Efficient off-the-vine seedling raising method for excellent camellia oleifera variety

Abstract

The application belongs to the technical field of plant tissue culture, and discloses a high-efficiency off-site seedling method for Camellia oleifera excellent varieties. The application optimizes basic medium setting, different hormone components and concentration level matching, takes Camellia oleifera micro-grafting stems as explants, forms complete plants through bud induction, proliferation culture and rooting culture, and obtains healthy Camellia oleifera seedlings which are in good growth, consistent in phenotype and good in genetic stability after being transplanted to a substrate. In actual production and application, the Camellia oleifera seedlings which are healthy, consistent and uniform can be produced on a large scale, and the stable and efficient propagation technology for Camellia oleifera excellent varieties is effectively solved, the efficient development and utilization of Camellia oleifera varieties is realized, and the application has great significance.

Description

Technical Field

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

[0002] Camellia oleifera Abel, belonging to the genus Camellia in the family Theaceae, is a small tree or shrub. It is one of the world's four major edible oil-bearing woody plants, alongside oil palm, olive, and coconut. Besides being a high-quality edible oil, camellia oil is traditionally used as a medicine to treat stomach aches and burns. It is also a high-quality raw material in the functional food, cosmetic, and pharmaceutical industries, and is used in the production of soap, margarine, lubricants, and rust inhibitors. The camellia shell, as an industrial raw material, is rich in hemicellulose, lignin, and cellulose, and can be used to produce a range of valuable products, including oligosaccharides, furfural, tea saponins, biochar, and activated carbon. Camellia oleifera possesses high edible and medicinal value and is a multifunctional tree species with significant industrial development potential, making it a promising candidate for comprehensive development. 'Cenruan No. 3' is a national-level superior camellia oleifera variety, exhibiting excellent comprehensive performance in terms of yield, resistance, and oil quality. Its oil yield per unit area can reach 938.55 kg / ha, far exceeding that of ordinary varieties. It also possesses strong resistance to pests, diseases, and adverse conditions, and its fruit has a high oil content and excellent oil quality. It has been listed as the primary and preferred variety for the development of the camellia oleifera industry in Guangdong Province. However, its seedling production capacity cannot meet production demand, restricting the further promotion of this variety. The root cause of this problem lies in the shortage of high-quality seedlings, leading to limited fruit yield. Camellia oleifera is a crop with unstable yields, and its offspring exhibit significant genetic variation. Currently, grafting is the main method for propagating camellia oleifera seedlings; however, seedling production costs are high, and long-term use of grafting may lead to aging and degeneration of trees, affecting the healthy development of the camellia oleifera industry. Selecting superior mother plants for tissue culture and large-scale seedling production to achieve asexual propagation of the superior variety is an important way to industrialize 'Cenruan No. 3'.

[0003] Current reports on the tissue culture of Camellia oleifera mainly present the following problems: the mother plant material used in tissue culture is not a mature, high-quality tree selected over a long period; the contamination rate during explant induction culture is high; the efficiency of establishing aseptic systems is low; buds are prone to browning, shedding, and even death during subculture; and the stem segment induction rate is low, limiting the efficiency of in vitro propagation. Furthermore, root induction efficiency is low, the cycle is long, and the success rate is not high. Huang Liya et al. used fresh, tender stem segments with buds from 'Cenruan No. 2' and 'Cenruan No. 3' as explants, and the highest induction survival rate was only 71% and 73%, respectively. Wang Yihong et al. induced buds from stem segments of 7-year-old Camellia oleifera mother trees of 'Cenruan No. 3', achieving an induction rate of only 53.6% and a rooting rate of 96.8%, but root growth was slow, taking approximately 60 days to reach 1 cm in length. Wu Youmei et al. induced rooting using aseptically propagated seedlings of 'Cenruan No. 2', achieving a rooting rate of 97.8% for single buds with 2-4 fully expanded leaves, but the average number of roots per plant was only 3.8. Guo Mengqing also optimized tissue culture conditions for 'Cenruan No. 3', achieving an average number of roots of 3.63, but the starting material was stem segments of seedlings grown from seed as explants. Neither method achieved efficient in vitro seedling propagation of superior Camellia oleifera varieties.

[0004] Therefore, using the perennial superior variety 'Cenruan No. 3' as the female parent, rejuvenation was achieved through micrografting, effectively reducing the endophytic load and improving the survival rate of explants. Further optimization of the basic culture medium design, hormone types, concentrations, and ratios led to the establishment of an efficient in vitro plant regeneration technology. This achieved the goals of robust and rapidly elongating proliferating buds, high proliferation rate, high rooting rate, robust rooted seedlings, and high transplant survival rate. The genetic stability of the regenerated plants was also verified. Large-scale asexual propagation will provide technical support for the propagation of high-quality seedlings needed for 'Cenruan No. 3' cultivation in my country, and is of great significance for improving the economic benefits of 'Cenruan No. 3'. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing technologies, the present invention aims to provide a highly efficient method for in vitro seedling cultivation of superior Camellia oleifera varieties. This method effectively overcomes the problem of exogenous contamination, achieving an explant survival rate of 84% and an induction rate as high as 88.89%. It effectively solves the technical problems of unstable growth, easy death, and slow elongation of axillary buds induced in vitro in mature superior trees due to their advanced physiological age. The method has a high proliferation coefficient of 4.56 and an average of 4.28 effective buds per clump. It breaks through the technical bottleneck of difficult rooting of tissue culture buds from superior Camellia oleifera varieties, achieving a rooting rate of 100% and an average of 10.23 roots per plant. The rooted seedlings grow vigorously, and the transplant survival rate reaches 98%. The method also has low cost for large-scale production.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for rapid propagation of Camellia oleifera seedlings via tissue culture includes the following steps:

[0008] (1) Explant preparation and treatment: Select semi-lignified branches from Camellia oleifera micro-grafted seedlings as explants. Micro-grafted seedlings refer to semi-lignified branches collected from superior individual trees and grafted onto the hypocotyl of Camellia oleifera seedlings. When the scion grows to the semi-lignified stage, it is grafted onto the hypocotyl of the Camellia oleifera seedling again. The entire process is repeated five times. Figure 1 This process yields rejuvenated material, which to some extent restores the vitality of the parent material and effectively reduces endophytic bacteria. Leaves are removed from the branches, but petioles are retained.

[0009] (2) Disinfection of explant surface;

[0010] (3) Bud induction: Sterilized and viable Camellia oleifera stem explants were transferred to an induction medium for culture. The induction differentiation medium was prepared by adding 0.5 mg / L 6-BA and 0.01-0.02 mg / L NAA, 30 g / L sucrose, and 5.8 g / L agar powder to modified MS basal medium, MS basal medium, 1 / 2 MS basal medium, and WPM basal medium, with a pH of 5.8. The culture was conducted at a temperature of 25 ± 2℃ and a light intensity of 60 μmol·m². -2 ·s -1 They were cultured in a culture room with a light exposure time of 12 h / d.

[0011] (4) Proliferation culture: The axillary buds obtained in the above steps were cut into 2-3 cm pieces and inoculated onto the proliferation medium for culture to obtain proliferated seedlings; the proliferation medium was modified MS basal medium supplemented with 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, 30 g / L sucrose, and 5.8 g / L agar powder, and repeatedly subcultured to expand the propagation, with a pH of 5.8; the temperature was 25 ± 2℃ and the light intensity was 60 μmol·m -2 ·s -1 They were cultured in a culture room with a light exposure time of 12 h / d.

[0012] (5) Rooting culture: When the proliferating seedlings obtained in step (4) grow to 2-3 cm, cut off 1.5 cm of the apical bud stem and inoculate it into the rooting medium for rooting culture; the rooting medium is made by adding 1.0-5.0 mg / L NAA, 5.8 g / L agar and 15 g / L sucrose to modified MS basal medium and 1 / 2 modified MS basal medium, with a pH of 5.8; the temperature is 25 ± 2℃ and the light intensity is 60 μmol·m -2 ·s -1 The plants were cultured in a culture room with a light duration of 12 h / d, and the rooting rate and average number of roots were recorded after 50 days.

[0013] In steps (3), (4), and (5), the modified MS basic culture medium formulation is as follows: 720 mg / L NH4NO3, 675 mg / L KNO3, 440 mg / L CaCl2·2H2O, 370 mg / L MgSO4·7H2O, 425 mg / L KH2PO4, 0.83 mg / L KI, 6.2 mg / L H3BO3, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2-EDTA·2H2O, 100 mg / L Myo-Inositol, 2 mg / L The medium consisted of Glycine, 0.1 mg / L Thiamin·HCl, 0.5 mg / L Nicotinic·acid, and 0.5 mg / L Pyridoxin·HCl, with the remainder being water, and a pH of 5.8. The 1 / 2 modified MS medium was prepared by halving the amounts of macroelements in the modified MS medium, while keeping the other components unchanged.

[0014] To better realize the present invention, the following steps are also included:

[0015] (6) Hardening off and transplanting: After 50 days of rooting culture, the tissue culture seedlings with healthy root systems were moved together with the bottles into the greenhouse (25℃, 80% relative humidity, 75% shading) for 7 days to harden off and adapt to the temperature, light intensity and diurnal rhythm of the greenhouse. The root culture medium was thoroughly washed off with running tap water. The small plants were then transplanted into a cultivation substrate made of peat moss, perlite and vermiculite in a volume ratio of 5:1:1 and placed in the greenhouse for cultivation under conventional greenhouse management conditions.

[0016] (7) Management of transplanted seedlings;

[0017] (8) Verification of genetic stability;

[0018] In step (1), the explant preparation and treatment uses semi-lignified branches of micro-grafted seedlings as explants. Micro-grafted seedlings refer to semi-lignified branches collected from superior individual trees and grafted onto the hypocotyl of Camellia oleifera seedlings. When the scion grows to the semi-lignified stage, it is grafted onto the hypocotyl of the Camellia oleifera seedlings again. The entire process is repeated five times to obtain rejuvenated material, which to some extent restores the vitality of the parent material and effectively reduces endophytic bacteria. The leaves of the semi-lignified branches of the explants are removed, the nodes are retained, and they are soaked and scrubbed with detergent solution (with Tween added), and then rinsed clean under running water.

[0019] In step (2), the surface disinfection of the explant is carried out by soaking the treated stem segments in a 1% sodium hypochlorite solution for 3-8 minutes, depending on the degree of lignification. After discarding the sodium hypochlorite solution, the stem segments are rinsed with sterile water 3-4 times. Then, the stem segments are soaked in a 0.1% mercuric chloride solution (with 3-4 drops of Tween added) for 1-4 minutes, with constant shaking. After discarding the mercuric chloride solution, the stem segments are rinsed with sterile water 5-6 times.

[0020] In step (3), the bud induction involves inoculating a stem segment with an axillary bud or apical bud into an induction culture medium after surface sterilization.

[0021] In step (4), the proliferation culture involves dividing the induced buds into 2-3 cm lengths after they differentiate into new bud clusters and then transferring them into a new proliferation culture medium to obtain proliferated seedlings.

[0022] In step (5), the rooting culture requires 7 days of dark culture before being transferred to a light intensity of 60 μmol·m⁻¹. 2 ·s -1 Cultivating under suitable conditions, especially in the dark for a short period, can reduce browning of the plants and promote root development.

[0023] In step (6), the seedling hardening and transplanting are carried out in the following steps: After 50 days of rooting culture, the rooted seedlings are placed in a greenhouse for 7-10 days, and then the tissue culture bottle is opened from half to full for 1 day. The seedlings are then taken out of the culture bottle, the culture medium adhering to the roots is washed off, and the seedlings are transplanted into a seedling container filled with substrate. After transplanting, the seedlings are thoroughly watered to settle the roots. The substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of 5:1:1. The cultivation substrate is first disinfected with a potassium permanganate solution with a mass-volume percentage concentration of 1‰ and then rinsed clean with water.

[0024] In step (7), the management of transplanted seedlings is as follows: after transplanting, cover the seedlings with a film to keep them moist. The transplanting environment is controlled at a temperature of 25℃, a shading degree of 75%, and a relative humidity of over 80%. After 10-15 days, the film and shade net can be gradually removed, and routine water and fertilizer management can be carried out.

[0025] In step (8), the genetic stability verification was performed by analyzing the genetic stability of the regenerated plants using simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers. Genomic DNA was extracted from leaf samples using a modified CTAB method, and all plant DNA samples were amplified and analyzed using 16 pairs of RAPD primers and 8 pairs of ISSR primers.

[0026] This invention optimizes the setup of different basic culture media and the ratio of different hormone components and concentrations to obtain mature stem explants. It also explores key aspects such as bud induction, proliferation culture, and rooting culture, ultimately achieving efficient regeneration of Camellia oleifera tissue culture, thus enabling its propagation and population expansion. Compared with existing technologies, this invention has the following advantages and effects: high explant survival rate (84%); rapid bud growth and high induction rate (88.89%), unaffected by seasons; high proliferation coefficient (4.56); high rooting rate (100%); well-developed root system (more than 10 roots per plant); robust rooted seedlings with a 98% transplant survival rate. In practical production applications, it allows for year-round large-scale rapid seedling production, producing robust and uniform Camellia oleifera seedlings. This technology will provide technical support and seedling guarantee for the resource protection, industrial development, and population expansion of Camellia oleifera in my country; through large-scale seedling production, it provides technical assurance for the promotion and application of Camellia oleifera. Attached Figure Description

[0027] Figure 1 These are micrografted seedlings of Camellia oleifera.

[0028] Figure 2 This describes the induction of camellia oleifera buds.

[0029] Figure 3 This describes the proliferation of camellia oleifera.

[0030] Figure 4 This is a description of the rooting status of the camellia oleifera.

[0031] Figure 5 The results of transplanting Camellia oleifera tissue culture seedlings 30 days after transplanting.

[0032] Figure 6 The results are for RAPD stability verification. M: DNA molecular weight standard (100-5000 bp); MP: mother plant; 1-18: DNA PCR amplification bands of 18 randomly selected regenerated plants transplanted to the field.

[0033] Figure 7 The results are for ISSR stability verification. M: molecular weight standard (100-5000 bp); MP: mother plant; 1-18: DNA PCR amplification band patterns of 18 randomly selected regenerated plants transplanted to the field. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Those skilled in the art can achieve the objectives of the present invention based on the above disclosure and the ranges of parameters. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions.

[0035] The modified MS medium in the following examples contains per liter of the following components: NH4NO3 720 mg, KNO3 675 mg, CaCl2·2H2O 425 mg, MgSO4·7H2O 370 mg, KH2PO4 170 mg, KI 0.83 mg, H3BO3 6.2 mg, MnSO4·4H2O 22.3 mg, ZnSO4·7H2O 8.6 mg, Na2MoO4·2H2O 0.25 mg, CuSO4·5H2O 0.025 mg, CoCl2·6H2O 0.025 mg, FeSO4·7H2O 27.8 mg, Na2-EDTA·2H2O 37.3 mg, Myo-Inositol 100 mg, Glycine 2 mg, Thiamin·HCl 0.1 mg, and Nicotinic acid 0.5 mg. mg, Pyridoxin·HCl 0.5 mg, balance water, pH 5.8. The 1 / 2 modified MS medium is prepared by halving the amount of macroelements in the modified MS medium, while keeping the other components unchanged.

[0036] (1) Preparation and treatment of explants: Micrografted seedling stem segments were used as explants. Semi-lignified branches collected from superior individual trees were selected and grafted onto the cotyledons of tea oil seedlings. When the buds grew to the semi-lignified stage, they were grafted onto the cotyledons of the seedlings again. The entire process was repeated 5 times to obtain rejuvenated material. Micrografting effectively restored the vitality of the parent material to a certain extent and effectively reduced endophytic bacterial contamination. After the last grafting, the leaves of the semi-lignified branches were removed, leaving the nodes. The branches were soaked and scrubbed with a detergent solution (with Tween added), and then the stem segments were rinsed clean under running water.

[0037] (2) Explant surface disinfection: Cut the branches into stem segments with 1-2 nodes. Depending on the degree of semi-lignification, disinfect with 1% sodium hypochlorite for 3-4 min, followed by disinfection with 0.1% mercuric chloride (containing 3-4 drops of Tween) for 1-4 min. After each disinfection, the sample needs to be rinsed with sterile water 5 times, and then about 0.5 cm of the wound end of the explant is cut off.

[0038] (3) Adventitious bud induction: Sterilized explants were cut into stem segments with one axillary or terminal bud using a sterile blade and inoculated into induction media. Modified MS medium, MS medium, 1 / 2 MS medium, and WPM medium were used as basal media, with 0.5 mg / L 6-BA + 0.01 mg / L NAA and 0.5 mg / L 6-BA + 0.02 mg / L NAA added, respectively. Sucrose 30 g / L and agar 5.8 g / L were added to the media, and the pH was adjusted to 5.8. The induction was carried out at a temperature of 25 ± 2℃ and a light intensity of 60 μmol·m⁻². -2 ·s -1 The plants were cultured in a culture room with a light exposure of 12 h / d, and the bud induction rate and growth status were recorded after 30 days.

[0039] The results showed that among the four basic culture media, the modified MS medium was the most suitable for inducing axillary buds in *Camellia oleifera*. Approximately 30 days after inoculation, the axillary buds of the explants germinated and grew rapidly, with bright green leaves. Figure 2 MS and WPM media showed low induction rates, slow growth, and large, thin new leaves that gradually died after 3-4 months. 1 / 2 MS medium also showed low induction rates, slow bud growth, and yellowish-green leaves. In the other three media, new buds easily turned brown and died within 2-3 months. The combined use of 6-BA and NAA induced adventitious buds in micrografted Camellia oleifera stem segments, but the growth of adventitious buds varied with different concentration ratios, and the induction rates also differed significantly (P<0.05). The modified MS + 0.5 mg / L BA + 0.02 mg / L NAA combination showed the best induction effect (Table 1), with an induction rate of 88.89%. The buds were robust, grew well, and were green, significantly better than other treatments. Under optimal conditions, the explants could maintain green leaves and robust buds for up to 2 months.

[0040] Table 1. Effects of different basal culture media and different concentrations of BA and NAA on shoot induction.

[0041]

[0042] Note: Different lowercase letters in the same column indicate significant differences (P<0.05).

[0043] (4) Proliferation culture

[0044] To optimize the proliferation medium, different concentrations of 6-BA and NAA were added to a modified MS medium. All media contained 30 g / L sucrose and 5.8 g / L agar, and the pH was adjusted to 5.8. Results showed that the combined use of 6-BA and NAA promoted the proliferation of adventitious buds in *Camellia oleifera*. With a constant 6-BA concentration, the proliferation coefficient initially increased and then decreased with increasing NAA concentration (Table 2). Excessive NAA concentration led to a decrease in the number of effective buds and vitrification, while 6-BA significantly increased the proliferation coefficient. The combination of 1.5 mg / L 6-BA + 0.2 mg / L NAA resulted in a proliferation coefficient of 4.56, with 4.28 effective buds per bud cluster (bud length ≥1.5 cm), resulting in a large number of buds, rapid growth, and robust stems. Figure 3 In summary, the optimal proliferation medium was modified MS medium + 1.5 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 5.8 g / L agar, with a proliferation coefficient of 4.65. All the buds were effective, long and vigorous.

[0045] Table 2 Effects of different plant hormones and their concentrations on bud proliferation

[0046]

[0047] Note: Different lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0048] (5) Rooting culture

[0049] When the buds reached 2-3 cm in length and were suitable for rooting, 1.5 cm stem segments with terminal buds were cut and inoculated into rooting medium. To screen for the optimal rooting medium, this experiment used modified MS and 1 / 2 modified MS as basal media, supplemented with different concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 mg / L) of NAA. After 50 days of culture, the rooting rate, average number of roots, and root growth were recorded. The results showed (Table 3) that seedlings cultured in modified MS medium produced more callus, but leaves turned yellow and fell off after 4 weeks. Low concentrations of NAA easily induced callus rather than rooting, and concentrations exceeding 5 mg / L reduced the effect or even inhibited rooting. 1 / 2 modified MS medium was more suitable for rooting than proliferation medium. The optimal rooting medium was 1 / 2 modified MS + 4.0 mg / L NAA, with a rooting rate of 100%, an average of 10.23 roots per plant, and vigorous growth. Figure 4 Rooting begins on day 15, and a well-developed root system suitable for transplanting is formed by day 50.

[0050] Table 3. Effects of different basic culture media and plant hormones and their concentrations on adventitious shoot rooting.

[0051]

[0052] Note: Different lowercase letters in the same column indicate significant differences between different treatments (P<0.05).

[0053] (6) Transplanting and management of tissue culture seedlings:

[0054] After transplanting the rooted seedlings to the greenhouse for 7 days, unscrew the bottle caps to allow them to gradually adapt to the greenhouse environment. One day later, remove the seedlings, wash off the culture medium adhering to the roots, and transplant them into seedling cups containing a mixture of peat moss, perlite, and vermiculite in a 5:1:1 volume ratio. The mixed substrate should be sterilized with a 1‰ potassium permanganate solution before use. Plant one seedling per cup, covering the roots with soil just deep, generally no more than 1 cm. Firmly press the substrate to ensure close contact between the seedling roots and the substrate. Water thoroughly after transplanting. Water thoroughly after transplanting and cover with plastic film and a 75% shade net to reduce water loss and solar radiation. Seven days after transplanting, spray with a 0.1% carbendazim or chlorothalonil solution to prevent fungal diseases. Also, control pests and prevent seedling rot. For the first 10-15 days after transplanting, water regularly during the day and spray the leaves every 3-4 hours. After 10-15 days, remove the plastic film and shade netting and carry out routine water and fertilizer management. 15 days after transplanting, spray with urea solution at a concentration of 1‰ (by weight and volume) every 10 days for 2 consecutive times. Thereafter, spray with compound fertilizer solution at a concentration of 1‰-2‰ (by weight and volume) once every 15 days until the seedlings reach the required size for transplanting.

[0055] (7) Genetic stability verification: PCR-based ISSR and RAPD molecular markers were used for evaluation. Genomic DNA was extracted from leaf samples using the cetyltrimethylammonium bromide (CTAB) method. All plant DNA samples were analyzed using 16 RAPD primers and 8 ISSR primers (Table 4). Primers were provided by BGI Genomics (Shenzhen). RAPD and ISSR DNA amplification was performed in a 20 µL reaction system containing: 1.0 µL template DNA (20 ng / µL), 10 µL 2×Taq PlusMaster Mix (Beijing Kangrun Biotechnology Co., Ltd.), 1.1 µL primers (10 µM), and 7.9 µL ddH2O. The RAPD amplification program was as follows: initial denaturation at 95℃ for 3 min; followed by denaturation at 94℃ for 1 min, annealing at 45℃ for 1 min, extension at 72℃ for 2 min, for a total of 40 cycles; and a final extension at 72℃ for 7 min. ISSR amplification was performed using a Bio-Rad thermal cycler with the following program: initial denaturation at 94℃ for 4 min; followed by denaturation at 94℃ for 30 s, annealing at 58℃ for 45 s, extension at 72℃ for 90 s, for a total of 38 cycles; and a final extension at 72℃ for 7 min. PCR amplification products were separated by electrophoresis on a 1.0% agarose gel containing 0.25 μg / mL ethidium bromide, labeled with 1.5% TAE buffer and 5000 bp DNA, and electrophoresed at 150 V for 20 min.

[0056] The results showed that 16 RAPD primers amplified 90 clear bands (Table 4). Figure 6 The size is 250-5000 bp, and each primer amplifies 4-9 bands; 8 ISSR primers amplify 39 bands (Table 5). Figure 7 The primers (100-3000 bp in size, 3-7 primers per primer) were used. Both RAPD and ISSR amplification products were monomorphic, and there was no genetic variation between the mother plant and the cloned plant. The results indicate that direct induction of shoot clusters can maximize the maintenance of genetic stability.

[0057] Table 4. Number and size range of fragments amplified by RAPD and ISSR primers in Camellia oleifera

[0058]

[0059] The above embodiments are preferred embodiments of this patent, but the embodiments of this patent are not limited to the above embodiments. The scope of protection of this invention is defined by the appended claims, and any modifications based on the claims of this patent are within the scope of protection of this invention.

Claims

1. A highly efficient method for the in vitro seedling raising of superior Camellia oleifera varieties, characterized in that, Includes the following steps: (1) Explant preparation and treatment: Select Camellia oleifera stem segments for micrografting propagation; (2) Disinfection of explant surface; (3) Bud induction: Cut and sterilize the Camellia oleifera micro-grafted seedlings and inoculate them into the induction medium for culture; the induction medium is a modified MS basic medium with 0.5 mg / L 6-BA, 0.01 ~ 0.02 mg / L NAA, 30 g / L sucrose, 5.8 g / L agar powder added, and the pH is 5.

8. (4) Proliferation culture: The induced buds were cut into 2-3 cm pieces and transferred to the proliferation medium to obtain proliferated seedlings; the proliferation medium was a modified MS basic medium with 1.0-2.0 mg / L 6-BA, 0.1-0.5 mg / L NAA, 30 g / L sucrose, and 5.8 g / L agar powder added, and the pH was 5.

8. (5) Rooting culture: Select 2-3 cm buds from the propagation seedlings, cut 1.5 cm stems with apical buds and inoculate them into the rooting culture medium to obtain rooted seedlings; the rooting culture medium is 1 / 2 modified MS basal medium with 1.0-5.0 mg / L NAA, 15 g / L sucrose and 5.8 g / L agar powder added, and the pH is 5.8; The modified MS medium contains per liter of the following components: NH₄NO₃ 720 mg, KNO₃ 675 mg, CaCl₂·2H₂O 440 mg, MgSO₄·7H₂O 370 mg, KH₂PO₄ 425 mg, KI 0.83 mg, H₃BO₃ 6.2 mg, MnSO₄·4H₂O 22.3 mg, ZnSO₄·7H₂O 8.6 mg, Na₂MoO₄·2H₂O 0.25 mg, CuSO₄·5H₂O 0.025 mg, CoCl₂·6H₂O 0.025 mg, FeSO₄·7H₂O 27.8 mg, Na₂-EDTA·2H₂O 37.3 mg, Myo-Inositol 100 mg, Glycine 2 mg, Thiamin·HCl 0.1 mg, and Nicotinic acid 0.5 mg. mg, Pyridoxin·HCl 0.5 mg, balance water, pH 5.

8. The 1 / 2 modified MS medium is prepared by halving the amount of macroelements in the modified MS medium, while keeping the other components unchanged; (6) Hardening off and transplanting: After the rooted seedlings are hardened off in the greenhouse, the seedlings are taken out, the culture medium stuck to the roots is washed off, and they are transplanted into the mixed substrate for tissue culture seedling transplanting management to obtain camellia seedlings. (7) Verification of genetic stability.

2. The method according to claim 1, characterized in that: The micrografting explants mentioned in step (1) refer to the collection of semi-lignified branches from individual trees of superior varieties, which are then grafted onto the hypocotyl of etiolated seedlings of Camellia oleifera. When the scion grows to the semi-lignified stage, it is grafted onto the hypocotyl of the etiolated seedlings of Camellia oleifera again. The entire process is repeated five times to obtain rejuvenated material, which to some extent restores the vitality of the parent material and effectively reduces endophytic bacteria. After the last grafting, the branches that have grown to the semi-lignified stage are stripped of leaves, leaving the petioles. They are then soaked and scrubbed with a detergent solution (with Tween added), scrubbed with sterile water, and finally rinsed clean under running water.

3. The method according to claim 1, characterized in that: The explants described in step (1) need to be rinsed with purified water to remove dust from the surface of the Camellia oleifera stem segments, soaked in detergent solution for 5 minutes, and then gently brushed with a soft brush in detergent solution to clean the seedling surface, and then rinsed clean with running water.

4. The method according to claim 1, characterized in that, The explant surface disinfection described in step (2) involves soaking the treated stem segments in a 1% sodium hypochlorite solution for 3-8 minutes, depending on their tenderness. After discarding the sodium hypochlorite solution, rinse the stem segments 3-4 times with sterile water. Then, soak the stem segments in a 0.1% mercuric chloride solution (with 3-4 drops of Tween added) for 1-4 minutes, shaking the stem segments continuously. After discarding the mercuric chloride solution, rinse the stem segments 5-6 times with sterile water.

5. The method according to claim 1, characterized in that, The bud induction described in step (3) involves inoculating a stem segment with one axillary bud or apical bud into an induction medium after surface sterilization.

6. The method according to claim 1, characterized in that, The proliferation culture described in step (4) involves differentiating the buds to form new bud clusters, cutting the tender bud clusters with a bud height ≥ 3 cm into stem segments of 2 to 3 cm, and then transferring them into a new proliferation culture medium to obtain proliferated seedlings.

7. The method according to claim 1, characterized in that, The culture conditions for steps (3) and (4) are: temperature 25 ± 2℃, light intensity 60 μmol·m -2 ·s -1 The culture was carried out in a culture room with a light exposure time of 12 h / d. The rooting culture described in step (5) requires 7 days of dark culture before being transferred to a light intensity of 60 μmol·m⁻¹. -2 ·s -1 Cultured under specific conditions.

8. The method according to claim 1, characterized in that, In step (6), the seedling hardening process involves placing 50-day-old rooted seedlings in a greenhouse for 7-10 days, then unscrewing the bottle cap to allow them to gradually adapt to the greenhouse environment, and transplanting them 1 day later. The mixed matrix is ​​prepared by mixing peat moss, perlite and vermiculite in a volume ratio of 5:1:1, packing the mixture into a container bag, and sterilizing it with a potassium permanganate solution with a mass-volume percentage concentration of 1‰ before use.

9. The method according to claim 1, characterized in that, In step (6), the transplanting management of tissue culture seedlings involves controlling the transplanting environment temperature at 25℃, 75% shading, and relative humidity above 80%. After transplanting, the seedlings are covered with a film to retain moisture. After 10 to 15 days, the film is removed and routine water and fertilizer management is carried out.

10. The method according to claim 1, characterized in that, In step (7), the genetic stability verification is performed by analyzing the genetic stability of regenerated plants that have been subcultured 12 times using simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) molecular markers. Genomic DNA was extracted from leaf samples using a modified CTAB method, and all plant DNA samples were amplified and analyzed using 16 pairs of RAPD primers and 8 pairs of ISSR primers.

Images