CN115633639B - Culture medium combination for tissue culture and rapid propagation of buddleia 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 buddleia plants and application thereof. The culture medium combination comprises an induction differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium, wherein the culture medium combination combines the induction differentiation culture medium with the proliferation subculture medium, combines the strong seedling with the rooting culture medium with the proliferation subculture medium, and combines the culture mediums of all the steps into one on the basis of ensuring the optimal conditions of induction differentiation, proliferation subculture, strong seedling and rooting, thereby simplifying the tissue culture procedure, reducing the production cost, simplifying the culture medium type and tissue culture steps of the buddleia plants, realizing the rapid propagation of the buddleia plants and obtaining buddleia plant seedlings in a short time.

Description

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

Background

The buddleia glandula (Buddleja delavayi) belongs to the figwort family, the buddleia genus and is a special plant in China. The plant is 1-6 m high shrub or small arbor, is produced in mountain forest or hillside shrub with the altitude of 2000-3000 m in Yunnan Bingchun, luxi and Tibet ink drop, forest and glossy ganoderma, etc. and has high ornamental value in garden.

Because of the narrow field distribution area of the buddleia glandula, the buddleia glandula is a typical wild plant with a very small population, and the seed is smaller, so that the acquisition is difficult, and the expansion propagation and protection significance of the tissue culture method is great. 2017 is listed in the list of threat species for higher plants in China, which is a kind of easily dangerous VU. However, there has been no report so far on the tissue culture and rapid propagation of buddleia glandulosa and related biotechnology, and therefore, there is an urgent need for a tissue culture and rapid propagation method for buddleia glandulosa.

Disclosure of Invention

The invention aims to provide a culture medium combination for tissue culture and rapid propagation of buddleia plants and application thereof, wherein the culture medium combination simplifies the culture medium type and steps of buddleia plant tissue culture, realizes rapid propagation of buddleia plants, and solves the problems of sporadic distribution, sparse population quantity and extinct extinction of buddleia plants.

The invention provides a culture medium combination for tissue culture and rapid propagation of buddleia 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, IAA 0.1.1-1.0 mg/L of 6-BA, 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 0.1-0.5mg/L, IAA 0.1.1-0.5 mg/L of 6-BA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein 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, IBA 0.1.1-0.5 mg/L IAA, 30g/L sucrose and 5.5-6.0 g/L agar, wherein the pH value is 5.8-6.4.

Preferably, the induced differentiation culture medium is based on an MS culture medium and further comprises 0.2-0.5 mg/L, IAA 0.5.5-1.0 mg/L of 6-BA, 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, IAA 0.2.2-0.5 mg/L of 6-BA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein 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, IBA 0.2.2-0.5 mg/L IAA, 30g/L sucrose and 5.5-6.0 g/L agar, wherein the pH value is 5.8-6.4.

Preferably, the induced differentiation medium is based on an MS medium, and further comprises 0.2mg/L, IAA 0.5.5 mg/L of 6-BA, 30g/L of sucrose and 5.0g/L of agar, wherein the pH value is 6.0;

the proliferation subculture medium is based on an MS culture medium, and further comprises 0.2mg/L, IAA 0.2.2 mg/L of 6-BA, 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 IAA0.2mg/L, IBA 0.2.2 mg/L, sucrose 30g/L and agar 5.0g/L, 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 soil 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 buddleia plant tissue culture.

Preferably, the buddleia plant comprises buddleia glandula.

The invention provides a tissue culture and rapid propagation method of buddleia glandulosa.

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

performing second culture on the lateral buds and the adventitious buds in a proliferation subculture medium to obtain a root-free Cong Miao;

and carrying out third culture on the unrooted Cong Miao in a strong seedling rooting culture medium to obtain the aseptic root seedling of buddleia glandulosa.

Preferably, the temperature of the first 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 45-60D;

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, wherein the culture time is 45-60D;

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, the culturing time is 30-45D.

Preferably, the method further comprises the step of carrying out fourth culture on the aseptic root seedlings of the buddleia adenophylla in a transplanting matrix to obtain buddleia adenophylla seedlings;

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

The beneficial effects are that:

the invention provides a culture medium combination for tissue culture and rapid propagation of buddleia plants, which comprises an induction differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium, wherein the culture medium combination combines the induction differentiation culture medium with the proliferation subculture medium, combines the strong seedling with the rooting culture medium into one, combines the culture mediums of all steps into one on the basis of ensuring the optimal conditions of induction and differentiation, proliferation and subculture, and strong seedling and rooting, not only simplifies the tissue culture procedure, but also reduces the production cost, simplifies the culture medium type and tissue culture steps of buddleia plants, realizes rapid propagation of buddleia plants, and can obtain buddleia plant seedlings in a short time.

Meanwhile, the invention also provides application of the culture medium combination in the tissue culture of the buddleia plant, and the application of the culture medium combination in the tissue culture of the buddleia plant has high induced differentiation rate and rooting rate, and the propagation period is only 45-60 d, so that the propagation rate of buddleia glandula is greatly improved. Meanwhile, the problems of sporadic field distribution, sparse population quantity and extinct buddleja plants including buddleja glandula can be solved; the genetic stability and consistency of the buddleia glandula are highly maintained; the method provides technical support for the protection and breeding, introduction and domestication, preservation and large-scale production of the species, and fills the gap of the development of the buddleia plant in biotechnology.

Drawings

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

FIG. 1 shows the axillary buds and adventitious buds of buddleia glandula in example 1;

FIG. 2 is a root-less Cong Miao of buddleia adenophylla in example 2;

FIG. 3 is a sterile root seedling of Zostera glandulosa in example 3.

Detailed Description

The invention provides a culture medium combination for tissue culture and rapid propagation of buddleia 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, IAA 0.1.1-1.0 mg/L of 6-BA, 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 0.1-0.5mg/L, IAA 0.1.1-0.5 mg/L of 6-BA, 30g/L of sucrose and 5.5-6.0 g/L of agar, wherein 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, IBA 0.1.1-0.5 mg/L IAA, 30g/L sucrose and 5.5-6.0 g/L agar, wherein the pH value is 5.8-6.4.

The induced differentiation culture medium of the invention takes an MS culture medium as a basic culture medium and also comprises 0.1-0.5mg/L, IAA 0.1.1-1.0 mg/L of 6-BA, 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, more preferably 0.2mg/L; the IAA concentration is 0.1 to 1.0mg/L, preferably 0.5 to 1.0mg/L, more preferably 0.5mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5-6.0 g/L, preferably 5.0g/L. The pH of the induced differentiation medium is 5.8-6.4, preferably 6.0. The hormone combination and the content in the induction differentiation culture medium can realize the one-step induction of adventitious buds of the buddleia plant, the induction culture medium and the differentiation culture medium are combined into a whole, the operation steps are simplified, the effect of the induction of the adventitious buds is good, and the induction rate of the adventitious buds and the average number of the induced adventitious buds are obviously improved.

The induced differentiation culture medium of the invention takes an MS culture medium as a basic culture medium and also comprises 0.1-0.5mg/L, IAA 0.1.1-0.5 mg/L of 6-BA, 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, more preferably 0.2mg/L; the IAA concentration is 0.1 to 0.5mg/L, preferably 0.2 to 0.5mg/L, more preferably 0.2mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5-6.0 g/L, preferably 5.0g/L. The pH of the induced differentiation medium is 5.8-6.4, preferably 6.0. The specific hormone combination and content in the proliferation subculture medium can realize the combination of proliferation and subculture of buddleia plants, simplify the operation steps, and the obtained rooting-free plant has higher proliferation multiple because of no glass seedling generation.

The seedling strengthening and rooting culture medium takes an MS culture medium as a basic culture medium and also comprises 0.1-0.5mg/L, IBA 0.1.0.1-0.5 mg/L IAA, 30g/L sucrose and 5.5-6.0 g/L 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 IAA concentration is 0.1 to 0.5mg/L, preferably 0.2 to 0.5mg/L, more preferably 0.2mg/L; the concentration of the sucrose is 30g/L; the concentration of the agar is 5.5-6.0 g/L, preferably 5.0g/L. The pH of the induced differentiation medium is 5.8-6.4, preferably 6.0. The specific hormone combination and content in the strong seedling rooting culture medium can realize the combination of strong seedling and rooting culture of 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 soil and raw rosewood; the volume ratio of the perlite, the humus soil and the raw rosewood is preferably (1-2): (1-2): (1 to 3), more preferably 1:2:3. the specific proportion of the perlite, the humus and the raw laterite increases the porosity of the matrix and the air permeability of the root, and also provides enough nutrition for the root; meanwhile, the laterite is soil deep in a soil layer, the bacteria carrying amount is less, the growth of aseptic seedlings is facilitated, and the transplanting survival rate of buddleia glandulosa can reach 95% by using the transplanting culture medium.

The culture medium combination provided by the invention combines the induction culture medium and the differentiation culture medium, and the proliferation culture medium and the secondary culture medium, and combines the strong seedling culture medium and the rooting culture medium, so that the operation steps of the tissue culture of the buddleia plant are simplified, the seedling formation is easy, the effective propagation rate is high, the rapid propagation of the buddleia plant is realized, the problems of sporadic distribution, sparse population number and endangered extinguishment of the buddleia plant are solved, the foundation is laid for comprehensive protection, development and continuous utilization of the buddleia plant and the preservation of germplasm resources, and the development blank of the buddleia plant in biotechnology is filled.

The invention also provides application of the culture medium combination in buddleia plant tissue culture. The buddleia plant of the present invention preferably comprises buddleia glandula. The invention carries out tissue rapid propagation culture on buddleia plants including buddleia glandula, greatly improves the propagation quantity and growth rate of buddleia glandula, and provides technical support for the protection and propagation, introduction and domestication, preservation and large-scale production of the species.

The invention provides a tissue culture and rapid propagation method of buddleia glandulosa.

The invention carries out first culture on the sterilized buddleia glandulosa explant in an induced differentiation medium to obtain lateral buds and adventitious buds. The size of the explant is preferably 1cm of a small stem segment with a node.

The buddleia glandulosa explant preferably comprises terminal buds and/or lateral buds of buddleia glandulosa, and more preferably terminal buds; the terminal buds and lateral buds are preferably tender terminal buds and lateral buds. The disinfection method of the buddleia glandulosa explant preferably comprises the following steps: soaking the buddleja glandulifolia explant for 3-5min by using an explant disinfectant with the volume percentage of 10%, and washing the soaked buddleja glandulifolia explant by using filtered water to obtain a clean buddleja glandulifolia explant; and (3) sequentially sterilizing the clean buddleia glandulifera explant by using an alcohol solution with the volume concentration of 75% and mercuric chloride with the mass concentration of 0.1%, and flushing with sterile water for 3-5 times to obtain the sterilized buddleia glandulifera explant. The source of the explant disinfectant is not particularly limited, and the explant disinfectant is conventional in the art, such as Wanjin disinfectant. The invention preferably washes the soaked buddleia glandulosa explant 3 times with filtered water. In the present invention, the disinfection treatment time of the alcohol solution is preferably 10s, and the treatment time of the mercuric chloride is preferably 5 to 7min, more preferably 6min. The present invention preferably further comprises rinsing 3 times with sterile water after sterilizing the clean buddleia glandulosa explant with 75% by mass alcohol solution. The sterilization method can enable the sterility of the buddleia glandulosa explant to reach more than 80%.

After the sterilized buddleia glandulosa explant is obtained, the sterilized buddleia glandulosa explant is subjected to first culture in an induced differentiation medium to obtain lateral buds and adventitious buds. The present invention preferably places the sterilized buddleia adenophora explant flat in the induced differentiation medium. When the explant is a terminal bud or an axillary bud, the inoculation amount of the sterilized buddleia adenophora explant is preferably 3-5 terminal buds or axillary buds/100 cm ² Further preferably 3 terminal buds or axillary buds per 100cm ² The method comprises the steps of carrying out a first treatment on the surface of the The temperature of the first culture according to the present invention is preferably 23 to 25℃and more preferably 25 ℃; the illumination intensity is preferably 1500-2000 LUX, more preferably 1600-1800LUX, and even more preferably 1800LUX; the illumination 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. Under the specific induction and differentiation culture conditions, the buddleia glandula induced and differentiated lateral buds grow well.

After the lateral buds are obtained, the lateral buds are transferred into a proliferation subculture medium for second culture, and the rootless seedlings are obtained. The temperature of the second culture of the invention is preferably 23-25 ℃, and more preferably 24-25 ℃; the illumination intensity is preferably 1500 to 2000LUX, more preferably 1600 to 1800LUX, and even more preferably 1800LUX; the illumination period is preferably (10 to 12) L: (12-14) D; further 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 proliferation subculture medium and culture conditions, the buddleless young buddless of buddleia glandulosa grow well.

After the rootless plantlet is obtained, the rootless Cong Miao is subjected to third culture in a strong seedling rooting culture medium to obtain the buddleja glandulosa aseptic rooting seedling. The temperature of the third culture is preferably 23-25 ℃, and more preferably 24-25 ℃; the illumination intensity is preferably 1500 to 2000LUX, more preferably 1600 to 1800LUX, and even more preferably 1800LUX; the illumination period is preferably (10 to 12) L: (12-14) D; further 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 strong seedling rooting culture condition, the buddleia glandulosa rooting rate is 100%, and the growth vigor is good.

After the aseptic root seedlings of the buddleia glandulosa are obtained, the aseptic root seedlings of the buddleia glandulosa are preferably subjected to seedling hardening. The seedling hardening is preferably carried out in a greenhouse. In the present invention, the shading rate of the seedling is preferably 70 to 85%, more preferably 75 to 80%, and even more preferably 75%; the temperature is preferably 23-25 ℃; even more preferably 24 ℃; the relative humidity of the air is preferably 40-50%, more preferably 40-45%; more preferably 45%; the time is preferably 7 to 10 days, more preferably 7 to 8 days, and still more preferably 8 days. The seedling hardening can improve the transplanting survival rate of buddleia glandulosa.

After the seedling hardening is completed, the aseptic root seedlings of the buddleia adenophora are preferably transplanted into a transplanting matrix for fourth culture, so that the buddleia adenophora seedlings are obtained. Before the transplanting, the invention preferably uses 800-1000 times carbendazim to spray the transplanting matrix and then completes disinfection. After the sterilization is completed, the method preferably further comprises the step of adjusting the pH of the transplanting matrix to 6.2-6.4, and then transplanting. The temperature of the fourth culture is preferably 23-25 ℃, and more preferably 24-25 ℃; the humidity of the substrate is preferably 50% to 60%, more preferably 50% to 55%, and even more preferably 55%; the relative humidity of air is preferably 70% to 80%, more preferably 70% to 75%, and even more preferably 75%. The specific transplanting conditions of the invention ensure the high survival rate of the transplanted buddleia glandulosa root seedlings, which can reach 95%.

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

The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.

Example 1

Induced differentiation Medium Screen

Taking young terminal buds or lateral buds of buddleia glandulosa as explants, soaking the young terminal buds or lateral buds in 5% of gold spray disinfectant for 3-5min, washing the buddleia glandulosa with running water, taking the buddleia glandulosa onto an ultra-clean bench, removing leaves, cutting the buddleia glandulosa into small stem segments with the size of 1cm, sterilizing the stem segments with the small stem segments with the size of 5-10 s with 75% alcohol, washing the stem segments with sterile water for 3 times, sterilizing the stem segments with 0.1% mercuric solution for 5-6 min, washing the stem segments with sterile water for 3-5 times, inoculating the stem segments into a prepared induction culture medium, and inserting 1 bud segment into each bottle;

the stem sections are respectively inoculated on a culture medium with the composition of MS+0.1-0.5 mg/L6-BA+0.1-1.0 mg/L IAA, and the culture medium also comprises 30g/L sucrose, 5g/L agar and pH value of 5.8. 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 buddleia glandulosa induction medium screening

Remarks: in the table "-" indicates no callus; "+" indicates a small number of calli "++" indicates a large number of calli.

As shown by the results in the table 1, the induction rate of the buddleia glandula lateral buds is 40% -60% when the buddleia glandula lateral buds are induced on MS+0.1-0.5 mg/L6-BA+0.1-1.0 mg/L IAA culture medium; wherein the induction effect of MS+0.2mg/L6-BA+0.5mg/L IAA is the best, and the induction condition is shown in figure 1. The invention preferably selects the components with small callus and high side bud or terminal bud induction rate as the induced differentiation culture medium.

Example 2

Proliferation subculture Medium selection

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

TABLE 2 proliferation Medium hormone screening

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

Proliferation coefficient = number of shoots of effective shoots/number of inoculated shoots;

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

Example 3

Screening of rooting culture medium for strong seedlings

The MS culture medium is taken as a basic culture medium for hormone screening of strong seedlings and rooting culture medium, and effective bud segments and cluster buds obtained by combining MS+0.2 mg/L6-BA+0.2 mg/L IAA in the embodiment 2 are taken as research objects, so that the strong seedling rooting culture medium is screened. 3 gradients are set for auxin IAA, namely 0.1mg/L,0.2mg/L and 0.5mg/L respectively, 3 gradients are set for IBA, namely 0.1mg/L,0.2mg/L and 0.5mg/L respectively, and proper strong seedling rooting culture mediums are screened. The rooting culture medium for each strong seedling also contains 30g/L of sucrose and 5g/L of agar, and the pH value is 6.0. The conditions of strong seedling and rooting culture are as follows: the light intensity is 1800LUX, the temperature is 23-25 ℃, and the illumination period is 10L:14D, the culture period is 30D. The statistical results of the strong seedlings and rooting culture for 30d are shown in Table 3.

TABLE 3 rooting medium hormone screening results

Remarks: in the table "-" indicates no callus; "+" indicates a small number of calli "++" indicates a large number of calli;

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

Example 4

Transplanting Medium Screen

Rooting aseptic seedlings of buddleia glandulosa obtained in the embodiment 3 by MS+0.5 mg/L6-BA+1.0 mg/L NAA culture medium are moved to a greenhouse for hardening seedlings with a shading rate of 75% -85% one week in advance. Transplanting the rooting aseptic seedlings after seedling hardening into different transplanting culture mediums, and screening proper transplanting culture mediums. The transplanting culture mediums for the test are respectively as follows: raw red soil, perlite, humus soil/raw red soil mixed matrix and perlite/humus soil/raw red soil mixed matrix, wherein the volume ratio of the humus soil to the raw red soil in the humus soil/raw red soil mixed matrix is 1:1, the volume ratio of perlite, humus soil and raw red soil in the perlite/humus soil/raw red soil mixed matrix is as follows: 1:2:3. spraying and mixing soil with 800-1000 times carbendazim, sealing and sterilizing for 7d with a plastic film, adjusting the pH value to 6.2-6.4, and transplanting. Spraying water in time after transplanting and covering a plastic film, 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 test was repeated 3 times. After 30d of transplanting, the transplanting survival rate of each treatment is counted. The detection results are shown in Table 4.

TABLE 4 screening of transplanting Medium

As can be seen from the results in Table 4, the mixed matrix of perlite, humus soil and raw red soil has the highest transplanting survival rate, and the survival rate after 30 days of transplanting reaches 95%; meanwhile, the shape of the transplanted buddleia adenophylla after flowering is kept consistent with that of a female parent, so that the genetic stability and consistency of the buddleia adenophylla are highly maintained by adopting the culture medium combination and the method provided by the invention.

According to the embodiment, the culture medium combination provided by the invention can be used for obtaining buddleia glandulosa 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 buddleia glandulosa is greatly improved, and a very effective propagation method is provided for the introduction, domestication, preservation, gardening and the commercial and scientific research value development and utilization of the buddleia glandulosa.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims (3)

1. The method for tissue culture and rapid propagation of buddleia glandulifolia is characterized in that a culture medium combination is adopted to culture buddleia glandulifolia to obtain aseptic root seedlings of buddleia glandulifolia;

the culture medium combination consists of an induced differentiation culture medium, a proliferation subculture medium and a strong seedling rooting culture medium;

the induced differentiation culture medium consists of an MS culture medium, 6-BA0.2mg/L, IAA 0.5.5 mg/L, sucrose 30g/L and agar 5.0g/L, and the pH value is 6.0;

the proliferation subculture medium consists of MS culture medium, 6-BA0.2mg/L, IAA0.2mg/L, sucrose 30g/L and agar 5.0g/L, and the pH value is 6.0;

the strong seedling rooting culture medium consists of an MS culture medium, IAA0.2mg/L, IBA0.2mg/L, sucrose 30g/L and agar 5.0g/L, and the pH value is 6.0;

the method for tissue culture and rapid propagation of buddleia glandulosa comprises the following steps:

performing first culture on the sterilized buddleia adenophylla explant in an induced differentiation medium to obtain lateral buds and adventitious buds;

performing second culture on the lateral buds and the adventitious buds in a proliferation subculture medium to obtain a root-free Cong Miao;

thirdly culturing the unrooted Cong Miao in a strong seedling rooting culture medium to obtain the aseptic root seedling of buddleia glandulosa;

the explant is a young terminal bud or a lateral bud of buddleia glandula;

the temperature of the first 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 45-60D;

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, wherein the culture time is 45-60D;

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, the culturing time is 30-45D.

2. The method of claim 1, further comprising fourth culturing the buddleja glandulifolia sterile rooting seedling in a transplanting medium to obtain buddleja glandulifolia seedling;

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

3. The method of claim 2, wherein the transplanting substrate is 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).