CN115633639A - Culture medium combination for tissue culture and rapid propagation of buddleja plants and application thereof - Google Patents

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium combination for tissue culture and rapid propagation of buddleja and application thereof. The culture medium combination comprises an induction differentiation culture medium, a proliferation successive transfer culture medium and a strong seedling rooting culture medium, wherein the culture medium combination integrates the induction and differentiation culture medium into one, integrates the proliferation and successive transfer culture medium into one, integrates the strong seedling and rooting culture medium into one, integrates the culture mediums of all the steps into one on the basis of guaranteeing the best states of induction and differentiation, proliferation and successive transfer, strong seedling and rooting effects, simplifies the culture medium type and tissue culture steps of the buddleia plants, not only simplifies the tissue culture procedure, but also reduces the production cost, realizes the rapid propagation of the buddleia plants, and can obtain buddleia plant seedlings in a short time.

Description

Culture medium combination for tissue culture and rapid propagation of buddleja plants and application thereof

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium combination for tissue culture and rapid propagation of buddleja and application thereof.

Background

The Buddleja delavayi belongs to Scrophulariaceae, buddleja, and is a unique plant in China. It is 1-6 m high shrub or small arbor, produced in mountain sparse forest or hillside shrub bush with elevation of 2000-3000 m in Yunnan Bingchuan, luxi and Tibet ink doff, linzhi, etc., and the conic cymbidium terminal grows from top or axillary, and has high ornamental value in garden.

The buddleja davidii wild distribution area is narrow, the population quantity is small, the buddleja davidii wild distribution area is a typical wild plant with a very small population, the seeds are small, the collection is difficult, and the tissue culture method has great significance in expanding propagation and protecting. The VU is listed in 'book of species threatened by higher plants in China' in 2017, which is a category of easily dangerous VU. However, there are no reports on the tissue culture rapid propagation of buddleia and related biotechnology so far, and therefore, a tissue culture rapid propagation method for buddleia is urgently needed.

Disclosure of Invention

The invention aims to provide a culture medium combination for tissue culture and rapid propagation of buddleja and application thereof, wherein the culture medium combination simplifies the types and the steps of culture mediums for tissue culture of buddleja, realizes rapid propagation of buddleja, and solves the problems of sporadic field distribution, rare population and imminent extinction of buddleja.

The invention provides a culture medium combination for tissue culture and rapid propagation of buddleja plants, which comprises an induced differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium;

the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-0.5mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of IAA, 0.1-0.5mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4.

Preferably, the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2-0.5 mg/L of 6-BA, 0.5-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein the pH value is 5.8-6.4;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2-0.5 mg/L of 6-BA, 0.2-0.5 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein the pH is 5.8-6.4;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2-0.5 mg/L of IAA, 0.2-0.5 mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4.

Preferably, the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5.0g/L of agar, and the pH value is 6.0;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2mg/L of 6-BA, 0.2mg/L of IAA, 30g/L of sucrose and 5.0g/L of agar, wherein the pH value is 6.0;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2mg/L IAA, 0.2mg/L IBA, 30g/L sucrose and 5.0g/L agar, wherein the pH value is 6.0.

Preferably, the culture medium combination further comprises a transplanting culture medium, wherein the transplanting culture medium comprises perlite, humus and raw rosewood; the volume ratio of the perlite to the humus to the raw rosewood is (1-2): (1-2): (1-3).

The invention also provides application of the culture medium combination in the technical scheme in the tissue culture of the buddleja plants.

Preferably, the buddleja plant comprises buddleja glandularis.

The invention provides a method for tissue culture and rapid propagation of buddleia officinalis, which adopts the culture medium combination of the technical scheme to culture buddleia officinalis to obtain buddleia officinalis aseptic rooted seedlings.

Preferably, the method comprises the following steps: carrying out first culture on the sterilized buddleia glandulifera explant in an induced differentiation culture medium to obtain lateral buds and adventitious buds;

carrying out secondary culture on the lateral buds and the adventitious buds in a proliferation subculture medium to obtain a rootless plantlet;

and (3) performing third culture on the rootless clump seedlings in a strong seedling rooting culture medium to obtain the buddleja glandularis aseptic rooting seedlings.

Preferably, the temperature of the first culture is 23 to 25 ℃, the illumination intensity is 1500 to 2000LUX, and the illumination period is (10 to 12) L: (12-14) D, culturing for 45-60 days;

the temperature of the second culture is 23-25 ℃, the illumination intensity is 1500-2000 LUX, and the illumination period is (10-12) L: (12-14) D, culturing for 45-60 days;

the temperature of the third culture is 23-25 ℃, the illumination intensity is 1500-2000 LUX, and the illumination period is (10-12) L: (12-14) D, wherein the culture time is 30-45D.

Preferably, the method further comprises the step of carrying out fourth culture on the buddleia glandulosa sterile rooted seedlings in a transplanting matrix to obtain buddleia glandulosa seedlings;

the fourth culture temperature is 23-25 ℃, the relative air humidity is 70-80%, and the relative matrix humidity is 50-60%.

Has the advantages that:

the invention provides a culture medium combination for the tissue culture and rapid propagation of buddleja plants, which comprises an induction and differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium, wherein the culture medium combination integrates the induction and differentiation culture medium, the proliferation and subculture culture medium and the strong seedling and rooting culture medium, and combines the culture mediums of all the steps on the basis of ensuring the best induction and differentiation, proliferation and subculture, strong seedling and rooting effects, so that the tissue culture procedure is simple, the production cost is reduced, the culture medium type and tissue culture steps of the buddleja plants are simplified, the rapid propagation of the buddleja plants is realized, and the buddleja plant seedlings can be obtained in a short time.

Meanwhile, the invention also provides the application of the culture medium combination in the tissue culture of the buddleja, the culture medium combination is applied to the tissue culture of the buddleja, the induced differentiation rate and the rooting rate are high, the propagation period is only 45-60 d, and the propagation rate of the buddleja glandularis is greatly improved. Meanwhile, the problems of sporadic distribution, rare population and imminent extinction of inebrietum plants including inebrietum glandulifera can be solved; the genetic stability and consistency of the buddleia officinalis are highly maintained; provides technical support for the protection and breeding, introduction and domestication, preservation and large-scale production of the species, and fills the blank of research and development of the buddleia plants on biotechnology.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.

FIG. 1 shows axillary buds and adventitious buds of Buddleja glandulifera in example 1;

FIG. 2 is a rootless clumped seedling of Buddleja glandulifera in example 2;

FIG. 3 is a diagram of a sterile rooted seedling of Buddleja glandulifera in example 3.

Detailed Description

The invention provides a culture medium combination for tissue culture and rapid propagation of buddleja plants, which comprises an induced differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium; the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4; the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-0.5mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4; the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of IAA, 0.1-0.5mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4.

The induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar. The concentration of 6-BA in the induced differentiation culture medium is 0.1-0.5mg/L, preferably 0.2-0.5 mg/L, and more preferably 0.2mg/L; the concentration of the IAA is 0.1-1.0 mg/L, preferably 0.5-1.0 mg/L, and more preferably 0.5mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5 to 6.0g/L, preferably 5.0g/L. The pH of the differentiation induction medium is 5.8-6.4, preferably 6.0. The hormone combination and the content in the induction differentiation medium can realize the one-step induction of adventitious buds by buddleia plants, the induction medium and the differentiation medium are combined into a whole, the operation steps are simplified, the effect of inducing the adventitious buds is good, and the induction rate of the adventitious buds and the average number of the induced adventitious buds are both obviously improved.

The induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-0.5mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar. The concentration of 6-BA in the induced differentiation culture medium is 0.1-0.5mg/L, preferably 0.2-0.5 mg/L, and more preferably 0.2mg/L; the concentration of the IAA is 0.1-0.5mg/L, preferably 0.2-0.5 mg/L, and more preferably 0.2mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5 to 6.0g/L, preferably 5.0g/L. The pH of the differentiation induction medium is 5.8-6.4, preferably 6.0. The specific hormone combination and content in the proliferation subculture medium can realize the integration of proliferation and subculture of the buddleja plants, the operation steps are simplified, the obtained rootless seedlings have no glass seedlings, and the multiplication times are high.

The strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of IAA, 0.1-0.5mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar. The concentration of 6-BA in the strong seedling rooting culture medium is 0.1-0.5mg/L, preferably 0.2-0.5 mg/L, and more preferably 0.2mg/L; the concentration of the IAA is 0.1-0.5mg/L, preferably 0.2-0.5 mg/L, and more preferably 0.2mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5 to 6.0g/L, preferably 5.0g/L. The pH of the differentiation induction medium is 5.8-6.4, preferably 6.0. The combination and the content of the specific hormones in the strong seedling rooting culture medium can realize the integration of strong seedling culture and rooting culture of the buddleja plants, simplify the operation steps, and have good rooting and strong seedling effects, and the rooting rate can reach 100%.

The culture medium combination preferably further comprises a transplanting culture medium, wherein the transplanting culture medium preferably comprises perlite, humus and raw rosewood; the volume ratio of the perlite to the humus to the raw rosewood is preferably (1-2): (1-2): (1 to 3), more preferably 1:2:3. according to the invention, the proportion of the perlite, the humus soil and the raw red soil is specified, so that the porosity of the matrix and the air permeability of the root are increased, and sufficient nutrition is provided for the root; meanwhile, laterite is soil deep in the soil layer, the bacteria carrying amount is less, the growth of aseptic seedlings is facilitated, and the transplanting survival rate of the buddleia glandularis can reach 95% by using the transplanting culture medium.

The culture medium combination provided by the invention integrates the induction culture medium and the differentiation culture medium into one, integrates the proliferation culture medium and the subculture culture medium into one, integrates the seedling strengthening culture medium and the rooting culture medium into one, simplifies the operation steps of tissue culture of the buddleia plants, is easy to grow seedlings, has high effective reproduction rate, realizes the rapid propagation of the buddleia plants, solves the problems of sporadic distribution, rare population quantity and imminent extinction of the buddleia plants in the field, lays a foundation for the comprehensive protection, development and continuous utilization of the buddleia plants and germplasm resource preservation, and fills the blank of research and development of the buddleia plants on biotechnology.

The invention also provides application of the culture medium combination in the technical scheme in the tissue culture of the buddleja plants. The buddleja plant of the present invention preferably comprises buddleja glandularis. The culture medium combination is used for carrying out tissue rapid propagation culture on the buddleia plants including the buddleia, so that the propagation quantity and the growth rate of the buddleia are greatly improved, and technical support is provided for the protection and propagation, introduction and domestication, preservation and scale production of the species.

The invention provides a method for tissue culture and rapid propagation of buddleia officinalis, which adopts the culture medium combination of the technical scheme to culture buddleia officinalis to obtain buddleia officinalis aseptic rooted seedlings.

The invention carries out the first culture of the disinfected inebria glandulifera explant in an induced differentiation culture medium to obtain lateral buds and adventitious buds. The size of the explant of the invention is preferably 1cm of small stem segments with nodes.

The buddleia glandularis explant preferably comprises terminal buds and/or side buds of buddleia glandularis, and further preferably terminal buds; the terminal and lateral buds are preferably young terminal and lateral buds. The method for disinfecting the buddleia officinalis explant preferably comprises the following steps of: soaking the buddleia adenophora explant for 3-5min by using an explant disinfectant with the volume percentage content of 10%, and washing the soaked buddleia adenophora explant by using filtered water to obtain a clean buddleia adenophora explant; and sequentially disinfecting the clean buddleia officinalis explant by using an alcohol solution with the volume concentration of 75% and mercuric chloride with the mass concentration of 0.1%, and washing with sterile water for 3-5 times to obtain the disinfected buddleia officinalis explant. The source of the explant disinfectant is not particularly limited, and the explant disinfectant can be prepared from the conventional explant disinfectant in the field, such as a Wanjin brand disinfectant. The invention preferably washes the soaked buddleia glandulosa explants 3 times with filtered water. In the present invention, the alcohol solution is preferably sterilized for 10 seconds, and the mercuric chloride is preferably treated for 5 to 7min, more preferably 6min. After the clean inebriant brevifolius explant is sterilized by using 75% alcohol solution by mass, the method preferably further comprises the step of washing the clean inebriant brevifolius explant by using sterile water for 3 times. The sterilization method of the invention can lead the aseptic rate of the buddleia explant to reach more than 80 percent.

After the sterilized buddleia adenophora explant is obtained, the sterilized buddleia adenophora explant is subjected to first culture in an induced differentiation culture medium to obtain a side bud and an adventitious bud. The sterilized buddleia adenophora explants are preferably placed in the differentiation induction culture medium flatly. When the explant is terminal bud or axillary bud, the inoculation amount of the sterilized buddleia glandulosa explant is preferably 3-5 terminal buds or axillary buds/100 cm ² More preferably 3 terminal buds or axillary buds/100 cm ² (ii) a The temperature of the first culture is preferably 23-25 ℃, and more preferably 25 ℃; the light intensity is preferably 1500 to 2000LUX, more preferably 1600 to 1800LUX, and still more preferably 1800LUX; the light period is preferably (10 to 12) L: (12 to 14) D, more preferably (11 to 12) L: (12 to 13) D, more preferably 12L:12D; the time is preferably 45 to 60 days, more preferably 50 to 55 days; more preferably 50d. In bookUnder the specific induction and differentiation culture conditions, the buddleia marina induction and differentiation lateral bud growth vigor is good.

After the lateral buds are obtained, the lateral buds are transferred to a multiplication subculture medium for secondary culture to obtain rootless plantlets. The temperature of the second culture is preferably 23-25 ℃, and more preferably 24-25 ℃; the light intensity is preferably 1500 to 2000LUX, more preferably 1600 to 1800LUX, and still more preferably 1800LUX; the light cycle is preferably (10 to 12) L: (12-14) D; more preferably (11 to 12) L: (12 to 13) D, more preferably 12L:12D; the time is preferably 40 to 60 days, more preferably 45 to 60 days, and still more preferably 50 days. Under the specific multiplication subculture medium and the specific culture conditions, the buddleia glandulosa rootless seedlings grow well.

After the rootless plantlets are obtained, the rootless clump plantlets are subjected to third culture in a strong seedling rooting culture medium to obtain the buddleia aseptic rooted plantlets. The temperature of the third culture is preferably 23-25 ℃, and more preferably 24-25 ℃; the light intensity is preferably 1500 to 2000LUX, more preferably 1600 to 1800LUX, and still more preferably 1800LUX; the light period is preferably (10 to 12) L: (12-14) D; more preferably (11 to 12) L: (12 to 13) D, more preferably 12L:12D; the time is preferably 30 to 45d, more preferably 30 to 40d, and still more preferably 35d. Under the specific condition of strong seedling rooting culture, the buddleia marina rooting rate is 100%, and the growth vigor is good.

After the buddleia glandularis aseptic rooting seedling is obtained, the buddleia glandularis aseptic rooting seedling is preferably hardened. The invention preferably carries out seedling exercising in a greenhouse. In the present invention, the light-shielding rate of the acclimatized seedling is preferably 70 to 85%, more preferably 75 to 80%, and still more preferably 75%; the temperature is preferably 23-25 ℃; even more preferably 24 ℃; the relative humidity of the air is preferably 40 to 50 percent, and more preferably 40 to 45 percent; more preferably 45%; the time is preferably 7 to 10d, more preferably 7 to 8d, and still more preferably 8d. The seedling hardening of the invention can improve the transplanting survival rate of buddleia.

After the hardening off is finished, the hardened off aseptic rooting seedlings of the buddleia areolata are preferably transplanted into a transplanting matrix for fourth cultivation, and buddleia areolata seedlings are obtained. Before the transplanting, the transplanting substrate is preferably sprayed with 800-1000 times of carbendazim to complete disinfection. After the sterilization is completed, the present invention preferably further comprises adjusting the pH of the transplanting substrate to 6.2 to 6.4, and then transplanting. The fourth culture temperature is preferably 23-25 ℃, and more preferably 24-25 ℃; the substrate humidity is preferably 50% to 60%, more preferably 50% to 55%, and still more preferably 55%; the relative humidity of the air is preferably 70% to 80%, more preferably 70% to 75%, and still more preferably 75%. The specific transplanting conditions of the method ensure the high survival rate of the transplanted buddleja davidii rooting seedlings, and the survival rate can reach 95%.

By adopting the method for the tissue culture and rapid propagation of the buddleia officinalis, the induction differentiation and rooting rate of the buddleia officinalis are high, the propagation period is only 45-60 d, the propagation rate of the buddleia officinalis is greatly improved, and technical support is provided for the protection and propagation, introduction and domestication, storage and scale production of the species.

In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Induced differentiation medium screening

Taking young terminal buds or side buds of buddleia hainanensis as explants, soaking the young terminal buds or side buds of buddleia hainanensis for 3-5min by using 5% of Wanjin spray disinfectant, washing the buds clean by running water, taking the buds on a super clean bench, removing leaves, cutting the buds into small stem sections with 1cm in size, disinfecting the small stem sections with 75% of alcohol for 5-10 s, washing the small stem sections with sterile water for 3 times, disinfecting the small stem sections with 0.1% of mercuric chloride solution for 5-6 min, washing the small stem sections with the sterile water for 3-5 times, inoculating the small stem sections into a prepared induction culture medium, and inserting 1 bud section into each bottle;

the stem segments are respectively inoculated on a culture medium with the components of MS +0.1-0.5 mg/L6-BA + 0.1-1.0 mg/L IAA, the culture medium also comprises 30g/L of cane sugar, 5g/L of agar and 5.8 of pHs. The light intensity is 1000-2000LUX, the temperature is 23-30 ℃, and the illumination time is 8-10 hours.

The statistical results are shown in Table 1.

TABLE 1 Alangium adenophorum induction medium screening

Remarking: in the table "-" indicates no callus; "+" indicates a small amount of callus "+" indicates more callus.

The results in Table 1 show that the induction rate of the buddleia side buds on MS +0.1-0.5 mg/L6-BA + 0.1-1.0 mg/L IAA culture medium reaches 40% -60%; the best induction effect of MS +0.2mg/L6-BA +0.5mg/L IAA is shown in figure 1. The invention preferentially selects the components with small callus and high side bud or terminal bud inductivity as the induced differentiation culture medium.

Example 2

Proliferation subculture medium screening

Taking an MS culture medium as a proliferation subculture medium, and taking lateral buds obtained by MS +0.2mg/L6-BA +0.5mg/L IAA in example 1 as a plant material screening basic culture medium, wherein the cytokinin 6-BA is provided with 3 gradients which are respectively 0.1mg/L,0.2mg/L and 0.5mg/L; 3 gradients of 0.1mg/L,0.2mg/L and 0.5mg/L are set for auxin IAA, and a uniform design method is adopted to screen a proper proliferation subculture medium. Each multiplication subculture medium also contains 30g/L of sucrose, 5.5g/L of agar and 5.8 of pHs. The conditions of proliferation and subculture are as follows: light intensity is 1800LUX, temperature is 25 ℃, and illumination period is 10L:24D, the culture period is 50D. The statistical results are shown in Table 2.

TABLE 2 proliferation Medium hormone screening

Remarking: in the table "-" indicates no callus; "+" indicates a small amount of callus; "+ +" indicates more callus.

Proliferation factor = number of shoots of active shoots/number of inoculated shoots;

as can be seen from Table 2, in MS culture medium +0.1-0.5 mg/L6-BA (6-benzylpurine) +0.1-0.5mg/L IAA, sucrose 30g/L + agar 5.5g/L, pH5.8, culture period 45-60 days, light intensity 1800LUX, average effective bud number (including apical bud) at 23-25 ℃ of 3-6, multiplication coefficient 3-6, no glass seedling, fast growth speed, plant height 5-7.0cm, stem thickness 1-2 mm. See fig. 2.

Example 3

Screening of strong seedling rooting culture medium

MS culture medium is used as basic culture medium for selecting strong seedling and rooting culture medium hormone, effective bud nodes and cluster buds obtained by combining MS, 0.2mg/L6-BA and 0.2mg/L IAA in example 2 are used as research objects, and the strong seedling rooting culture medium is selected. 3 gradients of 0.1mg/L,0.2mg/L and 0.5mg/L for auxin IAA and 3 gradients of 0.1mg/L,0.2mg/L and 0.5mg/L for IBA are set for IBA, and proper seedling and rooting culture media are screened. The rooting culture medium for each strong seedling also contains 30g/L of sucrose, 5g/L of agar and 6.0 of pH. The conditions of strong seedling and rooting culture are as follows: the light intensity is 1800LUX, the temperature is 23-25 ℃, the illumination period is 10L:14D, culture period 30D. The statistical results of the strong seedling and rooting culture for 30 days are shown in Table 3.

TABLE 3 rooting Medium hormone screening results

Remarking: in the table "-" indicates no callus; "+" indicates a small amount of callus "+" indicates more callus;

the results show that: in MS culture medium +0.1-0.5mg/L IAA (indoleacetic acid), MS culture medium +0.1-0.5mg/L IBA (indolebutyric acid) and MS culture medium +0.1-0.5mg/L IAA (indoleacetic acid) +0.1-0.5mg/L IBA (indolebutyric acid, sucrose 30g/L, agar 5g/L, pH 5.8-6.4, culture period 30 days, rooting rate reaches 100%, plant growth is obviously faster in MS +0.2mg/L IAA +0.2mg/L IBA, the number of roots is more, and there is no callus at the roots, see figure 3.

Example 4

Screening of transplanting medium

The buddleia glandulifera rooting aseptic seedling obtained by MS +0.5 mg/L6-BA +1.0mg/L NAA culture medium in the embodiment 3 is transferred to a greenhouse hardening seedling with the shading rate of 75-85% one week in advance. Transplanting the rooted aseptic seedling after hardening seedling into different transplanting culture media, and screening the proper transplanting culture media. The experimental transplanting culture media are respectively as follows: raw laterite, perlite, a humus soil/raw laterite mixed matrix and a perlite/humus soil/raw laterite mixed matrix, wherein the volume ratio of humus soil to raw laterite in the humus soil/raw laterite mixed matrix is 1:1, the volume ratio of the perlite to the humus soil to the raw laterite in the perlite/humus soil/raw laterite mixed matrix is as follows: 1:2:3. spraying 800-1000 times of carbendazim on the transplanting culture medium, mixing with soil, sealing and disinfecting for 7 days by using a plastic film, adjusting the pH value to 6.2-6.4, and then transplanting. Spraying water and covering a plastic film in time after transplanting, wherein the temperature of the greenhouse is 24-25 ℃, and the humidity of the matrix is 40-50%. The number of transplants per treatment was 20, and the experiment was repeated 3 times. And after 30d of transplanting, counting the transplanting survival rate of each treatment. The results are shown in Table 4.

TABLE 4 selection of transplanting Medium

The results in table 4 show that the transplanting survival rate of the mixed matrix of perlite, humus soil and raw laterite is the highest, and the survival rate after 30d transplanting reaches 95%; meanwhile, the shape of the transplanted buddleia marina after flowering is consistent with that of the female parent, and further, the culture medium combination and the method provided by the invention are adopted to highly maintain the genetic stability and consistency of the buddleia marina.

The embodiment can show that the culture medium combination provided by the invention can obtain the buddleia nitida seedlings in a short time, the induced differentiation rate is 60% in 45-60 days, the proliferation coefficient is 6 in 45-60 days, the rooting rate is 100%, the transplanting survival rate is 95%, the proliferation coefficient of the buddleia nitida is greatly improved, and a very effective propagation method is provided for introduction and domestication, preservation, gardening and utilization of the commercial and scientific research values of the species.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Claims (10)

1. A culture medium combination for tissue culture and rapid propagation of buddleja plants is characterized in that the culture medium combination comprises an induced differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium;

the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of 6-BA, 0.1-0.5mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein the pH is 5.8-6.4;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.1-0.5mg/L of IAA, 0.1-0.5mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4.

2. The culture medium combination according to claim 1, wherein the induced differentiation culture medium takes an MS culture medium as a basic culture medium, and further comprises 0.2-0.5 mg/L of 6-BA, 0.5-1.0 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2-0.5 mg/L of 6-BA, 0.2-0.5 mg/L of IAA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises 0.2-0.5 mg/L of IAA, 0.2-0.5 mg/L of IBA, 30g/L of sucrose and 5.5-6.0 g/L of agar, and the pH value is 5.8-6.4.

3. The culture medium combination according to claim 2, wherein the differentiation induction culture medium is a basic culture medium based on MS culture medium, and further comprises 0.2mg/L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5.0g/L of agar, and has pH of 6.0;

the proliferation subculture medium takes an MS culture medium as a basic culture medium, and also comprises 6-BA0.2mg/L, IAA0.2mg/L, 30g/L of sucrose and 5.0g/L of agar, wherein the pH is 6.0;

the strong seedling rooting culture medium takes an MS culture medium as a basic culture medium, and also comprises IAA0.2mg/L, IBA0.2mg/L, sucrose 30g/L and agar 5.0g/L, wherein the pH value is 6.0.

4. The culture medium combination according to any one of claims 1 to 3, further comprising a transplanting culture medium comprising perlite, humus soil and raw rosewood; the volume ratio of the perlite to the humus to the raw rosewood is (1-2): (1-2): (1-3).

5. Use of the culture medium combination of any one of claims 1 to 4 for tissue culture of buddleja.

6. The use of claim 5, wherein the buddleja plant comprises buddleja glandulosa.

7. A method for tissue culture and rapid propagation of buddleia, which is characterized in that buddleia is cultured by adopting the culture medium combination of any one of claims 1 to 4 to obtain buddleia aseptic rooting seedlings.

8. The method of claim 7, comprising the steps of: carrying out first culture on the sterilized buddleia glandulifera explant in an induced differentiation culture medium to obtain lateral buds and adventitious buds;

carrying out secondary culture on the lateral buds and the adventitious buds in a proliferation subculture medium to obtain a rootless plantlet;

and (3) performing third culture on the rootless clump seedlings in a strong seedling rooting culture medium to obtain the buddleja glandularis aseptic rooting seedlings.

9. The method according to claim 8, wherein the first culture is carried out at a temperature of 23 to 25 ℃, a light intensity of 1500 to 2000LUX, a light cycle of (10 to 12) L: (12-14) D, culturing for 45-60 days;

the temperature of the second culture is 23-25 ℃, the illumination intensity is 1500-2000 LUX, and the illumination period is (10-12) L: (12-14) D, culturing for 45-60 days;

the temperature of the third culture is 23-25 ℃, the illumination intensity is 1500-2000 LUX, and the illumination period is (10-12) L: (12-14) D, wherein the culture time is 30-45D.

10. The method according to any one of claims 7 to 9, further comprising subjecting the buddleia aseptic rooted seedlings to a fourth cultivation in a transplanting substrate to obtain buddleia seedlings;

the fourth culture temperature is 23-25 ℃, the relative air humidity is 70-80%, and the relative substrate humidity is 50-60%.

Images