Seed germination and tissue culture rapid propagation method of machilus sinensis

Abstract

The application belongs to the technical field of seedling culture and particularly relates to a seed germination and tissue culture and rapid propagation method of Phoebe sheareri. First, a seed germination method of Phoebe sheareri is provided, and the sterile seedlings are obtained by culturing the seeds of Phoebe sheareri in 1 / 2MS medium containing NAA and AC, so that the seed germination rate is increased to more than 95%, and the problems of great difficulty in seed storage, low seedling emergence rate and failure to meet the production demand of Phoebe sheareri are solved. Further, on the basis of the sterile seedlings obtained by the seed germination, a tissue culture and rapid propagation method of Phoebe sheareri is provided, different concentrations and types of growth hormones are added in different stages of adventitious bud induction culture, proliferation culture and rooting culture, so that the success rate of adventitious bud induction is more than 90%, the proliferation coefficient is more than 6.0, and the rooting rate is more than 96%, and the problem of easy browning of the explant induction culture is solved, and the survival rate and rapid propagation efficiency in the tissue culture and rapid propagation stage are greatly improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of seedling raising, and particularly relates to a seed germination and tissue culture and rapid propagation method of Phoebe hui. BACKGROUND

[0002] Phoebe hui is a plant of the genus Phoebe and mainly distributed in Sichuan, Chongqing and Hunan of China. Sichuan is the core distribution area of Phoebe hui. The wood of Phoebe hui is mostly yellow or light yellowish brown. The high-quality large-diameter wood is called “gold silk Phoebe wood” due to its golden yellow silk-like luster and is one of the optimal tree species of “gold silk Phoebe wood” recognized by the current wood industry, which has a broad market and application prospect in the precious wood industry. The latest research results of the Chinese Academy of Forestry show that the Forbidden City, a Chinese ancient building, is built mainly of Phoebe hui. Phoebe hui has extremely high economic, ecological and ornamental values, and the demand for seedlings is increasing. However, there are few good seedlings on the market, which cannot meet the market demand. Therefore, the research on the rapid propagation of Phoebe hui is of great significance. At present, Phoebe hui is mainly propagated by sowing and seedling raising. However, the seeds of Phoebe hui are difficult to store, the “large and small year” phenomenon is very serious, and the seedling variation is large, which may lose the excellent traits of the mother tree. In addition, the seed germination rate of Phoebe hui is low, which cannot meet the demand for high-quality seedlings in afforestation production. Moreover, there is no cutting propagation technology for Phoebe hui, which seriously restricts the production of Phoebe hui seedlings.

[0003] Based on the above problems, the prior art 1 (Phoebe hui plug seedling technology, forestry practical technology, 2012) discloses a method for plug seedling of Phoebe hui. The best seedling scheme for plug seedling of Phoebe hui is obtained through seed treatment, substrate selection and treatment, plug selection and greenhouse plug seedling management. However, the seed germination rate of the best scheme is only 54.9%, which is too low to meet the production demand. The prior art 2 (Physiological response of Phoebe hui seed dormancy and germination to low temperature stratification, Plant Science Reports, 2022) studies the effect of different low temperature treatments on the seed germination rate of Phoebe hui and analyzes the physiological and biochemical changes of low temperature stratification on the seed germination process of Phoebe hui. However, only theoretical research results are proposed, and no specific technical operation method is proposed. Therefore, a method for improving the seed germination rate and rapidly obtaining sterile seedlings and tissue culture and rapid propagation technology of Phoebe hui is needed in the field. SUMMARY

[0004] Based on the above problems, the present application uses Phoebe hui seeds as explants and adopts the bud occurrence pathway to establish a method for improving the seed germination rate of Phoebe hui and a tissue culture and rapid propagation technology, so as to rapidly obtain sterile seedlings of Phoebe hui and realize the factory propagation of the excellent genotype of Phoebe hui, thereby solving the practical problems such as the lack of good seedlings of Phoebe hui.

[0005] To achieve the above object, the first technical scheme of the present application discloses a seed germination method of Phoebe sheareri, comprising the following steps:

[0006] After the pretreated P. sheareri seeds are sterilized, the seeds are placed in 1 / 2MS medium containing NAA and AC for culture to obtain sterile seedlings.

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

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

[0009] The second technical scheme of the present application discloses a tissue culture and rapid propagation method of P. sheareri, comprising the following steps:

[0010] Adventitious bud induction culture: the cotyledon node of the sterile seedlings obtained by the seed germination method is used as an explant to be inoculated on an adventitious bud induction medium to obtain adventitious buds by bud induction;

[0011] Proliferation culture: the adventitious buds are cut and inoculated into a proliferation medium for proliferation culture to obtain proliferated buds;

[0012] Rooting culture: the proliferated buds with stems are cut and inoculated into a rooting medium for rooting induction culture to obtain tissue culture seedlings;

[0013] Seedling hardening: the tissue culture seedlings are subjected to seedling hardening treatment to obtain P. sheareri seedlings.

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

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

[0016] Further, the rooting medium is 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] Further, the rooting induction culture temperature is 25℃±2℃, the light intensity is 2500 lux, and the light duration is 12h / d.

[0018] Further, the seedling hardening is to move the culture bottle containing the tissue culture seedlings to the outdoor for strong light closed bottle seedling hardening, and then to natural light open bottle seedling hardening.

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

[0020] The application provides a seed germination method of Machilus sinensis, and the seed germination rate of Machilus sinensis is increased to more than 95% by culturing the seeds of Machilus sinensis in 1 / 2MS culture medium containing 0.01 mg / L NAA and 0.2 g / L AC to obtain sterile seedlings, so that the problem of Machilus sinensis seed preservation difficulty, low seedling emergence rate and failure to meet production needs is solved.

[0021] On the basis of the above-mentioned seed germination to obtain sterile seedlings, the application provides a tissue culture and rapid propagation method of Machilus sinensis, different concentrations and types of culture medium are added in different stages of adventitious bud induction culture, proliferation culture and rooting culture to regulate the growth, so that the success rate of adventitious bud induction in the adventitious bud induction culture stage is more than 90%, and the problem of easy browning of the explant induction culture is solved; the proliferation coefficient in the proliferation culture stage is more than 6.0, the rooting rate in the rooting culture stage is more than 96%, and the survival rate and rapid propagation efficiency in the tissue culture and rapid propagation stage are greatly improved. DETAILED DESCRIPTION

[0022] The technical solutions of the application will be described clearly and completely below in combination with the embodiments of the application. Obviously, the described embodiments are only some of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0023] Unless otherwise specified, the relative arrangement, numerical expressions and values of the components and steps set forth in these embodiments do not limit the scope of the application. The technology, methods and devices known to those skilled in the related art can not be discussed in detail, but should be considered as part of the authorized description under appropriate circumstances. In all examples shown and discussed here, any specific value should be interpreted as merely exemplary, rather than as a limitation. Therefore, other examples of the exemplary embodiments can have different values. It should be noted that similar reference numerals and letters represent similar items in the following drawings, so further discussion is not needed for the items once they are defined in one drawing.

[0024] For the convenience of understanding, the technical terms appearing in the specification are first explained.

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

[0026] The term "NAA" is naphthalene acetic acid;

[0027] The term "AC" is: activated carbon;

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

[0029] The term "IBA" is: indole-3-butyric acid.

[0030] In a first aspect, the application discloses a seed germination method of Machilus sinensis, comprising the following steps:

[0031] After sterilization, the pretreated Machilus sinensis seeds are placed in 1 / 2MS medium containing NAA and AC for culture to obtain sterile seedlings.

[0032] It can be understood that in the present embodiment, the obtaining of the pretreated Machilus sinensis seeds includes seed collection and treatment, which is a conventional method in the art. The application preferably comprises the following steps in the seed collection stage: collecting preferred Machilus sinensis trees (straight trunk, no pests and diseases, and large fruiting amount).

[0033] After collection, the Machilus sinensis seeds are pretreated. The pretreatment is a conventional scheme in the art, and the application preferably comprises the following steps: cutting off the outer seed coat with a small knife, washing clean with running water, selecting seeds with full seed kernels and no pests and diseases, peeling off the inner seed coat wrapped on the surface of the seed embryo with a scalpel (attention should be paid to not damaging the seed embryo to avoid inducing secondary metabolites during aseptic sowing and affecting the quality of aseptic sowing). Then, the seed coat removed seeds are washed under tap water for 10 minutes, soaked in water with an appropriate amount of detergent for 10 minutes, and the visible impurities on the surface of the seed embryo are removed with a soft brush. Finally, the seeds are washed under tap water for 24 hours.

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

[0035] In further embodiments, the optimal amount of growth hormone for the seed germination stage of Machilus sinensis is screened, so that the seed germination rate can reach more than 95% under the optimal amount, and the specific amount is as follows:

[0036] Example 1: Machilus sinensis seed germination experiment

[0037] Experimental method: the seed without seed coat was washed under tap water for 10 minutes, and then soaked in water with detergent for 10 minutes, and the visible impurities on the surface of the seed embryo were removed with a soft brush, and finally the seed was washed under tap water for 24 hours. The pre-treated Phoebe sheareri seed was placed on a clean bench and sterilized with 0.1% mercuric chloride, and the sterilization time was set as: 0.5min, 1min, 1.5min, 3min, 5min, 10min. After washing with sterile water for 6-8 times, it was inoculated into 1 / 2MS medium containing different NAA and AC concentration ratios, and the NAA concentration was set as 0.005mg / L, 0.01mg / L, 0.02mg / L, 0.03mg / L, 0.04mg / L, 0.05mg / L; the AC concentration was set as 0.2g / L. Each experimental treatment was repeated 5 times, 20 bottles were inoculated in each treatment, and the pollution and seed germination were observed and counted after 15-20d. The pollution statistics are shown in Table 1, and the seed germination statistics are shown in Table 2.

[0038] The experimental results are shown in Tables 1 and 2: from Table 1, it can be seen that different sterilization times have different effects on the pollution rate and the necrosis rate. When the sterilization time is 1.5min, the pollution rate of Phoebe sheareri seed is 5.34%, and the necrosis rate is 1.03%. By comprehensively comparing the effects of different sterilization times on the pollution rate and the necrosis rate, the best sterilization method for Phoebe sheareri seed is selected as: 0.1% mercuric chloride sterilization for 1.5min.

[0039] From Table 2, it can be seen that different NAA concentrations have a significant difference on the seed germination of Phoebe sheareri. The germination time of Phoebe sheareri seed decreases first and then increases with the increase of NAA concentration; the germination rate increases first and then decreases with the increase of NAA concentration. Therefore, low concentration of NAA promotes the seed germination of Phoebe sheareri, and high concentration of NAA inhibits the seed germination. When the NAA concentration is 0.01mg / L, the germination time is the shortest, and the germination rate is the highest. Therefore, the best medium formula for the seed germination of Phoebe sheareri is selected as: 1 / 2MS+0.01mg / L NAA+0.2g / L AC

[0040] Table 1 Effect of sterilization time on seed germination of Phoebe sheareri

[0041]

[0042] Table 2 Effect of NAA concentration on seed germination of Phoebe sheareri

[0043]

[0044] In the second aspect, the application discloses a tissue culture and rapid propagation method of Phoebe sheareri, which comprises the following steps:

[0045] Induction of adventitious buds: the cotyledon node of the aseptic seedling obtained by the seed germination method above is inoculated on the induction medium of adventitious buds to obtain the adventitious buds by bud induction;

[0046] Proliferation culture: the adventitious buds are inoculated on the proliferation medium to obtain the proliferated buds by proliferation culture;

[0047] Rooting culture: the proliferated buds with stems are cut and inoculated on the rooting medium to induce rooting culture, and the tissue culture seedlings are obtained;

[0048] Acclimatization: the tissue culture seedlings are acclimatized to obtain the Machilus japonica seedlings.

[0049] It should be noted that in the above tissue culture and rapid propagation method, the applicant adjusts the growth by adding different concentrations and types of culture media at different culture stages, so that the success rate of adventitious bud induction in the induction culture stage of adventitious buds reaches more than 90%, and the problem of easy browning of the explant induction culture is solved; 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%, greatly improving the survival rate and rapid propagation efficiency in the tissue culture and rapid propagation stage. The following examples will explain in detail the addition of growth hormones at different culture stages.

[0050] Example 2: Experiment of induction of adventitious buds

[0051] This experiment includes the selection of explant materials of the aseptic seedlings obtained in Example 1 and the selection of growth hormones and concentrations of the culture medium.

[0052] Test 1: Selection of explant materials

[0053] Experimental method: the aseptic seedlings obtained by germination in Example 1 are used as the source of explant materials, and MS medium is used as the basic medium. The selection of explant materials is carried out by adding 6-BA with a concentration of 2.0 mg / L and IBA with a concentration of 0.05 mg / L to the medium. The explant materials are derived from different parts of the aseptic seedlings, i.e. cotyledon node, epicotyl and stem segment with apical bud. This experiment has three treatments, each with 5 replicates, and 20 bottles are inoculated in each treatment.

[0054] The experimental results are shown in Table 3: from Table 3, it can be seen that when the basic medium and the hormone concentration are the same, the induction effects of adventitious buds of different explants are different, among which the average number of adventitious buds induced by cotyledon node is 6, the average number of adventitious buds induced by epicotyl is 3, and the average number of adventitious buds induced by stem segment with apical bud is 1. Obviously, compared with the above, the induction effect of adventitious buds by cotyledon node is better than that of epicotyl and stem segment with apical bud. The best explant material selected by this screening is the cotyledon node of the aseptic seedling.

[0055] Table 3: Induction effects of adventitious buds of different explants

[0056]

[0057] Medium selection for growth hormone and concentration selection

[0058] Experimental method: Cotyledon nodes selected by experiment 1 were used as explant material for the next experiment. MS was used as the basic medium to explore the differences in the effects of different concentrations of 6-BA and IBA on adventitious bud induction. The 6-BA concentrations were set at 1.0 mg / L, 2.0 mg / L, and 5.0 mg / L, and the IBA concentrations were set at 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 in 20 bottles with 5 replicates.

[0059] The experimental results are shown in Table 4. As shown in Table 4, different concentrations of 6-BA and IBA had significant differences in the induction effect of adventitious buds. The induction rate and growth status of adventitious buds were different under different hormone concentration ratios. When the 6-BA concentration was 2.0 mg / L and the IBA concentration was 0.05 mg / L, the induction effect of adventitious buds was the best, with an induction rate of 89.34%. The induced adventitious buds grew well, with large and leafy buds.

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

[0061]

[0062] Example 3 Proliferation culture experiment

[0063] Experimental method: When the adventitious buds grew to 1-2 cm, they were transferred to the proliferation medium for a completely randomized multi-factor design experiment to explore the effects of adding different types and concentrations of cytokinins or different types and concentrations of auxins to the medium on the proliferation of adventitious buds. The basic medium used in this experiment was MS, and the types of cytokinins added were 6-BA, TDZ, 2ip, and ZT, with cytokinin concentrations set at 0.5 mg / L, 1.0 mg / L, 2.0 mg / L, and 3.0 mg / L. The types of auxins added were IBA, NAA, and IAA, with auxin concentrations set at 0.01 mg / L, 0.05 mg / L, and 0.1 mg / L. Further, after screening the suitable cytokinins and auxins for the proliferation of adventitious buds of Phoebe parvifolia, an experiment was designed to explore the effects of cytokinin and auxin concentration ratios on the proliferation of adventitious buds. Each experimental treatment was inoculated in 20 bottles with 5 replicates.

[0064] The experimental results are shown in Tables 5-7. As shown in Table 5, the four kinds of cytokinins have different effects on the induction of adventitious buds. The 6-BA at a concentration of 3 mg / L has the best induction effect. The 6-BA at this concentration induces many tender green adventitious buds, the leaves are stretched, and the proliferation coefficient is as high as 5.83. As shown in Table 6, among the three kinds of auxins, the IBA at a concentration of 0.01 mg / L has a better growth effect than other kinds and concentrations. The proliferated adventitious buds are tender green, thick, the leaves are stretched, and the growth is better. Through the above screening experiments, the concentrations of 3.0 mg / L of 6-BA and 0.005 mg / L, 0.01 mg / L and 0.05 mg / L of IBA, which have a better effect on proliferation, are selected 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 the concentration of IBA. When the concentration of IBA is 0.01 mg / L, the proliferation coefficient is the largest and the number of effective buds is the most, and the growth of the adventitious buds is better. Therefore, through the above experiments, the proliferation medium suitable for the proliferation of Phoebe sheareri is selected as MS+3.0 mg / L NAA+0.01 mg / L IBA.

[0065] Table 5 Effects of different kinds and concentrations of cytokinins on the proliferation of adventitious buds

[0066]

[0067]

[0068] Table 6 Effects of different kinds and concentrations of auxins on the proliferation of adventitious buds

[0069]

[0070] Table 7 Proliferation effects under different hormone concentration ratios

[0071]

[0072] Example 4 Rooting culture experiment

[0073] Experimental method: The adventitious buds proliferated in the above experiment are used as materials. When the adventitious buds grow to 2-3 cm, the stem segments with buds of about 1.5 cm are cut and inoculated into the rooting medium. The rooting medium is 1 / 2MS medium as the basic medium, 0.2 g / L AC is added to the basic medium, and different concentrations of NAA and IBA are added, respectively. The concentration gradient is set as 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L and 0.5 mg / L. The best hormone type and concentration of the rooting medium are screened by a completely randomized multi-factor design experiment. 20 bottles are inoculated for each experimental treatment, and there are 5 replicates.

[0074] The experimental results are shown in Table 8: the experimental results of Table 8 show that the rooting rate gradually decreases with the increase of the concentration of NAA and IBA, thus it can be known that low concentration of auxin promotes the induction of roots, and high concentration of auxin inhibits the induction of roots. In addition, it can be known from Table 8 that the induction effect of auxin NAA on roots is better than that of IBA, and when the concentration of NAA is 0.01 mg / L, the rooting rate is as high as 96.5%, thus the best medium for inducing rooting can be screened as: 1 / 2MS+0.2 g / L AC+0.01 mg / L NAA.

[0075] Table 8: Different concentrations of IBA and NAA induce roots

[0076]

[0077] Finally, the tissue culture seedlings obtained by culture are subjected to hardening treatment, preferably the tissue culture seedlings obtained from the rooting culture medium and having transplanting conditions are subjected to hardening, the culture bottle is first moved to the outdoor shade shed for strong light closed bottle hardening for 15 days, the shading degree is 70%, then the culture bottle cover is opened, and the hardening is performed under natural light for 5 days, the shade shed is used to shade at the time of strong light in the middle of the day to avoid burning the seedlings, and the fine leaf nanmiao seedlings are obtained after the hardening is completed.

[0078] The above examples are only preferred examples of the present application, and the scope of the present application is not limited thereto, and the examples in the present application and the features in the examples can be combined with each other as long as there is no conflict. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed in the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for rapid propagation of *Machilus sieboldii* via tissue culture, characterized in that... Includes the following steps: Germination of Phoebe zhennan seeds: After sterilization, pretreated Phoebe zhennan seeds were cultured in 1 / 2 MS medium containing NAA and AC to obtain sterile seedlings; wherein the NAA concentration was 0.01 mg / L and the AC concentration was 0.2 mg / L; the Phoebe zhennan seeds were sterilized with 0.1% mercuric chloride for 1.5 min. Adventitious bud induction culture: Cotyledonary nodes of sterile seedlings were inoculated onto adventitious bud induction medium as explants to induce adventitious buds; Proliferation culture: Adventitious shoots are cut and inoculated into a proliferation medium for proliferation culture to obtain proliferating shoots; Rooting culture: Stem segments with buds that have proliferated are cut and inoculated into rooting medium for rooting induction culture to obtain tissue culture seedlings; Hardening off seedlings: Hardening off tissue culture seedlings to obtain small-leaved nanmu seedlings; The adventitious bud induction medium is 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; The proliferation medium is 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.

2. The tissue culture rapid propagation method according to claim 1, characterized in that, During the germination of the fine-leaved nanmu seeds, the culture temperature was 25℃±2℃, the light intensity was 2500 lux, the light duration was 12h / d, and the culture period was 15-20 days.

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

4. The tissue culture rapid propagation method according to claim 3, characterized in that, The rooting induction culture temperature is: 25℃±2℃, light intensity 2500 lux, light duration 12h / d.

5. The method for rapid propagation of *Machilus sieboldii* via tissue culture according to claim 1, characterized in that, The seedling hardening process involves moving the culture bottles containing tissue culture seedlings outdoors for hardening under strong light in a closed bottle, followed by hardening under natural light after opening the bottles.

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