Method for improving acclimatization and transplantation survival rate of camellia tissue culture seedlings

Abstract

The invention relates to a method for improving the survival rate of acclimatization and transplantation of camellia tissue culture seedlings. In the acclimatization process, the tissue culture seedlings are disinfected by a ferrous sulfate disinfectant and then are transplanted. The method successfully solves the problem of huge economic loss caused by discarding polluted camellia tissue culture seedlings which are difficult to breed or extremely precious, can realize normal growth of the tissue culture seedlings under the condition of bacterial colony pollution, and greatly improves the survival rate of the tissue culture seedlings after acclimatization and transplantation.

Description

Technical Field

[0001] This invention relates to the field of plants, particularly to plant regeneration through tissue culture technology, and specifically to a method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting. Background Technology

[0002] Camellia is one of my country's ten traditional famous flowers. Currently, the cultivation of new camellia varieties mostly adopts traditional hybridization breeding, and the propagation of specific varieties is mainly carried out through methods such as cuttings and grafting. In traditional hybridization breeding, it is necessary to select plump, disease-free, mature hybrid seeds for sowing and seedling cultivation. However, immature seeds produced by factors such as climate change and the fruit-setting ability of parent plants cannot be sown and cultivated, and these seeds contain a large number of new varieties. These seeds are often discarded or rot and die as a result of natural selection.

[0003] In the previous work of our team, such as the technologies disclosed in Chinese patent documents CN113303227A and CN115413578A, the mass propagation of new germplasm seedlings was achieved by constructing somatic cell embryonic clones using immature camellia seeds as explants. This technology enables the induction of sterile seedlings under sterile conditions through embryo rescue and somatic cell embryogenesis pathways. After hardening and transplanting, regenerated plants are formed, which greatly reduces the adverse effects of the natural environment on seed development and avoids the loss of germplasm resources caused by sowing and seedling failure. At the same time, it enables the rapid propagation of new varieties of seedlings. Furthermore, by hardening the seedlings in a loose and porous substrate under sterile conditions, the active absorption capacity of the fleshy roots of sterile seedlings for nutrients and water is gradually cultivated, solving the problem of difficult survival rates of camellia tissue culture seedlings after hardening and transplanting.

[0004] However, during the somatic embryo germination and seedling growth of camellia, the long culture period of about 6 months often leads to contamination by bacteria and fungi in the tissue culture seedlings. These contaminated seedlings are usually autoclaved to prevent the spread of contamination and need to be discarded. However, for slow-growing, sterile somatic embryonic seedlings of camellia, especially for new germplasm from distant hybridization, the already small number of seedlings becomes a major loss if contaminated. Therefore, finding a balance point for the symbiotic relationship between bacteria or fungi and tissue culture seedlings to continue the normal growth of tissue culture seedlings in a sterile environment has become a challenge.

[0005] The purpose of this invention is to overcome the shortcomings of the prior art. In the process of aseptic culture, chemical sterilization is carried out in a sterile environment on contaminated camellia tissue culture seedlings with intact roots and buds. This achieves the death of bacteria and fungi without affecting the growth of the tissue culture seedlings. In fact, it can even promote the further growth of the tissue culture seedlings, laying a good foundation for hardening and transplanting. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for improving the survival rate of camellia tissue culture seedlings during hardening and transplanting. In this method, the tissue culture seedlings are disinfected with ferrous sulfate disinfectant during the hardening process before transplanting.

[0007] Preferably, the ferrous sulfate disinfectant is FeSO4·7H2O with a concentration of 20-50 mg / L.

[0008] Most preferably, the ferrous sulfate disinfectant is FeSO4·7H2O with a concentration of 35 mg / L.

[0009] More preferably, the ferrous sulfate disinfectant is used for disinfection after bacterial or fungal contamination of the tissue culture seedlings is observed.

[0010] More preferably, the amount of ferrous sulfate disinfectant used on the bacterial-contaminated tissue culture seedlings is 5-10 mL each time, and it is used 1-2 times.

[0011] More preferably, the amount of ferrous sulfate disinfectant used on the fungal-contaminated tissue culture seedlings is 5-10 mL for the first time, and 1-5 mL each time thereafter until the contamination is eliminated.

[0012] Preferably, the method further includes transplanting tissue culture seedlings that have grown normally after the disinfection process after soaking and cleaning them in ferrous sulfate disinfectant of the same concentration.

[0013] More preferably, the culture conditions for the transplanted tissue culture seedlings are as follows: the main components of the culture medium are: V peat moss: V perlite in a ratio of 1:1 or 1:2, with the addition of 0.5-1.0 mg / L of auxin; pH value of 5.9-7.0; air humidity of not less than 90%; and a photoperiod of 16 h·d. -1 Dark culture for 8 hours and days -1 The light intensity is 50 μmol·m -2 ·s -1 The temperature is (25±2)℃

[0014] Furthermore, preferably, the method also includes continuing to check the transplanted tissue culture seedlings for bacterial or fungal contamination. Within one month after transplanting, the seedlings should be checked every two days, and then at least once a week for the next six months. If contamination is found, the seedlings should be disinfected again immediately according to the above disinfection conditions.

[0015] This invention also provides the application of ferrous sulfate in improving the survival rate of camellia tissue culture seedlings during hardening and transplanting. This invention has the following beneficial effects:

[0016] This invention fully utilizes the characteristics of camellias—their preference for iron-rich fertilizers and tolerance to acid—and, unlike the acid-resistant properties of culture media and bacterial / fungal colonies, kills bacteria or fungi, thus achieving sterilization and growth of camellia tissue-cultured seedlings. Camellias are acid-loving plants; in their natural wild environment, the pH of their soil can be as low as 4.1, with an optimal pH of 5.5-6.5 for growth. Generally, the optimal pH for bacteria is 6.5-7.5, and for fungi, it is 4.0-6.0. The suitable acidic environment provided by ferrous sulfate disinfectant kills bacteria or fungi without affecting, and may even promote, the growth of the camellia tissue-cultured seedlings themselves.

[0017] In existing technologies, ferrous sulfate is commonly used as an iron fertilizer in the cultivation and maintenance of camellias. Iron supplementation is necessary in camellia cultivation and maintenance, and iron salts with ferrous sulfate as the main component are often applied to reduce leaf yellowing. Simultaneously, because ferrous sulfate is acidic, it is often used to adjust soil pH. This invention unexpectedly discovered that, in addition to its use as a nutrient element and pH adjuster in tissue culture, ferrous sulfate can also act as a disinfectant to promote the growth of camellia tissue-cultured seedlings.

[0018] This invention fully utilizes the morphological changes of agar in MS medium to examine disinfection effectiveness and isolate contaminating bacteria and fungi. The amount of agar used in the medium is 7 g·L⁻¹. -1 After autoclaving, the pH value is 5.8, and it is in a solid state. When the pH value is higher than 5.8, the hardness of the agar increases; when the pH value is lower than 5.8, the agar tends to soften; when the pH value is 4.0, it cannot solidify at all and is in a liquid state.

[0019] In this invention, as the application rate of the appropriate concentration of ferrous sulfate disinfectant solution gradually increases, it does not damage the camellia tissue culture seedlings but inhibits and kills bacterial or fungal colonies, causing the agar to gradually change from a solid to a liquid state. Bacterial contamination typically appears as a liquid on the surface of MS medium, while the MS medium itself is solid. The bacteria are killed before the pH affects the medium's hardness and morphology, making bacterial contamination easy to observe and separate from the plant material. Contaminating fungi appear as hyphae; as the pH further decreases, the fungi gradually die, and the colonies gradually turn into a dark brown, soft, sticky substance. At this point, the medium has changed to a liquid state, making it easy to separate the hyphae from the tissue culture seedlings, thus allowing the colonies to be easily cleaned from the base of the seedlings.

[0020] This invention effectively and cost-effectively avoids the huge economic losses caused by the discarding of tissue culture seedlings obtained from camellia somatic embryonic clones that are difficult to propagate or extremely precious due to contamination. It enables the normal growth of tissue culture seedlings even under colony contamination, greatly improving the survival rate of tissue culture seedlings after hardening and transplanting. This is of great significance for promoting the breeding of plants that are difficult to propagate and has extremely high economic value. Under normal circumstances, the contamination rate of camellia tissue culture seedlings reaches 5%, and their transplant survival rate is no higher than 10%. After the disinfection treatment of this invention, the survival rate after hardening and transplanting can reach as high as 60%. Brief description of the attached figures

[0021] Figure 1 The image shows a photograph of a camellia tissue culture seedling contaminated with untreated fungi according to the present invention.

[0022] Figure 2 The image shows a photograph of the tea plantlets treated with ferrous sulfate in Example 2 of this invention, showing the effect of antibacterial test on promoting leaf growth.

[0023] Figure 3 The image shows photographs of camellia tissue culture seedlings that withered and died after an antibacterial test with carbendazim treatment, as a comparative example of the present invention. Detailed Implementation

[0024] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0025] Those skilled in the art will recognize that the Latin name for camellia is *Camellia japonica* L., also known as foreign tea, camellia, and late-blooming camellia, and it is a shrub or small tree belonging to the genus *Camellia* in the family Theaceae. Camellias commonly grow in forests on hills at altitudes of 300-1100 meters and near coastlines. They prefer sunlight and thrive in sparse forests; they prefer warm, humid environments and are intolerant of alkaline soils, preferring fertile, moist, well-drained, slightly acidic soils. Camellias have beautiful tree shapes, are evergreen, have large, brightly colored flowers, and a long flowering period, making them a commonly used and prized flowering tree for landscaping in late winter and early spring. Camellias have a long history of cultivation, with many horticultural varieties, and are one of the world's most famous ornamental flowering trees. In addition, camellias are also a valuable oil crop; their seeds can be pressed for oil, which is commonly used industrially for lubricating precision machinery and making rust-preventive coatings on metals. Camellia oil can also be used in cooking; its dried petals can be cooked and used in catering, and camellia leaves can be used as a substitute for tea for brewing. Camellia seeds, leaves, flowers, and roots can all be used in medicine. The seeds are the source of the Chinese medicinal herb Camellia seed, which has the effect of removing grease; the leaves are the source of the Chinese medicinal herb Camellia leaf, which has the effects of clearing heat and detoxifying, and stopping bleeding; the flowers are the source of the Chinese medicinal herb Camellia flower, which has the effects of cooling blood and stopping bleeding, dispersing blood stasis and reducing swelling, etc.

[0026] Those skilled in the art will understand that the term "tissue culture" used in this invention refers to a new asexual reproduction technique developed based on the theory of totipotency of plant cells. In a broad sense, plant tissue culture, also known as in vitro culture, refers to the technique of isolating suitable tissues, organs, cells, protoplasts, etc., from a plant and, under aseptic conditions, inoculating them onto a culture medium containing various nutrients and plant growth regulators to obtain regenerated complete plants or produce other economically valuable products. In a narrow sense, plant tissue culture refers to the culture of various plant tissues, such as cambium, parenchyma, mesophyll, and endosperm, to obtain regenerated plants. It also refers to the culture of callus tissue generated from various organs during the culture process, which then undergoes redifferentiation to form regenerated plants.

[0027] In this invention, the tissue culture utilizes immature embryos and / or cotyledons from immature hybrid seeds as explants, based on previous research by the inventors' team (see the techniques disclosed in Chinese patent documents CN113303227A and CN115413578A). The entire contents of these documents are incorporated into this invention as part of its content, and the details already disclosed in these documents will not be repeated. Of course, this invention is also applicable to camellia tissue culture seedlings obtained through other tissue culture techniques.

[0028] In plant tissue culture, MS medium is commonly used. Designed by Murashige and Skoog in 1962 for tobacco cell culture, MS medium is characterized by its high concentration of inorganic salts and ions, making it a relatively stable ion-balanced solution. Its high nitrate content and suitable nutrient quantity and ratio meet the nutritional and physiological needs of plant cells, thus making it widely applicable. It is used as the basic medium for rapid propagation in most plant tissue cultures. MS solid medium can be used to induce callus tissue and for the culture of embryos, stem segments, shoot tips, and anthers. Its liquid medium is highly successful for cell suspension culture. The quantity and ratio of inorganic nutrients in MS medium are sufficient to meet the nutritional and physiological needs of plant cells. Therefore, under normal circumstances, it is not necessary to add organic additives such as amino acids, casein hydrolysate, yeast extract, and coconut juice. Compared with the basic components of other culture media, MS medium has a high content of nitrates, potassium, and ammonium, which is its significant characteristic. While the formulations of different MS medium types vary slightly, the basic cost is the same, and these products are all commercially available.

[0029] The MS culture medium used in this invention has the following formulation:

[0030] element Concentration (mg / L) element Concentration (mg / L) <![CDATA[KNO3]]> 1900 <![CDATA[CuSO4·5H2O]]> 0.025 <![CDATA[NH4NO3]]> 1650 <![CDATA[CoCl2·6H2O]]> 0.025 <![CDATA[KH2PO4]]> 170 <![CDATA[FeSO4·7H2O]]> 27.8 <![CDATA[MgSO4·7H2O]]> 370 <![CDATA[Na2·EDTA·H2O]]> 37.3 <![CDATA[CaCl2·2H2O]]> 440 glycine 2 <![CDATA[H3BO3]]> 6.2 Thiamine hydrochloride 0.1 <![CDATA[ZnSO4·7H2O]]> 8.6 niacin 0.5 <![CDATA[MnSO4·H2O]]> 16.9 Pyridoxine hydrochloride 0.5 <![CDATA[Na2Mo4·2H2O]]> 0.25 Inositol 100 KI 0.83

[0031] The ferrous sulfate used in this invention is an inorganic compound with the chemical formula FeSO4. The crystalline hydrate of ferrous sulfate is a heptahydrate, FeSO4·7H2O, at room temperature. Ferrous sulfate can be used to produce iron salts, iron oxide pigments, mordants, water purifiers, preservatives, disinfectants, etc. The MS medium used in this invention contains 27.8 mg / L of FeSO4·7H2O as an iron provider. The ferrous sulfate used as a disinfectant in this invention is prepared and applied separately.

[0032] Although this article uses camellia tissue culture seedlings as an example to verify the effectiveness of the related technology of the present invention, those skilled in the art will understand that the basic principles of plant tissue culture are the same, and therefore the technology of the present invention is also applicable to the tissue culture of other plants.

[0033] Unless otherwise specified in this invention, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if the technologies, instruments, equipment, and materials used in this invention are specifically specified, it does not mean that this invention can only use these technologies, instruments, equipment, and materials, but merely represents the preferred solution of this invention. Those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0034] Example 1: Obtaining Camellia Tissue Culture Seedlings

[0035] Based on the technical solution disclosed in Chinese patent document CN115413578A, which is the previous work of the inventor's team, camellia tissue culture seedlings were obtained through the following steps:

[0036] S1. Sterilization of explants: Take the immature embryo and / or cotyledons from immature hybrid seeds as explants and inoculate them into embryo rescue medium.

[0037] S2, induction of embryonic callus;

[0038] S3. Proliferation of embryogenic callus and differentiation of somatic embryos: Embryogenic callus was inoculated into proliferation medium to differentiate into embryogenic material with cotyledon-shaped embryos that were not easily separated.

[0039] S4. Maturation and Germination: Embryogenic materials are inoculated into germination medium and cultured to form hardened seedlings;

[0040] S5. Hardening off or rooting: The hardened-off material is cultured in a loose, porous sterile substrate to obtain tissue culture seedlings. Example 2: Disinfection of contaminated camellia tissue culture seedlings

[0041] 1. Preparation of disinfectants

[0042] The concentration to be prepared is 20-50 mg / L. -1 Ferrous sulfate (FeSO4·7H2O) is used as a disinfectant.

[0043] The concentration to be prepared is 20-50 mg / L. -1 Carbendazim was used as a control for disinfectants.

[0044] Observation of contamination in tissue culture seedlings

[0045] Tissue culture seedlings often exhibit bacterial and fungal contamination, which can occur from the initial hardening and rooting process until the later stages of seedling growth. When contamination is observed, the contaminated tissue culture bottles should be promptly removed and placed in an artificial climate chamber away from a sterile area for separate cultivation.

[0046] Bacterial contamination typically appears as a liquid on the surface of MS medium, while the MS medium itself is solid, making bacterial contamination easy to observe. Fungal contamination typically appears as filamentous distribution on the surface of MS medium, while the MS medium becomes liquid at low pH levels that kill fungi, also making fungal contamination easy to observe.

[0047] Figure 1 The image shows a photograph of a tissue culture seedling contaminated with fungi.

[0048] 3. Ferrous sulfate disinfection test on contaminated tissue culture seedlings

[0049] (1) Bacterial contamination: When liquid contamination is observed, add 5-10 ml of the pre-prepared ferrous sulfate disinfectant to evenly cover the surface of the MS medium, 1-2 times, until the liquid contamination on the medium completely disappears. Since the bacterial colonies are liquid, they are easy to separate from the plant material. When the bacteria are completely dead, stop adding the disinfectant. At this time, the MS medium under the pH conditions will be solid.

[0050] (2) Fungal contamination: When the hyphae first appear in the early stage of contamination, add 5-10 ml of the pre-prepared ferrous sulfate disinfectant to evenly cover the surface of MS medium. Then check every 2 days. Each time, add 1-5 ml of disinfectant depending on the contamination situation until the colonies are observed to turn into a blackish-brown viscous substance and no more filamentous fungi grow. At this time, the medium under the pH conditions has become liquid.

[0051] When bacteria and fungi are killed or their growth slows down, tissue culture seedlings continue to absorb nutrients from the culture medium and begin to sprout or root. Depending on the growth status of the plant material, they can be transplanted immediately or cultured for a period of time before transplanting.

[0052] The specific experimental plan is shown in Table 1 below:

[0053] Table 1. Test protocol for ferrous sulfate disinfectant

[0054]

[0055] When ferrous sulfate disinfectant at a concentration of 20 mg / L was used in test samples 1 and 4, a certain inhibitory effect on bacteria and fungi was observed, but it was poor. Bacterial growth on the culture medium was completely inhibited, but secondary fungal contamination was very likely to occur on the leaves and branches of the tissue culture seedlings. If secondary contamination occurred, the application time of ferrous sulfate needed to be extended again, requiring more than 2 months to kill all the fungi. When the contamination was completely inhibited, the survival rate of the tissue culture seedlings was less than 10%.

[0056] When ferrous sulfate disinfectant at a concentration of 50 mg / L was used in test samples 3 and 6, the disinfectant was observed to inhibit bacteria and fungi. When bacterial and fungal contamination was initially controlled, the seedlings should be transplanted in time to reduce the application of ferrous sulfate disinfectant. Otherwise, it may cause significant damage to the camellia tissue culture seedlings, such as yellow spots and drying of the leaves.

[0057] When using ferrous sulfate disinfectant at a concentration of 35 mg / L in test samples 2, 5, and 7, the inhibitory effects on bacteria and fungi were good. Bacteria were completely killed after one application, minimizing the risk of secondary fungal contamination. Fungi were completely controlled after about one month of application, at which point the colonies were dark brown and soft-sticky, the agar was thick, and new leaves began to sprout from the camellia tissue culture seedlings. The treatment effectively inhibited bacteria, degraded bacterial colonies, and promoted the growth of camellia tissue culture seedlings, achieving a survival rate of approximately 60%. Figure 2 The photograph shows how ferrous sulfate disinfectant inhibits bacteria and promotes leaf growth in camellia tissue culture seedlings.

[0058] 4. Carbendazim control test on contaminated tissue culture seedlings

[0059] According to the scheme in Table 1 above, a control experiment was conducted on contaminated tissue culture seedlings under the same conditions, using the same concentration and dosage of carbendazim for disinfection.

[0060] The experimental results showed that, regardless of the scheme, none of the control experiments could simultaneously achieve effective antibacterial activity and maintain or promote leaf growth in camellia tissue culture seedlings. Figure 2 The image shows photographs of camellia tissue culture seedlings that withered and died after an antibacterial test with carbendazim treatment.

[0061] Example 3: Subsequent culture and disinfection of camellia tissue culture seedlings after disinfection

[0062] 1. Cleaning of tissue culture seedlings

[0063] Prepare warm water with a temperature not exceeding 50℃. Select tissue culture seedlings that have grown well after being disinfected with ferrous sulfate according to Example 2. Place them in the warm water to wash away the sticky bacterial residue and culture medium on the roots. After letting them stand and drain, soak them in the same concentration of disinfectant for 3-5 minutes, and then transplant them.

[0064] 2. Transplanting

[0065] The cleaned tissue culture seedlings were planted in a substrate supplemented with auxin (0.5-1.0 mg / L). -1 To promote the differentiation and elongation of adventitious roots, the air humidity should not be lower than 90%. The main components of the substrate are: V peat moss: V perlite in a ratio of 1:1 or 1:2, with a pH value of 5.9-7.0.

[0066] The culture environment is as follows: light duration is 16 hours per day. -1 Dark culture for 8 hours and days -1 The light intensity is 50 μmol·m －2 ·s －1 The temperature is (25±2)℃.

[0067] 3. Sterilization after transplanting

[0068] Within one month after transplanting, check the growth of the tissue culture seedlings every two days. If fungal contamination occurs, add 1-5 ml of ferrous sulfate disinfectant of the same concentration until the contamination is completely eliminated.

[0069] Because camellias are slow-growing plants, especially tissue culture seedlings, their growth is particularly slow in the first six months after transplanting. Therefore, the growth status of tissue culture seedlings should be checked at least once a week, and any contamination should be disinfected immediately.

[0070] In summary, this invention successfully solves the problem of huge economic losses caused by the disposal of contaminated or extremely precious camellia tissue culture seedlings that are difficult to propagate. It can enable the normal growth of tissue culture seedlings under colony contamination conditions and greatly improve the survival rate of tissue culture seedlings after hardening and transplanting.

[0071] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting, characterized in that, The tissue culture seedlings were disinfected with ferrous sulfate disinfectant during the hardening-off process before transplanting.

2. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 1, characterized in that, The ferrous sulfate disinfectant is FeSO4·7H2O with a concentration of 20-50 mg / L.

3. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting, as described in claim 2, is characterized in that... The ferrous sulfate disinfectant is FeSO4·7H2O with a concentration of 35 mg / L.

4. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 3, characterized in that, The ferrous sulfate disinfectant was used for disinfection after bacterial or fungal contamination of the tissue culture seedlings was observed.

5. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 4, characterized in that, The amount of ferrous sulfate disinfectant used for the bacterially contaminated tissue culture seedlings is 5-10 mL each time, used 1-2 times.

6. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 4, characterized in that, The dosage of ferrous sulfate disinfectant used for tissue culture seedlings contaminated with fungi is 5-10 mL initially, followed by 1-5 mL each time until the contamination is eliminated.

7. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting, as described in any one of claims 1-6, is characterized in that... It also includes transplanting tissue culture seedlings that have grown normally after the aforementioned disinfection after soaking and cleaning them in the same concentration of ferrous sulfate disinfectant.

8. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 7, characterized in that, The culture conditions for the transplanted tissue culture seedlings were as follows: the main components of the culture medium were: V peat moss: V perlite in a ratio of 1:1 or 1:2, with the addition of 0.5-1.0 mg / L of auxin; pH value of 5.9-7.0; air humidity of not less than 90%; and a photoperiod of 16 h·d. -1 Dark culture for 8 hours and days -1 The light intensity is 50 μmol·m －２ ·s －1 The temperature is (25 ± 2)℃.

9. The method for improving the survival rate of camellia tissue culture seedlings after hardening and transplanting as described in claim 8, characterized in that, This also includes continuing to check the transplanted tissue culture seedlings for bacterial or fungal contamination. Within one month after transplanting, the seedlings should be checked every two days, and then at least once a week for the next six months. If contamination is found, the seedlings should be disinfected again immediately according to the above disinfection conditions.

10. Application of ferrous sulfate in improving the survival rate of Camellia tissue culture seedlings after hardening and transplanting.

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