Phoebe tenuifolia seed germination and tissue culture rapid propagation method

Abstract

The invention belongs to the technical field of seedling raising, and particularly relates to a phoebe tenuifolia seed germination and tissue culture rapid propagation method. According to the method for germinating the phoebe davidii seeds, the phoebe davidii seeds are cultured through a 1 / 2 MS culture medium of NAA and AC to obtain aseptic seedlings, the seed germination rate is increased to 95% or above, and the problems that the phoebe davidii seeds are difficult to preserve, the seeding emergence rate is low, and production requirements cannot be met are solved. According to the method, growth hormones with different concentrations and types are added in different stages of adventitious bud induction culture, multiplication culture and rooting culture, so that the adventitious bud induction success rate reaches 90% or above, the multiplication coefficient reaches 6.0 or above, and the rooting rate reaches 96% or above; meanwhile, the problem of easy browning of explant induction culture is solved, and the survival rate and the rapid propagation efficiency of the tissue culture rapid propagation stage are greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of seedling cultivation, and in particular relates to a method for germination and tissue culture rapid propagation of Machilus tenuifolia seeds. Background Art

[0002] Phoebe hui is a plant of the genus Phoebe, mainly distributed in Sichuan, Chongqing, Hunan and other places in my country. Sichuan is the core distribution area of ​​Phoebe hui. Phoebe hui wood is mostly yellow or light yellow-brown in color. High-quality large-diameter wood is called "golden silk nanmu" because of its golden silk-like luster. It is one of the best tree species recognized by the current timber industry as "golden silk nanmu" and has broad market and application prospects in the precious timber industry. The latest research results of the Chinese Academy of Forestry show that the Forbidden City, an ancient building in my country, was built with Phoebe hui as the main wood material. Due to the extremely high economic, ecological and ornamental value of Phoebe hui, the demand for seedlings is increasing. However, there are very few high-quality seedlings on the market, which is difficult to meet the market demand. Therefore, the research on the rapid propagation of Phoebe hui is of great significance. At present, Phoebe cinnamomi is mainly propagated through sowing and seedling cultivation. However, Phoebe cinnamomi seeds are difficult to store, the "big year and small year" phenomenon is very serious, and the seedlings vary greatly, which may lose the excellent traits of the mother tree. In addition, the emergence rate of Phoebe cinnamomi seed sowing and seedling cultivation is low, which cannot meet the demand for high-quality seedlings in afforestation production, and there is currently no cutting propagation technology for Phoebe cinnamomi. All these have seriously restricted the production of Phoebe cinnamomi seedlings.

[0003] Based on the above problems, prior art 1 (Phnom Penh plug seedling raising technology, Forestry Practical Technology, 2012) discloses a method for Phenix oxyphylla plug seedling raising, through seed treatment, substrate selection and treatment, plug selection, greenhouse plug seedling management and other technical measures, the best seedling raising scheme for Phenix oxyphylla plug seedling raising is screened, but the seed germination rate of the best scheme is only 54.9%, which is too low to meet production needs; prior art 2 (Physiological response of Phenix oxyphylla seed dormancy and germination to low temperature stratification, Journal of Plant Sciences, 2022) studies the effects of different low temperature treatments on the germination rate of Phenix oxyphylla seeds, and analyzes the physiological and biochemical changes of low temperature stratification in the germination process of Phenix oxyphylla seeds, but only theoretical research results are proposed, and no specific technical operation methods are proposed. Based on this, a method for improving the seed germination rate and quickly obtaining sterile seedlings of Phenix oxyphylla and tissue culture rapid propagation technology is needed in this field. Summary of the invention

[0004] Based on the above problems, this application uses Macaoia seeds as explants and adopts the bud development approach to establish a method for improving the germination rate of Macaoia seeds and tissue rapid propagation technology, so as to quickly obtain sterile seedlings of Macaoia, realize the factory breeding of excellent genotypes of Macaoia, and solve the practical problems such as the lack of high-quality and strong seedlings of Macaoia.

[0005] To achieve the above-mentioned purpose, the first technical solution of the present application discloses a method for germinating Machilus philadelphus seeds, comprising the following steps:

[0006] The pretreated Machilus tenuifolia seeds were sterilized and then cultured in a 1 / 2MS medium containing NAA and AC to obtain sterile seedlings.

[0007] Furthermore, the concentration of NAA is 0.01 mg / L, and the concentration of AC is 0.2 mg / L.

[0008] Preferably, the culture temperature is 25°C ± 2°C, the light intensity is 2500 lux, the light duration is 12h / d, and the culture is for 15-20d.

[0009] The second technical solution of the present application discloses a method for rapid propagation of Machilus philadelphus through tissue culture, comprising the following steps:

[0010] Adventitious bud induction culture: the cotyledonary nodes of the sterile seedlings obtained by the seed germination method according to any one of claims 1 to 3 are inoculated as explants on an adventitious bud induction medium to obtain adventitious buds from bud induction;

[0011] Proliferation culture: cutting the adventitious buds and inoculating them into the proliferation medium for proliferation culture to obtain the proliferating buds;

[0012] Rooting culture: cutting the stem segments with proliferating buds and buds into rooting medium for rooting induction culture to obtain tissue culture seedlings;

[0013] Seedling hardening: Harden the tissue culture seedlings to obtain the Machilus philadelphus seedlings.

[0014] Furthermore, the adventitious bud induction medium is an MS medium containing 6-BA and IBA, wherein the concentration of 6-BA is 2 mg / L and the concentration of IBA is 0.05 mg / L.

[0015] Furthermore, the proliferation medium is an MS medium containing 6-BA and IBA, wherein the concentration of 6-BA is 3 mg / L and the concentration of IBA is 0.01 mg / L.

[0016] Furthermore, the rooting medium is a 1 / 2MAS medium containing NAA and activated carbon, wherein the NAA is 0.01 mg / L and the activated carbon is 0.2 g / L.

[0017] Furthermore, the rooting induction culture temperature is 25°C±2°C, the light intensity is 2500lux, and the light duration is 12h / d.

[0018] Furthermore, the seedling hardening is obtained by moving the culture bottle containing the tissue culture seedlings outdoors for hardening in a closed bottle under strong light and then hardening in an open bottle under natural light.

[0019] The present invention has the following beneficial effects:

[0020] The present application provides a method for germinating Machilus philadelphus seeds, wherein sterile seedlings are obtained by culturing Machilus philadelphus seeds in a 1 / 2MS culture medium containing 0.01 mg / L NAA and 0.2 g / L AC, thereby increasing the seed germination rate to more than 95%, thereby solving the problems of Machilus philadelphus seeds being difficult to preserve and having a low sowing germination rate that cannot meet production needs.

[0021] Based on the above-mentioned seed germination to obtain sterile seedlings, the present application proposes a method for tissue culture and rapid propagation of Machilus philadelphus, by adding culture media of different concentrations and types in different stages of adventitious bud induction culture, proliferation culture, and rooting culture to adjust the growth conditions, so that the adventitious bud induction success rate in the adventitious bud induction culture stage reaches more than 90%, and at the same time solves the problem that explant induced culture is prone to browning; the proliferation coefficient in the proliferation culture stage reaches more than 6.0, and the rooting rate in the rooting culture stage reaches more than 96%, which greatly improves the survival rate and rapid propagation efficiency in the tissue culture rapid propagation stage. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specifically stated, the relative arrangement of the parts and steps set forth in these embodiments, numerical expressions and numerical values ​​do not limit the scope of the application. The technology, method and equipment known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology, method and equipment should be considered as a part of the authorization specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0024] To facilitate understanding, the technical terms appearing in the specification are first explained.

[0025] The term "MS medium" means: Murashige and Skoog medium, wherein 1 / 2MS medium is 1 / 2Murashige and Skoog medium;

[0026] The term "NAA" stands for: naphthylacetic acid;

[0027] The term “AC” stands for: activated carbon;

[0028] The term "6-BA" stands for: 6-benzylaminopurine;

[0029] The term "IBA" stands for: indolebutyric acid.

[0030] In a first aspect, the present application discloses a method for germinating Machilus philadelphus seeds, comprising the following steps:

[0031] The pretreated Machilus tenuifolia seeds were sterilized and then cultured in a 1 / 2MS medium containing NAA and AC to obtain sterile seedlings.

[0032] It can be understood that in this embodiment, obtaining pre-treated Machilus philadelphus seeds includes collecting and processing seeds, which is a conventional method in the field. The present application is preferably: in the seed collection stage, preferably Machilus philadelphus superior trees (straight trunks, free of diseases and pests, and large fruit yields) are collected.

[0033] After collection, the seeds of Phoebe philodendron are pretreated. The pretreatment is a conventional solution in this field. The preferred method in this application is to remove the outer seed coat with a knife, rinse with running water, select seeds with full seeds and no pests and diseases, and use a scalpel to peel off the inner seed coat wrapped on the surface of the embryo (be careful not to damage the embryo, so as not to induce the production of secondary metabolites during sterile sowing and affect the quality of sterile sowing). Subsequently, the seeds with the seed coat removed are placed under tap water for 10 minutes, soaked in clean water with an appropriate amount of detergent for 10 minutes, and the visible debris on the surface of the embryo is removed with a soft brush, and finally placed under tap water for 24 hours.

[0034] In a preferred embodiment, the culture temperature of the present application is 25°C ± 2°C, the light intensity is 2500 lux, the light duration is 12h / d, and the culture is 15-20d.

[0035] In a further embodiment, the present application screens the optimal dosage of growth hormone in the germination stage of Machilus tenuifolia seeds, so that the seed germination rate can reach more than 95% under the optimal dosage, specifically:

[0036] Example 1: Experiment on germination of Machilus philadelphica seeds

[0037] Experimental method: rinse the seeds with the seed coat removed under running water for 10 minutes, add an appropriate amount of detergent and soak them in clean water for 10 minutes, and use a soft brush to remove visible debris on the surface of the embryo, and finally rinse them under running water for 24 hours. The pretreated seeds of Phoebe nanmu were placed on a clean bench and sterilized with 0.1% mercuric chloride, where the sterilization time was set to: 0.5min, 1min, 1.5min, 3min, 5min, 10min. After rinsing with sterile water 6 to 8 times, they were inoculated in 1 / 2MS medium containing different NAA and AC concentration ratios for culture, where the NAA concentration was set to 0.005mg / L, 0.01mg / L, 0.02mg / L, 0.03mg / L, 0.04mg / L, 0.05mg / L; the AC concentration was set to 0.2g / L. Each experimental treatment was repeated 5 times, and 20 bottles were inoculated in each treatment. The contamination situation and seed germination situation were observed and counted after 15-20 days. The contamination situation statistics are shown in Table 1, and the seed germination situation statistics are shown in Table 2.

[0038] The experimental results are shown in Table 1 and Table 2: As can be seen from Table 1, different sterilization times have different effects on contamination rate and necrosis rate. When the sterilization time is 1.5 minutes, the contamination rate of the seeds of Machilus thunbergii is 5.34%, and the necrosis rate is 1.03%. The effects of different sterilization times on contamination rate and necrosis rate are comprehensively compared, and the best sterilization method for Machilus thunbergii seeds is screened out as: 0.1% mercuric chloride sterilization for 1.5 minutes.

[0039] As can be seen from Table 2, different NAA concentrations have significant differences in the effects on the germination of Machilus chinensis seeds. The germination time of Machilus chinensis seeds shows a trend of first decreasing and then increasing with the increase of NAA concentration; the germination rate shows a trend of first increasing and then decreasing with the increase of NAA concentration. It can be seen that low concentrations of NAA promote the germination of Machilus chinensis seeds, and high concentrations of NAA inhibit seed germination. When the NAA concentration is 0.01 mg / L, the germination time is the shortest and the germination rate is the highest. The best medium formula for Machilus chinensis seed germination is screened out as: 1 / 2MS+0.01mg / LNAA+0.2g / LAC

[0040] Table 1 Effect of disinfection and sterilization time on germination of Machilus philadelphica seeds

[0041]

[0042] Table 2 Effect of NAA concentration on germination of Machilus philadelphica seeds

[0043]

[0044] In a second aspect, the present application discloses a method for rapid propagation of Machilus philadelphus through tissue culture, comprising the following steps:

[0045] Adventitious bud induction culture: the cotyledon nodes of the sterile seedlings obtained by the above seed germination method are inoculated as explants onto the adventitious bud induction medium to obtain adventitious buds from the buds;

[0046] Proliferation culture: cutting the adventitious buds and inoculating them into the proliferation medium for proliferation culture to obtain the proliferating buds;

[0047] Rooting culture: cutting the stem segments with proliferating buds and buds into rooting medium for rooting induction culture to obtain tissue culture seedlings;

[0048] Seedling hardening: Harden the tissue culture seedlings to obtain the Machilus philadelphus seedlings.

[0049] It should be noted that in the above-mentioned tissue culture rapid propagation method, the applicant adjusts the growth conditions by adding culture medium of different concentrations and types at different culture stages, so that the success rate of adventitious bud induction in the adventitious bud induction culture stage reaches more than 90%, and at the same time solves the problem that the explant induction culture is easy to brown; the proliferation coefficient in the proliferation culture stage reaches more than 6.0, and the rooting rate in the rooting culture stage reaches more than 96%, which greatly improves the survival rate and rapid propagation efficiency of the tissue culture rapid propagation stage. The following examples describe in detail the addition of growth hormones at different culture stages.

[0050] Example 2 Adventitious bud induction culture experiment

[0051] This experiment involves the selection of explant material for the sterile seedlings obtained in Example 1 and the selection of growth hormone and concentration in the culture medium.

[0052] Experiment 1: Selection of explant materials

[0053] Experimental method: The sterile seedlings germinated in the experiment of Example 1 were used as the source of explant materials, and MS medium was used as the basic medium. In this experiment, 2.0 mg / L 6-BA and 0.05 mg / L IBA were added to the medium to screen the explant materials. The explant materials were derived from different parts of the sterile seedlings, namely, the cotyledon nodes, epicotyls, and stem segments with terminal buds of the sterile seedlings. This step of the experiment had three treatments, each with 5 replicates, and each treatment was inoculated with 20 bottles.

[0054] The experimental results are shown in Table 3: It can be seen from Table 3 that when the basic culture medium and hormone concentrations are the same, the adventitious bud induction effects of different explants are different, among which the average number of adventitious buds induced by the cotyledonary node is 6, the average number of adventitious buds induced by the epicotyl is 3, and the average number of adventitious buds induced by the stem segment with buds is 1. Obviously, in comparison, the effect of inducing adventitious buds in the cotyledonary node is better than that of the epicotyl and the stem segment with terminal buds. The best explant material screened out is the cotyledonary node of the sterile seedling.

[0055] Table 3 Effect of adventitious bud induction of different explants

[0056]

[0057] Experiment 2: Growth hormone and concentration selection in culture medium

[0058] Experimental method: Through experiment 1, cotyledon nodes were selected as explant materials for the next test. This experiment used MS as the basic culture medium to explore the difference in the induction effect of 6-BA and IBA at different concentrations on adventitious buds. The 6-BA concentrations were set to 1.0 mg / L, 2.0 mg / L, and 5.0 mg / L, and the IBA concentrations were set to 0.01 mg / L, 0.05 mg / L, and 1.0 mg / L for a completely randomized multi-factor design experiment. Each experimental treatment was inoculated with 20 bottles and 5 replicates.

[0059] The experimental results are shown in Table 4. It can be seen from Table 4 that different concentration ratios of 6-BA and IBA have significant differences in the induction effect on adventitious buds. The induction rate and growth conditions of adventitious buds are different under different hormone concentration ratios. Among them, when the 6-BA concentration in the culture medium is 2.0 mg / L and the IBA concentration is 0.05 mg / L, the induction effect on adventitious buds is best, and the induction rate of adventitious buds is 89.34%. The induced adventitious buds grow well, with large buds and many leaves.

[0060] Table 4 Effects of different hormone concentration ratios on adventitious bud induction

[0061]

[0062] Example 3 Proliferation culture experiment

[0063] Experimental method: When the adventitious buds grow to 1cm-2cm, they are transferred to the proliferation medium and a completely randomized multi-factor design experiment is conducted to explore the effect of adding different types and concentrations of cytokinins or different types and concentrations of auxins to the culture medium on the proliferation of adventitious buds. In this step, MS is used as the basic culture medium. The types of cytokinins added are 6-BA, TDZ, 2ip, and ZT. The cytokinin concentrations are set to 0.5mg / L, 1.0mg / L, 2.0mg / L, and 3.0mg / L; the types of auxins added are IBA, NAA, and IAA, and the auxin concentrations are set to 0.01mg / L, 0.05mg / L, and 0.1mg / L. Further, after the cytokinins and auxins suitable for the proliferation of adventitious buds of Phoebe styloides are screened out through experiments, the experiment is then designed to explore the effect of the concentration ratio of cytokinins and auxins on the proliferation of adventitious buds. Each experimental treatment is inoculated with 20 bottles and 5 replicates.

[0064] The experimental results are shown in Tables 5 to 7. Table 5 shows that the four cytokinins have different induction effects on adventitious buds, among which 3 mg / L 6-BA has the best induction effect. The adventitious buds induced by 6-BA at this concentration are numerous and tender green, with stretched leaves and a proliferation coefficient of up to 5.83. As shown in Table 6, among the three auxins, IBA with a concentration of 0.01 mg / L has a better growth effect than other types and concentrations. Its proliferated adventitious buds are tender green, thick, with stretched leaves, and have good growth. Through the screening of the above experiments, we selected 6-BA with a concentration of 3.0 mg / L and IBA with concentrations of 0.005 mg / L, 0.01 mg / L, and 0.05 mg / L, which have a better effect on the proliferation effect, for further experiments. The experimental results are shown in Table 7. When the concentration of 6-BA is fixed at 3.0 mg / L, the proliferation coefficient and the number of effective buds increase first and then decrease with the increase of IBA concentration. When the concentration of IBA is 0.01 mg / L, the proliferation coefficient is the largest and the number of effective buds is the largest, and the adventitious bud growth is better. Therefore, through the above experiments, the proliferation medium suitable for the proliferation of adventitious buds of Phoebe philodendron was selected as

[0065] MS+3.0mg / LNAA+0.01mg / LIBA.

[0066] Table 5 Effects of different types and concentrations of cytokinins on adventitious bud proliferation

[0067]

[0068]

[0069] Table 6 Effects of different types and concentrations of auxin on adventitious bud proliferation

[0070]

[0071] Table 7 Proliferation effect under different hormone concentration ratios

[0072]

[0073] Example 4 Rooting culture experiment

[0074] Experimental method: The adventitious buds proliferated from the above experiment were used as materials. When the adventitious buds grew to 2cm-3cm, a 1.5cm stem segment with buds was cut and inoculated into the rooting medium. The rooting medium used 1 / 2MS medium as the basic medium, and 0.2g / LAC was added to the basic medium. NAA and IBA of different concentrations were added to the medium. The concentration gradient was set to 0.01mg / L, 0.05mg / L, 0.1mg / L, 0.2mg / L, and 0.5mg / L. The experiment adopted a completely randomized multi-factor design experiment to screen out the hormone type and concentration of the best rooting medium. Each experimental treatment was inoculated with 20 bottles and 5 replicates.

[0075] The experimental results are shown in Table 8: The experimental results in Table 8 show that the rooting rate shows a trend of gradually decreasing with the increase of NAA and IBA concentrations. It can be seen that low concentrations of auxin promote root induction, while high concentrations of auxin inhibit root induction. In addition, it can be seen from Table 8 that the root induction effect of auxin NAA is better than that of IBA. When the concentration of NAA is 0.01 mg / L, the rooting rate is as high as 96.5%. Therefore, the best root induction medium can be screened as: 1 / 2MS+0.2g / LAC+0.01mg / LNAA.

[0076] Table 8 Different concentrations of IBA and NAA induced root

[0077]

[0078] Finally, the tissue culture seedlings obtained by culture are subjected to seedling hardening treatment, preferably the tissue culture seedlings obtained on the rooting medium and meeting the transplanting conditions are hardened, first the culture bottle is moved to an outdoor shade shed for strong light closed bottle hardening for 15 days with a shading degree of 70%, then the lid of the culture bottle is opened and the seedlings are hardened under natural light for 5 days, and the shade shed is used for shading when the light is strong at noon to avoid burning the seedlings, and after the hardening is completed, the Machilus philadelphus seedlings are obtained.

[0079] The above embodiments are only preferred embodiments of the present application, and the scope of implementation of the present application is not limited thereto. The embodiments and features in the embodiments of the present invention can be arbitrarily combined with each other without conflict. Any technician familiar with the technical field can easily think of changes or replacements within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. A method for germinating Machilus philadelphus seeds, characterized in that: The steps include: The pretreated Machilus tenuifolia seeds were sterilized and then cultured in a 1 / 2MS medium containing NAA and AC to obtain sterile seedlings.

2. The seed germination method according to claim 1, characterized in that: The concentration of NAA is 0.01 mg / L, and the concentration of AC is 0.2 mg / L.

3. The seed germination method according to claim 1, characterized in that: The culture temperature is 25°C ± 2°C, the light intensity is 2500 lux, the light duration is 12h / d, and the culture is 15-20d.

4. A method for rapid propagation of Machilus tenuifolia by tissue culture, characterized in that: The steps include: Adventitious bud induction culture: the cotyledonary nodes of the sterile seedlings obtained by the seed germination method according to any one of claims 1 to 3 are inoculated as explants on an adventitious bud induction medium to obtain adventitious buds from bud induction; Proliferation culture: cutting the adventitious buds and inoculating them into the proliferation medium for proliferation culture to obtain the proliferating buds; Rooting culture: cutting the stem segments with proliferating buds and inserting them into the rooting medium for rooting induction culture to obtain tissue culture seedlings; Seedling hardening: Harden the tissue culture seedlings to obtain the Machilus philadelphus seedlings.

5. The tissue culture rapid propagation method according to claim 4, characterized in that: The adventitious bud induction medium is an MS medium containing 6-BA and IBA, wherein the concentration of 6-BA is 2 mg / L and the concentration of IBA is 0.05 mg / L.

6. The tissue culture rapid propagation method according to claim 4, characterized in that: The proliferation medium contains 6-BA and IBA MS medium, in which the 6-BA concentration was 3 mg / L and the IBA concentration was 0.01 mg / L.

7. The tissue culture rapid propagation method according to claim 4, characterized in that: The rooting medium is a 1 / 2MAS medium containing NAA and activated carbon, wherein the NAA content is 0.01 mg / L and the activated carbon content is 0.2 g / L.

8. The tissue culture rapid propagation method according to claim 7, characterized in that: The rooting induction culture temperature is 25°C±2°C, the light intensity is 2500 lux, and the light duration is 12h / d.

9. The tissue culture and rapid propagation method of Machilus tenuifolia according to claim 4, characterized in that: The seedling hardening is obtained by moving the culture bottle containing the tissue culture seedlings outdoors for strong light closed bottle hardening and then opening the bottle for natural light hardening.

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