Method for large-scale tissue culture propagation of camellia nitidissima

Abstract

The application discloses a tissue culture large-scale propagation method of Camellia meiocarpa. The method comprises the following steps: obtaining aseptic seedlings of Camellia meiocarpa, cluster bud proliferation culture, single plantlet culture of adventitious buds, bottle-out root preparation culture and bottle-out root culture. The composition of the cluster bud proliferation culture medium and the plantlet culture medium is optimized, the cluster bud proliferation and the formation of the adventitious buds are promoted, and the proliferation coefficient reaches 8.18. In addition, the bottle-in and bottle-out root culture scheme is combined, so that the rooting rate and the survival rate are significantly improved, and the rooting rate reaches 90%. The tissue culture method has important significance in the protection and population recovery of Camellia meiocarpa, and provides technical support for the protection and sustainable utilization of the endangered species.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a high-efficiency in-vitro tissue culture propagation method suitable for Camellia granthamiana Sealy. BACKGROUND

[0002] Camellia granthamiana Sealy is a small evergreen tree of the Camellia L. genus of the Theaceae family, and is only distributed in Guangdong and Hong Kong. The flower growth position of Camellia granthamiana Sealy is different from that of the common Camellia, each flower is born on a branch, the petals are white and flat, and the flower looks like an ice tray, which is a rare gem in Camellia flower viewing. Camellia granthamiana Sealy is listed as a vulnerable (VU) species in the IUCN, and was selected into the List of Key Protected Wild Plants in Guangdong Province (the first batch) in March 2023, and is a rare tree species unique to China. Camellia granthamiana Sealy has high scientific research value and ornamental value, is a rare wild tetraploid species in the Camellia L. genus, and has important significance in the research field of evolution and development of the Camellia L. genus. Due to its high value and large market demand, the quantity of seeds is small, the propagation is difficult, and the tissue culture propagation technology is not mature enough, so it is difficult to carry out large-scale production of seedlings to meet market demand. At present, although the leaf blades of Camellia granthamiana Sealy have been successfully induced into callus, there are still great difficulties in realizing differentiation of the callus into adventitious buds, the tissue culture system is not perfect, and a high-efficiency and stable in-vitro propagation system suitable for the species has not been formed, which limits effective preservation and large-scale utilization of resources of Camellia granthamiana Sealy.

[0003] Camellia granthamiana Sealy has extremely high scientific research and ornamental value, and as a rare wild tetraploid species in the Camellia L. genus, it plays an important role in the research field of evolution and development of the Camellia L. genus. However, the huge market demand brought by its high value faces a severe propagation dilemma: the quantity of naturally seeded seeds is small and the propagation is difficult, and the artificial tissue culture propagation technology is not mature enough. At present, although cotyledons can be successfully induced into somatic embryos, there are problems such as low somatic embryo seedling rate and large variation. Therefore, the tissue culture system has not been perfect so far, and there is a lack of a high-efficiency and stable in-vitro propagation technology system. Ultimately, the above problems seriously limit the large-scale production of Camellia granthamiana Sealy seedlings, hinder effective preservation of resources, and lead to difficulty in meeting market demand. SUMMARY

[0004] The present application provides a Camellia granthamiana Sealy tissue culture rapid propagation method, which successfully realizes large-scale rapid propagation of Camellia granthamiana Sealy seedlings, and provides an efficient production mode for industrial production of Camellia granthamiana Sealy seedlings.

[0005] To achieve the above object, the technical scheme adopted by the present application is as follows:

[0006] A method for large-scale tissue culture propagation of Camellia macrocarpa includes the following steps:

[0007] (1) Obtaining sterile seedlings: Select large-bracted white camellia fruits, peel off the pericarp to obtain seeds, place them on a clean bench for sterilization, dry them, and then inoculate them into seed germination medium. After induction germination culture, sterile seedlings are obtained. Then, on this medium, use stem segments with nodes for 5 subcultures to obtain a certain number of sterile seedlings as subsequent experimental materials. The seed germination and the first 5 subcultures were all carried out on MS + 0.1-0.5 mg / L 6-BA + 20-30 g / L sucrose + 4.5-5.0 g / L agar, with a pH of 5.8-6.0.

[0008] (2) Proliferation culture of clustered buds: Select adventitious seedlings with good growth, cut the adventitious seedlings into stem segments with nodes, and insert them into the proliferation culture medium; the proliferation culture medium is 2 / 3MS + 0.8-1.0mg / L 6-BA + 0.08-0.1mg / L NAA + 0.0075-0.01mg / L TDZ + 20-30g / L sucrose + 4.5-5.0g / L agar + 250-350mg / L hydrolyzed casein, with a pH of 5.8-6.0;

[0009] (3) Culture of vigorous single adventitious buds: Select vigorous single plants from the proliferated adventitious buds and inoculate them into a seedling strengthening medium for culture; the seedling strengthening medium is WPM + 0.5-1.0 mg / L 6-BA + 0.5-1.0 mg / L NAA + 2.0-3.0 mg / L GA3 + 20-30 g / L sucrose + 4.5-5.0 g / L agar, with a pH of 5.8-6.0;

[0010] (4) In vitro rooting preparation culture: After one and a half months of strong seedling culture, select healthy sterile seedlings, retain the terminal bud, cut off the lower leaves, retain 2-3 healthy leaves, cut off 0.4-0.6cm from the bottom of each plant to leave a wound, and insert them into the rooting preparation culture medium (to enhance the endogenous auxin content of the cuttings to promote rooting); the rooting preparation culture medium is 1 / 2MS + 1.0-1.5mg / L NAA + 0.8-1.0mg / L IBA + 20-30g / L sucrose + 4.5-5.0g / L agar, with a pH of 5.8-6.0;

[0011] (5) External rooting: After the adventitious seedlings have been induced to root for 5-10 days in the rooting preparation culture, they are taken out of the tissue culture bottle with tweezers, soaked in 800-1200mg / L IBA + 200-300mg / L NAA solution for 10-20 minutes, and then transplanted. The lower end of the sterile seedling is then obliquely cut and inserted into the perlite.

[0012] Preferably, in step (1), the seed germination medium is MS + 0.1 mg / L 6-BA + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8.

[0013] Preferably, in step (2), the proliferation medium consists of 2 / 3 MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / L LTDZ + 30 g / L sucrose + 4.8 g / L agar + 300 mg / L hydrolyzed casein, with a pH of 5.8.

[0014] Preferably, in step (3), the seedling culture medium is WPM + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 3.0 mg / L GA3 + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8.

[0015] Preferably, in step (4), the rooting preparation medium is 1 / 2MS + 1.5 mg / L NAA + 1.0 mg / L IBA + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8.

[0016] Preferably, in step (5), the concentration of IBA is 1000 mg / L and the concentration of NAA is 250 mg / L.

[0017] Preferably, in step (1), the seed disinfection steps are as follows: first, gently wipe the seed surface with an alcohol cotton ball 2-4 times, then soak it in 75% alcohol for 30-45 seconds, rinse it with sterile water 2-3 times, then disinfect it with 0.1% mercuric chloride for 4-6 minutes, rinse it with sterile water 3-5 times, and then air dry it.

[0018] Preferably, in steps (1)-(5), the cultivation conditions are a temperature of 25±2℃, a light intensity of 1500-2500lx, and a light duration of 12-16 hours / day.

[0019] This invention achieves large-scale, efficient propagation of *Camellia dabaoensis* by systematically optimizing the tissue culture technology system. Specific beneficial effects include:

[0020] 1. Significantly improved proliferation efficiency: The optimized bud proliferation medium resulted in a proliferation coefficient as high as 8.18, an average original bud height of 1.93cm, an average new bud length of 0.82cm, and robust and uniform buds, effectively breaking through the bottleneck of low proliferation coefficient in traditional methods;

[0021] 2. Significantly improved seedling quality: After optimization of the seedling culture medium, the seedling height can reach 4.63cm, with more leaves and vigorous plant growth, laying a good foundation for subsequent transplanting;

[0022] 3. Significantly improved rooting and survival rates: The innovative combination of in-bottle and out-of-bottle rooting techniques results in a rooting rate exceeding 90%, significantly higher than conventional methods. This effectively reduces production costs and provides reliable technical support for industrialization and promotion. Attached Figure Description

[0023] Figure 1 : Large-bracted white camellia bush bud proliferation.

[0024] Figure 2 Cultivation of robust seedlings from single adventitious buds of Camellia dabaoensis.

[0025] Figure 3 Rootless seedlings culture via exogenous rooting.

[0026] Figure 4 Effects of different concentrations of auxin on in-bottle rooting culture of Camellia dabaoensis (60 days). Detailed Implementation

[0027] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0029] Example 1

[0030] A method for large-scale tissue culture propagation of Camellia macrocarpa includes the following steps:

[0031] (1) Obtaining aseptic seedlings of Camellia macrocarpa:

[0032] Select large-bracted white camellia fruits, peel off the pericarp to obtain seeds, and sterilize them on a clean bench. First, gently wipe the seed surface with an alcohol swab 2-4 times, then soak in 75% alcohol for 30-45 seconds, rinse 2-3 times with sterile water, then sterilize with 0.1% mercuric chloride solution for 4-6 minutes, rinse 3-5 times with sterile water, and air dry. After inoculation, inoculate into MS medium containing 0.1 mg / L 6-BA, 30 g / L sucrose, and 4.8 g / L agar at pH 5.8. After induced germination culture, sterile seedlings are obtained. Then, on this medium, use stem segments with nodes for 5 subcultures to obtain a certain number of sterile seedlings as subsequent experimental materials. The culture conditions are: temperature 25±2℃, light intensity 2000 lx, and light duration 14 hours / day.

[0033] (2) Propagation and cultivation of clustered buds of Camellia macrocarpa:

[0034] Select healthy, adventitious seedlings of *Camellia macrocarpa*, cut them into stem segments with nodes, and insert them into a proliferation medium. Cultivate the seedlings at a temperature of 25±2℃, a light intensity of 2000 lx, and a light duration of 14 hours / day. The proliferation medium consisted of 2 / 3 MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / L TDZ + 30 g / L sucrose + 4.8 g / L agar + 300 mg / L hydrolyzed casein, with a pH of 5.8. Figure 1 ).

[0035] (3) Cultivation of vigorous seedlings from single adventitious buds of Camellia dabaoensis:

[0036] Vigorous individual plants were selected from the proliferating adventitious buds and inoculated into a seedling strengthening medium. The plants were cultured at a temperature of 25±2℃, a light intensity of 2000 lx, and a light duration of 14 hours / day. The seedling strengthening medium consisted of WPM + 0.5 mg / L IBA + 0.5 mg / L NAA + 3.0 mg / L GA3 + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8. Figure 2 ).

[0037] (4) Preparation of exogenous rooting culture of Camellia macrocarpa in bottles:

[0038] Because the initial experiments showed a 0% rooting rate in tissue culture bottles, the experimental method was improved by using in-bottle propagation to strengthen seedlings before external rooting via cuttings. After one and a half months of cultivation, healthy, sterile seedlings were selected, retaining the terminal bud, removing the lower leaves, leaving 2-3 strong leaves, and cutting off 0.5cm from the bottom of each plant to create a wound. These seedlings were then inserted into a rooting preparation medium for 7 days (to enhance the endogenous auxin content of the cuttings and promote rooting). The rooting preparation medium consisted of 1 / 2 MS + 1.5 mg / L NAA + 1.0 mg / L IBA + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8. The cultivation conditions were: temperature 25 ± 2℃, light intensity 2000 lx, and light duration 14 hours / day.

[0039] (5) Dabao white camellia rooting outside the bottle:

[0040] After 7 days of rooting preparation culture, the adventitious seedlings were removed from the tissue culture bottle with tweezers and soaked in a 1000 mg / L IBA + 250 mg / L NAA solution for 15 minutes before transplanting. The lower end of the sterile seedling was then obliquely cut and inserted into perlite. The rooting rate could reach up to 90%. Figure 3 ).

[0041] Example 2

[0042] 1. Following the method of Example 1, using healthy adventitious seedlings of Camellia dabaoensis as material, the adventitious seedlings were cut into stem segments with nodes and inserted into different proliferation media (containing 30 g / L sucrose, 4.8 g / L agar, and pH 5.8 in addition to the components listed in Table 1 below), and cultured at a temperature of 25 ± 2 °C, a light intensity of 2000 lx, and a light duration of 14 hours / day. The effects of different media on the proliferation of clustered buds of Camellia dabaoensis were evaluated, and the results are shown in Table 1.

[0043] Table 1. Effects of different culture media on the proliferation of clustered buds of *Camellia macrocarpa*.

[0044]

[0045]

[0046] Comparing the k values, the optimal combination of levels in Table 1 is 2 / 3MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.01 mg / LTDZ.

[0047] 2. Following the method in Example 1, using healthy adventitious seedlings of Camellia dabaoensis as material, the adventitious seedlings were cut into stem segments with nodes and inserted into the following proliferation medium containing different concentrations of TDZ (2 / 3 MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0-0.02 mg / L TDZ + 30 g / L sucrose + 4.8 g / L agar, pH 5.8). The medium was cultured at a temperature of 25 ± 2℃, a light intensity of 2000 lx, and a light duration of 14 hours / day to evaluate the effect of TDZ concentration on the proliferation of clustered buds of Camellia dabaoensis. The results are shown in Table 2.

[0048] Table 2 Effects of TDZ concentration on the proliferation of buds in *Camellia macrocarpa* (Note: Table 2 is based on the optimal combination obtained in Table 1, with different TDZ concentration gradients set).

[0049]

[0050]

[0051] Taking into account both the proliferation coefficient and the growth of the buds, Table 2 shows that 2 / 3MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / L TDZ is the optimal culture medium for the proliferation of clustered buds of Camellia macrocarpa.

[0052] 3. Following the method in Example 1, using healthy adventitious seedlings of Camellia dabaoensis as material, the adventitious seedlings were cut into stem segments with nodes and inserted into the following proliferation medium containing different organic additives and their concentrations (2 / 3MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / L TDZ + 30 g / L sucrose + 4.8 g / L agar + 0-300 mg / L organic additives, pH 5.8). The medium was cultured at a temperature of 25 ± 2℃, a light intensity of 2000 lx, and a light duration of 14 hours / day to evaluate the effect of organic additives on the proliferation of clustered buds of Camellia dabaoensis. The results are shown in Table 3.

[0053] Table 3. Effects of organic additives on the proliferation of clustered buds of *Camellia macrocarpa* (Note: Table 3 is based on the results in Table 2 with the addition of organic additives).

[0054]

[0055] Table 3 shows that the optimal organic additive is hydrolyzed casein, with an optimal concentration of 300 mg / L. Combining Tables 1, 2, and 3, the optimal culture medium for the proliferation of large-bracted white tea shoots is 2 / 3 MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / L TDZ + 300 mg / L hydrolyzed casein.

[0056] Example 3

[0057] Following the method in Example 1, vigorous individual plants were selected from the proliferated adventitious buds and inoculated into a seedling-strengthening medium containing different plant growth regulators and their concentrations (WPM + 0-1.0 mg / L 6-BA + 0.5-2.0 mg / L NAA + 3.0 mg / L GA3 + 30 g / L sucrose + 4.8 g / L agar, pH 5.8). The medium was cultured at a temperature of 25 ± 2 °C, a light intensity of 2000 lx, and a light duration of 14 hours / day. The effects of different plant growth regulators on the cultivation of vigorous seedlings from individual adventitious buds of Camellia dabryana were evaluated, and the results are shown in Table 4.

[0058] Table 4. Effects of different plant growth regulators on the cultivation of vigorous seedlings from adventitious buds of Camellia macrocarpa.

[0059]

[0060] Table 4 shows that the most suitable culture medium for robust seedlings of Camellia dabaoensis is WPM + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 3.0 mg / L GA3.

[0061] Example 4

[0062] Following the method in Example 1, after the adventitious seedlings were induced to root for 7 days in the rooting preparation culture, they were removed from the tissue culture bottle with tweezers, soaked in a solution of 0-1000 mg / L IBA + 0-250 mg / L NAA for 15 minutes, and then transplanted. The lower end of the sterile seedling was then obliquely cut and inserted into perlite. The effects of different types and concentrations of auxin on the exogenous rooting culture of rootless seedlings were evaluated, and the results are shown in Table 5.

[0063] Table 5. Effects of different types and concentrations of auxin on exogenous rooting culture of rootless seedlings.

[0064]

[0065] Table 5 shows that using a rooting solution of 250 mg / L NAA + 1000 mg / L IBA is the most effective way to promote external rooting of rootless seedlings in bottles.

[0066] Example 5

[0067] *Camellia macrocarpa*, belonging to the Theaceae family, is extremely difficult to root. In preliminary experiments, this invention induced rooting in *Camellia macrocarpa* through tissue culture. Sterile *Camellia macrocarpa* seedlings, after one and a half months of cultivation, were sterile, retaining the terminal bud, with the lower leaves removed, leaving 2-3 healthy leaves. A 0.5cm cut was made at the bottom of each plant, leaving a wound. These seedlings were then inserted into rooting medium (MS + 0-1.0 mg / L auxin + 30 g / L sucrose + 4.8 g / L agar, pH 5.8) and cultured at 25±2℃, 2000 lx light intensity, and 14 hours / day. The results showed that AM treatment failed to induce rooting in rootless *Camellia macrocarpa* seedlings, only showing slight differences in growth. Figure 4 ).

[0068] Table 6. Effects of different concentrations of auxin on rooting culture of Camellia dabaoensis.

[0069]

[0070]

[0071] The above detailed description is a specific description of the embodiments of the present invention. These embodiments are not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included in the patent scope of this case.

Claims

1. A method for large-scale tissue culture propagation of Camellia macrocarpa, characterized in that, Includes the following steps: (1) Obtaining sterile seedlings: Select large-bracted white camellia fruits, peel off the pericarp to obtain seeds, place them on a clean bench for sterilization, dry them, and then inoculate them into seed germination medium. After induction germination culture, sterile seedlings are obtained. Then, on this medium, a certain number of sterile seedlings are obtained by subculturing five times using stem segments with nodes as subsequent experimental materials. The seed germination and the first five subcultures were all carried out on MS + 0.1 mg / L 6-BA + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.

8. (2) Proliferation culture of clustered buds: Select adventitious seedlings with good growth, cut the adventitious seedlings into stem segments with nodes, and insert them into the proliferation culture medium; the proliferation culture medium is 2 / 3MS + 1.0 mg / L 6-BA + 0.1 mg / L NAA + 0.0075 mg / LTDZ + 30 g / L sucrose + 4.8 g / L agar + 300 mg / L hydrolyzed casein, with a pH of 5.8; (3) Culture of vigorous single adventitious buds: Select vigorous single plants from the proliferated adventitious buds and inoculate them into a seedling culture medium; the seedling culture medium is WPM + 0.5 mg / L 6-BA + 0.5 mg / L NAA + 3.0 mg / L GA3 + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8; (4) In vitro rooting preparation culture: After one and a half months of strong seedling culture, select healthy sterile seedlings, retain the terminal bud, cut off the lower leaves, retain 2-3 healthy leaves, cut off 0.4-0.6 cm of the lower part of each plant to leave a wound, and insert them into the rooting preparation culture medium; the rooting preparation culture medium is 1 / 2MS + 1.5 mg / L NAA + 1.0 mg / L IBA + 30 g / L sucrose + 4.8 g / L agar, with a pH of 5.8; (5) External rooting: After the adventitious seedlings have been induced to root for 5-10 days in the rooting preparation culture, they are taken out of the tissue culture bottle with tweezers, soaked in 1000 mg / L IBA + 250 mg / L NAA solution for 10-20 min and then transplanted. The lower end of the sterile seedling is then obliquely cut and inserted into the perlite. In steps (1)-(5), the cultivation conditions are a temperature of 25±2℃, a light intensity of 1500-2500lx, and a light duration of 12-16 hours / day.

2. The method according to claim 1, characterized in that, In step (1), the seed disinfection steps are as follows: first, gently wipe the seed surface with an alcohol cotton ball 2-4 times, then soak it in 75% alcohol for 30-45 seconds, rinse it with sterile water 2-3 times, then disinfect it with 0.1% mercuric chloride for 4-6 minutes, rinse it with sterile water 3-5 times, and then air dry it.

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