoot germination rate, and induced shoot growth indicates that MS + 1.0 mg / L 6-benzylaminopurine (6-BA) + 0.1 mg / L gibberellin (GA3) + 0.1 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) is the optimal culture medium for inducing submerged camphor zygotic embryos.

[0048] Table 1. Effects of different culture media on adventitious shoot induction in submerged camphor zygote embryos

[0049] Example 3: Effects of different culture media on adventitious shoot proliferation After the zygotic embryos developed into seedlings, they were cultured for adventitious shoot proliferation. Suitable components of the adventitious shoot proliferation culture medium were screened. Based on Example 2, the effects of different concentrations of growth regulators 6-benzylaminopurine (6-BA), diethylaminoethanol hexanoate (DA-6), and naphthaleneacetic acid (NAA) on adventitious shoot proliferation were investigated. Since the shoot induction effect varied significantly under different treatment conditions, this example used an orthogonal experiment to study the effect of their combinations on adventitious shoot proliferation. Initially, two shoots were placed in each bottle, followed by four shoot clusters per bottle, with five bottles per treatment, and three subcultures were performed. Because the proliferation coefficient increased with the number of subcultures, and tended to stabilize after the third subculture, the average proliferation coefficient after each subculture cycle (30 days) was calculated, and the growth of adventitious shoots was observed.

[0050] Shoot proliferation coefficient = Number of shoots after one subculture cycle / Number of inoculated shoots.

[0051] The cotyledons of the germinated plumules were removed, and the hypocotyl (the point where the cotyledon meets the stem) was inoculated into an adventitious bud proliferation medium. After three subcultures under the same conditions, the effects of different media on adventitious bud proliferation were found to be significantly different (P<0.05), as shown in Table 2. In treatment B1, no growth regulator was added, and the germinating buds showed high growth but did not induce the differentiation of new adventitious buds. After three subcultures, leaf drop and withering occurred. When 6-BA or DA-6 was used alone in combination with NAA, the number of adventitious buds was low, and it was difficult to form bud clusters on the hypocotyl, with a proliferation rate of only 2.0-3.0%. When 6-BA, DA-6 and NAA were used in combination, the number of adventitious buds significantly increased. In particular, when 6-BA was higher than 1.0 mg / L, the hypocotyl swelled, and adventitious buds could be directly differentiated on its tissue blocks. When the concentration of DA-6 reaches above 0.1 mg / L, the number of buds increases, adventitious buds grow small, stems and leaves become transparent and even callus formation occurs, and leaves become brittle, turning yellow, black and falling off in the later stages. This may be because high concentrations of DA-6 easily lead to excessive activation of plant cell division and swelling, causing the rate of thin-walled cell division to far exceed the rate of cell wall lignification. Cells only increase in volume without increasing the thickness and toughness of the cell wall, resulting in thinner cell walls and increased intracellular water content, causing the cells to appear hydrated and transparent. When the dosage of DA-6 is controlled at 0.05 mg / L, the differentiation-promoting effect is significant, but the vitrification rate is low. Low concentrations of NAA can promote growth, while high concentrations inhibit growth. When the NAA concentration is 0.1 mg / L, a significant increase in callus tissue is observed, and with increasing subcultures, it easily accumulates, resulting in soft, juicy, blackish callus tissue with no effect. Based on experimental data and the growth performance of the seedlings, the suitable culture medium composition for the proliferation of adventitious buds of submerged camphor trees is MS + 2.0 mg / L 6-benzylaminopurine (6-BA) + 0.05 mg / L diethylaminoethanol hexanoate (DA-6) + 0.05 mg / L naphthaleneacetic acid (NAA).

[0052] Table 2. Effects of different concentrations of growth regulator combinations on adventitious shoot proliferation.

[0053] Note: "+" indicates that adventitious buds can be induced, the more "+" signs there are, the better the induction effect, and "-" indicates that adventitious buds cannot be induced.

[0054] Example 4: Effects of different additives on the prevention of yellowing and browning during the propagation of submerged camphor trees During the adventitious bud induction process, with the increase of subcultures, the callus tissue at the base of the bud gradually increases, and tissue metabolites infiltrate the culture medium, resulting in browning and yellowing of the tissue. This manifests as weak seedlings, shortened internodes, inhibited transpiration, curled or drooping leaves, yellowing and leaf drop, and even inhibition of bud growth. To prevent browning and yellowing, in addition to the suitable proliferation medium (MS + 2.0 mg / L 6-BA + 0.05 mg / L DA-6 + 0.05 mg / L NAA) used in Example 3, different concentrations of glutathione (GSH) and coconut juice were added. Five bottles were used for each treatment, and the yellowing status and leaf growth of the seedlings were observed after a single subculture cycle (30 days). Images of the first, second, and third subculture seedlings after the combined treatments of Example 3 B8 and Example 4 C6 are shown below. Figure 2 B in Figure 2 C and Figure 2 As shown in D in the diagram.

[0055] As shown in Table 3, treatment 6, with the addition of 2 mg / L glutathione and 40 ml / L coconut juice, significantly improved the physiological state of the budding seedlings in the propagation bottle. The leaf surface changed from dull yellow-green to glossy light green, the number of new leaves increased significantly, and the leaves changed from narrow and small to wide and large, with an average leaf width of over 2.04 cm. The stems grew longer and thicker, with an average seedling height of over 3.41 cm. It had a significant effect in preventing yellowing and leaf drop. The stems grew uniformly, and the internodes were normal. This may be due to the important nutritional supplementation role played by the organic and mineral-rich coconut juice in cell metabolism and growth during the cultivation process. Low concentrations of glutathione can reduce browning in submerged camphor trees. It can scavenge free radicals produced by metabolism and inhibit the activity of polyphenol oxidase, thereby preventing browning. However, excessively high concentrations of glutathione have adverse effects on seedling growth. For example, in treatments C7-C9, the stems showed basal swelling and shortened internodes, with minimal change in the overall growth status of the seedlings. Therefore, adding 2 mg / L glutathione and 40 ml / L coconut juice to the culture medium for the propagation of submerged camphor trees can alleviate metabolic accumulation or damage caused by stress during subculture and ensure the normal growth of the subcultured seedlings.

[0056] Table 3. Effects of different additives on yellowing and browning during the proliferation of *Cinnamomum camphora*.

[0057] Example 5: Effects of different growth regulators on rooting of submerged camphor tissue culture seedlings after transplanting After proliferation culture, intact branches approximately 3 cm tall were selected from adventitious bud clusters, cut flat, and transferred to rooting medium. Rooting culture was conducted using 1 / 2 MS as the basal medium, with different ratios of NAA and IBA added, employing an orthogonal experiment. Eight branches were inserted into each bottle, with five bottles inoculated for each treatment, and the results were replicated three times. Rooting rate of the tissue culture seedlings was calculated after 30 days. Images of rooted seedlings from treatments D1-D9 are shown below. Figure 3 As shown in AI, remove and wash the seedlings, then transplant them into 8 cm × 10 cm container bags. The substrate consisted of red soil, perlite, coconut coir, rice husks, and wood ash in a volume ratio of 6:1:1:1:1. After planting, water thoroughly, spray with a 1000-fold diluted 50% carbendazim solution, cover with a film to retain moisture, and remove the film as needed depending on the weather. Observe the seedling growth and calculate the transplant survival rate after one month, using the emergence of new leaves from the terminal bud as the criterion. Images of rooted seedlings treated with D5 15 days after transplanting are shown below. Figure 2 As shown in F in the diagram.

[0058] As shown in Table 4, the rooting rate of seedlings differed significantly under different treatments (p<0.05). Without growth regulators on the basis of 1 / 2MS, the buds and stems could not develop roots. Under treatment D5 (1 / 2MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / L AC), the rooting initiation time was the shortest, with roots directly induced from the stem cortex. Images of rooted seedlings under treatment D5 are shown below. Figure 2 As shown in E, after 7 days, the epidermis around the stem base begins to swell, exposing white root primordia; after 12 days, the root system grows to 0.5-1 cm, with evenly distributed roots at the base of the single bud stem. The roots are white, fleshy, and robust, with more than 10 roots per bud, averaging 5 roots per bud. Both plant growth regulators (NAA and IBA) and activated carbon (AC) are indispensable. Without the physical adsorption and bottom darkening effect of activated carbon, root induction time can be longer than 10 days, resulting in weak roots with severe unidirectional root deviation, and intermittent silvery-white nodules appearing on some roots. During root induction in treatments D7, D8, and D9, excessive activated carbon adsorbed the plant growth regulators, inhibiting their induction of roots, leading to thin, uneven root growth and a reduced number of roots. Considering both the quantity and quality of roots, the suitable culture medium for the successful transplantation of submerged camphor tissue culture seedlings is 1 / 2MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / L AC.

[0059] Table 4. Effects of different combinations of growth regulators on adventitious shoot rooting.

[0060] The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A zygotic embryo-based in-vitro rapid propagation and browning prevention seedling raising method for Cinnamomum subalatum, characterized in that, Includes the following steps: (1) Explant collection: Submerged camphor seeds were collected, dewaxed and degreased, and the seed coat was broken under sterile conditions. The endosperm was removed and the zygotic embryo was taken out as the explant. (2) Adventitious shoot initiation induction: The explants obtained in step (1) were inoculated into the initiation induction medium and cultured to obtain adventitious shoots; the initiation induction medium was based on MS medium, with the addition of 6-BA at a mass concentration of 1.0-2.0 mg / L, GA3 at a mass concentration of 0.05-0.2 mg / L and 2,4-D at a mass concentration of 0.05-0.2 mg / L. (3) Anti-vitrification and anti-browning culture of adventitious buds: The epicotyl part of the adventitious buds that have sprouted in step (2) with the cotyledons removed is cut off and inoculated into the proliferation medium for subculture to obtain adventitious bud clusters; the proliferation medium is based on MS medium, with the addition of 6-BA at a mass concentration of 1.0-3.0 mg / L, DA-6 at a mass concentration of 0.02-0.08 mg / L and NAA at a mass concentration of 0.02-0.1 mg / L, and glutathione at a mass concentration of 1.0-5.0 mg / L and coconut juice at a mass concentration of 20-50 ml / L. (4) Rooting induction culture: Single buds are cut from the adventitious bud clusters in step (3) and inoculated into the rooting medium for induction; the rooting medium is based on 1 / 2MS medium, with the addition of 0.05-0.2 mg / L NAA, 0.5-1.5 mg / L IBA and 0.1-0.5 g / L activated carbon. (5) Transplanting of strong seedlings: Transplant the tissue culture seedlings after the root system at the base of the seedlings from step (4) has grown to 0.5-1cm.

2. The method according to claim 1, wherein the method is characterized by the following steps: The submerged camphor seeds mentioned are those collected from August to November.

3. The method according to claim 2, wherein the method is characterized by, The submerged camphor seeds were collected in mid-September.

4. The method according to claim 1, wherein the method is characterized in that, The dewaxing and degreasing process described in step (1) involves soaking the submerged camphor seeds in warm water at 40-50℃ containing detergent for 12-24 hours. After the waxy outer skin softens, it is rubbed off. Then, the seeds are repeatedly rinsed and filtered with clean water until the seed coat is free of wax, lignin, and tannins, thus obtaining submerged camphor seeds with the seed coat removed.

5. The method according to claim 1, wherein the method is characterized in that, The initiation culture medium formulation described in step (2) includes MS + 1.0 mg / L 6-BA + 0.1 mg / L GA3 + 0.1 mg / L 2,4-D.

6. The method according to claim 1, wherein the method is characterized by the following steps: The proliferation medium formula described in step (3) includes MS + 2.0 mg / L 6-BA + 0.05 mg / L DA-6 + 0.05 mg / L NAA + 2 mg / L GSH + 40 ml / L coconut juice.

7. The method according to claim 1, wherein the method is characterized by the following steps: The rooting medium formulation in step (4) consists of 1 / 2 MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / LAC.

8. The method according to claim 1, wherein the method is characterized by the following steps: The environmental conditions for steps (2) to (4) are 25±2℃, the light conditions are 2700-3000 lux, and the light duration is 14-16 h / d.

9. A culture medium for rapid propagation and anti-browning of submerged camphor trees in vitro based on zygotic embryos, characterized in that, The culture medium for rapid propagation and browning prevention of submerged camphor trees based on zygotic embryos includes an initiation and induction medium, a proliferation medium, and a rooting medium. The initiation and induction medium consists of MS + 1.0 mg / L 6-BA + 0.1 mg / L GA3 + 0.1 mg / L 2,4-D. The proliferation medium consists of MS + 2.0 mg / L 6-BA + 0.05 mg / L DA-6 + 0.05 mg / L NAA + 2 mg / L glutathione + 40 ml / L coconut juice. The rooting medium consists of 1 / 2 MS + 0.1 mg / L NAA + 1.0 mg / L IBA + 0.2 g / L LAC.

10. The application of the zygotic embryo-based in vitro rapid propagation and anti-browning seedling culture medium of *Cinnamomum camphora* as described in claim 9 in the propagation of *Cinnamomum camphora*.

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