CN110663552B - A Tissue Culture Rapid Propagation Method of Diantong - Google Patents

Abstract

The invention provides a tissue culture and rapid propagation method of Diantong, which relates to the technical field of plant tissue culture. The tissue culture rapid propagation method of the invention combines induction and differentiation culture into one, proliferation and subculture into one, strong seedlings and rooting culture into one, and the culture method is simplified. The tissue culture and rapid propagation method of the invention can solve the problems of few natural resources of T. sinensis, and achieve mass reproduction, preservation and sustainable utilization in a short time, fill the research gap of T. sinensis in biotechnology, and provide comprehensive development and availability of T. sinensis. Sustained utilization lays the foundation.

Description

A Tissue Culture Rapid Propagation Method of Diantong

technical field

The invention belongs to the technical field of plant tissue culture, and in particular relates to a tissue culture and rapid propagation method of Diantong.

Background technique

Craigia yunnanensis is a deciduous tree with a height of 6 to 20 meters. It is mainly distributed in the subtropical hot and dry climate regions and mountain forests at an altitude of 500 to 1000 meters. The capsule has thin papery wings. Endemic to Southwest China. Diantong is one of the important tree species of the genus Diantong, which is of great value in the study of floristic geography and the application of precious tree species. In 2017, it was listed as an endangered EN C2a; D species in the "List of Threatened Species of Higher Plants in China"; it was also listed as an endangered species by the International Union for Conservation of Nature (IUCN) [EN]. At present, there are two main factors that seriously threaten the wild survival of the Diantong population: one is to plant a large number of grass and fruits for economic benefits, which seriously encroach on the Diantong habitat; It is a typical species with very small populations, resulting in a narrow distribution area and a serious threat to its survival.

At present, aseptic rapid propagation in biotechnology has become an important means for the production of seedlings such as Chinese medicinal materials, flowers, endangered species, and economic forest fruits. But so far, there is no report on T. yunnanensis excellent individual plant tissue culture and fast propagation and related biotechnology in the prior art.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a kind of tissue culture fast propagation method of Diantong, to solve the problems that Diantong has few natural resources and can not achieve mass reproduction, preservation and continuous utilization in a short time.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a tissue culture and rapid propagation method of T. dianthus, comprising the following steps: (1) using the sterilized young tissue of T. dianthus as an explant, and inoculating the explant on a co-culture medium for inducing differentiation Induction and differentiation co-cultivation is carried out to separate differentiated buds; the induction and differentiation co-culture medium takes MS medium as the basic medium, and also includes: 1.0 mg/L 6-BA, 0.5 mg/L IAA, 30 g/L sucrose and 5g/L agar;

(2) inoculating the differentiated buds on the proliferation subculture co-culture medium to carry out proliferation subculture co-culture to obtain seedlings; the proliferation subculture co-culture medium takes MS medium as the basic medium, and also includes: 0.8 mg/ L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;

(3) the seedling is inoculated on the strong seedling and rooting co-culture medium, and the strong seedling and rooting co-cultivation is obtained, and the Tung tung tissue culture seedling is obtained; The strong seedling and rooting co-medium is a basic medium with MS medium, and also comprises: 0.8 mg/L of IAA, 0.8 mg/L of IBA, 30 g/L of sucrose and 5 g/L of agar.

Preferably, the young Tung Tung tissue in step (1) includes terminal buds or lateral buds of Tung syringae; the method for disinfection includes: soaking in soapy water with a mass-to-volume ratio of 1% for 10 min, and after rinsing with running water, using Disinfect with 75% alcohol solution by volume for 10s, rinse with sterile water for 3 times, then disinfect with 0.1% mercuric solution by volume for 5 minutes, and wash with sterile water for 3 to 5 times.

Preferably, the light intensity of the induced differentiation co-culture in step (1), the proliferation and subculture co-culture in step (2) and the strong seedling rooting co-culture in step (3) are all 1500Lux, and the temperature is 23-30 °C.

Preferably, the culture period of the induction and differentiation co-culture in step (1) is 30 days; the culture period of the proliferation and subculture co-culture in step (2) is 60 days; the culture period of the strong seedling rooting co-culture in step (3) is 30d.

Preferably, after obtaining the Tung tung tissue culture seedlings in step (3), it also includes hardening and transplanting.

Preferably, the shading rate during seedling hardening is 75-85%.

Preferably, the transplanted substrate comprises the following components: perlite, humus and laterite, and the volume ratio of the perlite, humus and laterite is 1:2:3.

Preferably, after the transplanting, the cultivation temperature is 20-30° C., the shading degree is 75-85%, the air humidity is 50-60%, and the substrate humidity is 75-85%.

Compared with the prior art, the present invention has the following beneficial effects: (1) the present invention establishes an effective T. yunnanensis tissue culture and rapid propagation method, solves its narrow distribution, the difficulty of introduction and domestication, and fills up the biological technology of T. yunnanensis. R&D blank;

(2) the present invention adopts the T. dianthus tissue culture rapid propagation method, which is easy to grow into seedlings, simplifies the breeding steps, and has a high effective reproduction rate, which is of great significance for preserving and expanding the T. yunnanensis population;

(3) the present invention highly maintains the genetic stability and consistency of excellent individual plants of T. tungensis through the tissue culture and fast propagation method of T. tungensis, and lays a foundation for the comprehensive development and continuous utilization of T. tungensis;

(4) Diantong tung bred by the tissue culture fast propagation method of the present invention, the induced differentiation rate in 30 days is 92%, the proliferation coefficient in 60 days is 4.7, the rooting rate in 30 days is 91%, and the transplant survival rate is 90%, It has greatly improved the reproduction coefficient of T. yunnanensis, and provided a very effective reproduction method for the introduction, domestication, preservation, gardening and scientific research value of this species.

Description of drawings

Fig. 1 is a diagram of T. yunnanensis tissue culture induced differentiation in Example 1 of the present invention;

Fig. 2 is the proliferation and subculture diagram of T. dianthus tissue culture in the embodiment of the present invention 1;

Fig. 3 is the rooting diagram of T. yunnanensis tissue cultured strong seedlings in Example 1 of the present invention.

Detailed ways

The invention provides a tissue culture and rapid propagation method of T. dianthus, comprising the following steps: (1) using the sterilized young tissue of T. dianthus as an explant, and inoculating the explant on a co-culture medium for inducing differentiation Induction and differentiation co-cultivation is carried out to separate differentiated buds; the induction and differentiation co-culture medium takes MS medium as the basic medium, and also includes: 1.0 mg/L 6-BA, 0.5 mg/L IAA, 30 g/L sucrose and 5g/L agar;

(2) inoculating the differentiated buds on the proliferation subculture co-culture medium to carry out proliferation subculture co-cultivation to obtain seedlings; the proliferation subculture co-culture medium takes MS medium as the nodal medium, and also includes: 0.8 mg /L of 6-BA, 0.5mg/L of IAA, 30g/L of sucrose and 5g/L of agar;

(3) the seedling is inoculated on the strong seedling and rooting co-culture medium, and the strong seedling and rooting co-cultivation is obtained, and the Tung tung tissue culture seedling is obtained; The strong seedling and rooting co-medium is a basic medium with MS medium, and also comprises: 0.8 mg/L of IAA, 0.8 mg/L of IBA, 30 g/L of sucrose and 5 g/L of agar.

According to the tissue culture rapid propagation method of the present invention, the sterilized young tissue of T. yunnanensis is used as an explant, and the explant is inoculated on an induced differentiation co-culture medium for induced differentiation and co-cultivation, and the differentiated buds are divided; The induction-differentiation co-culture medium takes MS medium as the basic medium, and also includes: 1.0 mg/L 6-BA, 0.5 mg/L IAA, 30 g/L sucrose and 5 g/L agar. The young tissue of T. sinensis in the present invention preferably includes a terminal bud or a lateral bud of T. sinensis. The disinfection method of the present invention preferably includes: soaking in soapy water with a mass-volume ratio of 1% for 10 minutes, rinsed with running water, disinfecting with an alcohol solution with a volume-percentage of 75% for 10s, and rinsing with sterile water for 3 times Then, disinfect with 0.1% mercuric chloride solution by volume for 5 minutes, and wash with sterile water for 3 to 5 times. The present invention has no particular limitation on the preparation method of the induction-differentiation co-culture medium. Preferably, the above components are mixed uniformly and obtained after high temperature and high pressure sterilization. The pH value of the induction-differentiation co-culture medium is preferably 5.8.

In the present invention, when the induced differentiation co-culture medium is used for induced differentiation and co-culture, the light intensity is preferably 1500 Lux, the temperature is preferably 23-30° C., and the culture period is preferably 30 d. In the present invention, the terminal buds or lateral buds begin to germinate after one week of co-cultivation through the induction and differentiation.

After the differentiated buds are differentiated, the present invention inoculates the differentiated buds on the proliferative subculture co-culture medium to obtain the seedlings; the multiplication subculture co-culture medium takes the MS medium as the basic medium, and further comprises: : 0.8mg/L 6-BA, 0.5mg/L IAA, 30g/L sucrose and 5g/L agar. In the proliferation subculture co-cultivation of the present invention, the proliferation is carried out in the manner of the occurrence of lateral buds, and the lateral buds grown on the seedlings are re-inoculated on the proliferation subculture co-culture medium for the proliferation subculture co-culture. The light intensity of the proliferation and subculture co-culture in the present invention is preferably 1500 Lux, and the temperature is preferably 23-30°C. The culture period of the proliferation subculture co-culture in the present invention is preferably 60 d. In the present invention, after the proliferation and subculture for 60 days, the growth height reaches 4.8 cm, and the average reproduction multiple is 4.7.

After the seedlings are obtained, the present invention inoculates the seedlings on the strong seedlings and rooting co-culture medium to carry out the strong seedlings and rooting co-cultivation to obtain the Tung tung tissue culture seedlings; Also included: 0.8 mg/L IAA, 0.8 mg/L IBA, 30 g/L sucrose, and 5 g/L agar. The light intensity of the strong seedling rooting co-cultivation of the present invention is preferably 1500 Lux, and the temperature is preferably 23-30°C. The culture period of the strong seedling rooting co-cultivation of the present invention is preferably 30 d.

In the present invention, after obtaining the T. dianthus tissue culture seedlings, it preferably also includes hardening and transplanting. The seedling hardening of the present invention is preferably performed in a greenhouse with a shading rate of 75-85% for one week. In the present invention, the tissue culture seedlings that have undergone the hardening are transplanted, and the transplanted substrate preferably includes the following components: perlite, humus and laterite, and the volume of the perlite, humus and laterite The ratio is 1:2:3. Before use, the substrate of the present invention preferably also includes sterilizing with 800 times of carbendazim, sealing and sterilizing with plastic film for 7 days, and adjusting the pH to 5.8. The transplanting method of the present invention is preferably to take out the hardened tissue culture seedlings, wash the culture medium of the roots, transplant them into the sterilized substrate, spray water in time and cover with plastic film to keep moisture, and the temperature of the greenhouse is 20～30℃ , shading rate of 75 to 85%, air humidity of 60 to 70%, substrate humidity of 65 to 75%, and survival rate of 90% after 30 days. In the transplanting matrix of the present invention, the looseness of the matrix and the air permeability of the roots are increased, and sufficient nutrition is also provided for the roots. Meanwhile, the laterite is the deep soil of the soil layer, and the amount of bacteria is less, which is beneficial to the growth of sterile seedlings.

Below in conjunction with embodiment, the tissue culture fast propagation method of Diantong provided by the present invention is described in detail, but they can not be construed as limiting the protection scope of the present invention.

Example 1

Take the young terminal buds and lateral buds of Diantong as explants, soak them in 1% soapy water for 10 minutes, rinse them with running water, put them on the ultra-clean table, cut them into stem segments with a single node, use 75% Alcohol disinfection for 10 s, rinsed with sterile water for 3 times, disinfected with 0.1% mercuric solution for 5 minutes, washed with sterile water for 3 to 5 times, and inoculated into the prepared co-culture medium for induced differentiation of Tung tung, 1 per bottle Festival.

1. The effect of different induction media on the induction rate

The stem segments were inoculated in the following medium: ①MS+0.1mg/L 6-BA+0.5mg/L NAA+sucrose 30g/L+agar 5g/L, pH5.8, ②MS+0.5mg/L 6-BA+0.5 mg/L NAA+sucrose 30g/L+agar 5g/L, pH5.8, ③MS+0.8mg/L 6-BA+0.5mg/L NAA+sucrose 30g/L+agar 5g/L, pH5.8, ④MS+1mg/L 6-BA+0.5mg/L NAA+sucrose 30g/L+agar 5g/L, pH5.8, ⑤MS+2mg/L 6-BA+0.5mg/L NAA+sucrose 30g/L+agar 5g/L, pH5.8. The light intensity is 1000LUX, the temperature is 23～30℃, and the illumination time is 10 hours. After 30 days, the statistical induction rate is shown in Table 1:

Table 1 Effects of different media on the induction of fine stems of T. yunnanensis

As can be seen from Table 1, on the medium ④, the induction rate reached 92% (as shown in Figure 1).

2. The effect of different proliferation and subculture co-culture medium on proliferation and subculture

The obtained differentiated buds were inoculated on the proliferation subculture co-culture medium for proliferation subculture, and the hormone content in the proliferation subculture co-culture medium was set as: cytokinin 6-BA, and the concentration range was (0.5mg/ L, 0.8mg/L, 1mg/L), the auxin concentrations were (0.1mg/L, 0.5mg/L, 0.8mg/L), and each of the proliferation subcultures contained: sucrose 30g/L+agar 5g/L, pH5.8. Statistics are carried out after 60d, and the results are shown in Table 2. For convenience, the contents of sucrose and agar are not shown in Table 2:

Table 2 Effects of different media on proliferation and subculture co-culture

It can be seen from Table 2 that MS medium+0.8mg/L 6-BA+0.5mg/L NAA+sucrose 30g/L+agar 5g/L, pH 5.8, the culture period is 60 days, the light intensity is 1500LUX, and the temperature is 23-30 ℃, the average number of effective bud nodes (including terminal buds) is 4.7, the leaves of the plant are stretched and the growth rate is fast, the plant height is about 5.1 cm, and the stem diameter is about 3 mm (as shown in Figure 2).

3. Effects of different media on rooting co-cultivation of strong seedlings

The above-mentioned seedlings were inoculated on different strong seedlings and rooting co-culture mediums for strong seedlings and rooting co-cultivation, and different hormone contents were set respectively: IAA (0.5mg/L, 0.8mg/L, 1.0mg/L), IBA (0.5mg/L) /L, 0.8mg/L, 1.0mg/L), and each of the strong seedling rooting co-culture medium contains: sucrose 30g/L+agar 5g/L, pH5.8. Statistics are performed after 30d, and the results are shown in the table 3, the contents of sucrose and agar are not shown in Table 3 for the convenience of comparison:

Table 3 Effects of different media on rooting rate

As can be seen from Table 3, MS medium+0.8mg/L IAA+0.8mg/L IBA+sucrose 30g/L+agar 5g/L, pH 5.8, the culture period was 30 days, and the rooting rate reached 91% (as shown in Figure 3) .

4. The effect of different substrates on the survival rate of transplanting

The rooting bottle seedlings after culturing for 30 days need to be moved to a greenhouse with a shading rate of 75-85% for a week in advance, and then the transplanting substrates in Table 4 are used for transplanting, and the survival rate after 30 days is counted. The results are shown in the table. 4 shows:

Table 4 Influence of transplanting medium on transplanting survival rate

It can be seen from Table 4 that in the transplanting substrate (the volume ratio is perlite: humus: red soil = 1:2:3), the transplanting survival rate reached 90% after 30 days.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (5)

1. a tissue culture fast propagation method of T. dianthus, is characterized in that, may further comprise the steps: (1) with the young tissue of T. yunnanensis after disinfection as explant, described explant is inoculated in induced differentiation co-cultivation The induction and differentiation co-culture was carried out on the base, and the differentiated buds were divided; the induction and differentiation co-culture medium consisted of MS medium, 1.0 mg/L 6-BA, 0.5 mg/L IAA, 30 g/L sucrose and 5 g/L The agar is composed; the young tissue of T. tungensis includes terminal buds or lateral buds of T. tungensis; the method of disinfection includes: soaking in soapy water with a mass volume ratio of 1% for 10 minutes, rinsed with running water, and then using the volume percentage Sterilize 75% alcohol solution for 10s, rinse with sterile water for 3 times, then disinfect with 0.1% mercuric solution by volume for 5 minutes, and wash with sterile water for 3 to 5 times; the culture of the induced differentiation co-culture The period is 30d; (2) inoculating the differentiated buds on the proliferation subculture co-culture medium for proliferation subculture to obtain seedlings; mg/L of IAA, 30g/L of sucrose and 5g/L of agar; the culture period of the proliferation subculture co-culture is 60d; (3) the seedlings are inoculated on the strong seedlings and rooting co-culture medium to carry out the strong seedlings and rooting co-cultivation, to obtain Tung tung tissue culture seedlings; 0.8mg/L of IBA, 30g/L of sucrose and 5g/L of agar; the culture period of the strong seedling rooting co-culture is 30d; The light intensity of the induced differentiation co-culture in step (1), the proliferation and subculture co-culture in step (2) and the strong seedling rooting co-culture in step (3) are all 1500Lux, and the temperature is 23-30°C. 2. tissue culture rapid propagation method according to claim 1, is characterized in that, after obtaining described Tung tung tissue culture seedling in step (3), also comprises hardening seedling and transplanting. 3 . The tissue culture rapid propagation method according to claim 2 , wherein the shading rate during seedling hardening is 75-85%. 4 . 4. tissue culture fast propagation method according to claim 2 is characterized in that, the matrix of described transplanting comprises following components: perlite, humus and raw red soil, described perlite, humus and raw red soil The volume ratio is 1:2:3. 5. tissue culture fast propagation method according to claim 2 is characterized in that, the cultivation temperature after described transplanting is 20～30 ℃, shading degree is 75～85%, air humidity is 50～60%, matrix Humidity is 75-85%.

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