CN111631137A - Culture medium and culture method for tissue culture of Sorbus sophorae - Google Patents

Abstract

The invention relates to the technical field of tissue culture, in particular to a culture medium and a culture method for Sorbus sophorae. The culture medium is an induction culture medium, the basic culture medium of the induction culture medium is A, 7g of agar, 25g of cane sugar, 0.3-0.6mg of IBA, 0.1-0.3mg of 6-BA, 0.5-1.2mg of NAA and pH of 5.6-6 are added into 1L of A; a, 2.5-2.8g/L Huabao No. 1, 1.1-1.2g/L of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, wherein the solvent is water. The invention also provides a culture method of the Sorbus sophorae bunge tissue, which is matched with an induction culture medium for use, can obviously improve the induction rate of the Sorbus sophorae bunge explant, can finally obtain a great number of high-quality tissue culture seedlings of the Sorbus sophorae bunge, and lays a solid foundation for the mass propagation of the good varieties of the Sorbus sophorae bunge.

Description

Culture medium and culture method for tissue culture of Sorbus sophorae

Technical Field

The invention relates to the technical field of tissue culture, in particular to a culture medium and a culture method for Sorbus sophorae.

Background

Sorbus sophorae is an ancient Chinese catalpa variety, and the trunk of the Chinese Sorbus sophorae is very similar to that of the Sorbus sophorae, so the Chinese Sorbus sophorae is called as Sorbus sophorae. The Chinese scholartree is noble, simple and practical, and is a good tree species capable of transmitting wealth to offspring. Sophora japonica Makino is a high-quality and rare wood facing the seed culture in the north of the Yangtze river, grows in the Shandong peninsula and the Lu-Wen south, and is slow and rare. The general adult trees are 60 years to 80 years old. The wood has the characteristics of clear texture, fine and uniform structure, decay resistance, no deformation, no cracking and no peculiar smell. According to the records of the history of the Ming and Qing dynasties, Sorbus pohuashanensis is the preferred wood for making appliances at that time and is appreciated by the world population. The invented furniture made of wood not only possesses the characteristics of practicality, appreciation and value-keeping increment of all redwood furniture, but also possesses the quality which can not be reached by redwood due to soil and water inadequacy, desiccation, deformation and slotting. In addition, the rosewood furniture in the current market has mixed fish eyes, and the authenticity is difficult to distinguish. Antique furniture made of Sorbus pohuashanensis is popular among middle-aged and elderly people and successful people due to the characteristics of firmness, durability, nature, environmental protection and the like.

Sorbus sophorae is a precious variety of mountain ash trees and has other uses. Ancient books are also documented. For the medical purpose, Ben Cao gang mu states that pounding catalpa leaves to affected sores and swelling, boiling and washing pus and blood. "the decoction of the root and bark of catalpa bungei can be used for treating baldness, fistula and swelling by external application; eating, recorded in the Ming Dynasty abalone mountain's wild vegetable Bo Lu's book, the food recipe, fried flowers and oil-salt mixed food. Or sun drying, frying, and parching; used as feed, the sushi of songshi (food rough talk) is described in: the Tung catalpa bungei is immediately fat and easy to raise when being used for feeding pigs in leaves. From the ancient books, it can be seen how important the catalpa bungei plays a role in the life and production of ancient workers in China.

With the development of ecological forestry and the requirement of market economy, the mass propagation of improved varieties of Sorbus pohuashanensis becomes a problem to be solved urgently at present. Although the purpose of propagation can be basically achieved in tissue culture, the conventional induction medium of a combination of MS medium and phytohormone has the problem of low induction rate.

Disclosure of Invention

One of the purposes of the invention is to provide a culture medium for tissue culture of Sorbus sophorae, wherein the culture medium is an induction culture medium, the basic culture medium of the induction culture medium is A, 7g of agar, 25g of cane sugar, 0.3-0.6mg of IBA, 0.1-0.3mg of 6-BA, 0.5-1.2mg of NAA and pH of 5.6-6 are added in each 1L of A;

a, 2.5-2.8g/L Huabao No. 1, 1.1-1.2g/L of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, wherein the solvent is water.

The invention also aims to provide a method for culturing Sorbus Sophora by using the culture medium, which comprises the following steps:

s1, collecting shoots of Sorbus pohuashanensis newly germinated in spring, cutting the shoots into 2-4cm stem segments with axillary buds, and sterilizing to obtain sterile explants;

s2, inoculating the sterile explant of S1 to the induction medium of claim 1 for 25-30d to germinate new tender shoots;

s3, transferring the new burgeons germinated in the S2 to a proliferation culture medium for continuous culture, promoting proliferation to obtain cluster buds, and carrying out rooting culture;

s4, selecting and inoculating 2-3cm buds proliferated in S3 into a rooting culture medium for culture;

s5, hardening seedlings for 5-7 days after 3-4 roots grow out from the seedlings of S4;

s6, after hardening, washing off the residual culture medium at the base of the rooted seedling, transplanting the seedling to a culture medium consisting of perlite, vermiculite and turf, and performing moisture-keeping culture at a humidity of 80-90% and a temperature of 25-30 ℃.

Further, in S1, the sterilizing step specifically includes:

washing stem with axillary bud with sterile water for 2-3min, sequentially soaking in 70% alcohol for 10-30s, 2g/100mL sodium hypochlorite for 10-15min, and washing with sterile water for 2-3 min.

Further, in S3, the basic culture medium of the proliferation culture medium is B, 7g of agar, 25g of sucrose, 0.3-0.6mg of IBA, 0.1-0.3mg of 6-BA, 0.5-1.2mg of NAA and pH5.6-6 are added in each 1L of B;

b, 1.8-2g/L Huabao No. 1, 1.1-1.2g/L of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, wherein the solvent is water.

Furthermore, the basic culture medium of the rooting culture medium is C, 7g of agar, 25g of cane sugar, 1g of activated carbon, 50g of potato juice, 0.8-1mg of IBA, 0.5-0.6mg of NAA and 5.6-5.8 of pHs are added in each 1L of C;

c, 0.8-1g/L of Huabao No. 1, 1.1-1.2g of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, and the solvent is water.

Furthermore, in S6, the mass ratio of the perlite to the vermiculite to the turf is 1:1: 1.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts a self-made basic culture medium to replace the conventional MS culture medium, adopts the Huabao No. 1 and the high-preservation multi-dimensional element tablet to optimize the design required by the Sorbus sophorae, has reasonable compatibility, provides sufficient nutrient elements for tissue culture of the Sorbus sophorae, can improve the induction rate and the multiplication coefficient by matching with a plant regulator, has high robustness of the obtained cluster buds, can root 100 percent after the cluster buds are subjected to rooting culture, further obtains tissue culture seedlings of the Sorbus sophorae with large quantity and good quality, and lays a solid foundation for the mass propagation of the good varieties of the Sorbus sophorae.

Detailed Description

The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples can be commercially available unless otherwise specified.

Example 1

Preparation of the Medium

According to the formula of table 1, Huabao No. 1 (product number EH0001, produced by Shanghai Yika), the high-preservation multi-dimensional element tablet (national standard of medicine H10950026, produced by Heibei pharmaceutical Co., Ltd.) and inositol (produced by Hebei Ruiyang Biotechnology Co., Ltd.) are respectively weighed, dissolved in distilled water, stirred and dissolved, agar, sucrose and plant regulator are continuously added, the pH of the induction culture medium is adjusted to 5.8, the pH of the propagation culture medium is adjusted to 5.7, the pH of the rooting culture medium is adjusted to 5.8, the mixture is subpackaged into tissue culture bottles, sealed and sterilized, and cooled and solidified to obtain the culture medium combination, the comparison group 1 adopts MS culture medium as a basic culture medium, the concentration of the agar in each group is 7g/L, the concentration of the sucrose is 25g/L, the concentrations of the potato juice in the propagation culture medium and the rooting culture medium in each group are 50g/L, and the concentration of the activated carbon in the rooting culture medium in each group is 1g/L, the remaining compositional differences are shown in table 1.

TABLE 1 Medium composition and concentration

Example 2

Method for culturing Sorbus Sophora tissue culture seedling by using induction, proliferation and rooting culture medium described in Table 1

S1, collecting shoots of the Sorbus sophorae bunge newly germinated in spring, cutting the shoots into 2-4cm stem sections with axillary buds, and disinfecting to obtain sterile explants; the disinfection step specifically comprises:

washing stem with axillary bud with sterile water for 2-3min, sequentially soaking in 70% alcohol for 10-30s, 2% sodium hypochlorite for 10-15min, and washing with sterile water for 2-3 min;

s2, inoculating the sterile explant of S1 to an induction culture medium for culture for 25-30d to ensure that a new bud germinates;

s3, transferring the new burgeons germinated in S2 to a proliferation culture medium for continuous culture to promote proliferation, obtaining cluster buds, repeatedly proliferating for multiple generations in such a way, and performing rooting culture after the required number is reached;

s4, selecting and inoculating 2-3cm buds proliferated in S3 into a rooting culture medium for culture;

s5, hardening seedlings for 5-7 days after 3-4 roots grow out from the seedlings of S4;

s6, after hardening, washing off residual culture medium at the base of the rooting seedling, transplanting the seedling to a culture medium consisting of perlite, vermiculite and turf, wherein the mass ratio of the perlite to the vermiculite to the turf is 1:1:1, and carrying out moisture-preserving culture at the humidity of 80-90% and at the temperature of 25-30 ℃.

The culture effects of the combinations of the culture media used in the experimental groups 1 to 3 and the control groups 1 to 3 are shown in Table 1.

TABLE 2 Effect of cultivation

In the table, "+" indicates that the number of cluster buds is small, and the cluster buds are thin and weak and grow slowly. "+ + +" indicates that the cluster buds are less numerous and grow more slowly, and "+ + + + +" indicates that the cluster buds are more numerous and robust.

The above experimental results show that:

as can be seen from the comparison between the control group 1 and the experimental group 2, the adoption of Huabao 1, the abundant multi-dimensional element tablet and inositol as the induction culture medium, the multiplication culture medium and the rooting culture medium of the basic culture medium can induce the generation of a large amount of cluster buds, and the cluster buds can all root after rooting culture to obtain the tissue culture seedlings of the Sorbus sophorae with high quantity and quality.

As can be seen from the comparison between the experimental group 1-3 and the control group 2-3, the Huabao No. 1, the high-preservation multi-vitamin tablet and the inositol have high induction rate, multiplication coefficient and rooting rate within a certain concentration range, and the multiple shoots are robust, and when the induction rate, the multiplication coefficient and the rooting rate are reduced and the robustness of the multiple shoots is reduced after the range is exceeded.

It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims (6)

1. The culture medium for the tissue culture of Sorbus sophorae is characterized in that,

the culture medium is an induction culture medium, the basic culture medium of the induction culture medium is A, 7g of agar, 25g of cane sugar, 0.3-0.6mg of IBA, 0.1-0.3mg of 6-BA, 0.5-1.2mg of NAA and pH of 5.6-6 are added into 1L of A;

a, 2.5-2.8g/L Huabao No. 1, 1.1-1.2g/L of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, wherein the solvent is water.

2. The method for culturing Sorbus Sophora japonica by using the culture medium of claim 1, comprising the steps of:

s1, collecting shoots of Sorbus pohuashanensis newly germinated in spring, cutting the shoots into 2-4cm stem segments with axillary buds, and sterilizing to obtain sterile explants;

s2, inoculating the sterile explant of S1 to the induction medium of claim 1 for 25-30d to germinate new tender shoots;

s3, transferring the new burgeons germinated in the S2 to a proliferation culture medium for continuous culture, promoting proliferation to obtain cluster buds, and carrying out rooting culture;

s4, selecting and inoculating 2-3cm buds proliferated in S3 into a rooting culture medium for culture;

s5, hardening seedlings for 5-7 days after 3-4 roots grow out from the seedlings of S4;

s6, after hardening, washing off the residual culture medium at the base of the rooted seedling, transplanting the seedling to a culture medium consisting of perlite, vermiculite and turf, and performing moisture-keeping culture at a humidity of 80-90% and a temperature of 25-30 ℃.

3. The method according to claim 2, wherein in S1, the sterilizing step is specifically:

washing stem with axillary bud with sterile water for 2-3min, sequentially soaking in 70% alcohol for 10-30s, 2g/100mL sodium hypochlorite for 10-15min, and washing with sterile water for 2-3 min.

4. The method according to claim 3, wherein the minimal medium of the proliferation medium in S3 is B, 7g of agar, 25g of sucrose, 0.3-0.6mg of IBA, 0.1-0.3mg of 6-BA, 0.5-1.2mg of NAA, pH 5.6-6;

b, 1.8-2g/L Huabao No. 1, 1.1-1.2g/L of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, wherein the solvent is water.

5. The method of claim 4, wherein in S4, the minimal medium of the rooting medium is C, 7g of agar, 25g of sucrose, 1g of activated carbon, 50g of potato juice, 0.8-1mg of IBA, 0.5-0.6mg of NAA, pH5.6-5.8 are added to each 1L of C;

c, 0.8-1g/L of Huabao No. 1, 1.1-1.2g of high-preservation multi-vitamin tablets and 0.1-0.15g/L of inositol, and the solvent is water.

6. The method according to claim 5, wherein in S6, the mass ratio of the perlite to the vermiculite to the turf is 1:1: 1.