A method for rapid propagation and efficient regeneration of 'Fengdan' by tissue culture

Abstract

The present invention relates to a method for rapid tissue culture propagation and efficient regeneration of 'Feng Dan', belonging to the technical field of rapid peony cultivation. The method addresses at least one of the problems of low rapid propagation and regeneration efficiency, poor rooting rate and proliferation rate, poor root system condition, and poor callus induction and differentiation effects of 'Feng Dan' in the prior art. The present invention discloses a method for rapid tissue culture propagation and efficient regeneration of 'Feng Dan', comprising S1: initiation culture, S2: proliferation culture, and S3: rooting culture; wherein the initiation culture medium used in step S1 comprises NN69 and IBA; and the proliferation culture medium used in step S2 comprises NN69, CPPU, and IBA. The present invention innovatively utilizes NN69 minimal culture medium and a CPPU+IBA growth regulator combination for peony cultivation, constructs a peony tissue callus regeneration system, and optimizes the culture conditions for tissue culture propagation. This method results in a high proliferation coefficient and high seedling success rate for tissue culture seedlings, low cost, and high returns, enabling factory-scale production of 'Feng Dan' seedlings.

Description

Technical Field

[0001] The present invention relates to the technical field of rapid peony cultivation, and in particular to a method for rapid tissue culture propagation and efficient regeneration of 'Fengdan'. Background Art

[0002] Peony is a perennial deciduous shrub of the genus Paeonia (Paeonia L.) in the family Paeoniaceae. It possesses both high ornamental and economic value and is often called the national flower. Oil-producing peonies, with their high fruit set and oil content, are cultivated as oil crops. They combine ornamental, medicinal, and edible uses. The peony seed oil produced contains a variety of unsaturated fatty acids, trace elements, and bioactive substances, with health benefits such as lowering blood lipids and blood sugar and enhancing immunity. 'Fengdan' (P. ostii 'Fengdan'), one of the main cultivated varieties of oil-producing peonies, is characterized by abundant flowers, few suckers, high seed production, high oil yield, wide adaptability, and robust growth.

[0003] Currently, the propagation of 'Fengdan' still relies primarily on seed propagation, which suffers from a long reproductive cycle and is subject to seasonal and environmental constraints. Furthermore, the offspring exhibit significant variability in traits, making it difficult to maintain the superior traits of the parent plant, resulting in a loss of its high oil yield and quality. 'Fengdan' can also be propagated by cuttings, grafting, or division. However, cuttings pose challenges such as low rooting rates and perishable material. Grafting is often limited by the compatibility of the rootstock and scion, their growth cycle, and the season. Division leads to a prolonged seedling acclimatization period, weak growth, and often exposes root wounds, which can lead to infection and rot.

[0004] Compared to the above traditional methods, which suffer from low propagation coefficients, long seedling growth cycles, and uneven quality, the establishment of a tissue culture rapid propagation and efficient regeneration technology system for 'Fengdan' has the advantages of being unrestricted by seasons and the environment, shortening the breeding cycle, and maintaining the excellent quality of the parent plants, which is conducive to its factory production. Researchers in related fields have conducted some exploration of tissue culture rapid propagation and efficient regeneration methods for 'Fengdan', but the following shortcomings still exist:

[0005] (1) The induction rate, proliferation rate, and rooting rate of tissue culture seedlings were low, resulting in low rapid propagation, regeneration efficiency, and proliferation coefficient of 'Fengdan';

[0006] (2) The tissue culture seedlings of 'Fengdan' have browning problems, which affect the growth and development of the tissue culture seedlings, resulting in low rooting rate, poor rooting state, and low survival rate;

[0007] (3) The regeneration system is single, focusing on direct organogenesis of axillary or terminal buds, which affects the regeneration efficiency and proliferation coefficient;

[0008] (4) Tissue culture seedlings grow slowly and have a long cultivation cycle.

[0009] In conclusion, it is necessary to develop a method for rapid tissue culture propagation and efficient regeneration of 'Danfeng'. Summary of the Invention

[0010] In view of the above analysis, the embodiments of the present invention aim to provide a method for rapid propagation and efficient regeneration of 'Fengdan' by tissue culture, so as to solve at least one of the following problems: the induction rate, proliferation rate, and rooting rate of existing tissue culture seedlings are low, resulting in low rapid propagation and regeneration efficiency and proliferation coefficient of 'Fengdan'; the browning problem of 'Fengdan' tissue culture seedlings affects the growth and development of tissue culture seedlings, resulting in low rooting rate, poor rooting state, and low survival rate; the regeneration system is single, the tissue culture seedlings grow slowly, and the cultivation cycle is long.

[0011] The present invention discloses a method for rapid propagation and efficient regeneration of 'Fengdan' tissue culture, characterized in that the method comprises S1 initiation culture, S2 proliferation culture and S3 rooting culture;

[0012] The start-up culture medium used in step S1 includes NN69 and IBA; the proliferation culture medium used in step S2 includes NN69, CPPU and IBA.

[0013] The above method specifically includes the following steps:

[0014] S1: preparing a priming culture medium comprising NN69+IBA; inoculating axillary stem segment explants with leaves sterilized with mercuric chloride and alcohol onto the priming culture medium for priming culture to obtain primary axillary buds and primary leaves; wherein the primary leaves are induced and differentiated to obtain differentiated adventitious buds;

[0015] S2 proliferation culture: preparing a proliferation culture medium comprising NN69+IBA+CPPU+PVP; inoculating primary axillary buds or differentiated adventitious buds onto the proliferation culture medium for proliferation culture to obtain unrooted tissue culture seedlings;

[0016] S3 Rooting culture: Prepare a rooting culture medium and use a one-step rooting method or a two-step rooting method to perform rooting culture on the unrooted tissue culture seedlings to obtain complete tissue culture seedlings.

[0017] Specifically, the IBA concentration in the start-up culture medium is 0.1-0.2 mg / L, preferably 0.1 mg / L.

[0018] Specifically, the IBA concentration in the proliferation culture medium is 0.1-0.2 mg / L, the CPPU concentration is 0.2-0.6 mg / L, and the PVP concentration is 4 mg / L; preferably, the IBA concentration is 0.1 mg / L, and the CPPU concentration is 0.4 mg / L.

[0019] Furthermore, the specific operations of the primary leaf induction and differentiation are:

[0020] S1-1 Induction culture: prepare an induction medium, which includes NN69+IBA+CPPU+PVP, inoculate the primary leaves onto the induction medium, perform induction culture, and obtain callus tissue;

[0021] S1-2 Differentiation culture: Prepare a differentiation medium comprising NN69+IBA+CPPU+PVP, inoculate the callus tissue onto the differentiation medium, perform differentiation culture, and obtain differentiated adventitious buds.

[0022] Preferably, the IBA concentration in the induction medium is 0.3 mg / L, the CPPU concentration is 0.9 mg / L, and the PVP concentration is 3 mg / L; and the IBA concentration in the differentiation medium is 0.2 mg / L, the CPPU concentration is 0.2 mg / L, and the PVP concentration is 3 mg / L.

[0023] Furthermore, the method further comprises a step of S2.5 strong seedling cultivation, which is arranged after S2 proliferation cultivation and before S3 rooting cultivation;

[0024] The specific operation is that after completing the S2 proliferation culture, the unrooted tissue culture seedlings are exposed to light and cultured to strengthen the seedlings, so as to obtain strong and elongated unrooted tissue culture seedlings for subsequent rooting culture.

[0025] Furthermore, the illumination conditions are as follows: the LED light source combination is a mixture of full-spectrum white light and red light W:R=7:1, and the illumination intensity is W=26.25 μmol·m -2 s -1 , R = 3.75 μmol·m -2 s -1 , where W and R represent full-spectrum white light and red light, respectively; or, full-spectrum white light 50 μmol·m -2 s -1 .

[0026] Furthermore, the specific operation of the one-step rooting method in step S3 is to inoculate the unrooted tissue culture seedlings into the rooting medium for rooting culture, and the composition of the rooting medium is NN69 + 3mg·L -1 CaCl2 or NN69+3mg·L -1 CaCl2+0.2mg·L -1 IBA.

[0027] Furthermore, the specific operations of the two-step rooting method in step S3 are:

[0028] The unrooted tissue culture seedlings were cold-treated and inoculated into a root formation medium for culture. The root formation medium consisted of NN69 + 3 mg·L-1 CaCl2+0.2mg·L -1 IAA, tissue culture seedlings with initial rooting were obtained;

[0029] The tissue culture seedlings with initial rooting were transferred to a root elongation medium for culture. The root elongation medium composition was NN69 + 6 mg·L -1 CaCl2 or NN69+6mg·L -1 CaCl2+0.2mg·L -1 IBA, and finally obtain complete tissue culture seedlings.

[0030] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects:

[0031] 1. This invention uses bud-bearing stem segments of the 'Feng Dan' plant as explants and successfully obtains 'Feng Dan' plantlets through tissue culture regeneration through priming (including induction and differentiation), proliferation, and rooting. Simultaneously, a systematic 'Feng Dan' plantlet regeneration system is established through an indirect organogenesis pathway involving leaf callus induction and differentiation. This system offers advantages such as a high tissue culture plantlet proliferation coefficient, high seedling success rate, low cost, and high returns. By utilizing plant tissue culture technology to increase the speed and scale of its propagation, it enables factory-scale production of 'Feng Dan' seedlings.

[0032] The present invention optimizes the type of 'Fengdan' basic culture medium, and uses NN69 for the cultivation of peony plants for the first time, breaking through the dilemma of using only two major categories of MS and WPM. Peony is a plant of the genus Paeonia, Paeonia itself is a herbaceous genus, while peony is a shrub (woody) with relatively rich woody tissue. Therefore, the culture medium suitable for the genus Paeonia in the prior art is often not effective for cultivating peonies. The NN69 culture medium in the present invention is commonly used as a basic culture medium for fruit trees such as the genus Pyrus. The present invention pioneered the use of it in the peony cultivation system for the first time, and further developed a combination culture medium type suitable for peonies (especially Fengdan) based on it as a matrix; the NN69 matrix plays a role in increasing the differentiation rate and reducing the browning rate during the cultivation process, and also provides good culture conditions for subsequent proliferation and rooting.

[0033] This invention uses the growth regulator IBA for initiation and a combination of IBA and CPPU for other steps, breaking away from the traditional approach of selecting growth regulators from among several major categories, such as 6-BA, KT, TDZ, and ZT. During the development process, researchers overcame the following technical difficulties through theoretical deduction and experimental demonstration:

[0034] (1) Different explants or organs of 'Fengdan' respond differently to growth regulators. For example, the experimental data from the S1 start-up culture show that the NN69 medium supplemented with IBA has the highest proliferation coefficient, which is better than the combination of IBA+CPPU. Therefore, in the research and development of this link, it is easy to draw the wrong conclusion that the combination of IBA+CPPU is not suitable for the cultivation of 'Fengdan'. However, from the experimental data of the subsequent steps, it can be seen that for primary leaves, differentiated adventitious buds, primary axillary buds and callus tissue, the effect of the combination of IBA+CPPU is better than that of IBA alone.

[0035] (2) For different explants or organs of 'Fengdan', the optimal concentration ranges of the same growth regulator combination vary greatly. For example, the experimental data in S2 proliferation culture show that the combination of medium and low concentrations of IBA+CPPU has a higher proliferation coefficient for axillary buds or adventitious buds, while the combination of high concentrations is harmful to the proliferation effect. This leads to the misleading conclusion that the combination of medium and low concentrations of IBA+CPPU is more suitable for the cultivation of 'Fengdan' in other links of speculation or design.

[0036] However, for the induction and differentiation in step S1, medium and low concentrations of IBA combined with higher concentrations of CPPU have better induction effects on primary leaves, and medium and low concentrations of IBA combined with lower concentrations of CPPU have better differentiation effects on callus tissue; that is to say, for organs or explants with different degrees of differentiation or morphology, the effect curves and inflection points of the growth regulator combination are different, and there are large differences in the optimal concentration combination and the preferred range, which are difficult to predict and speculate.

[0037] (3) The two-step rooting method in the rooting culture step breaks the traditional thinking and achieves a higher rooting rate (more than 70%), which can effectively solve the problems of low rooting rate, poor root system condition, and low transplant survival rate of peony tissue culture seedlings.

[0038] 2. The present invention establishes a callus regeneration system for tree peony tissue (corresponding to the induction and differentiation in step S1), providing a foundation for a genetic transformation system. Numerous studies have failed to demonstrate favorable induction and differentiation effects on 'Fengdan' callus, reducing the proliferation coefficient and the establishment of a genetic transformation system. This study not only provides a formula for callus induction and differentiation, but also achieves excellent induction and differentiation rates, significantly optimizing the tree peony regeneration system. Furthermore, by utilizing primary leaves for further proliferation, the proliferation coefficient is effectively increased.

[0039] 3. This study studies the lighting conditions during tissue culture and provides new ideas and options for cultivation. Currently, there is a lack of exploration of culture conditions for 'Fengdan' tissue culture, and most people use traditional LED artificial light sources. This study first discovered that 50μmol·m -2 s -1Full-spectrum white light irradiation can strengthen stems and leaves and increase the proliferation coefficient; mixing red light and full-spectrum white light with a W:R ratio of 7:1 can make test tube seedlings have a main stem and significantly tend to form complete plants. Therefore, the present invention provides a technical solution for selecting different lighting culture conditions according to different needs.

[0040] In the present invention, the above-mentioned technical solutions can be combined with each other to achieve more preferred combinations. Other features and advantages of the present invention will be described in the following description, and some advantages will become apparent from the description or be learned through practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like parts throughout the drawings.

[0042] Figure 1 This is a flow chart of the tissue culture rapid propagation and efficient regeneration method for 'Fengdan';

[0043] Figure 2 Figure a is a photograph of the stem segment explant with axillary buds 2-3 in experimental group 2 (14d), and Figure b is a photograph of the stem segment explant with axillary buds 2-1 in experimental group 2 (14d);

[0044] Figure 3 In the figure, Figure a is a photo of 3-5 axillary buds in experimental group 3 (30 days), and Figure b is a photo of 3-4 axillary buds in experimental group 3 (30 days);

[0045] Figure 4 In the figure, Figure a is a photo of the callus tissue of 4-1-6 in experimental group 4 (30 days), and Figure b is a photo of the callus tissue of 4-1-3 in experimental group 4 (30 days);

[0046] Figure 5 In the figure, Figure a is a photo of the adventitious buds of 4-2-4 in experimental group 4 (30 days), and Figure b is a photo of the adventitious buds of 4-2-1 in experimental group 4 (30 days);

[0047] Figure 6 These are scanning electron microscope photos of the 4-2-4 adventitious buds in experimental group 4, where Figure a is a 10-day scanning electron microscope photo, Figure b is a 15-day scanning electron microscope photo, and Figure c is a 20-day scanning electron microscope photo;

[0048] Figure 7 Figure a is a photo of the 5-2 cultured buds in experimental group 5 (30 days), and Figure b is a photo of the 5-3 cultured buds in experimental group 5 (30 days);

[0049] Figure 8Figure a is a diagram showing the rooting status of 6-1 tissue culture seedlings in experimental group 6 (60d), Figure b is a diagram showing the rooting status of 6-2 tissue culture seedlings in experimental group 6 (60d), and Figure c is a diagram showing the rooting status of 6-4 tissue culture seedlings in experimental group 6 (60d). DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, and are not used to limit the scope of the present invention.

[0051] The researchers of this invention have previously conducted a thorough analysis of the deficiencies in existing tissue culture, rapid propagation, and regeneration technologies for tree peonies, particularly 'Fengdan', by consulting literature and conducting extensive theoretical analysis. They have taken the lead in identifying or further clarifying the deficiencies in existing technologies and their corresponding causes, and have creatively proposed possible solutions or technical routes, as detailed below:

[0052] (1) The current culture medium types for rapid propagation and regeneration of 'Fengdan' are limited, focusing on two main categories: MS and WPM. More culture medium types suitable for tree peonies have not been developed. The culture medium type is directly related to the nutritional supply of the tissue culture seedlings. Excess or deficiency of certain elements will affect the growth of the peony tissue culture seedlings during the induction, proliferation, and rooting stages, limiting the rapid propagation and regeneration efficiency of 'Fengdan'.

[0053] (2) The current tissue culture rapid propagation and regeneration technology for 'Fengdan' uses a limited variety of growth regulators, especially cytokinins, which are concentrated in the categories of 6-BA, KT, TDZ, and ZT. No new growth regulators suitable for the peony itself have been tried. Different plants respond differently to different hormones, which can affect the induction rate, proliferation rate, and rooting rate of peony tissue culture seedlings, limiting the efficiency and proliferation coefficient of 'Fengdan' rapid propagation and regeneration.

[0054] (3) The existing 'Fengdan' tissue culture rapid propagation and regeneration technology has problems with browning and low rooting rate in peony tissue culture seedlings, which affects seedling formation. Phenolic substances are easily accumulated during the peony tissue culture process, and browning affects the growth and development of tissue culture seedlings and even affects the survival rate. The rooting stage is a crucial step in the propagation system, which is related to whether it can become a complete tissue culture seedling. Currently, the focus of peony tissue culture is on the improvement of basic culture medium and the use of growth regulators, which often leads to problems such as low rooting rate, poor root system condition, and low transplant survival rate.

[0055] (4) The existing tissue culture rapid propagation and regeneration technology for 'Feng Dan' is limited in its regeneration system, focusing on direct organogenesis of axillary or terminal buds, and lacking research on callus tissue. The indirect organogenesis pathway of tree peony refers to the process in which the explant first undergoes dedifferentiation induction to produce callus tissue, which then differentiates into adventitious buds or adventitious roots, thus forming a complete plant. However, a large number of studies have failed to achieve good induction and differentiation effects on 'Feng Dan' callus tissue, which has reduced the proliferation coefficient and the construction of the genetic transformation system.

[0056] (5) Neglect of culture conditions, especially light quality. Currently, traditional LED artificial light sources are mostly used in the tissue culture of 'Fengdan'. These light sources have a wide wavelength range, and the proportion of light energy suitable for photosynthesis is relatively small. Different light quality combinations directly affect the growth and biomass accumulation of tissue culture seedlings. The lack of exploration of different light culture conditions for tree peonies is not conducive to the accumulation of dry matter in tissue culture seedlings, resulting in slow growth and a long cultivation cycle.

[0057] In response to the above problems, after obtaining sterile explants, the present invention provides a 'Fengdan' tissue culture rapid propagation and efficient regeneration technology system, and conducts new explorations in aspects such as culture medium type, cytokinin type, and culture conditions, thereby significantly improving the proliferation rate and rooting rate. At the same time, an indirect organogenesis pathway for callus induction and differentiation is successfully constructed.

[0058] The present invention discloses a method for rapid propagation and efficient regeneration of 'Fengdan' tissue culture, characterized in that the method comprises S1 initiation culture, S2 proliferation culture and S3 rooting culture;

[0059] The start-up culture medium used in step S1 includes NN69 and IBA; the proliferation culture medium used in step S2 includes NN69, CPPU and IBA.

[0060] The above method specifically includes the following steps:

[0061] S1: preparing a priming culture medium comprising NN69+IBA; inoculating axillary stem segment explants with leaves sterilized with mercuric chloride and alcohol onto the priming culture medium for priming culture to obtain primary axillary buds and primary leaves; wherein the primary leaves are induced and differentiated to obtain differentiated adventitious buds;

[0062] The purpose of this step is to obtain axillary buds or adventitious buds suitable for subsequent proliferation culture. The induction and differentiation steps enable the primary leaves to be used for subsequent proliferation culture, making full use of the organs of the explant, helping to improve proliferation efficiency and shorten the growth or industrial production cycle;

[0063] S2 proliferation culture: preparing a proliferation culture medium comprising NN69+IBA+CPPU+PVP; inoculating primary axillary buds or differentiated adventitious buds onto the proliferation culture medium for proliferation culture to obtain unrooted tissue culture seedlings;

[0064] The purpose of this step is to "proliferate buds with buds" and greatly increase the multiplication coefficient;

[0065] S3 Rooting culture: Prepare a rooting culture medium and use a one-step rooting method or a two-step rooting method to perform rooting culture on the unrooted tissue culture seedlings to obtain complete tissue culture seedlings.

[0066] Specifically, the IBA concentration in the priming culture medium is 0.1-0.2 mg / L, preferably 0.1 mg / L. For priming culture, the addition of IBA alone is more effective than the addition of CPPU alone or the combination of CPPU and IBA. Lower IBA concentrations are more conducive to explant induction. When the IBA concentration in the culture medium is 0.1 mg / L, the induction rate can reach 75.6±6.8%.

[0067] Specifically, the IBA concentration in the proliferation culture medium is 0.1-0.2 mg / L, the CPPU concentration is 0.2-0.6 mg / L, and the PVP concentration is 4 mg / L; preferably, the IBA concentration is 0.1 mg / L and the CPPU concentration is 0.4 mg / L. For proliferation culture, unlike the start-up culture, the combined proliferation effect of CPPU+IBA is significantly better than that of using IBA and CPPU alone, and according to experimental data, it is easy to obtain that the proliferation coefficient is the highest when a lower concentration of IBA is used in combination with a medium concentration of CPPU. In addition, since browning is more serious in axillary bud proliferation culture, a higher concentration of PVP is added to inhibit the occurrence of browning. PVP, an anti-browning agent, mainly prevents the oxidation of phenolic substances or inhibits the synthesis of phenolic substances by inhibiting the activity of PPO (polyphenol oxidase) and AL (phenylalanine ammonia lyase), thereby preventing the axillary buds or adventitious buds from browning during proliferation culture.

[0068] Furthermore, the specific operations of the primary leaf induction and differentiation are:

[0069] S1-1 Induction culture: prepare an induction medium, which includes NN69+IBA+CPPU+PVP, inoculate the primary leaves onto the induction medium, perform induction culture, and obtain callus tissue;

[0070] S1-2 Differentiation culture: Prepare a differentiation medium comprising NN69+IBA+CPPU+PVP, inoculate the callus tissue onto the differentiation medium, perform differentiation culture, and obtain differentiated adventitious buds.

[0071] Preferably, the IBA concentration in the induction medium is 0.3 mg / L, the CPPU concentration is 0.9 mg / L, and the PVP concentration is 3 mg / L. Experimental data show that the induction effect of the combination of IBA and CPPU is significantly better than that of either alone. The induction effect first increases and then decreases with the IBA concentration, with the inflection point at an IBA concentration of 0.3 mg / L. For the experimental group with an IBA concentration of 0.3 mg / L, the combination with a higher concentration of CPPU has a better induction effect. It is worth emphasizing that analysis of different groups with the same IBA concentration shows that the induction effect is not positively correlated with the CPPU concentration. For example, when the IBA concentration is 0.4 mg / L, the induction effect is negatively correlated with the CPPU concentration, making the experimental results difficult to predict.

[0072] Preferably, the differentiation medium contains 0.2 mg / L IBA, 0.2 mg / L CPPU, and 3 mg / L PVP. Experimental data clearly demonstrate that the combination of IBA and CPPU significantly outperforms either alone, with the optimal differentiation effect being achieved with a moderate IBA concentration combined with a lower CPPU concentration.

[0073] Furthermore, the method further comprises a step of S2.5 strong seedling cultivation, which is arranged after S2 proliferation cultivation and before S3 rooting cultivation;

[0074] The specific operation is that after completing the S2 proliferation culture, the unrooted tissue culture seedlings are exposed to light and cultured to strengthen the seedlings, so as to obtain strong and elongated unrooted tissue culture seedlings for subsequent rooting culture.

[0075] It is worth mentioning that the purpose of this step is to allow the buds to grow longer and stronger, laying a good foundation for subsequent rooting culture, helping to increase the rooting rate, improve the growth state, and increase the survival rate.

[0076] Furthermore, the illumination conditions are as follows: the LED light source combination is a mixture of full-spectrum white light and red light W:R=7:1, and the illumination intensity is W=26.25 μmol·m -2 s -1 , R = 3.75 μmol·m -2 s -1 , where W and R represent full-spectrum white light and red light, respectively;

[0077] The tissue culture seedlings grown under the mixed full-spectrum white light: red light = 7:1 irradiation conditions did not show a significant increase in proliferation coefficient, but the stem length was significantly longer and had a distinct main trunk. It can be observed that the stem was purple-red, with complete leaves developed at the top, and no callus growth or vitrification at the base. It has shown a tendency to develop into a complete plant, making it more suitable for rooting culture.

[0078] or full spectrum white light 50 μmol·m -2 s -1 The tissue culture seedlings developed well under the illumination conditions of full-spectrum white light. Not only were they taller than the tissue culture seedlings treated with fluorescent light, there was no obvious vitrification phenomenon, no callus tissue developed at the base, the stems and leaves were strong, and the proliferation coefficient was significantly improved.

[0079] It is worth noting that, in specific implementation, technicians can choose different seedling culture conditions according to actual needs.

[0080] Furthermore, the specific operation of the one-step rooting method in step S3 is to inoculate the unrooted tissue culture seedlings into the rooting medium for rooting culture, and the composition of the rooting medium is NN69 + 3mg·L -1 CaCl2 or NN69+3mg·L -1 CaCl2+0.2mg·L -1 IBA.

[0081] It is worth noting that during the rooting culture process of the one-step rooting method, it was observed that the rootless seedlings in the rooting medium under the irradiation of mixed red light and full-spectrum white light gradually took root regardless of whether the IBA growth regulator was added. The medium without IBA began to take root at the end of the culture period (about 55 days), with a rooting rate of 40%. The average number of roots of the rooted tissue culture seedlings was 1.8 roots per plant, with an average root length of 3.1 cm, and the root system was in good condition. -1 In IBA rooting medium, roots began to form around 45 days old, with a rooting rate of 60%. The average number of roots per plant was 2.7, and the average root length was 6.1 cm.

[0082] Furthermore, the specific operations of the two-step rooting method in step S3 are:

[0083] The unrooted tissue culture seedlings were cold-treated and inoculated into a root formation medium for culture. The root formation medium consisted of NN69 + 3 mg·L -1 CaCl2+0.2mg·L -1 IAA, tissue culture seedlings with initial rooting were obtained;

[0084] The tissue culture seedlings with initial rooting were transferred to a root elongation medium for culture. The root elongation medium composition was NN69 + 6 mg·L -1 CaCl2 or NN69+6mg·L -1 CaCl2+0.2mg·L -1 IBA, and finally obtain complete tissue culture seedlings.

[0085] It is worth noting that the two-step rooting method can significantly improve the rooting rate of tissue culture seedlings and reduce the quality requirements for rootless seedlings. -1 IBA root elongation medium can make rootless seedlings with a height of 2.5 to 3.0 cm take root, with a rooting rate of 70%. Rooting begins around 35 days after incubation. The average number of roots per rooted seedling is 1.2, and the average root length is 1.6 cm.

[0086] Experimental Group 1 - Screening of Basic Culture Medium

[0087] The growth of explants is affected by the basal culture medium. Three culture media were selected for this experiment: NN69, MS, and WPM. Thirty stem explants with leaf axils were inoculated in each medium. After two weeks, the stem segments were observed for growth, and the bud induction rate and browning rate were calculated.

[0088] The treatment results are detailed in Table 1. As can be seen from the table, MS medium had the lowest induction rate, only 22.2%, while NN69 medium had the highest, reaching 66.7%, three times that of MS medium. However, there was no significant difference between the induction rate of NN69 and WPM medium. The browning rate was highest in MS medium, reaching 64.4%, significantly higher than the other two media. WPM had the lowest browning rate of 35.6%, but the difference was not significant compared to NN69. Overall, NN69 is a more suitable medium for inducing etiolated stem segments in tissue culture of 'Fengdan'.

[0089] Table 1 Effects of different basic culture media on stem segment induction

[0090]

[0091] Experimental Group 2 - Design and Screening of Start-up Culture Medium Formula

[0092] Different concentrations of IBA and CPPU were added to the basic culture medium NN69 for a total of 6 treatments, as detailed in Table 2-1. 30 explants were inoculated in each treatment, repeated 3 times, and the bud induction rate was calculated after two weeks of culture.

[0093] Table 2-1 Start-up training program

[0094]

[0095] Different concentrations of IBA and CPPU were added to improve the efficiency of inducing adventitious buds from the yellowed leaf axils in treatment A. The bud induction rate was calculated after two weeks of cultivation. The treatment results are shown in Table 2-2. -1At 4 hr (treatment groups 2-3), the axillary bud induction rate reached a maximum of 75.6%, which was significantly higher than that of other treatments except treatment groups 2-5. At this time, the axillary buds isolated were in good condition and grew vigorously (e.g. Figure 2 a), so NN69+IBA 0.1 mg·L was selected -1 The culture medium is the optimal starting medium for axillary stem segments.

[0096] Table 2-2 Effects of different growth regulator combinations on primary culture

[0097]

[0098] Experimental Group 3 - Design and Screening of Proliferation Culture Medium Formula

[0099] After 45 days of initiation of culture (in this experiment, the 2-3 treatment groups were continued), the axillary buds with good separation status on the stem segments were cultured for proliferation. Different concentrations of IBA and CPPU were added to the culture medium. Due to the serious browning of the axillary buds during proliferation culture, a higher concentration of PVP (4 mg·L) was added to each treatment group. -1 See Table 3-1 for details. Each treatment group was inoculated with 20 explants. After 30 days of culture, the axillary bud proliferation coefficient was calculated using the following formula. It is worth noting that, based on theoretical analysis, proliferation is primarily influenced by cytokinins, specifically CPPU, which promote cell division and thus further proliferation. IBA is an auxin that generally promotes growth and rooting, so a treatment group supplemented with only IBA was not established at this stage.

[0100] Table 3-1 Subculture Program

[0101]

[0102] This experimental group added a higher concentration of PVP (4 mg·L -1 ) as an anti-browning agent. Table 3-2 shows the effects of different hormone combinations on axillary bud proliferation culture. The results showed that treatment group 3-5 had the highest proliferation coefficient, tissue culture seedlings grew well, and the fronds developed well and had a tendency to transform into plants (such as Figure 3 After comprehensive consideration, NN69+IBA0.1mg·L was selected. -1 +CPPU 0.4mg·L -1 +PVP 4mg·L -1 As a culture medium for axillary bud proliferation.

[0103] Table 3-2 Effects of different hormone combinations on axillary bud proliferation culture

[0104]

[0105] Experimental Group 4 - Design and Screening of Culture Media for Induction and Differentiation

[0106] 4-1 Primary callus induction

[0107] Cut the green leaves that have expanded after the start of culture and inoculate them into the induction medium. Add different concentrations of IBA and CPPU according to the method in Table 4-1. Each treatment group is added with PVP 3mg·L -1 Each bottle of culture medium was inoculated with 3 to 4 leaves, and 30 bottles were inoculated per treatment group, with three replicates. After 30 days of culture, the callus growth was observed, and the callus induction rate and browning rate of the leaves were calculated.

[0108] It's worth noting that callus induction and differentiation in woody plants is extremely difficult, and the researchers' success in inducing and differentiating primary leaves from the 'Fengdan' variety is truly remarkable. The entire process involves both dedifferentiation and redifferentiation, encompassing stages such as cell division, proliferation, development, and growth. Therefore, synergistic treatment with cytokinins and auxins yields superior results. After extensive research, it was found that nearly all plant induction and differentiation protocols utilize both auxins in combination, with no examples of either auxin used alone. Therefore, separate treatments using either IBA or CPPU were not included in this experiment.

[0109] Table 4-1 Callus induction test plan

[0110]

[0111] Different concentrations of IBA and CPPU were added to NN69 medium to screen out the most suitable concentration of callus induction growth regulator. The results of each treatment are shown in Table 4-2. When the CPPU concentration was 0.9 mg·L -1 When the concentration of IBA increased, the induction rate first increased and then decreased. -1 The inflection point was reached at 4-1-6, and the differences among the three treatments were significant. Therefore, the highest callus induction rate was achieved in 4-1-6 (67.8%), i.e., the culture medium was NN69 + IBA 0.3 mg·L -1 +CPPU0.9mg·L -1 +PVP 3mg·L -1 The callus induction rate was the highest when the callus was induced. Morphological observation showed that the callus structure was full of granular protrusions and the color was light green (such as Figure 4 a).

[0112] Table 4-2 Effects of different treatments on leaf callus induction

[0113]

[0114] 4-2 Adventitious bud differentiation of callus

[0115] After the leaf callus formed, it was planted in a medium containing different concentrations of IBA and CPPU according to the method in Table 4-3 to induce differentiation to produce adventitious buds. Each treatment group was inoculated with 30 bottles, repeated 3 times, and PVP 3 mg·L was added. -1 After 30 days of culture, the growth of adventitious buds was observed, and the differentiation rate and browning rate of adventitious buds were calculated.

[0116] Table 4-3 Callus differentiation test plan

[0117]

[0118] Different concentrations of IBA and CPPU were added to NN69 medium to screen out the most suitable concentration of growth regulator for adventitious bud differentiation. The results of each treatment are shown in Table 4-4. When the CPPU concentration was 0.2 mg·L -1 When the concentration of IBA increased, the callus differentiation rate showed a trend of first increasing and then decreasing, and the 4-2-4, that is, when the IBA concentration was 0.2 mg·L -1 The differentiation rate reached the inflection point at 54.4%, which was significantly higher than 4-2-1 (38.9%) and 4-2-7 (14.1%). Therefore, the optimal formula of callus differentiation medium is NN69 + IBA 0.2 mg·L -1 +CPPU 0.2mg·L -1 +PVP 3mg·L -1 , the highest differentiation rate was 54.4% (e.g. Figure 5 a).

[0119] Table 4-4 Effects of different treatments on leaf callus induction

[0120]

[0121] In the optimal callus differentiation formula NN69+IBA 0.2mg·L -1 +CPPU 0.2mg·L -1 +PVP 3mg·L -1 On this basis, scanning electron microscopy was used to observe the surface structure of the callus tissue of the 'Fengdan' leaf during the culture period to analyze the callus differentiation process from a microscopic perspective and to supplement the feasibility of the formula. It was found that after 10 days of culture, most of the surface cell tissues had not yet formed, and a small part had a tendency to develop into single spherical protrusions or large, vaguely shaped protrusions, with large areas of mucous membrane substances (such as Figure 6 After 15 days of culture, distinct independent spherical protrusions were observed, and the mucosal material attached to the surface was broken (as shown in a). Figure 6b); After 20 days of culture, the surface of the callus tissue was basically covered by cells that developed into spherical bodies. Due to the inconsistent growth rate, some spherical embryos had developed into cylindrical shapes. The growth was vigorous and the tissue structure was full. The entire development process was transformed from a few fuzzy block protrusions at the beginning to a single clear outline protrusion, and finally to a full long cylindrical column, becoming the precursor tissue of the adventitious bud (as shown in Figure 6 c).

[0122] Experimental Group 5 - Design and Screening of Light Quality Ratios for Strong Seedling Culture

[0123] The common fluorescent lamp was used as a control, and the light intensity was 30 μmol·m -2 s -1 Two light quality ratio treatments were set up: 5-2, full spectrum white light, light intensity of 50 μmol·m -2 s -1 ; 5-3, mixed red light and full spectrum white light are configured in a ratio of 7:1, and the light intensity is 30μmol·m -2 s -1 Specific treatments are detailed in Table 5-1. After 30 days of cultivation, the stem length of each treatment was counted to screen the best light quality ratio.

[0124] Table 5-1 Light quality ratio scheme of LED light processing

[0125]

[0126] Note: W and R represent full spectrum white light and red light respectively.

[0127] Under the illumination conditions of 5-2, i.e., full-spectrum white light, the tissue culture seedlings developed well, with an average stem length of up to 1.84 cm. Not only were they taller than the tissue culture seedlings treated with fluorescent light (average stem length 1.40 cm), but they also showed no obvious vitrification phenomenon, no callus tissue developed at the base, and strong stems and leaves. In particular, the proliferation coefficient was significantly improved (e.g., Figure 7 a).

[0128] The tissue culture seedlings grown under the irradiation conditions of 5-3, i.e., mixed full spectrum white light: red light = 7:1, did not show a significant increase in proliferation coefficient, but their stems were significantly longer, with an average stem length of 2.27 cm, and a distinct main trunk. It can be observed that the obvious difference from other treatments is that the stems are purple-red, with complete leaves developed at the top, and no callus growth or vitrification at the base. They have shown a tendency to develop into complete plants and are more suitable for rooting culture (e.g. Figure 7 b).

[0129] Experimental Group 6-Optimal Rooting Culture Method

[0130] Take tissue culture seedlings and inoculate them into rooting medium according to the method in Table 6-1. Treat 10 plants in each group. After 60 days of culture, count the rooting rate, number of roots and root length of the two rooting methods to screen the optimal rooting method and hormone concentration.

[0131] Table 6-1 Explant pretreatment methods

[0132]

[0133] In the rooting culture process of the one-step rooting method, it was observed that the rootless seedlings in the rooting medium under the irradiation of mixed red light and full-spectrum white light gradually took root regardless of whether the IBA growth regulator was added. However, the medium without IBA only began to take root at the end of the culture period (about 55 days), with a rooting rate of 40%. The average number of roots of the rooted tissue culture seedlings was 1.8 roots per plant, the average root length was 3.1 cm, and the root system was in good condition (such as Figure 8 a). Adding 0.2 mg·L -1 In IBA rooting medium, roots began to form around 45 days later, with a rooting rate of 60%. The average number of roots per plant was 2.7, and the average root length was 6.1 cm (e.g. Figure 8 b).

[0134] The two-step rooting method can significantly improve the rooting rate of tissue culture seedlings and reduce the quality requirements for rootless seedlings. Figure 8 c It can be seen that 0.2 mg·L -1 IBA root elongation medium can make rootless seedlings with a height of 2.5 to 3.0 cm take root, with a rooting rate of 70%. Rooting begins after about 35 days of cultivation. The average number of roots per plant is 1.2, and the average root length is 1.6 cm (e.g. Figure 8 c).

[0135] This method uses bud-bearing stem segments of the 'Feng Dan' plant as explants and successfully obtains 'Feng Dan' plantlets through tissue culture regeneration through induction, proliferation, and rooting. Simultaneously, a systematic 'Feng Dan' plantlet regeneration system is established through an indirect organogenesis pathway involving leaf callus induction and differentiation. This system offers advantages such as a high tissue culture seedling proliferation coefficient, high seedling success rate, low cost, and high returns. By utilizing plant tissue culture technology to increase the speed and scale of propagation, it enables factory-scale production of 'Feng Dan' seedlings.

[0136] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for rapid propagation and efficient regeneration of 'Fengdan' tissue culture, characterized in that: The method comprises S1 initiation culture, S2 proliferation culture and S3 rooting culture; the method specifically comprises the following steps: S1: preparing a priming culture medium comprising NN69+IBA; inoculating axillary stem segment explants with leaves sterilized with mercuric chloride and alcohol onto the priming culture medium for priming culture to obtain primary axillary buds and primary leaves; wherein the primary leaves are induced and differentiated to obtain differentiated adventitious buds; the IBA concentration in the priming culture medium is 0.1-0.2 mg / L; S2 proliferation culture: preparing a proliferation medium comprising NN69+IBA+CPPU+PVP; inoculating the differentiated adventitious buds onto the proliferation medium for proliferation culture to obtain unrooted tissue culture seedlings; the proliferation medium comprises an IBA concentration of 0.1-0.2 mg / L, a CPPU concentration of 0.2-0.6 mg / L, and a PVP concentration of 4 mg / L; S3 rooting culture: preparing a rooting medium, and performing rooting culture on the unrooted tissue culture seedlings using a one-step rooting method or a two-step rooting method to obtain complete tissue culture seedlings; The specific operations of the primary leaf induction and differentiation in step S1 are as follows: S1-1 Induction culture: Prepare an induction medium comprising NN69+IBA+CPPU+PVP, inoculate the primary leaf blade onto the induction medium, perform induction culture, and obtain callus tissue; the induction medium contains 0.3 mg / L IBA, 0.9 mg / L CPPU, and 3 mg / L PVP; S1-2 Differentiation culture: Prepare a differentiation medium comprising NN69+IBA+CPPU+PVP, inoculate the callus onto the differentiation medium, perform differentiation culture, and obtain differentiated adventitious buds; the differentiation medium comprises an IBA concentration of 0.2 mg / L, a CPPU concentration of 0.2 mg / L, and a PVP concentration of 3 mg / L.

2. The method according to claim 1, characterized in that The method further comprises a step of S2.5 seedling cultivation, which is arranged after S2 proliferation cultivation and before S3 rooting cultivation; The specific operation is that after completing the S2 proliferation culture, the unrooted tissue culture seedlings are exposed to light and cultured to strengthen the seedlings, so as to obtain strong and elongated unrooted tissue culture seedlings for subsequent rooting culture.

3. The method according to claim 2, characterized in that The lighting conditions are: (1) The LED light source is a mixture of full-spectrum white light and red light (W:R=7:1), and the light intensity is W=26.25 μmol·m -2 s -1 , R = 3.75 μmol·m -2 s -1 , where W and R represent full-spectrum white light and red light, respectively; or, (2) Full spectrum white light 50 μmol·m -2 s -1 .

4. The method according to claim 1, wherein The specific operation of the one-step rooting method in step S3 is to inoculate the unrooted tissue culture seedlings into the rooting medium for rooting culture. The composition of the rooting medium is "NN69 + 3mg·L - 1 CaCl2" or "NN69+3mg·L -1 CaCl2+0.2mg·L -1 IBA”.

5. The method according to claim 1, wherein The specific operations of the two-step rooting method in step S3 are: The unrooted tissue culture seedlings were cold-treated and inoculated into a root formation medium for culture. The root formation medium composition was NN69 + 3 mg·L -1 CaCl2+0.2mg·L -1 IAA, tissue culture seedlings with initial rooting were obtained; The tissue culture seedlings that had initially taken root were transferred to a root elongation medium for culture. The root elongation medium consisted of "NN69 + 6 mg L -1 CaCl2" or "NN69+6mg·L -1 CaCl2+0.2mg·L -1 IBA", and finally obtained complete tissue culture seedlings.

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