Method for inducing multiple shoots, increasing nodes, increasing height and proliferating camellia nitidissima

Abstract

A method for inducing shoot proliferation and simultaneously increasing internode length and height in Camellia oleifera is proposed. Mature, plump Camellia oleifera seeds are selected, and the seed coat is removed to use them as explants. When the shoot length reaches ≥4.0 cm after initial shoot induction culture, the initial shoots are cut into three types: terminal buds, segments, and hypocotyls, and inoculated into three different subculture media for proliferation culture. This proliferation method can shorten internode length and increase the number of nodes while inducing shoot proliferation, significantly improving the proliferation coefficient. Furthermore, this proliferation culture method does not involve callus induction, avoiding potential variations caused by callus differentiation and maximizing the preservation of the superior traits of the parent plant. This not only lays the foundation for the industrialized seedling production of Camellia oleifera through tissue culture, but the hypocotyl proliferation method also lays the foundation for the construction of an Agrobacterium-mediated genetic transformation system.

Description

Technical Field

[0001] This invention belongs to the field of Camellia oleifera tissue culture and seedling technology, and particularly relates to a method for inducing clustered buds while increasing the number of nodes and height to propagate fragrant Camellia oleifera. Background Technology

[0002] Camellia osmantha Ye CX, Ma JL et Ye H is a new species of camellia discovered in Nanning in 2012. It is an evergreen shrub, about 3.5 meters tall, with light red bark, ovate or obovate-elliptic leaves, and white flowers. Two to three flowers grow in clusters in the leaf axils, emitting a fragrance. Each flower has 6 to 8 or more petals and a flowering period of 4 to 5 months. The fruit matures in September and October, with a reddish-brown pericarp that is spherical or nearly obovate. Camellia osmantha is not easily damaged by low or high temperatures, prefers sunlight and a warm, humid climate, but can also grow well in partial shade. It can grow normally in acidic or slightly acidic red and yellow-red soils. It not only produces abundant fruit with a high seed yield, early fruiting, and good oil quality, but also grows quickly, is highly resistant, has an attractive tree shape, a long flowering period, and fragrant flowers. It can be used as both an oil crop and an ornamental tree, showing great development potential.

[0003] Currently, the common propagation methods for Camellia oleifera include seed sowing, cutting, and grafting. Traditional propagation mainly relies on seed sowing, but this method suffers from severe genetic differentiation, resulting in inconsistent quality and low-level superior varieties. This leads to mixed stand varieties, a high proportion of inferior plants, and significant differences in yield per plant. Applying superior clones of Camellia oleifera for afforestation can significantly increase yield. Currently, asexual propagation of Camellia oleifera mainly relies on grafting and cutting. Incomplete removal of sprouts during grafting can greatly affect the maintenance of superior varieties, and yield is closely related to the number of cuttings. While cutting is simple and easy to operate, the seedling growth period is relatively delayed, taking approximately two years. Conventional cutting and grafting propagation coefficients are low and highly susceptible to seasonal influences, far from meeting production needs. To fully utilize the superior germplasm resources of Camellia oleifera, improve its productivity, and meet the market demand for seedlings, the most effective and economical approach is to establish a tissue culture propagation system for superior clones of Camellia oleifera suitable for production. A mature tissue culture propagation system not only maintains the superior traits of the parent plant but is also unaffected by seasonal or other objective factors, allowing for the production of a large number of high-quality seedlings in a short period. Furthermore, tissue culture lays the foundation for further utilizing modern biotechnology to improve Camellia oleifera varieties and accelerate the breeding process. Therefore, conducting research on the tissue culture of superior clones of Camellia oleifera has significant production and scientific research value.

[0004] Currently, no research on tissue culture of Camellia oleifera has been reported. Previous studies on tissue culture of common Camellia oleifera have yielded some results, but these cannot be directly applied to Camellia oleifera. This is because comparative studies on tissue culture of common Camellia oleifera have revealed significant differences in optimal culture schemes and cultivation difficulties among different clones. For example, Chinese patents CN 110583269 A, CN 113767850 A, CN 103461143 B, CN107743870B, and CN 103039362 B have proposed culture schemes and proliferation effects for different Camellia oleifera strains “Haida,” “Minyu,” “Qianbi,” “Huashuo,” “Huangshan,” and “Changlin,” respectively, showing extremely significant differences. The optimal culture medium and plant growth regulators vary considerably, with the highest proliferation coefficient ranging from only 4.09 to as high as 12.6. Other literature reports on tissue culture of common Camellia oleifera generally suffer from low proliferation coefficients and easy browning of culture materials. Camellia oleifera and Camellia oleifera belong to different species. Due to differences in their endogenous hormone content and secondary metabolites, different species exhibit varying sensitivities and adaptability to different plant growth regulators, resulting in more significant differences in growth habits and nutritional requirements. Referring to publicly reported optimal Camellia oleifera culture protocols, tissue culture of Camellia oleifera failed to achieve the expected results, and the optimal conditions for each stage of plant tissue culture still require extensive experimental screening. A crucial step in propagating seedlings using plant tissue culture technology is subculture, where the proliferation coefficient determines whether the tissue culture technology can be applied to industrial production. The proliferation coefficient is negatively correlated with the production cost of tissue-cultured seedlings; increasing the proliferation coefficient helps reduce production costs. During their research on Camellia oleifera tissue culture, the inventors discovered that Camellia oleifera also faces problems such as difficulty in inducing callus sprouting, low proliferation coefficient, and easy browning of plant materials during subculture of Camellia oleifera, severely hindering the application and promotion of its asexual tissue culture technology. Therefore, there is an urgent need to find a high-efficiency propagation method for Camellia oleifera to improve the propagation coefficient, so as to lay the foundation for the high-quality and high-volume asexual seedling cultivation of Camellia oleifera using tissue culture technology. This is of great strategic significance for accelerating the development of the Camellia oleifera industry and promoting the rural economy. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for efficient propagation of Camellia oleifera tissue culture through direct organogenesis, characterized by a high propagation coefficient, no browning of propagating buds, and high efficiency.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: After the initial bud induction culture of Camellia oleifera, the composition and ratio of the culture medium are optimized and adjusted to obtain a subculture proliferation culture medium formula suitable for different tissue types. Then, with the aid of appropriate shearing and culture methods, a method is provided to induce clustered buds and simultaneously increase the number of nodes and height of Camellia oleifera for proliferation.

[0007] This invention is implemented as follows: a method for inducing clustered shoots while simultaneously increasing node size and height in the propagation of Camellia oleifera, comprising the preparation of an initial shoot induction medium, initial shoot induction culture, preparation of a subculture proliferation medium, and subculture proliferation culture. The specific operational steps are as follows:

[0008] (1) Preparation of initial bud induction medium: The initial induction medium formula is modified WPM + BA 0.5-1.0 mg·L -1 +NAA 0.1~0.2mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The culture container was a 240ml straight-mouth glass bottle with a height of 9.5cm, a diameter of 6.6cm, and a mouth diameter of 6.3cm. 50mL of culture medium was added to each bottle, and the bottles were autoclaved at 121℃ for 20min.

[0009] (2) Initial bud induction culture: Mature and plump Camellia oleifera seeds were taken, the seed coat was removed and used as explants. After routine sterilization, they were inoculated onto the primary induction medium for initial bud induction culture.

[0010] (3) Preparation of subculture proliferation medium: The formula of subculture proliferation medium ① is modified WPM + 2-IP 1.0~2.0mg·L -1 +NAA 1.6~1.8mg·L -1 +IAA 0.5~1.5mg·L -1 +BR 0.3~0.5mg·L -1 +PP33 3.0~6.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The subculture proliferation medium ② was formulated with modified WPM + 2-IP at a concentration of 1.0–2.0 mg / L. -1 +NAA 0.6~0.8mg·L -1 +IAA 0.5~1.5mg·L -1 +PP33 3.0~6.0g·L -1 +CPPU 0.03~0.05mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1The subculture proliferation medium ③ has a modified WPM + 2-IP formulation of 0.6–0.8 mg / L. -1 +NAA 0.6~0.8mg·L -1 +IAA 1.5~2.5mg·L -1 +PP33 3.0~6.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 In a clean environment, after preparing the proliferation culture medium according to the above-described formula, boil it for 10 minutes, adjust the pH value to 5.8-6.0, and dispense it into culture containers. The culture containers are 650mL small-mouth glass bottles with a height of 14cm, a diameter of 9.5cm, and a mouth diameter of 5.3cm. Add 100mL of culture medium to each bottle and autoclave at 121℃ for 20 minutes.

[0011] (4) Subculture of shoots: When the initial shoot length is ≥4.0cm, the initial shoots are cut into three types: ① terminal shoot; ② segment; ③ hypocotyl, and inoculated into three different subculture media for proliferation culture. After 50-60 days of culture, the base swells and differentiates into cluster shoots. The cluster shoots are 4.5-8.0cm high and contain 5-12 buds and 6-8 stem nodes. The cluster shoots are cut into terminal shoots, segments and small cluster shoots, and inoculated into three different subculture media for proliferation culture again.

[0012] Preferably, the modified WPM culture medium described in steps (1) and (3) has the following composition:

[0013] <![CDATA[KNO3]]> <![CDATA[600mg·L -1 ]]> <![CDATA[NH4NO3]]> <![CDATA[400mg·L -1 <!-- 2 -->]]> <![CDATA[KH2PO4]]> <![CDATA[170mg·L -1 ]]> <![CDATA[CaCl2·2H2O]]> <![CDATA[96mg·L -1 ]]> <![CDATA[MgSO4·7H2O]]> <![CDATA[370mg·L -1 ]]> <![CDATA[Ca(NO3)2]]> <![CDATA[556mg·L -1 ]]> <![CDATA[MnSO4·H2O]]> <![CDATA[22.3mg·L -1 ]]> <![CDATA[ZnSO4·7H2O]]> <![CDATA[8.6mg·L -1 ]]> <![CDATA[CuSO4·5H2O]]> <![CDATA[0.025mg·L -1 ]]> <![CDATA[H3BO3]]> <![CDATA[6.2mg·L -1 ]]> <![CDATA[Na2MoO4·2H2O]]> <![CDATA[0.25mg·L -1 ]]> Thiamine hydrochloride <![CDATA[0.5mg·L -1 ]]> Pyridoxine hydrochloride <![CDATA[0.5mg·L -1 ]]> niacin <![CDATA[0.5mg·L -1 ]]> Inositol <![CDATA[100mg·L -1 ]]> glycine <![CDATA[2mg·L -1 ]]> <![CDATA[Na2-EDTA]]> <![CDATA[37.3mg·L -1 ]]> FeSO4 <![CDATA[27.8mg·L -1 ]]>

[0014] Preferably, the initial bud cutting in step (4) is divided into three types of cutting methods: apical bud, segment, and hypocotyl. The three types of cutting methods are: keeping the apical bud with 1 to 2 stem nodes, the segment with 1 to 2 stem nodes, the hypocotyl with 0.5 to 1 cm of radicle at the bottom and 0.5 cm of stem segment at the top.

[0015] Preferably, the cutting method for dividing the clustered buds into terminal buds, segments and small clustered buds in step (4) is as follows: cut off the tall buds of the clustered buds ≥1cm, cut off the terminal buds separately, retain 1 to 2 stem nodes, make the cut 0.2 to 0.5cm below the stem node, cut the remaining stem nodes 0.2 to 0.5cm below the stem node, divide them into segments containing 1 to 2 stem nodes, and cut the clustered buds after removing the buds ≥1cm into small clustered buds of 2 to 3 short buds or bud buds.

[0016] Preferably, the culture medium schemes for the different types of plant tissues inoculated in step (4) are as follows: the apical bud is inoculated into subculture proliferation medium ①, the segment is inoculated into subculture proliferation medium ②, and the hypocotyl and small bud clusters are inoculated into subculture proliferation medium ③.

[0017] Preferably, the method for inoculating different types of plant materials into the culture medium in step (4) is as follows: the terminal bud and the segment are vertically inserted into the culture medium, keeping the stem node just in contact with the culture medium, and the hypocotyl and the small bud are placed directly on the surface of the culture medium.

[0018] Preferably, the initial bud induction culture method in step (2) is as follows: dark culture at 24-26℃ for the first 20 days after inoculation, and light culture at 26-28℃, 1500-2500 LX, and 12-14h light per day from the 21st day after inoculation; the proliferation culture method in step (4) is as follows: dark culture at 24-26℃ for the first 15 days after inoculation, and light culture at 26-28℃, 1500-2500 LX, and 12-14h light per day from the 16th day after inoculation.

[0019] The advantages of this invention compared to the prior art are:

[0020] (1) Like most Camellia oleifera plants, Camellia oleifera contains a certain amount of tea saponins. During tissue culture, its metabolism is particularly vigorous, producing a large amount of hydrogen peroxide. Endogenous tea saponins will reduce the activity of catalase in cells to a certain extent, so that the excessive hydrogen peroxide produced during bud growth cannot be cleared in time, resulting in varying degrees of inhibition of bud growth. In addition, excessively high levels of growth regulators will also stimulate the activity of polyphenol oxidase in Camellia oleifera explants, thereby accelerating browning or even necrosis of explants. Therefore, adding appropriate types and concentrations of plant growth regulators is an important basis for the successful proliferation of Camellia oleifera buds. This invention, after extensive experimental screening, utilizes appropriate culture media for different tissue parts of Camellia oleifera to induce clustered buds while allowing buds to grow up to 8cm in length, with 6-8 segments on each bud. This shortens the internode length and increases the number of nodes. The top, segments, and clustered buds of each bud can all be used as propagation material, making full use of the culture materials from all parts, greatly improving material utilization, and significantly increasing the propagation coefficient, with an average propagation coefficient of 12.87. A large amount of material for propagation can be obtained in the same amount of time, laying a solid foundation for the industrialized seedling production of Camellia oleifera through tissue culture. Furthermore, the propagated single buds can be directly used as scions for grafting, alleviating the current shortage of grafting scions for bud-rootstock seedlings, and has high economic, social, and ecological benefits.

[0021] (2) The method of subculture of Camellia oleifera of the present invention does not involve callus induction. Instead, it carries out subculture through direct organogenesis by bud-to-bud propagation. This process omits the callus induction process and avoids the possible variations caused by callus differentiation in the indirect pathway. This method can maintain the excellent traits of the parent plant to the greatest extent and also shortens the culture time cycle.

[0022] (3) The subculture proliferation medium obtained by the optimized screening in this invention improves the concentration of KNO3 in the WPM medium and adds 400 mg·L⁻¹. -1 The NH4NO3 appropriately increased the content of nitrate nitrogen, ammonium nitrogen and potassium, resulting in robust sub-buds, appropriately sized leaves, normal color, and no browning, yellowing or withered leaves or excessive leaf growth in the propagation of Camellia oleifera.

[0023] (4) The subculture proliferation medium obtained by the optimized screening of this invention has appropriate amounts of BR, PP33 and CPPU added to the apical bud and segment proliferation medium; BR is brassinolide, which promotes the elongation of plant stems and cell division, promotes the germination of lateral buds, and can induce bud differentiation; PP33 paclobutrazol can effectively shorten the internodes, increase the number of nodes under a certain height, and at the same time strengthen the stem, avoiding the proliferation of buds being too thin and weak; CPPU chlorpyrifos can accelerate cell division, promote cell expansion and elongation, and improve the plant growth rate. The above components can play a synergistic role, so that different parts of Camellia oleifera can complete a large-scale proliferation in about 50 days.

[0024] (5) In the subculture stage of this invention, a 650mL small-mouth bottle with a height of 14cm and a diameter of 9.5cm is used, so that the growing proliferating buds have enough space to grow and avoid the restriction of growth caused by using small bottles.

[0025] (6) The most commonly used method for plant genetic system transformation is Agrobacterium-mediated transformation, which involves infecting plant leaves, hypocotyls, petioles, radicles, and other organs. Hypocotyls are embryogenic organs that develop from zygotes, exhibiting strong embryogenicity, high vitality, and differentiation and regeneration capabilities, and thus hold great promise for plant genetic system transformation. The hypocotyl induction and proliferation scheme proposed in this invention can provide a possibility for realizing the genetic transformation of Camellia oleifera, and is of great significance for genetic engineering breeding and variety improvement. Attached Figure Description

[0026] Figures 1-4 This refers to the tissue culture subcultured seedlings of Camellia oleifera (tea oil tea) after 60 days of propagation in Example 1. Figure 1 : Apical bud proliferation; Figure 2 Segmental proliferation; Figure 3 Hypocotyl proliferation; Figure 4 (Small bud proliferation).

[0027] Figure 5 For Comparative Example 1, the tissue culture subcultured seedlings of Camellia oleifera were cultured for 60 days (all materials were placed in subculture proliferation medium ①).

[0028] Figure 6 For Comparative Example 2, the tissue culture subcultured seedlings of Camellia oleifera were cultured for 60 days (all materials were placed in subculture proliferation medium ②).

[0029] Figure 7 For Comparative Example 3, the tissue culture subcultured seedlings of Camellia oleifera were cultured for 60 days (all materials were placed in subculture proliferation medium ③).

[0030] Figure 8 The initial buds of Camellia oleifera stem segments were induced for 30 days as a control example.

[0031] Figures 9-12 To compare the results of tissue culture subcultured seedlings of Camellia oleifera (comparative example 5) after 60 days of propagation. Figure 9 : Apical bud proliferation; Figure 10 Segmental proliferation; Figure 11 Hypocotyl proliferation; Figure 12 (Small bud proliferation).

[0032] Figures 13-16 To compare the tissue culture subcultured seedlings of Camellia oleifera (comparative example 6) after 60 days of propagation. Figure 13 : Apical bud proliferation; Figure 14 Segmental proliferation; Figure 15 Hypocotyl proliferation; Figure 16 (Small bud proliferation).

[0033] Figures 17-20 For Comparative Example 7, the tissue culture subcultured seedlings of Camellia oleifera after 60 days of propagation ( Figure 17 : Apical bud proliferation; Figure 18 Segmental proliferation; Figure 19 Hypocotyl proliferation; Figure 20 (Small bud proliferation). Detailed Implementation

[0034] The following embodiments are intended to provide a more comprehensive understanding of the present invention by those skilled in the art. These embodiments are only a portion of, and not all, of the invention, and are not intended to limit the invention in any way. However, those skilled in the art should understand that various changes in form and detail can be made without departing from the scope defined by the claims.

[0035] In the following examples, the composition of the modified WPM medium is as follows:

[0036] <![CDATA[KNO3]]> <![CDATA[600mg·L -1 ]]> <![CDATA[NH4NO3]]> <![CDATA[400mg·L -1 ]]> <![CDATA[KH2PO4]]> <![CDATA[170mg·L -1 ]]> <![CDATA[CaCl2·2H2O]]> <![CDATA[96mg·L -1 ]]> <![CDATA[MgSO4·7H2O]]> <![CDATA[370mg·L -1 ]]> <![CDATA[Ca(NO3)2]]> <![CDATA[556mg·L -1 ]]> <![CDATA[MnSO4·H2O]]> <![CDATA[22.3mg·L -1 ]]> <![CDATA[ZnSO4·7H2O]]> <![CDATA[8.6mg·L -1 ]]> <![CDATA[CuSO4·5H2O]]> <![CDATA[0.025mg·L -1 ]]> <![CDATA[H3BO3]]> <![CDATA[6.2mg·L -1 ]]> <![CDATA[Na2MoO4·2H2O]]> <![CDATA[0.25mg·L -1 ]]> Thiamine hydrochloride <![CDATA[0.5mg·L -1 ]]> Pyridoxine hydrochloride <![CDATA[0.5mg·L -1 ]]> niacin <![CDATA[0.5mg·L -1 ]]> Inositol <![CDATA[100mg·L -1 ]]> glycine <![CDATA[2mg·L -1 ]]> <![CDATA[Na2-EDTA]]> <![CDATA[37.3mg·L -1 ]]> FeSO4 <![CDATA[27.8mg·L -1 ]]>

[0037] Example 1

[0038] A method for inducing clustered buds while simultaneously increasing node height and propagating fragrant camellia oleifera, the operation steps are as follows:

[0039] (1) Preparation of initial bud induction medium: The initial induction medium formula is modified WPM + BA 1.0 mg·L -1 +NAA 0.2 mg·L -1 + 30g / L of sucrose -1+4.5g / L agar -1 The culture container was a 240ml straight-mouth glass bottle with a height of 9.5cm, a diameter of 6.6cm, and a mouth diameter of 6.3cm. 50mL of culture medium was added to each bottle, and the bottles were autoclaved at 121℃ for 20min.

[0040] (2) Initial bud induction culture: Mature and plump Camellia oleifera seeds were taken, and the seed coat was removed as explants. After routine sterilization treatment (soaking in 75% alcohol for 1 min and then soaking in 0.1% mercuric chloride for 20 min), they were inoculated onto the primary induction medium for initial bud induction culture. For the first 20 days after inoculation, they were cultured in the dark at 24-26℃. From the 21st day after inoculation, they were cultured in the light at 26-28℃, 1500-2500 LX, and 12-14 h of light per day.

[0041] (3) Preparation of subculture proliferation medium: The formula of subculture proliferation medium ① is modified WPM + 2-IP 2.0 mg·L -1 +NAA 1.6 mg·L -1 +IAA 0.5 mg·L -1 +BR 0.3mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The subculture proliferation medium ② has the following formula: modified WPM + 2-IP 2.0 mg·L⁻¹ -1 +NAA 0.8 mg·L -1 +IAA 1.5 mg·L -1 +PP33 6.0g·L -1 +CPPU 0.03 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The subculture proliferation medium ③ has the following formula: modified WPM + 2-IP 0.6 mg·L⁻¹ -1 +NAA 0.8 mg·L -1 +IAA 1.5mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 In a clean environment, after preparing the proliferation culture medium according to the above-described formula, boil it for 10 minutes, adjust the pH value to 5.8-6.0, and dispense it into culture containers. The culture containers are 650mL small-mouth glass bottles with a height of 14cm, a diameter of 9.5cm, and a mouth diameter of 5.3cm. Add 100mL of culture medium to each bottle and autoclave at 121℃ for 20 minutes.

[0042] (4) Subculture of shoots: When the initial shoot length is ≥4.0cm, the initial shoots are cut into three types: ① terminal shoot (the terminal shoot contains 1 to 2 stem nodes, and the cut is made 0.2 to 0.5cm below the stem node); ② segment (containing 1 to 2 stem nodes, and the cut is made 0.2 to 0.5cm below the stem node); ③ hypocotyl (containing 0.5cm to 1cm radicle at the bottom and 0.5cm stem segment at the top). The terminal shoot is inoculated into subculture medium ①, the segment into subculture medium ②, and the hypocotyl into subculture medium ③ for proliferation culture. After 50–60 days of culture, the base swells to induce differentiation into clustered shoots, which are 4.5–8.0 cm tall and contain 5–12 buds and 6–8 stem nodes. The clustered shoots are then divided into terminal buds, segments, and small clusters (after removing buds ≥1 cm, the remaining portion is divided into 2–3 short buds or bud buds). The cutting method for terminal buds and segments, and the use of the proliferation medium, are the same as before. The small clusters are inoculated into subculture proliferation medium ③, with the terminal buds and segments inserted vertically into the medium, ensuring the stem nodes are just in contact with the medium. The hypocotyls and small clusters are placed directly on the surface of the medium. For the first 15 days after inoculation, the plants are cultured in the dark at 24–26℃. From day 16 onwards, they are cultured in the light at 26–28℃, 1500–2500 LX, with 12–14 hours of light per day.

[0043] Example 2

[0044] A method for inducing clustered buds while simultaneously increasing node height and promoting the growth of fragrant flowering camellia, the operation steps are as follows:

[0045] The steps in this example are the same as in Example 1, the difference being the culture medium formulation used. Specifically, the initial induction medium formulation is a modified WPM + BA 0.5 mg·L⁻¹. -1 +NAA 0.1 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ① is modified WPM + 2-IP 1.0 mg·L⁻¹. -1 +NAA 1.8 mg·L -1 +IAA 1.5mg·L -1 +BR 0.3 mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ② is modified WPM+2-IP 1.0 mg·L⁻¹. -1 +NAA 0.6 mg·L -1 +IAA 0.5mg·L -1 +PP33 3.0g·L -1+CPPU 0.05mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ③ is modified WPM + 2-IP 0.8 mg·L⁻¹. -1 +NAA 0.6 mg·L -1 +IAA 2.5mg·L -1 +PP33 3.0~6.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 .

[0046] Example 3

[0047] A method for inducing clustered buds while simultaneously increasing node height and promoting the growth of fragrant flowering camellia, the operation steps are as follows:

[0048] The steps in this example are the same as in Example 1, the difference being the culture medium formulation used. Specifically, the initial induction medium formulation is a modified WPM + BA 0.5 mg·L⁻¹. -1 +NAA 0.2 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ① is modified WPM + 2-IP 2.0 mg·L⁻¹. -1 +NAA 1.8 mg·L -1 +IAA 1.5mg·L -1 +BR 0.5 mg·L -1 +PP33 6.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ② is modified WPM+2-IP 1.0 mg·L⁻¹. -1 +NAA 0.8 mg·L -1 +IAA 0.5mg·L -1 +PP33 6.0g·L -1 +CPPU 0.03 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ③ is modified WPM + 2-IP 0.8 mg·L⁻¹. -1 +NAA 0.8 mg·L -1 +IAA 2.5mg·L -1 +PP33 3.0~6.0g·L -1 + 30g / L of sucrose -1+4.5g / L agar -1 .

[0049] To verify the effectiveness of the invention, the inventors conducted the following comparative experiments.

[0050] Comparative Example 1:

[0051] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0052] The steps in this example are the same as those in Example 1, except that in step (3), when preparing the subculture medium, the subculture medium formula is the same as that in Example 1, which is the modified WPM+2-IP 2.0 mg·L⁻¹. -1 +NAA 1.6 mg·L -1 +IAA 0.5 mg·L -1 +BR 0.3mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 In step (4), the apical bud, segment, hypocotyl and bud cluster are all inoculated into this culture medium for proliferation culture.

[0053] Comparative Example 2:

[0054] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0055] The steps in this example are the same as those in Example 1, except that in step (3), when preparing the subculture medium, the subculture medium formula is the same as that in Example 1, which is the modified WPM+2-IP 2.0 mg·L⁻¹. -1 +NAA 0.8 mg·L -1 +IAA 1.5mg·L -1 +PP33 6.0g·L -1 +CPPU 0.03 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 In step (4), the apical bud, segment, hypocotyl and bud cluster are all introduced into this culture medium for proliferation culture.

[0056] Comparative Example 3:

[0057] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0058] The steps in this example are the same as those in Example 1, except that in step (3), when preparing the subculture medium, the subculture medium formula is the same as that in Example 1, which is the modified WPM+2-IP 0.6 mg·L⁻¹. -1 +NAA 0.8 mg·L -1 +IAA 1.5 mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 In step (4), the apical bud, segment, hypocotyl and bud cluster are all introduced into this culture medium for proliferation culture.

[0059] Comparative Example 4:

[0060] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0061] The steps in this example are the same as those in Example 1. The difference is that in step (2), clean, disease-free stem segments with buds are selected as explants for initial bud induction culture. After routine sterilization treatment (soaking in 75% alcohol for 30 seconds and then soaking in 0.1% mercuric chloride for 10 minutes), they are inoculated onto the primary induction medium for initial bud induction.

[0062] Comparative Example 5:

[0063] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0064] This example follows the same steps as Example 1, except that in steps (1) and (3), when preparing the culture medium, the modified WPM medium used as the basic medium in the initial bud induction medium formula and the subculture proliferation medium formulas ①, ②, and ③ is replaced with WPM medium. That is, the initial induction medium formula is WPM + BA 1.0 mg·L⁻¹. -1 +NAA 0.2 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The subculture proliferation medium ① has the following formula: WPM + 2-IP 2.0 mg·L⁻¹ -1 +NAA 1.6 mg·L -1 +IAA 0.5 mg·L -1 +BR 0.3mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The subculture proliferation medium ② has the following formula: WPM + 2-IP 2.0 mg·L⁻¹ -1 +NAA 0.8 mg·L -1+IAA 1.5mg·L -1 +PP33 6.0g·L -1 +CPPU 0.03 mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium ③ has the following formula: WPM + 2-IP 0.6 mg·L⁻¹ -1 +NAA 0.8 mg·L -1 +IAA 1.5mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 .

[0065] The composition of WPM medium is as follows:

[0066]

[0067]

[0068] Comparative Example 6:

[0069] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0070] The steps in this example are the same as those in Example 1, except that in step (3), when preparing the subculture proliferation medium, BR, PP33, and CPPU are not added to the subculture proliferation medium. That is, the subculture proliferation medium formula ① is modified WPM + 2-IP 2.0 mg·L -1 +NAA 1.6 mg·L -1 +IAA 0.5mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture proliferation medium formula ② is modified WPM+2-IP 2.0 mg·L⁻¹. -1 +NAA 0.8 mg·L -1 +IAA 1.5mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Subculture medium ③ has the following formulation: WPM + 2-IP 0.6 mg·L⁻¹ -1 +NAA 0.8 mg·L -1 +IAA 1.5 mg·L -1 +PP33 3.0g·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 .

[0071] Comparative Example 7:

[0072] A method for propagating fragrant camellia oleifera, the operation steps are as follows:

[0073] This example follows the same steps as Example 1, except that in steps (1) initial bud induction medium and (3) subculture proliferation medium, the medium formula used is the optimal initial bud induction medium formula disclosed in Chinese Patent CN 113767850 A "A method for promoting Camellia oleifera proliferation and rooting and its application" (i.e., MS+BA 3mg·L). -1 +NAA 0.1 mg·L -1 ++ sucrose 30g·L -1 +4.5g / L agar -1 ) and the optimal proliferation medium formulation (i.e., 1 / 2 MS + BA 3 mg·L) -1 +ZT 0.5mg·L -1 +IAA 0.1 mg·L -1 +Spermine 40mg·L -1 +Vb2 2.5mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 ).

[0074] Results of propagation implementation:

[0075] The *Camellia oleifera* was cultured using the treatment methods of Examples 1-3 and Comparative Examples 1-7 of this invention. The experimental results within 60 days were observed and statistically analyzed. The treatment results were compared and calculated as follows: Induction rate (%) = Number of budding explants / Number of inoculated explants × 100; Proliferation coefficient = Number of buds that have grown to a height (≥1cm) / Total number of buds at the time of inoculation. The results are shown in Table 1.

[0076] Table 1: Induction of proliferation effect of the examples

[0077]

[0078]

[0079] The induced proliferation effects of the three embodiments are shown in Table 1. As can be seen from Table 1, Embodiment 1 is the optimal embodiment, exhibiting the highest initial shoot induction rate, the highest number of shoots obtained from different types of tissue materials, the most nodes, the highest proliferation coefficient, and the best shoot condition. Figure 1 As shown.

[0080] Proliferation comparison effect:

[0081] Comparative Examples 1-3 primarily investigated whether the same culture medium could achieve the same proliferation effect when different types of plant tissues were subcultured. Compared with Example 1, under the same conditions, Comparative Examples 1-3 (with similar effects) Figures 2-4 During the proliferation stage, the three types of plant tissues were inoculated into only one type of culture medium, and the results are shown in Table 2.

[0082] Table 2: Comparison of proliferation effects between Example 1 and Comparative Examples 1-3

[0083]

[0084]

[0085] As shown in Table 2, when the segments, hypocotyls, and small bud clusters were all inoculated with the suitable proliferation medium ① for the terminal bud, the bud height was inconsistent, the number of nodes was significantly reduced, the internode spacing was longer, and the proliferation coefficient decreased by 6.53, 6.44, and 10.50, respectively. Furthermore, there were fewer proliferating buds, the leaves were slightly yellow, and the bud condition was significantly worse than in Example 1. When the terminal bud, hypocotyl, and small bud clusters were all inoculated with the suitable proliferation medium ② for the segments, both the bud height and the number of nodes were significantly reduced, and the proliferation coefficient decreased by 4.61, 9.14, and 11.85, respectively. The buds were shorter, the callus tissue at the base was large, dark brown, and had withered leaves. When the terminal bud and segments were inoculated with the suitable proliferation medium ③ for the hypocotyls and small bud clusters, both the bud height and the number of nodes were significantly reduced, the internode spacing was longer, and the proliferation coefficient decreased by 8.39 and 6.44, respectively. The leaves were larger, the internode spacing was longer, and the bud condition was significantly less uniform than in Example 1. The reason for this may be that different plant tissues have varying meristematic abilities and different sensitivities to hormones, making it difficult to achieve ideal proliferation results when using the same culture medium for different plant materials. This invention designs different proliferation culture media for different types of tissue materials, and screens and optimizes them to obtain the optimal culture medium scheme suitable for different plant tissues.

[0086] Comparative Example 4 mainly examines whether stem segments can be used as explants in this invention. The induced proliferation effect is shown in Table 3.

[0087] Table 3: Comparison of the induced proliferation effects between Example 1 and Comparative Example 4

[0088]

[0089]

[0090] As shown in Table 3, the results of Comparative Example 4 compared with Example 1 show that, under the same conditions, Comparative Example 4, which used stem segments as explant material in the initial induction culture, exhibited a significantly lower induction rate and initial bud height, with an induction rate of only 55.12% and an initial bud height of only 3.28 cm. In contrast, Example 1 had an induction rate of 90.21% and an initial bud height of 6.41 cm, making it unsuitable as an explant material for induction. However, the proliferation effect during subculture was not significantly different from that of Example 1, indicating that the proliferation effect of the present invention is not significantly related to the source of the explant. As long as the initial bud is induced, the proliferation method of the present invention can be used for efficient proliferation. However, considering the low induction rate of stem segments as explants, seeds are used as explant material in this invention.

[0091] Comparative Example 5 mainly investigated the effect of the basal culture medium on the subculture proliferation effect. The modified WPM medium of this invention was compared with the ordinary WPM medium, and the results are shown in Table 4.

[0092] Table 4: Comparison of proliferation effects between Example 1 and Comparative Example 5

[0093]

[0094] As shown in Table 4, the results indicate that, compared with Example 1, under the same conditions, Comparative Example 5, which directly used WPM as the basic culture medium during the proliferation stage, showed a significant decrease in proliferation efficiency. Regarding the proliferation cycle, compared with Example 1, Comparative Example 5 showed a 2-day, 10-day, and 10-day extension in the proliferation cycle of the other three culture materials, except for the apical bud. In terms of the proliferation coefficient, the average proliferation coefficient of Comparative Example 5 was 8.68 lower than that of Example 1. Regarding bud height, the average bud height of Comparative Example 5 was 1.76 cm lower than that of Example 1. Regarding the number of nodes, the average number of nodes of Comparative Example 5 was 2.57 lower than that of Example 1. Regarding seedling condition, the seedlings of Comparative Example 5 had larger leaves, larger internodes, and exhibited withered leaves and blackened bases, indicating a worse condition than that of Example 1. In summary, the comparative results demonstrate that this invention innovatively uses a modified WPM culture medium, increases the concentration of KNO3, and adds 400 mg·L⁻¹. -1 The addition of NH4NO3 appropriately increased the content of nitrate nitrogen, ammonium nitrogen, and potassium, resulting in stronger sub-buds, more suitable leaf size, and normal color in the propagation of Camellia oleifera, without browning, yellowing of dead leaves, or excessive leaf growth. At the same time, it significantly improved the propagation effect.

[0095] Comparative Example 6 mainly investigated the effects of BR, PP33, and CPPU in the culture medium formulation of this invention on the proliferation effect. The results compared with Example 1 are shown in Table 5.

[0096] Table 5: Comparison of proliferation effects between Example 1 and Comparative Example 6

[0097]

[0098] As shown in Table 5, under the same conditions, the proliferation effect was significantly reduced in Comparative Example 3 without the addition of BR, PP33, and CPPU to the subculture medium. Comparing the proliferation effects of the four culture materials, Comparative Example 6, compared to Example 1, had an average proliferation cycle extended by 5 days, a bud height decreased by 2.62 cm, a node number decreased by 2.31 nodes, and a proliferation coefficient decreased by 8.24. Furthermore, the seedling condition of Comparative Example 6 was inconsistent with that of Example 1, with some seedlings being too tall and others too short, exhibiting large, blackened callus tissue at the base. This indicates that the addition of appropriate amounts of BR, PP33, and CPPU to the culture medium in this invention is of great significance for the proliferation and growth of Camellia oleifera. BR is brassinolide, which promotes stem elongation and cell division, promotes lateral bud germination, and can induce bud differentiation. PP33 paclobutrazol can effectively shorten internodes, increasing the number of nodes at a given height, while also strengthening the stem and preventing the proliferating buds from being too thin and weak. CPPU chlorpyrifos can accelerate cell division, promote cell expansion and elongation, and increase the plant growth rate. The above components can work synergistically, enabling different types of Camellia oleifera tissues to complete a large-scale proliferation in about 55 days.

[0099] Comparative Example 7 primarily investigated whether existing Camellia oleifera tissue culture techniques are suitable for the efficient propagation of Camellia oleifera. Compared with Example 1, under the same conditions, Comparative Example 7 used the publicly reported culture medium formula with the best propagation effect in ordinary Camellia oleifera for induction and propagation culture, and the results are shown in Table 6.

[0100] Table 6: Comparison of proliferation effects between Example 1 and Comparative Example 7

[0101]

[0102] The induction medium used in Comparative Example 7 was MS + BA 3 mg·L⁻¹. -1 +NAA 0.1 mg·L -1 ++ sucrose 30g·L -1 +4.5g / L agar -1 The induction medium formula used in Example 1 of this invention is WPM + BA 0.5-1.0 mg·L⁻¹. -1 +NAA 0.1~0.2mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 The comparison reveals that the main differences between the two lie in the concentrations of the basal culture medium and cytokinin. Example 1 used WPM medium, while Comparative Example 7 used MS basal medium. The BA concentration used in Example 1 was only 0.5–1.0 mg / L.-1 In contrast, the concentration of BA used in Comparative Example 7 was 3 mg·L⁻¹. -1 The culture medium used for subculture proliferation in Comparative Example 7 was 1 / 2 MS + BA 3 mg·L⁻¹. -1 +ZT 0.5mg·L -1 +IAA 0.1 mg·L -1 +Spermine 40mg·L -1 +Vb2 2.5mg·L -1 + 30g / L of sucrose -1 +4.5g / L agar -1 Example 1 optimized different culture media for different types of plant tissues, and each culture medium formula was completely different from the formula in Comparative Example 7. Although the published data showed that the proliferation coefficient of common Camellia oleifera "Huashuo" using this formula could reach 12.6, the proliferation coefficient of Fragrant Camellia oleifera was only 3.21 at most, and the average proliferation coefficient was only 2.36. The average bud height was 6.05 cm lower than that of Example 1, the number of nodes was reduced by 4.72 nodes, and the proliferation cycle was extended by 6.5 days. Moreover, compared with Example 1, the seedlings of Comparative Example 7 had yellow leaves, fewer proliferating buds, and larger and blackened callus tissue at the bottom. The proliferation effect was significantly lower than that of Example 1, indicating that Fragrant Camellia oleifera and common Camellia oleifera belong to different species. Different species have different sensitivities and adaptability to different types of plant growth regulators due to differences in their endogenous hormone content and secondary metabolites. Their growth habits and nutritional requirements are more significantly different. Referring to the optimal Camellia oleifera culture scheme reported in the publication, tissue culture of Fragrant Camellia oleifera could not achieve the expected results. The culture and formula of this invention are suitable for the proliferation culture of Fragrant Camellia oleifera.

[0103] The above analysis proves that Example 1 has the best propagation effect. This shows that the efficient propagation method disclosed in this invention can shorten the length of internodes and increase the number of nodes while inducing the growth of clustered buds. It can obtain a large amount of propagation material in the same time period, significantly improve the propagation coefficient, and has good economic, social and ecological benefits. It is of great significance for promoting the development of the Camellia oleifera industry.

Claims

1. A method for inducing clustered buds while simultaneously increasing node height and promoting the growth of fragrant flowering camellia, characterized in that: The process includes preparing the initial bud induction medium, initial bud induction culture, preparing the subculture proliferation medium, and subculture bud proliferation culture; the main operational steps are as follows: (1) Preparation of initial bud induction medium: The initial induction medium formula is modified WPM + BA 0.5~1.0 mg·L -1 +NAA 0.1~0.2 mg·L -1 + 30 g·L sucrose -1 +4.5 g·L agar -1 The culture container was a 240 ml straight-mouth glass bottle with a height of 9.5 cm, a diameter of 6.6 cm, and a mouth diameter of 6.3 cm. 50 mL of culture medium was added to each bottle, and the bottles were autoclaved at 121 ℃ for 20 min. (2) Initial bud induction culture: Mature and plump Camellia oleifera seeds were taken, the seed coat was removed and used as explants, and after routine sterilization, they were inoculated onto the primary induction medium for initial bud induction culture. (3) Preparation of subculture proliferation medium: The formula of subculture proliferation medium ① is modified WPM + 2-IP 1.0~2.0 mg·L -1 +NAA 1.6~1.8 mg·L -1 + IAA 0.5~1.5 mg·L -1 +BR 0.3~0.5 mg·L -1 +PP33 3.0~6.0 g·L -1 + 30 g·L sucrose -1 +4.5 g·L agar -1 The subculture proliferation medium ② was formulated with modified WPM + 2-IP 1.0~2.0 mg·L⁻¹. -1 +NAA 0.6~0.8 mg·L -1 +IAA 0.5~1.5 mg·L -1 +PP33 3.0~6.0 g·L -1 +CPPU 0.03~0.05mg·L -1 + 30 g·L sucrose -1 +4.5 g·L agar -1 The subculture proliferation medium ③ has a modified WPM + 2-IP formulation of 0.6~0.8 mg·L⁻¹. -1 +NAA 0.6~0.8 mg·L -1 +IAA 1.5~2.5 mg·L -1 +PP33 3.0~6.0 g·L -1 + 30 g·L sucrose -1 +4.5 g·L agar -1 In a clean environment, after preparing the proliferation culture medium according to the above-described formula, boil it for 10 minutes, adjust the pH value to 5.8-6.0, and dispense it into culture containers. The culture containers are 650 mL small-mouth glass bottles with a height of 14 cm, a diameter of 9.5 cm, and a mouth diameter of 5.3 cm. Add 100 mL of culture medium to each bottle and autoclave at 121 ℃ for 20 minutes. (4) Subculture of shoots: When the initial shoot length is ≥4.0 cm, the initial shoot is cut into three types: terminal shoot, segment and hypocotyl, and inoculated into three subculture culture media respectively for proliferation culture; after 50-60 days of culture, the base swells and induces differentiation of cluster shoots; when the cluster shoots are 4.5-8.0 cm high and contain 5-12 shoots and 6-8 stem nodes, the cluster shoots are cut into terminal shoots, segments and small cluster shoots, and inoculated into three subculture culture media again for proliferation culture; The modified WPM culture medium has the following composition: The above describes the process of cutting the initial buds into three types: terminal buds, segments, and hypocotyls. The corresponding culture media for each type of plant tissue are as follows: terminal buds are inoculated into subculture proliferation medium ①, segments are inoculated into subculture proliferation medium ②, and hypocotyls and small bud clusters are inoculated into subculture proliferation medium ③.

2. The method for inducing clustered buds and simultaneously increasing the number of nodes and height of fragrant camellia oleifera according to claim 1, characterized in that: The initial bud cutting method described in step (4) is divided into three types: terminal bud, segment, and hypocotyl. The method is as follows: the terminal bud contains 1 to 2 stem nodes, the segment contains 1 to 2 stem nodes, the hypocotyl contains 0.5 to 1 cm of radicle at the bottom and 0.5 cm of stem segment at the top.

3. The method for inducing clustered buds while simultaneously increasing node height and propagating fragrant camellia oleifera according to claim 1, characterized in that: The cutting method for dividing the clustered buds into terminal buds, segments and small clustered buds in step (4) is as follows: cut off the tall buds of the clustered buds ≥1 cm, cut off the terminal buds separately, retain 1~2 stem nodes, make the cut 0.2~0.5 cm below the stem node, cut the remaining stem nodes 0.2~0.5 cm below the stem node, divide them into segments containing 1~2 stem nodes, and cut the clustered buds after removing the buds ≥1 cm into small clustered buds of 2~3 short buds or bud buds.

4. The method for inducing clustered buds while simultaneously increasing node height and propagating fragrant camellia oleifera according to claim 1, characterized in that: The method for inoculating different types of plant materials into the culture medium in step (4) is as follows: the terminal bud and the segment are vertically inserted into the culture medium, keeping the stem node just in contact with the culture medium, and the hypocotyl and the small bud are placed directly on the surface of the culture medium.

5. The method for inducing clustered buds while simultaneously increasing node height and propagating fragrant camellia oleifera according to claim 1, characterized in that: The initial bud induction culture method described in step (2) is as follows: dark culture at 24~26 ℃ for the first 20 days after inoculation, and light culture at 26~28 ℃, 1500~2500 LX, and 12~14 h of light per day from the 21st day after inoculation; the proliferation culture method described in step (4) is as follows: dark culture at 24~26 ℃ for the first 15 days after inoculation, and light culture at 26~28 ℃, 1500~2500 LX, and 12~14 h of light per day from the 16th day after inoculation.

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