CN115968786A - Culture medium and culture method for tea tree tissue culture - Google Patents
Culture medium and culture method for tea tree tissue culture Download PDFInfo
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Abstract
The invention discloses a culture medium and a culture method for tea tree tissue culture, and belongs to the technical field of plant tissue culture. The components of the culture medium comprise: 0.3-0.7mg/L quercetin, 5-15g/L sodium silicate solution, 1-4g/L ammonium tartrate solution, 1-3g/L ferric citrate solution, 3-7mg/LIBA, 0.3-0.7mg/LNAA, 0.3-0.7g/L active carbon, 3-6g/L agar and 15-25g/L sucrose. The method solves the problem of low rooting rate of the tea tissue culture seedling in the prior art, obviously improves the growth characteristic of the tea tissue culture seedling, enables the seedling to grow strongly, can avoid the phenomena of browning and germ infection of the tea tissue culture seedling, and provides a foundation for the realization of researches on commercial production and the like.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a culture medium and a culture method for tea tree tissue culture.
Background
Tea leaves move from China to the world and have long become one of the important varieties in the beverage market in the world in three thirds of the day. The world tea market competition is increasingly sharp, and new operation modes are continuously appeared in all major tea production and consumption countries since the 90 s of the 20 th century. The tea contains catechin, cholestenone, caffeine, inositol, folic acid, pantothenic acid and other components, can improve the health of human bodies, and a tea drink-tea is known as one of the three major drinks in the world, so that tea tree planting has great economic value. However, the conventional breeding of tea trees is time-consuming and labor-consuming, and has long growth period and low fruiting rate which is only 3-15%. Mainly because tea trees are perennial plants, the polyphenol substance content is high and the like, the cultured callus is easy to brown and difficult to differentiate, and genes cannot be expressed in sequence. The sexual propagation quality is unstable, the seed seedling is easy to cause variety mixing and degeneration, the production of high-quality tea is limited, and the yield, quality and benefit of tea are influenced.
Tissue culture has become an important propagation means in the current agriculture and forestry seedling production, compared with the conventional asexual propagation method, the tissue culture has the advantages of high propagation speed, annual production, good product consistency and the like, and particularly, the tissue culture can reflect the advantages of certain varieties with scarce seed sources. Compared with cuttage seedlings, the tea tree tissue culture seedlings are not limited by seasons and are more suitable for industrial seedling culture, however, the rooting rate, the rooting number, the growth and development states of the tea tree tissue culture seedlings cultured by the existing plant tissue culture method and the like are not ideal, and the transfer culture or transplantation of the tea tree tissue culture seedlings is influenced.
Disclosure of Invention
The invention aims to provide a culture medium and a culture method for tea tree tissue culture, which aim to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a culture medium for tea tree tissue culture, which comprises the following components: 0.3-0.7mg/L quercetin, 5-15g/L sodium silicate solution, 1-4g/L ammonium tartrate solution, 1-3g/L ferric citrate solution, 3-7mg/LIBA, 0.3-0.7mg/LNAA, 0.3-0.7g/L active carbon, 3-6g/L agar and 15-25g/L sucrose.
Further, the components of the culture medium comprise 0.5mg/L of quercetin, 10g/L of sodium silicate solution, 3g/L of ammonium tartrate solution, 2g/L of ferric citrate solution, 5mg/LIBA, 0.5mg/LNAA, 0.5g/L of activated carbon, 4g/L of agar and 20g/L of sucrose.
The invention also provides an application of the culture medium in tea tree tissue culture.
The invention also provides a culture method for tea tree tissue culture, which comprises the steps of sterilizing tea tree tooth seedlings, inoculating the tea tree tooth seedlings into the culture medium for culture, and transplanting the tea tree tooth seedlings after the tea tree tooth seedlings take roots.
Further, the disinfection treatment comprises ethanol solution disinfection treatment and mercury chloride disinfection treatment.
Further, the culturing step includes a light treatment using a light quality consisting of blue light and red light in a light quantity ratio of 0.8.
Further, the illumination intensity of the illumination treatment is 1500lux, and the time is 14h per day.
Further, the culture temperature of the culture is 25 ℃.
Further, the culture medium adopted for transplanting comprises a culture medium with a volume ratio of 5:2:1:1 garden soil, perlite, vermiculite and turf.
The invention discloses the following technical effects:
the quercetin flavonol compound with various biological activities is specifically adopted as the culture medium component, so that browning reaction possibly caused by the action of polyphenol oxidase on phenolic substances in plants can be obviously reduced, and the effect of resisting bacterial infection is achieved; sodium silicate is used as a silicon fertilizer, so that the disease-resistant and stress-resistant effects of plants are improved, the plants grow strongly, and the pH value of soil can be adjusted; ammonium tartrate is adopted to supplement nutrient components which are missing in plants, and meanwhile, the ammonium tartrate is more suitable for putting in the acidic soil which is suitable for tea tree planting; the ferric citrate is adopted and absorbed by the roots of plants, so that not only can iron be supplemented, but also the pH value of the growth environment of the plants can be adjusted, the rooting rate and the rooting number of the tea trees are effectively improved, and the rooting rate can reach 82.67 percent at most. Meanwhile, the blue light and the red light are adopted to illuminate the tea trees, so that the tea trees grow more robustly, and the plant height, stem thickness and leaf size are obviously higher than those of the plants which are not subjected to blue light and red light illumination treatment. Lays a foundation for the transfer culture or transplantation of the tea tree tissue culture seedlings and subsequent commercialization.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
(1) Seed selection and disinfection
Selecting tea tree tooth seedlings with height of more than 3cm after subculture, washing the tea tree tooth seedlings with running water, soaking the tea tree tooth seedlings in 70 vol ethanol solution for 30s, soaking the tea tree tooth seedlings in 0.05% mercuric chloride sterilized water for 2min, washing the tea tree tooth seedlings with sterile water for 3 times, and inoculating the tea tree tooth seedlings into a strong seedling rooting culture medium, wherein the strong seedling rooting culture medium is 1/2MS +5mg/LIBA +0.5mg/LNAA +0.5g/L active carbon +4.0g/L agar +20g/L sucrose +0.5mg/L quercetin +10g/L sodium silicate solution +3g/L ammonium tartrate solution +2g/L ferric citrate solution, and the pH value is 5.2; wherein the mass fraction of sodium silicate in the sodium silicate solution is 20%, the mass fraction of ammonium tartrate in the ammonium tartrate solution is 30%, and the mass fraction of ferric citrate in the ferric citrate solution is 15%.
(2) Strong seedling rooting culture
When the tea tree seedlings are subjected to rooting culture, the culture temperature is 25 ℃, the illumination time is 14 hours per day, light quality with the light quantity ratio of blue light to red light of 0.8. After 5 weeks of culture, the tea plants after rooting were transplanted when the root length reached an average of 1.2 cm.
(3) Transplanting
Cleaning the culture medium on the rooted tea plant, planting in a nutrition cup or a hole tray containing a culture medium, wherein the humidity is 80%, inoculating a membrane for ventilation after new roots and leaves are sent out, and culturing for 50 days to obtain the rooted seedling of the tea plant. Wherein the culture medium comprises a volume ratio of 5:2:1:1 garden soil, perlite, vermiculite and turf.
Example 2
(1) Seed selection and disinfection
Selecting tea tree seedlings with the height of more than 3cm after subculture, washing the tea tree seedlings with running water, soaking the tea tree seedlings in 70 vol ethanol solution for 30s, soaking the tea tree seedlings in 0.05% mercuric chloride sterilized water for 2min, washing the tea tree seedlings with sterile water for 3 times, and inoculating the tea tree seedlings into a strong seedling rooting culture medium, wherein the strong seedling rooting culture medium is 1/2MS +3mg/LIBA +0.3mg/LNAA +0.3g/L activated carbon +3g/L agar +15g/L sucrose +0.3mg/L quercetin +5g/L sodium silicate solution +1g/L ammonium tartrate solution +1g/L ferric citrate solution, and the pH value is 5.2; wherein the mass fraction of sodium silicate in the sodium silicate solution is 20%, the mass fraction of ammonium tartrate in the ammonium tartrate solution is 30%, and the mass fraction of ferric citrate in the ferric citrate solution is 15%.
(2) Strong seedling rooting culture
When the tea tree seedlings are subjected to rooting culture, the culture temperature is 25 ℃, the illumination time is 14 hours per day, light quality with the light quantity ratio of blue light to red light of 0.9. After 5 weeks of culture, transplanting when the root length of the rooted tea plant reaches an average of 1.2 cm;
(3) Transplanting
Cleaning the culture medium on the rooted tea plant, planting in a nutrition cup or a hole tray containing a culture medium, wherein the humidity is 75%, inoculating a membrane for ventilation after new roots and leaves are sent out, and culturing for 50 days to obtain the rooted seedling of the tea plant. Wherein the culture medium comprises a volume ratio of 2:2:1:1 garden soil, perlite, vermiculite and turf.
Example 3
(1) Seed selection and disinfection
Selecting tea tree seedlings with height of more than 3cm after subculture, washing the tea tree seedlings with running water, soaking the tea tree seedlings in 70 vol ethanol solution for 30s, soaking the tea tree seedlings in 0.05% mercuric chloride sterilized water for 2min, washing the tea tree seedlings with sterile water for 3 times, and inoculating the tea tree seedlings into a strong seedling rooting culture medium, wherein the strong seedling rooting culture medium is 1/2MS +7mg/LIBA +0.7mg/LNAA +0.7g/L active carbon +6g/L agar +25g/L sucrose +0.7mg/L quercetin +15g/L sodium silicate solution +4g/L ammonium tartrate solution +3g/L ferric citrate solution, and the pH value is 5.2; wherein the mass fraction of sodium silicate in the sodium silicate solution is 20%, the mass fraction of ammonium tartrate in the ammonium tartrate solution is 30%, and the mass fraction of ferric citrate in the ferric citrate solution is 15%.
(2) Strong seedling rooting culture
When the tea tree seedlings are subjected to rooting culture, the culture temperature is 25 ℃, the illumination time is 14 hours per day, light quality with the light quantity ratio of blue light to red light of 0.9. After 5 weeks of culture, transplanting when the root length of the rooted tea plant reaches an average of 1.2 cm;
(3) Transplanting
Cleaning the culture medium on the rooted tea plant, planting in a nutrition cup or a hole tray containing a culture medium, wherein the humidity is 75%, inoculating a membrane for ventilation after new roots and leaves are sent out, and culturing for 50 days to obtain the rooted seedling of the tea plant. Wherein the culture medium comprises a volume ratio of 2:2:1:1 garden soil, perlite, vermiculite and turf.
Comparative example 1
The difference from example 1 is that quercetin was replaced with catechin in the components in the strong seedling rooting medium used in step (1).
Comparative example 2
The difference from example 1 is that the components in the strong seedling rooting medium used in step (1) do not contain quercetin.
Comparative example 3
The difference from example 1 is that the components in the strong seedling rooting medium used in step (1) do not contain ferric citrate solution, quercetin, sodium silicate solution and ammonium tartrate solution.
Comparative example 4
The difference from the example 1 is that the tissue culture seedling is treated by using a common incandescent lamp as the supplementary light during the illumination in the step (2), wherein the power of the incandescent lamp is 22W, and the illumination intensity is 1500lux.
Comparative example 5
The difference from example 1 is that the culture medium used in step (3) is vegetable garden soil.
The rooting rates of examples 1 to 3 and comparative examples 1 to 5 were counted, and the results are shown in Table 1:
TABLE 1 Effect of different Medium treatments on rooting of tissue culture seedlings of tea plants
The effect of the different treatments of examples 1-3 and comparative examples 1-5 on the growth and development of tea was counted and the results are shown in Table 2:
TABLE 2 Effect of different treatments on the growth and development of tea plants
Note: the data from the above examples and comparative examples are taken from the mean of tea plant plants.
The effect of the different treatments of examples 1-3 and comparative examples 1-2 on tea tree browning was counted and the results are shown in table 3:
TABLE 3 Effect of different treatments on browning of tea Tree
According to tables 1, 2 and 3, the rooting rate of the tissue culture seedling of the tea tree cultured by adopting the specific strong seedling rooting culture medium in the example 1 can reach 82.67%, and compared with the conventional culture, the parameters of the plant height, the stem thickness and the like of the cultured tea tree plant are obviously superior to those of the tea tree plant obtained by the conventional culture. The method solves the problem of low rooting rate of the tea tree tissue culture seedling in the prior art, obviously improves the growth characteristic of the tea tree tissue culture seedling, and can avoid browning phenomenon and germ infection phenomenon of the tea tree tissue culture seedling.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (9)
1. A culture medium for tea plant tissue culture, wherein the components of the culture medium comprise: 0.3-0.7mg/L quercetin, 5-15g/L sodium silicate solution, 1-4g/L ammonium tartrate solution, 1-3g/L ferric citrate solution, 3-7mg/LIBA, 0.3-0.7mg/LNAA, 0.3-0.7g/L active carbon, 3-6g/L agar and 15-25g/L sucrose.
2. The culture medium according to claim 1, wherein the components of the culture medium comprise 0.5mg/L quercetin, 10g/L sodium silicate solution, 3g/L ammonium tartrate solution, 2g/L ferric citrate solution, 5mg/L IBA, 0.5mg/L NAA, 0.5g/L activated carbon, 4g/L agar and 20g/L sucrose.
3. Use of a culture medium according to any one of claims 1-2 in tea plant tissue culture.
4. A method for culturing tea tree tissue, comprising the steps of sterilizing tea tree seedlings, inoculating the sterilized tea tree seedlings to the culture medium according to any one of claims 1 to 2, culturing the sterilized tea tree seedlings, and transplanting the cultured tea tree seedlings after the seedlings have rooted.
5. The culture method according to claim 4, wherein the sterilization treatment comprises an ethanol solution sterilization treatment and a mercuric chloride sterilization treatment.
6. The culture method according to claim 4, wherein the step of culturing comprises light irradiation treatment using light quality treatment consisting of blue light and red light in a light quantity ratio of 0.8.
7. The culture method according to claim 6, wherein the light treatment is performed at a light intensity of 1500lux for 14h per day.
8. The method according to claim 4, wherein the culture temperature is 25 ℃.
9. The culture method according to claim 4, wherein the culture medium adopted for transplanting comprises garden soil, perlite, vermiculite and turf in a volume ratio of 5.
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