A method for in vitro rapid propagation of camellia sinensis var. assamica cv. honghua stem segments

Abstract

The application discloses a kind of quick propagation methods of safflower oil tea stem segments in vitro, and relates to the field of in vitro quick propagation technology, the present application uses excellent safflower oil tea, and the semi-lignified stem segment of current year is collected as explant in mid-April-early May, and establishes in vitro efficient breeding system.The present application finds that Hyponex is used as the basic culture medium of safflower oil tea in vitro culture, and is added with growth regulator 6-BA and IBA, so that cluster shoots can be quickly obtained, and after multiple subcultures, callus induced at the base of single bud can be differentiated to form cluster adventitious bud cluster, and after periodic culture, rootless plants are developed.The rootless plants are immersed in rooting agent after rooting pretreatment, are cultivated into seedling bags to generate root system, and a seedling raising method for obtaining complete regenerated plants is provided.The method is a kind of efficient and stable quick propagation technology and can be applied to seedling raising production.

Description

Technical Field

[0001] This invention relates to the field of in vitro rapid propagation technology, specifically to a method for in vitro rapid propagation of Camellia oleifera stem segments. Background Technology

[0002] Camellia chekiangoleosa Hu., belonging to the Camellia genus of the Theaceae family, is an evergreen shrub or small tree. It is a unique high-quality edible oil and excellent ornamental tree species in China. Due to its dark green, glossy leaves, beautiful shape, and vibrant flowers, Camellia chekiangoleosa oil has a higher oleic acid content than ordinary camellia oil, making it suitable for use as a cosmetic raw material and also a high-quality health-promoting edible plant oil with extremely high nutritional and economic value. It can help alleviate the supply and demand imbalance of grain and oil in my country, necessitating vigorous development of the camellia oil industry. The planting area and cultivation scope are continuously expanding, leading to a rapid demand for camellia seedlings. Since industry development prioritizes seed production, selecting superior varieties and innovating efficient propagation methods have become paramount for the healthy, sustainable, and long-term development of the camellia oil industry.

[0003] Although there have been previous research reports on the tissue culture of Camellia oleifera, some of which used tender branches as explants to obtain only callus tissue, and some of which screened methods to promote the growth of adventitious buds, the proliferation effect was not clear and had not achieved stable and rapid propagation to form a complete production line. The technology still needs to be further optimized. This method uses stem segments to directly induce organogenesis for propagation, which is a simple in vitro organ culture. This method can fully preserve the superior genetic traits of the maternal parent. The basic culture medium used is a nitrogen, phosphorus, and potassium synthetic fertilizer called "Huabao," which has been used in the rapid propagation of orchids. However, this is the first time it has been used as a basic culture medium for the in vitro propagation of Camellia oleifera. It was discovered that nitrogen, phosphorus, and potassium in Huabao serve as basic nutrients for the rapid propagation of Camellia oleifera. The method of obtaining regenerated plants by forming an efficient rapid propagation system through a single superior individual has not been reported before. The materials used in this rapid propagation technique are superior Camellia oleifera varieties that have been selected and identified by researchers over a long period of time. Axillary buds are induced from the stem organs of superior Camellia oleifera plants. Aseptic buds form callus tissue at the base, which then differentiates into organs and finally develops into regenerated plants. This method has the advantages of low material consumption, high regeneration rate, fast propagation speed, strong repeatability, and large quantity. It can also lay a solid foundation for genetic transformation and somatic cell hybridization research on Camellia oleifera. In vitro rapid propagation of Camellia oleifera is not only a method to produce a large number of seedlings in a short period of time by utilizing limited high-quality resources, but also provides the most effective way to preserve and cultivate Camellia oleifera germplasm resources in a targeted manner, and is conducive to promoting the rapid development of Camellia oleifera in the tea oil industry. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a method for rapid in vitro propagation of Camellia oleifera stem segments.

[0005] The technical solution of the present invention is as follows:

[0006] A method for rapid in vitro propagation of Camellia oleifera stem segments includes the following steps:

[0007] S1: Select semi-lignified stem segments of the current year from superior Camellia oleifera trees, culture them in a starter culture medium, and induce axillary bud germination;

[0008] S2: After the axillary buds germinate, the axillary buds are transferred to the proliferation medium to induce adventitious buds;

[0009] S3: Take stems and branches with adventitious buds and transfer them to a rooting pretreatment medium. After the culture is completed, transfer them to a substrate bag to harden off the seedlings and cultivate them to obtain rooted Camellia oleifera seedlings.

[0010] As a preferred embodiment of the present invention, in step S1, the initiation culture medium is Hyponex basic culture medium.

[0011] As a preferred embodiment of the present invention, in step S2, the proliferation medium is: 0-1.0 mg / L 6-BA and 0-0.5 mg / L IBA added to 1-3 g / L Hyponex basic medium.

[0012] As a preferred embodiment of the present invention, in step S2, the proliferation medium consists of 1.0 mg / L 6-BA and 0.2 mg / L IBA added to 1.5 g / L Hyponex basal medium.

[0013] As a preferred embodiment of the present invention, in step S3, the rooting pretreatment culture medium is: 0.2-1.0 mg / L NAA, 0.5-2.0 mg / L IBA, and 0-3.0 mg / L rooting powder added to Hyponex basic culture medium.

[0014] As a preferred embodiment of the present invention, the Hyponex basic culture medium specifically comprises N, P, and K in a ratio of 7:6:19.

[0015] As a preferred embodiment of the present invention, in step S3, the substrate bag comprises 60-70 wt% carbonaceous soil, 8-12 wt% perlite, 8-12 wt% coconut coir, 8-12 wt% rice husk, and 3-7 wt% wood ash.

[0016] As a preferred embodiment of the present invention, the conditions during the cultivation process in steps S1-S3 are: pH 5.6-5.8, and cultivation temperature 23±2℃.

[0017] As a preferred embodiment of the present invention, the light conditions during the cultivation process in steps S1-S3 are: light intensity of 2200-2700 lx and light duration of 12-18 h / d.

[0018] As a preferred embodiment of the present invention, the starting medium, the proliferation medium and the rooting pretreatment medium further include 28-32 g / L sucrose and 6-8 g / L carrageenan.

[0019] The beneficial effects of this invention are:

[0020] (1) The method provided by this invention uses stem segments as explants and employs biotechnology to establish an in vitro rapid propagation system, which maximizes the preservation of the excellent traits of the parent plant and allows its excellent characteristics to be fully utilized. It enables rapid asexual reproduction in a short period of time, promoting the rational, sufficient, rapid, and long-term sustainable use of excellent new species resources. It can provide high-quality seedlings for the construction of large-scale camellia oleifera afforestation and low-yield improvement.

[0021] (2) This invention cultivates Camellia oleifera seedlings through stem segment in vitro rapid propagation method. It only uses semi-lignified branches and uses organogenesis to maintain the genetic benefits of superior varieties. The whole process can be artificially controlled in the laboratory and is not limited by season or environment. The system is successfully constructed with strong stability, high propagation coefficient and uniform seedlings. It not only solves the technical problem of rapid propagation of superior Camellia oleifera varieties, but also accelerates the process of promoting and applying Camellia oleifera in production, and lays a solid material foundation for the industrial development and utilization of Camellia oleifera. Attached Figure Description

[0022] Figure 1 This is a photograph of a Camellia oleifera stem segment during its initial induction process.

[0023] Figure 2 Photograph of aseptic buds of Camellia oleifera;

[0024] Figure 3 A photograph of a specimen from the propagation culture of Camellia oleifera 'Safflower'.

[0025] Figure 4 These are propagated seedlings after three generations;

[0026] Figure 5 The diagram shows the control group of proliferation culture in different culture media, where (a) is 1.0 g / L and (b) is 3.0 g / L.

[0027] Figure 6 Camellia oleifera seedlings after two months of hardening off and transplanting;

[0028] Figure 7 To facilitate large-scale seedling cultivation of Camellia oleifera through tissue culture. Detailed Implementation

[0029] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0030] The following specific experiments serve as embodiments of the present invention:

[0031] It should be noted that:

[0032] The culture medium for the three stages of aseptic bud induction, proliferation, and rooting pretreatment also contained 30 g / L sucrose and 7.0 g / L carrageenan, with a pH of 5.6-5.8, a culture temperature of 23±2℃, a light intensity of 2500 lx, and a photoperiod of 16 h / d.

[0033] (1) Induction of aseptic buds from Camellia oleifera explants

[0034] A method for inducing axillary buds using Camellia oleifera stem segments as explants includes the following steps:

[0035] In mid-April to early May, cut the current year's semi-lignified stems and branches from the improved variety of Camellia oleifera. After wrapping them in a damp towel and bringing them back, use scissors to remove the leaves from the stems and branches and cut the stems and branches into small sections with buds.

[0036] After scrubbing the surface of the budded stem segments with cleaning solution, rinse off the cleaning solution with clean water and wipe the stem segments with 75% ethanol. Repeat 3-4 times.

[0037] After wiping, place it in an empty bottle for reference. Figure 1 Seal the bottle opening with gauze and rinse under running water for 2 hours;

[0038] After rinsing, add 75 vol% ethanol for 1 minute to disinfect, then add 20 vol% sodium hypochlorite for 15 minutes to disinfect. After disinfection, pour out the liquid in the bottle and rinse with sterile water 5-6 times.

[0039] After rinsing, remove the sterilized stem segments with buds from the bottle, blot dry on sterilized filter paper, fix the stem segments with tweezers, and cut off both ends and petioles of the stem segments with a blade, dividing them into segments with 1-2 axillary buds. Inoculate these segments into the starter culture medium, which consists of five basic culture media produced by Beijing Kangbeis Technology Co., Ltd.: Hyponex (specifically including N, P, K in a 7:6:19 ratio), MS, B5, WPM, and White. Without adding any growth regulators, the explant induction was observed. The results (as shown in Table 1) revealed that only stem segments in Hyponex medium maintained green stems and branches, and the axillary bud germination rate reached 75% (reference). Figure 3 The B5, WPM, and White media were mostly dried up and dead, while MS media had a few buds. The induced axillary buds were small and the leaves were yellowish, indicating that the N:P:K content in Hyponex was suitable for the in vitro growth of Camellia oleifera.

[0040] Table 1. Effects of basic culture medium on induction of axillary bud sprouting in Camellia oleifera.

[0041]

[0042]

[0043] (2) Propagation culture of Camellia oleifera

[0044] Wait until the axillary buds of the leaves in the above-mentioned starter culture medium sprout 2-3 leaves (reference). Figure 2 When the leaves are fully extended and the stems reach 2 cm, the explants are cut off and transferred to the proliferation medium in the bottle (see reference). Figure 3 The proliferation medium used Hyponex (1.0, 1.5, 2.0, 3.0 g / L) as the basic medium, with different ratios of 6-benzylaminopurine (6-BA) (0, 0.2, 0.5, 1.0 mg / L) and indolebutyric acid (IBA) (0, 0.1, 0.2, 0.5 mg / L) added. A three-factor, four-level orthogonal experimental design L16(43) was used. Four axillary buds were placed in each bottle, with five bottles per treatment, and the culture was repeated three times. After 120 days, the growth of aseptic buds that dedifferentiated and formed adventitious bud clusters was recorded. The results are shown in Table 2.

[0045] Table 2 Effects of different proliferation media on adventitious shoot induction

[0046]

[0047]

[0048] The results showed that 1.5 mg / L Hyponex per liter of culture medium was used as the basal medium, and the combination of 1.0 mg / L 6-BA and 0.2 mg / L IBA growth regulators was most suitable for the proliferation and growth of Camellia oleifera. The leaves were smooth, leathery with soft wavy texture, wide leaves and thick stems, with clusters of purplish-red adventitious buds at the base. (Reference) Figure 4 (a) Hyponex showed curling at a level of 2.0 g / L, reference [reference missing]. Figure 5 In (b), Hyponex showed signs of nutrient overload at 3.0 g / L, with older leaves curling, younger leaves turning scorched, and terminal buds withering. (See reference) Figure 5 In (a), nutrient deficiency was observed in plants treated with 1.0 g / L Hyponex, resulting in dull, small, and yellowish leaves. It was found that 1.5 g / L Hyponex was sufficient for the propagation and culture of Camellia oleifera. (Reference) Figure 4 (b) in the middle.

[0049] (3) Pretreatment for stem and branch rooting

[0050] Using half the amount of Hyponex as the basal medium, different ratios of naphthaleneacetic acid (NAA) (0, 0.2, 0.5, 1.0 mg / L), indolebutyric acid (IBA) (0, 0.5, 1.0, 2.0 mg / L), and rooting powder ABT (0, 1.0, 2.0, 3.0 mg / L) were added in a 3-factor, 4-level completely randomized design. Eight regenerated shoots were inserted into each bottle. After 30 days, the shoots were removed, the medium was washed off, and the shoots were dipped in 1000 ppm ABT solution and transplanted into substrate bags (6cm × 8cm). The substrate consisted of 65 wt% carbon soil, 10 wt% perlite, 10 wt% coconut coir, 10 wt% rice husk, and 5 wt% wood ash. After planting, the seedlings were thoroughly watered, sprayed with carbendazim fungicide, covered with film to retain moisture, and then uncovered. Two months later, the growth of the seedlings was observed, and the transplant survival rate was calculated based on the appearance of new leaves. The results are shown in Table 3.

[0051] Table 3. Pretreatment, hardening, and transplanting of regenerated buds from Camellia oleifera.

[0052]

[0053]

[0054] The results showed that inoculation with the rooting medium resulted in:

[0055] Rooting pretreatment was performed using a medium containing 1 / 2 Hyponex + NAA 0.5 mg / L + IBA 1.0 mg / L + ABT 1.0–2.0 g / L + 5–10 g / L. After transplanting and hardening off for two months, the survival rate reached 91%. (Reference) Figure 6 .

[0056] In summary, this invention uses only stem segments to directly induce organogenesis for propagation, which is a simple in vitro organ culture. This method can fully preserve the superior genetic traits of the parent plant. It has been found that nitrogen, phosphorus, and potassium in the plant are essential nutrients for the rapid propagation of Camellia oleifera. No other method has been reported for obtaining regenerated plants through a single superior individual to form an efficient rapid propagation system. The rapid propagation material of this invention is a superior Camellia oleifera variety selected and identified by researchers over a long period. Axillary buds are induced from the stem organs of superior Camellia oleifera plants. Aseptic buds form callus tissue at the base, which then differentiates into organs, eventually developing into regenerated plants. This provides a method for mass propagation of seedlings in a short period. (Reference) Figure 7 It has the advantages of low material consumption, high regeneration rate, fast propagation speed, strong repeatability, and large quantity.

[0057] Meanwhile, the method provided by this invention offers the most effective way to preserve and cultivate Camellia oleifera germplasm resources, and is conducive to promoting the rapid development of Camellia oleifera in the tea oil industry. Camellia oleifera is an oil-bearing economic forest species that integrates edible, medicinal, ornamental, and greening functions. It has extremely high ecological, economic, and social value and a broad market prospect. At present, Camellia oleifera is being vigorously developed in 15 regions across the country suitable for planting, which requires a large number of high-quality Camellia oleifera seedlings. The method provided by this invention can solve the problem of demand for Camellia oleifera seedlings.

[0058] This invention utilizes new seed resources for asexual seedling cultivation, increasing the types of varieties available for camellia oleifera afforestation. It promotes the development of new varieties and new business models in the camellia oleifera industry, overcomes the impact of fertilization obstruction caused by different external environments and genetic factors, and facilitates large-scale production in a short period. It can provide a large number of standardized seedlings for the large-scale development of the camellia oleifera industry, effectively promote the application of red-flowered camellia oleifera in the camellia oil industry, avoid the problem of uneven quality of camellia oleifera seedlings available in the camellia oleifera industry, and enhance the enterprise's ability to resist market risks.

[0059] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various other corresponding changes and modifications based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for rapid in vitro propagation of Camellia oleifera stem segments, characterized in that, Includes the following steps: S1: Select semi-lignified stem segments of the current year from superior Camellia oleifera trees, culture them in a starter culture medium, and induce axillary bud germination; S2: After the axillary buds germinate, the axillary buds are taken and transferred to the proliferation medium to induce adventitious buds; S3: Take the stems and branches with adventitious buds and transfer them to the rooting pretreatment medium. After the culture is completed, transfer them to the substrate bag to harden the seedlings and cultivate them to obtain rooted Camellia oleifera seedlings. In step S1, the starting medium is Hyponex basic medium; In step S2, the proliferation medium is: 0.5-2.0 mg / L 6-BA and 0.05-0.2 mg / L IBA added to 1-3 g / L Hyponex basic medium; In step S3, the rooting pretreatment medium is: Hyponex basic medium with 0.2-1.0 mg / L NAA, 0.5-2.0 mg / L IBA, and 0-3.0 mg / L rooting powder added.

2. The method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1, characterized in that, In step S2, the proliferation medium consists of 1.0 mg / L 6-BA and 0.2 mg / L IBA added to 1.5 g / L Hyponex basal medium.

3. A method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1 or 2, characterized in that, The Hyponex basal medium specifically comprises N, P, and K in a 7:6:19 ratio.

4. The method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1, characterized in that, In step S3, the substrate bag comprises 60-70 wt% carbonaceous soil, 8-12 wt% perlite, 8-12 wt% coconut coir, 8-12 wt% rice husk, and 3-7 wt% wood ash.

5. The method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1, characterized in that, The conditions for the cultivation process in steps S1-S3 are: pH 5.6-5.8 and cultivation temperature 23±2 ℃.

6. The method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1, characterized in that, The light conditions during the cultivation process in steps S1-S3 are as follows: light intensity of 2200-2700 lx and light duration of 12-18 h / d.

7. The method for rapid in vitro propagation of Camellia oleifera stem segments according to claim 1, characterized in that, The initiation medium, proliferation medium, and rooting pretreatment medium also contain 28-32 g / L sucrose and 6-8 g / L carrageenan.

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