A tissue culture rapid propagation method of magnolia acuminata

Abstract

The application discloses a rapid tissue culture and propagation method of Magnolia biondii, which comprises the following steps: taking the axillary bud wrapped in the flower bud and the stem segment thereof as an explant, and culturing the aseptic explant through induction, proliferation and rooting to obtain a tissue culture plant. The method successfully realizes the rapid tissue culture and propagation of the Magnolia biondii by selecting the axillary bud with the stem segment in the flower bud as the explant and combining the steps of disinfection, induction, proliferation and rooting. The method not only significantly improves the propagation efficiency and reduces the pollution rate, but also ensures the genetic stability of the tissue culture seedlings, and provides an effective technical means for the commercial production and ornamental value popularization of the Magnolia biondii.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant tissue culture, and relates to a tissue culture rapid propagation method of Magonlia wufengensis. BACKGROUND

[0002] Magonlia wufengensis I.Y.Ma et L.R.Wang, as a deciduous tree of Magnoliaceae, is well-known for its large and colorful flowers, and is an ideal flowering tree species in landscape and urban greening due to its high ornamental value. The natural distribution area of Magonlia wufengensis is relatively narrow, mainly in the mountainous area of 1000-1500 meters above sea level in Wufeng County, Hubei Province, which makes its germplasm resources scarce and faces the threat of habitat destruction.

[0003] The applicant has been committed to the research on grafting propagation technology of Magonlia wufengensis, and the grafting propagation technology has been mature, but there are certain limitations.

[0004] Firstly, grafting requires a large number of scions and rootstocks, which limits the propagation speed and scale.

[0005] Secondly, the grafting process has high technical requirements and is easily affected by seasons and environmental conditions, resulting in low propagation efficiency.

[0006] In addition, the cycle of grafting propagation is long, and it takes several years from rootstock culture to scion survival, which is not conducive to rapid expansion of planting area.

[0007] In order to solve the above problems, the applicant considers tissue culture technology as a new direction for the propagation of Magonlia wufengensis. Although tissue culture technology has achieved certain success in other plants, the existing technical system has not fully adapted to the special physiological and growth needs of Magonlia wufengensis in its rapid propagation. In the process of realizing the present application, the inventors found that there is at least one of the following technical problems in the tissue culture of Magonlia wufengensis: difficult selection of explants, high contamination rate, low proliferation efficiency, trait separation, easy vitrification and browning.

[0008] Therefore, developing a tissue culture rapid propagation method suitable for the characteristics of Magonlia wufengensis has important practical significance for improving the propagation efficiency, expanding the planting area, and protecting and utilizing this rare plant resource. SUMMARY

[0009] Therefore, the present application aims to provide a tissue culture rapid propagation method of Magonlia wufengensis, which significantly improves the propagation efficiency and survival rate of Magonlia wufengensis by optimizing the selection of explants and tissue culture measures, while maintaining its genetic stability, and provides technical support for the protection and commercial planting of Magonlia wufengensis.

[0010] The inventors provide a technical solution to solve the above technical problems through long-term exploration and attempts, and multiple experiments and efforts, and provide a rapid propagation method of Magnolia rostrata tissue culture.

[0011] Further, the flower bud is a flower bud in which flower bud differentiation is completed at the top of a branch.

[0012] Further, the axillary bud is provided with soft villi.

[0013] Further, the Magnolia rostrata is 'Jiao Hong 1' Magnolia rostrata.

[0014] Further, the diameter of the flower bud is greater than or equal to 1 cm.

[0015] Further, in mid-September, the flower buds of a healthy, well-grown, and pest-free perennial grafted mother tree are picked.

[0016] Further, when picking the flower buds, the leaves are removed, and the lower part of the flower bud is retained with a 5 cm long branch.

[0017] The friendship effect of the present application is that the applicant finds that the Magnolia rostrata has a different characteristic from common plants, i.e., 1-2 axillary buds in the flower bud, by deeply studying the growth habit and tissue culture characteristics of the Magnolia rostrata, and selects the explant based on the characteristic, which breaks through the limitation of traditional tissue culture methods in Magnolia rostrata propagation. After trying dormant buds, stem segments with dormant buds, single buds, and immature seeds in January-March, the inventors failed to obtain stable tissue culture seedlings. However, by selecting the stem segment axillary bud in the flower bud as the explant, and combining the steps of disinfection, induction culture, proliferation culture, and rooting culture, the inventors successfully realized the rapid propagation of Magnolia rostrata tissue culture. This method not only significantly improves the propagation efficiency and reduces the contamination rate, but also ensures the genetic stability of the tissue culture seedlings, and provides an effective technical means for the commercial production and ornamental value promotion of Magnolia rostrata.

[0018] According to one embodiment of the rapid propagation method of Magnolia rostrata tissue culture, the step of obtaining the sterile explant is as follows: first, disinfect the flower bud, remove the outer bracts after disinfection, retain 1-2 axillary buds and their stem segments wrapped in the bracts, and cut off the remaining stem segments and flower buds.

[0019] Further, the flower bud is retained during disinfection, and the branch below the flower bud is removed.

[0020] Further, the disinfection procedure is: 2% washing powder is soaked in warm water to clean the brush surface, and then is washed by flowing water for 30 minutes, and then is dried by filter paper, and then is soaked in 75% ethanol for 30 seconds, and then is washed by distilled water for 3 times; 0.15% HgCl2 for 10 minutes, 3 drops of Tween reagent per 50ml, and then is washed by distilled water for 6 times, and then is soaked in 8% NaClO for 10 minutes, and then is washed by distilled water for 5-6 times.

[0021] The present application has the following advantages: the sterile explant obtaining step is designed carefully, the flower bud is disinfected thoroughly, the outer bracts are removed and 1-2 axillary buds and the stems thereof are reserved, the rest stems and flower buds are removed, the sterile state of the explant and the quality of the propagation material are ensured, the disinfection effect is improved obviously by the multi-step disinfection procedure of 2% washing powder, 75% ethanol, 0.15% HgCl2 and 8% NaClO, the explant pollution rate is reduced, the subsequent induction culture, proliferation culture and rooting culture are laid a solid foundation, the efficient, healthy and rapid propagation of the Magnolia sieboldii is realized, and the genetic stability and growth activity of the tissue culture seedlings are ensured.

[0022] According to one embodiment of the Magnolia sieboldii tissue culture rapid propagation method, the induction culture uses an induction medium, and the induction medium comprises MS basic medium and 6-BA, NAA and KT.

[0023] Further, the concentration of 6-BA in the induction medium is 1.5-2.5 mg / L.

[0024] Preferably, the concentration of 6-BA in the induction medium is 2.0 mg / L.

[0025] Further, the concentration of NAA in the induction medium is 0.1-0.2 mg / L.

[0026] Preferably, the concentration of NAA in the induction medium is 0.15-0.2 mg / L.

[0027] Further, the concentration of KT in the induction medium is 0.15-0.25 mg / L.

[0028] Preferably, the concentration of KT in the induction medium is 0.2 mg / L.

[0029] According to one embodiment of the Magnolia sieboldii tissue culture rapid propagation method, the induction culture is:

[0030] The explant is inserted into the induction medium, and initial culture is carried out at 22-26 DEG C, and light intensity is 2000-3000 Lx; after 3-5 days, the stem segments without contamination are transferred into new induction medium, and culture is continued for 21-28 days until new shoots grow out.

[0031] The induction medium provided by the application successfully realizes effective induction of the Magnolia biondii explant, promotes rapid germination of new shoots, and the germination rate reaches 91.67%, and the survival rate reaches 66.67%, which is significantly higher than that of other induction media. The application ensures healthy growth of the new shoots, lays a good foundation for subsequent proliferation culture and rooting culture, and significantly improves the efficiency and success rate of the whole tissue culture process.

[0032] According to one embodiment of the Magnolia biondii tissue culture rapid propagation method, the proliferation culture uses a proliferation medium, and the proliferation medium comprises a MS basic medium and ADS, IBA and TDZ.

[0033] Further, the concentration of ADS in the proliferation medium is 2.5-7.5 mg / L.

[0034] Preferably, the concentration of ADS in the proliferation medium is 5.0 mg / L.

[0035] Further, the concentration of IBA in the proliferation medium is 0.1-1.0 mg / L.

[0036] Preferably, the concentration of IBA in the proliferation medium is 0.4 mg / L.

[0037] Further, the concentration of TDZ in the proliferation medium is 0.1-0.4 mg / L.

[0038] Preferably, the concentration of TDZ in the proliferation medium is 0.4 mg / L.

[0039] According to one embodiment of the Magnolia biondii tissue culture rapid propagation method, the proliferation culture is as follows:

[0040] The new shoots obtained by induction culture are inoculated into the proliferation medium, and culture is carried out at 22-26 DEG C, and light intensity is 2000-3000 Lx for 28-30 days.

[0041] The Magnolia biondii tissue culture rapid propagation method provided by the application has the beneficial effects that: in the proliferation culture stage, through the proliferation medium prepared with care and the optimized culture conditions, the proliferation coefficient is significantly improved, and the highest coefficient can reach 3, which means that each new shoot can produce up to three new plants, greatly improving the propagation efficiency and material utilization rate of Magnolia biondii, and providing strong technical support for rapid propagation and large-scale production of Magnolia biondii.

[0042] According to one embodiment of the rapid propagation method of Magnolia biondii through tissue culture, the rooting culture uses a rooting culture medium, and the rooting culture medium comprises MS basic medium and BNOA.

[0043] According to one embodiment of the rapid propagation method of Magnolia biondii through tissue culture, the rooting culture is:

[0044] The tissue culture seedlings obtained through the proliferation culture are transferred to the rooting culture medium, and after dark culture for 4-6 days, they are transferred to light, and then continue to be cultured under the condition of light intensity of 2000-3000 Lx for 21-28 days; the temperature is 18-22℃.

[0045] Further, the concentration of BNOA in the rooting culture medium is 2.0-8.0 mg / L.

[0046] Preferably, the concentration of BNOA in the rooting culture medium is 6.0 mg / L.

[0047] Further, the rooting culture selects healthy tissue culture seedlings with a height of more than 4 cm.

[0048] The beneficial effects of the present application are:

[0049] The present application selects BNOA as a key hormone to induce healthy and complete root system of 'Jiao Hong 1' Magnolia biondii, not only provides a feasible solution for Magnolia biondii which has difficulty in rooting, but also lays an experimental foundation for further optimizing the rooting culture condition and improving the rooting rate, and has important practical significance for realizing the complete tissue culture cycle of Magnolia biondii and improving the propagation success rate. The experiment shows that the use of BNOA is the key factor for the qualitative change of the rooting of 'Jiao Hong 1' Magnolia biondii tissue culture seedlings, and the selection of healthy tissue culture seedlings with a height of more than 4 cm is the key factor for the quantitative change of the rooting of 'Jiao Hong 1' Magnolia biondii tissue culture seedlings. BRIEF DESCRIPTION OF DRAWINGS

[0050] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor.

[0051] Figure 1 is a comparison chart of 'Jiao Hong 1' flower buds before and after removing the outer bracts. Figure 1 In the figure, a is a branch with complete flower buds, and b is a branch with flower buds after removing the outer bracts.

[0052] Figure 2 is a photo of each stage in the tissue culture process of 'Jiao Hong 1'.Figure 2 In the formula, a is an induction culture stage, b is a proliferation culture stage, and c and d are rooting culture stages.

[0053] Figure 3 is a result diagram of tissue culture of a stem segment with buds of 'Jiao Hong 1'.

[0054] Figure 4 is a result diagram of tissue culture of a single bud of 'Jiao Hong 1'.

[0055] In the diagram, the labels are as follows: 10, flower bud; 11, axillary bud; 12, top flower bud; 20, lateral bud; and 30, new root. DETAILED DESCRIPTION

[0056] The following will be described in combination with specific embodiments.

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present application in combination with the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application but not all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application. Therefore, the following detailed description of the embodiments of the present application is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application.

[0058] In the present embodiment, the abbreviations of the plant hormones are as follows: ADS (adenine sulfate), IBA (indole-3-butyric acid), TDZ (thidiazuron), BNOA (2-naphthoxyacetic acid), 6-BA (6-benzylaminopurine), NAA (1-naphthaleneacetic acid), and KT (kinetin).

[0059] In the present embodiment, the red magnolia is 'Jiao Hong 1' red magnolia. 'Jiao Hong 1' is the first variety of red magnolia artificially selected and bred. It has very high ornamental value because of its large and colorful flowers and various morphologies, and is an ideal ornamental tree species. Before the present application, grafting seedling was the only propagation way for 'Jiao Hong 1', and grafting is the most perfect technology so far in research, but there are problems such as few sources of scions, large occupation of land for rootstock culture, long time consumption, and the like.

[0060] There is no relevant research on rapid propagation of 'Jiao Hong 1' Magnolia liliiflora, which is not conducive to its mass propagation. The existing tissue culture technology of Magnolia focuses on two species, M. denudata and M. liliflora, but M. liliiflora is naturally distributed in a narrow area of Wufeng County, Hubei Province, at an altitude of 1000-1500 meters, which is not only the preservation (or origin) of many ancient plants, but also the differentiation place of many plants. M. liliiflora has great differences in growth habit and morphological construction compared with M. denudata and M. liliflora. The existing technical system has low reference value in the cultivation of M. liliiflora. In the process of asexual propagation of 'Jiao Hong 1' M. liliiflora, it is found that the commonly used hormones of Magnolia have no obvious effect on promoting the regeneration of M. liliiflora.

[0061] In this embodiment, the flower bud refers to the early stage of flower development, at which time each part of the flower (such as petals, stamens and pistils) is still tightly wrapped together and has not yet unfolded. See Figure 1 the flower bud 10. It should be noted that in the process of implementing the present application, the lateral bud under the already blooming flower cannot be selected. The wrapped bud is soft and covered with soft hair, see Figure 1 the axillary bud 11, and the surface of the bud after the bract falls off is leathery and smooth, see Figure 1 the lateral bud 12. It should be noted that the axillary bud 11 and the lateral bud 12 in this embodiment are only a distinction of buds in different positions, and from the perspective of botany, they both belong to axillary buds. After the bract falls off, the axillary bud 11 will grow into the lateral bud 12 in normal growth. More specifically, the axillary bud 11 is a lateral bud that is in the process of growth but has not yet completely dormant.

[0062] The axillary bud 11 is wrapped by the bract, which is a unique physiological phenomenon of Magnolia plants - the applicant has not found this feature in other non-Magnolia plants. Moreover, this feature has been ignored by researchers in plant tissue culture. In botany, flower buds (or flower buds) and axillary buds are usually two different structures: flower buds refer to buds that will develop into flowers, while axillary buds are lateral buds located in leaf axils, which can develop into new branches and leaves.

[0063] The method for rapid propagation of Magnolia liliiflora described in this embodiment has the following specific steps:

[0064] Selection of explants:

[0065] See Figure 1 , in mid-September, select a healthy, well-grown, and pest-free perennial grafted mother tree, and remove the leaves, with the flower bud at the top of the branch being differentiated into a flower bud (diameter ≥ 1 cm) and the lower part of the flower bud being retained for 5 cm in length.

[0066] Disinfection of explants:

[0067] Bud was reserved, lower branches of the bud were removed, and disinfection treatment was carried out: 2% washing powder was soaked in warm water to clean and brush the surface; the surface was washed with running water for 30 min, and water was absorbed by filter paper; 75% ethanol was immersed for 30 s, and distilled water was washed for 3 times; 0% HgCl2 was immersed for 10 min, and 3 drops of Tween reagent were added to 50 ml of distilled water, and the mixture was washed for 6 times; 8% NaClO was immersed for 10 min, and distilled water was washed for 5-6 times.

[0068] The disinfection effects of different disinfectant combinations are not the same, and specific examples are shown in Table 1.

[0069] Table 1 shows the disinfection effects of different disinfectant combinations on the 'Jiao Hong 1' Magnolia × alba explants.

[0070]

[0071]

[0072] Table 1 shows the comparison of the disinfection effects of different disinfectant combinations on the 'Jiao Hong 1' Magnolia × alba explants. By using different disinfectant combinations to treat the explants, the most effective disinfection method was determined to reduce the contamination rate during the primary culture (induction culture). The experimental results show that the use of 75% ethanol and 0.15% mercury, combined with other disinfectants such as vinegar, NaClO and LClO2, can significantly reduce the contamination rate of the explants, and the contamination rate of treatment 2 is the lowest, which is 0.83%, and it is the best disinfection scheme. This disinfection scheme can ensure the sterile state of the explants, thereby improving the success rate of tissue culture.

[0073] The selection of different explant materials also affects the contamination rate. Figure 3 The control treatment of the lateral bud with stem segment culture of 'Jiao Hong 1' Magnolia × alba is shown to illustrate the challenges faced when using mature branches as explants.

A

B

[0074] The present application selects the stem axillary bud in the bud as the explant. These stem segments are wrapped in the bud, and the protective effect of the bud effectively reduces the contact with external pollution sources, thereby reducing the risk of contamination. This method not only improves the success rate of disinfection, but also helps to protect the axillary buds and stem segments from external adverse factors, providing them with a relatively stable and suitable growth environment, thereby improving the efficiency of induction and the overall success rate of culture.

[0075] When the explant is a single lateral bud, such as Figure 4The contamination rate was very low; after disinfection with 75% ethanol for 30 seconds and 0.15% mercuric chloride for 3 minutes, the contamination rate was 6.66%, but it was basically impossible to induce germination.

[0076] Initial induction culture:

[0077] Remove the outer bracts from the sterilized explant, such as Figure 1 As shown in Figure b, axillary bud 11 is exposed. Then, the top flower bud 12 is removed, and the stem segment with axillary bud 11 is inserted into MS basal medium (i.e., induction medium) supplemented with plant hormones 2.0 mg / L 6-BA + 0.15 mg / L NAA + 0.2 mg / L KT. Initial culture is carried out at 22–26℃ and a light intensity of 2000–3000 Lx. After 3–5 days, the uncontaminated tissue culture material is transferred to a new induction medium and cultured for another 21–28 days until new shoots emerge. See also... Figure 2 Figure a in the middle.

[0078] In contrast, during the initial induction culture stage, such as Figure 4 As shown, although the contamination rate induced by single lateral bud regeneration is extremely low, these buds largely fail to induce sprouting. While they initially swell and some even develop individual leaves, their growth subsequently stagnates, eventually leading to browning and death of the internal tissues. Furthermore, single bud culture often exhibits vitrification due to the lack of nutrient support from stem segments, which not only affects normal bud growth but also increases the difficulty of culture. Therefore, after weighing factors such as bud activity, initial nutrient supply, and contamination rate, selecting axillary bud stem segments from flower buds as explants is the most suitable approach. Flower buds provide necessary protection for axillary buds and the attached stem segments, reducing the risk of contamination, while the attached stem segments provide the nutrients needed for initial growth of the axillary buds, thereby improving the success rate of tissue culture and ensuring healthy plant growth.

[0079] During induction culture, different hormone ratios significantly affected the germination rate and survival rate of explants, as shown in Table 2.

[0080] Table 2. Effects of hormone ratios on explant germination rate and survival.

[0081]

[0082] Table 2 shows that the hormone ratio has a significant effect on the germination rate and survival rate of explants. The experiment changes the concentration combination of 6-BA (6-benzylaminopurine), NAA (naphthalene acetic acid) and KT (6-furfuryladenine) to observe the effect of different hormone treatments on the germination and survival of explants. The results show that appropriate auxin and cytokinin ratio helps to promote the survival of explants. However, too high concentration or inappropriate hormone combination may cause growth inhibition or tissue necrosis. Therefore, optimizing the hormone ratio is crucial to improve the germination rate and survival rate of explants, which not only affects the success rate of initial culture, but also lays a good foundation for subsequent proliferation and rooting culture.

[0083] Proliferation culture:

[0084] The new shoots that grow out are transferred to MS medium containing growth regulators ADS 2.5-7.5 mg / L, IBA 0.1-1.0 mg / L and TDZ 0.1-0.4 mg / L, and cultured at 22-26°C with light intensity of 2000-3000 Lx for 28-30 days. See Figure 2 Figure b in the middle.

[0085] In the proliferation culture, different hormone ratios have a significant effect on the proliferation rate and growth status of explants, see Table 3.

[0086] Table 3 Effect of hormone ratio on the proliferation rate and growth status of explants

[0087]

[0088] According to the subculture proliferation data in Table 3, the hormone ratio has a significant effect on the proliferation rate and growth status of 'Jiao Hong 1' Magnolia × alba explants. The experiment adjusts the concentration of ADS (adenosine sulfate), IBA (3-indolebutyric acid) and TDZ (thidiazuron) to explore the effect of different hormone combinations on proliferation. The results show that as the concentration of ADS increases, the proliferation rate increases, but too high a concentration (such as 7.5 mg / L) does not bring higher proliferation rate, but may cause changes in growth status, such as leaf deformation. At the same time, the concentration of IBA and TDZ also has an important influence on the proliferation rate and growth status, and appropriate concentration of IBA and TDZ can promote the formation of multiple shoots, such as the combination of 5.0 mg / L ADS, 0.5 mg / L IBA and 0.4 mg / L TDZ, which can obtain the highest proliferation rate (3.00), and the morphology of shoots and leaves remains normal without deformation. This shows that the hormone ratio needs to be finely controlled to ensure that the explants not only increase in number during the proliferation process, but also maintain a healthy growth status, laying a good foundation for subsequent rooting and transplanting survival.

[0089] Rooting culture:

[0090] The tissue culture seedlings obtained by proliferation culture are transferred to rooting medium, which is MS basic medium with the addition of BNOA 2.0-8.0 mg / L, and after 5 days of dark culture, the medium is transferred to light with an intensity of 2000-3000 Lx for continued culture for 21-28 days. The temperature is 18-22°C. See Figure 2 Fig. 3 shows new roots 30 in the medium.

[0091] Different hormone ratios have a significant effect on the rooting rate of the tissue culture seedlings, as shown in Table 4.

[0092] Table 4 Effect of hormone ratios on rooting culture

[0093]

[0094] According to Table 4, different hormone ratios have a significant effect on the rooting rate of 'Jiao Hong 1' Magnolia liliflora tissue culture seedlings. By changing the concentrations of NAA (naphthalene acetic acid), IBA (3-indolebutyric acid), and BNOA (2-naphthoxyacetic acid), the experiment explores the effects of these growth regulators on rooting. The results show that when only BNOA is used as a hormone, the rooting rate is 10%, which indicates that BNOA plays a key role in promoting rooting. In contrast, the use of NAA or IBA alone, or a combination of these hormones, does not induce rooting, with a rooting rate of 0%. This finding reflects the importance of BNOA in the rooting medium and reveals that selecting the appropriate hormone and its concentration during the rooting culture stage is crucial for improving the rooting rate. In addition, these results suggest that specific hormone combinations and concentrations may be required during tissue culture to optimize rooting effects and improve the rooting success rate and subsequent survival rate of transplanted seedlings.

[0095] In analyzing the rooting results, the inventors found that the height of the tissue culture seedlings entering the rooting culture stage is one of the key factors affecting the rooting rate. According to the statistical data, all the rooted tissue culture seedlings are taller than 4 cm. Therefore, during the subculture stage, it is recommended to select healthy tissue culture seedlings with a height of more than 4 cm for rooting culture, while those that do not reach 4 cm during the division are continued for subculture. To verify this analysis result, the inventors selected 3 healthy tissue culture seedlings with a height of 4-5 cm, which were vigorous in growth, had no obvious external damage or diseases on the leaves, and had a bottom of more than 1 cm without leaf height. They were cultured in the same dish for rooting culture, and all the materials rooted normally, with a rooting induction rate of 100%, Figure 2 Fig. 4 shows the actual situation of the 28th day of rooting culture, and the height of the selected tissue culture seedlings for rooting culture is directly proportional to the rooting speed.

[0096] The method for cultivating 'Jiao Hong 1' Magnolia × alba seedlings has low pollution rate, cluster proliferation, and the obtained aseptic seedlings have less virus, white and thick roots, and are not easy to fall off. The tissue culture method of the 'Jiao Hong 1' Magnolia × alba is simple in operation, short in time, low in cost and high in survival rate. The tissue culture method can keep the excellent characteristics of the original variety, and lays a foundation for breeding and industrial production of good varieties.

[0097] Using immature seeds to induce regeneration, although immature seeds can induce healthy embryonic precursors and can be continuously cultured, there is a key defect: since 'Jiao Hong 1' Magnolia × alba is the result of natural and artificial selection, using seeds for propagation will lead to segregation of traits. This means that plants obtained through sexual reproduction of seed materials may exhibit different characteristics from the original variety, thereby affecting their ornamental value. Especially for 'Jiao Hong 1' Magnolia × alba, its ornamental value mainly lies in the color and morphology of flowers, and trait segregation may lead to new plants that cannot maintain their unique flower viewing characteristics. In contrast, using axillary buds in flower buds for tissue culture is a method of asexual reproduction, which can more effectively maintain the genetic characteristics of the mother plant and ensure that the ornamental value of new plants is consistent with the original variety. Therefore, although seed induction may be a viable propagation method in some cases, using axillary buds in flower buds for tissue culture is a more suitable choice for rapid propagation and variety protection of 'Jiao Hong 1' Magnolia × alba.

[0098] In the description of the present application, it should be understood that "-" and "~" represent the range of the same of two numerical values, and the range includes the endpoints. For example: "A-B" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0099] In the description of the present application, the term "and / or" herein is merely a description of the association relationship of the associated objects, which means that there can be three relationships, for example, A and / or B, which can represent the three cases of A alone, A and B together, and B alone.

[0100] In the description of the present application, the values related to time, temperature, ratio and mass, etc. can be based on actual measurement, equipment standard parameters, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0101] In the description of the present application, the term "about" or "approximately" is used to express the approximate value of a numerical value or interval, allowing for a certain error to ensure the flexibility and practicability of the description, while remaining within an acceptable error range, and the maximum error range is not more than 10% of the corresponding numerical value or numerical range.

[0102] The above merely describes the preferred embodiments of the present application, and it should be pointed out that the above preferred embodiments should not be regarded as a limitation to the present application, and the protection scope of the present application should be defined by the scope of the claims. For those skilled in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

Claims

1. A rapid propagation method of Magnolia acuminata tissue culture, characterized in that, 1-2 axillary buds and the stem segments where the axillary buds grow are taken as explants, the sterile explants are induced, proliferated and rooted to obtain tissue culture plants; The sterile explants are obtained by the following steps: sterilizing the flower buds by treating with 75% ethanol for 30s, 0.15% mercury for 10 min and 8% NaClO for 10 min, removing the outer bracts and retaining the axillary buds and the stem segments where the axillary buds grow, and cutting off the rest of the stem segments and flower buds; The induction culture uses a medium composed of MS basic medium, 6-BA 2.0 mg / L, NAA 0.1-0.2 mg / L and KT 0.2 mg / L; The proliferation culture uses a medium composed of MS basic medium, ADS 5.0 mg / L, IBA 0.4 mg / L and TDZ 0.4 mg / L; The rooting culture uses a medium composed of MS basic medium and BNOA 6.0 mg / L.

2. The rapid propagation method of Magnolia acuminata tissue culture according to claim 1, characterized in that, The flower bud is a flower bud where flower bud differentiation is completed at the top of a branch.

3. The rapid propagation method of Magnolia acuminata tissue culture according to claim 2, characterized in that, The axillary bud is provided with soft villi.

4. The rapid propagation method of Magnolia acuminata tissue culture according to claim 1, characterized in that, The induction culture is as follows: The explants are inserted into the induction medium and initially cultured at 22-26°C under light intensity of 2000-3000 Lx; after 3-5 days, the uncontaminated stem segments are transferred into new induction medium and cultured for 21-28 days until new shoots grow.

5. The rapid propagation method of Magnolia acuminata tissue culture according to claim 1, characterized in that, The proliferation culture is as follows: The new shoots obtained by induction culture are inserted into the proliferation medium and cultured at 22-26°C under light intensity of 2000-3000 Lx for 28-30 days.

6. The rapid propagation method of Magnolia acuminata tissue culture according to claim 1, characterized in that, The rooting culture is as follows: The tissue culture seedlings obtained by proliferation culture are transferred into the rooting medium, dark cultured for 4-6 days, then transferred into light under light intensity of 2000-3000 Lx and cultured for 21-28 days at 18-22°C.

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