Preparation method of houttuynia cordata planting seedlings

文档序号：1910103 发布日期：2021-12-03 浏览：7次中文

阅读说明：本技术 一种鱼腥草种植苗制备方法 (Preparation method of houttuynia cordata planting seedlings ) 是由谌金吾杨占南李琳琪叶海涛罗世琼侯彪郭显会李膳利陆兰芳于 2021-09-16 设计创作，主要内容包括：本发明提供一种鱼腥草种植苗制备方法,包括：制备桔青霉孢子悬浊液,提取鱼腥草的组织,对所述组织进行无菌化处理得到鱼腥草的外植体,将所述外植体置于所述桔青霉孢子悬浊液中并震荡培养,得到带菌外植体,将所述带菌外植体移入无菌培养基中,培养得到带菌幼苗,培养所述带菌幼苗,得到鱼腥草种植苗。本发明实施例提供一种鱼腥草种植苗制备方法,通过将鱼腥草外植体与桔青霉真菌孢子悬浊液混合并在无菌条件下培养,使得桔青霉真菌能够与鱼腥草外植体充分共生,得到的种植苗在生产种植过程中根系发达,植株长势旺盛。(The invention provides a preparation method of houttuynia cordata planting seedlings, which comprises the following steps: preparing penicillium citrinum spore suspension, extracting tissue of houttuynia cordata, performing aseptic treatment on the tissue to obtain an explant of the houttuynia cordata, placing the explant in the penicillium citrinum spore suspension and performing shake culture to obtain a bacterium-carrying explant, transferring the bacterium-carrying explant into an aseptic culture medium, culturing to obtain a bacterium-carrying seedling, and culturing the bacterium-carrying seedling to obtain a houttuynia cordata planting seedling. The embodiment of the invention provides a preparation method of a houttuynia cordata planting seedling, wherein a houttuynia cordata explant and a penicillium citrinum fungal spore suspension are mixed and cultured under an aseptic condition, so that penicillium citrinum fungi and the houttuynia cordata explant can fully symbiotic, and the obtained planting seedling has a developed root system and vigorous plant growth in a production and planting process.)

1. The preparation method of the houttuynia cordata planting seedlings is characterized by comprising the following steps:

preparing penicillium citrinum spore suspension;

extracting tissue of the houttuynia cordata, and performing aseptic treatment on the tissue to obtain an explant of the houttuynia cordata;

placing the explant in the penicillium citrinum spore suspension, and performing shake culture at the temperature of 16-30 ℃ for not less than 0.5 hour to obtain a bacterium-carrying explant;

transferring the explant with the bacteria into a sterile culture medium, and culturing to obtain seedlings with the bacteria;

and culturing the bacteria-carrying seedlings to obtain the houttuynia cordata planting seedlings.

2. The method for preparing houttuynia cordata seedlings according to claim 1, wherein the spore concentration of the penicillium citrinum spore suspension is 10²CFU/ml to 10⁶CFU/ml。

3. The method for preparing houttuynia cordata seedlings according to claim 2, wherein the spore concentration of the penicillium citrinum spore suspension is 10³CFU/ml。

4. The method for preparing houttuynia cordata seedlings according to claim 1, wherein the period of shake culture is 0.5 to 2 hours.

5. The method for preparing houttuynia cordata seedlings according to claim 1, wherein the step of culturing the seedlings with bacteria to obtain houttuynia cordata seedlings comprises the following steps:

preparing sterile culture soil;

and (3) planting the seedlings with bacteria into the sterile culture soil, and culturing for 12-16 days under the condition of avoiding direct irradiation of strong light to obtain the houttuynia cordata planting seedlings.

6. The method for preparing houttuynia cordata seedlings according to claim 5, wherein the step of planting the seedlings with bacteria into the sterile culture soil comprises the following steps:

selecting the bacteria-carrying seedlings with the plant heights of 3cm to 7 cm;

and (3) planting the seedlings with the bacteria into the sterile culture soil, wherein the planting depth is 2-3 cm.

7. The method for preparing houttuynia cordata seedlings according to claim 5, wherein the specific steps of preparing the sterile culture soil comprise:

selecting yellow soil with the organic matter content of 500-2000 g/kg, the clay grain silicon-aluminum rate of 2.0-2.5 and the silicon-iron-aluminum rate of 1.8-2.2, drying and crushing the yellow soil, and performing steam sterilization for not less than 60 minutes to obtain the sterile culture soil.

8. The method for preparing houttuynia cordata thunb seedlings according to claim 5, wherein the culture for 12 to 16 days under the condition of avoiding direct irradiation of strong light comprises the following specific steps:

culturing for 12-16 days under the condition of avoiding direct irradiation of strong light, and irrigating the germ-carrying seedlings with spore concentration not lower than 10 every other day²CFU/ml of Penicillium citrinum spore suspension enables the average Penicillium citrinum spore inoculated to each bacterium-carrying seedling to be not less than 20000CFU, and the houttuynia cordata planting seedling is obtained.

9. The method for preparing houttuynia cordata seedlings according to claim 1, wherein the step of extracting the tissue of houttuynia cordata comprises the following steps:

extracting at least one of the root tip, stem segment, stem tip, young leaf and young embryo of houttuynia cordata as the tissue.

10. The method for preparing a seedling of houttuynia cordata Thunb as claimed in any one of claims 1 to 8, wherein the sterile medium is selected from 1/2MS medium or MS medium.

Technical Field

The invention relates to the technical field of houttuynia cordata seedling culture, and particularly relates to a preparation method of a houttuynia cordata seedling.

Background

The houttuynia cordata is one of the traditional Chinese medicines, can be used for treating lung carbuncle, purulent vomiting, phlegm heat, cough and asthma and other symptoms, and is also a favorite vegetable in southwest regions. With the continuous improvement of living standard, the residents have higher pursuit on the yield and the quality of the houttuynia cordata.

However, due to the nature of houttuynia cordata and the limitations of traditional breeding techniques, the yield of houttuynia cordata in a unit area of land is still not high, and the problem that the yield of houttuynia cordata cannot meet the needs of the public still exists, and how to improve the yield of houttuynia cordata needs to be solved urgently. Therefore, a preparation method of the houttuynia cordata planting seedlings is provided.

Disclosure of Invention

The invention aims to provide a preparation method of houttuynia cordata seedlings, which can at least partially overcome the defects in the prior art.

According to one aspect of the invention, the preparation method of the houttuynia cordata seedlings comprises the following steps:

preparing penicillium citrinum fungal spore suspension;

extracting tissue of the houttuynia cordata, and performing aseptic treatment on the tissue to obtain an explant of the houttuynia cordata;

placing the explant in the penicillium citrinum spore suspension, and performing shake culture at the temperature of 16-30 ℃ for not less than 0.5 hour to obtain a bacterium-carrying explant;

transferring the explant with bacteria into a sterile culture medium, and culturing to obtain the houttuynia cordata seedling with bacteria;

and culturing the houttuynia cordata seedling with bacteria to obtain the houttuynia cordata planting seedling.

Preferably, the spore concentration of the penicillium citrinum spore suspension is 102CFU/ml to 106 CFU/ml.

Preferably, the spore concentration of the penicillium citrinum spore suspension is 103 CFU/ml.

Preferably, the time of the shake culture is 0.5 to 2 hours.

Preferably, the culturing the houttuynia cordata seedling with bacteria to obtain the houttuynia cordata planting seedling comprises the following steps:

preparing sterile culture soil;

and (3) planting the houttuynia cordata seedling with bacteria into the sterile culture soil, and culturing for 12-16 days under the condition of avoiding direct strong light to obtain the houttuynia cordata seedling.

Preferably, the step of implanting the houttuynia cordata seedling with bacteria into the sterile culture soil comprises the step of selecting the houttuynia cordata seedling with bacteria with the height of 3-7 cm;

and (3) planting the houttuynia cordata seedling with bacteria into the sterile culture soil, wherein the planting depth is 2-3 cm. .

Preferably, the preparing of the sterile culture soil includes:

Preferably, the culturing for 12 to 16 days under the condition of avoiding direct irradiation of strong light to obtain the houttuynia cordata planting seedlings comprises the following steps:

culturing for 12-16 days under the condition of avoiding direct irradiation of strong light, and irrigating the houttuynia cordata seedling with bacteria at a spore concentration of not less than 10 every other day²CFU/ml of Penicillium citrinum spore suspension enables the Penicillium citrinum fungal spores inoculated to each houttuynia cordata seedling with bacteria to be not lower than 20000CFU, and the houttuynia cordata seedling is obtained.

Preferably, the extracting the explant of the houttuynia cordata plant comprises:

and extracting at least one of germinated seeds and plant stem tips of the houttuynia cordata to serve as the explant.

Preferably, the sterile medium is selected from 1/2MS medium or MS medium.

The embodiment of the invention provides a preparation method of a houttuynia cordata planting seedling, wherein a houttuynia cordata explant and a penicillium citrinum fungal spore suspension are mixed and cultured under an aseptic condition, so that penicillium citrinum fungi and the houttuynia cordata explant can fully symbiotic, and the obtained planting seedling has a developed root system and vigorous plant growth in a production and planting process.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a flow chart for preparing houttuynia cordata seedlings according to the present invention;

FIG. 2 is a flow chart of culturing seedlings with bacteria into houttuynia cordata seedlings according to the present invention;

FIG. 3 is experimental data of experimental examples 1 to 4 according to the present invention and comparative example 1.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. For convenience of description, only portions related to the invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.

The Penicillium citrinum in the present invention is a fungus of the order chitinales, family californiaceae, named Penicillium citrinum by latin name. The spores mainly refer to fungus reproduction organs from penicillium citrinum hyphae, and the specific acquisition mode can be to culture the penicillium citrinum fungi by using a culture medium, scrape the hyphae after the hyphae grow out, and obtain the spores through the steps of dilution, filtration and the like, or can be spores obtained through air filtration and other methods under the field condition, and the spores belong to the fungus reproduction organs of the penicillium citrinum.

The planting seedling in the invention mainly refers to a seedling body which has certain plant height and activity and can be planted in suitable soil to obtain a mature plant of the houttuynia cordata, so that the houttuynia cordata seedling body can be conveniently packaged and transported while keeping some special shapes, and is beneficial to large-scale popularization.

The Houttuynia cordata in the invention is a plant with a scientific name of Houttuynia cordiata Thunb in Houttuynia of Saururaceae of Piperales.

The invention provides a preparation method of houttuynia cordata seedlings, as shown in figure 1, comprising the following steps:

s101: preparing penicillium citrinum spore suspension;

s102: extracting tissue of the houttuynia cordata, and performing aseptic treatment on the tissue to obtain an explant of the houttuynia cordata;

s103: placing the explant in penicillium citrinum spore suspension, and performing shake culture at 16-30 ℃ for not less than 0.5 h to obtain a bacterium-carrying explant;

s104: transferring the explant with bacteria into a sterile culture medium, and culturing to obtain the seedling with bacteria of houttuynia cordata;

s105: culturing the houttuynia cordata seedling with bacteria to obtain the houttuynia cordata seedling.

In the step S101, penicillium citrinum spore suspension is preparedThe liquid can be obtained by culturing Penicillium citrinum in culture medium known in the art such as potato glucose agar culture medium or glucose culture medium, scraping off mycelia after growing, mixing with sterile water, filtering with warp cloth, and collecting filtrate to obtain Penicillium citrinum spore suspension, or dissolving and centrifuging culture containing Penicillium citrinum. The spore concentration of Penicillium citrinum spore suspension is not less than 10²CFU/ml, preferably 10²CFU/ml to 10⁶CFU/ml, more preferably 10³CFU/ml. The proper spore concentration of the penicillium citrinum spore suspension is beneficial to planting enough spore quantity on the explant in the subsequent explant bacterium carrying process, and the interference of the future growth of the explant caused by the overhigh spore concentration is avoided. Through experimental research, 10 is found²CFU/ml to 10⁶CFU/ml is a better range of spore concentration of the penicillium citrinum suspension.

In the step S102, the tissue from which the houttuynia cordata is extracted may preferably be the root tip, stem segment, stem tip, young leaf or young embryo from which the houttuynia cordata is extracted, and these parts have strong plant cell differentiation capability, which is beneficial for the formation of complete seedlings by the subsequent culture. The method for performing the sterilization treatment on the tissue of the houttuynia cordata in the treatment step S102 may be sterilization by using alcohol, mercuric chloride or bleaching powder, and the specific sterilization operation is well known to those skilled in the art and will not be described herein again. The extraction method in step S102 may be cutting, picking, pinching, or the like, which is well known to those skilled in the art to extract the tissue of the houttuynia cordata, and is not described herein.

It should be noted that the processing S101 and the processing S102 may be interchanged without having a sequential execution order, and fig. 1 is only a flow processing step of a preferred implementation manner, and the sequential order of the processing S101 and the processing S102 is not limited. In the implementation mode shown in fig. 1, the time for the extracted tissue of the houttuynia cordata to wait for shake culture can be reduced, and the activity of the tissue of the houttuynia cordata can be improved.

In the treatment S103, the shake culture time of the explant in the Penicillium citrinum spore suspension can be preferably 0.5-2 hours, so that the culture time is sufficient and the probability of damage of the explant caused by too long shake culture time is reduced.

In the process S104, the sterile medium used may be an MS medium designed by Murashige and Skoog in 1962 and known to those skilled in the art and containing major elements such as ammonium nitrate, potassium nitrate, calcium chloride, magnesium sulfate, potassium dihydrogen phosphate, and the like, and trace elements such as potassium iodide, boric acid, magnesium sulfate, and the like, or an 1/2MS medium with half of the content of each element in the MS medium, so that the explant with bacteria can be cultured to the seedling with bacteria relatively quickly, and the operations such as controlling temperature and humidity, supplementing electrolyte, and the like during the culture process are plant cloning or plant tissue culture operations known to those skilled in the art, and will not be described herein again.

In the process S105, the purpose of culturing the seedlings with bacteria to the seedlings for planting is to culture the seedlings with lower plant height in the culture medium to have a certain plant height and activity, and to be planted in suitable soil to obtain the seedling body of the mature plant of houttuynia cordata, which can be conveniently packaged and transported while maintaining some special shapes.

The specific implementation manner of processing the seedlings with bacteria cultured in S105 to obtain the planted seedlings can be as follows as shown in fig. 2:

s1051: preparing sterile culture soil;

s1052: and (3) planting the seedlings with the bacteria into sterile culture soil, and culturing for 12-16 days under the condition of avoiding direct irradiation of strong light to obtain the houttuynia cordata planting seedlings.

One way of implementing the preparation of the culture soil of the bacteria in the process of S1051 may be: selecting yellow soil with the organic matter content of 500-2000 g/kg, the clay grain silicon-aluminum rate of 2.0-2.5 and the silicon-iron-aluminum rate of 1.8-2.2, drying, crushing, and performing steam sterilization for no less than 60 minutes to obtain the sterile culture soil. Experiments prove that the yellow soil meeting the coefficient can better promote the growth of the seedlings with bacteria into the houttuynia cordata planting seedlings meeting the conditions through the fertility, the water storage performance and the air permeability

In the treatment S1051, preferably, seedlings with bacteria with plant heights of 3cm to 7 cm can be selected, so that the culture survival rate is higher; transplanting the seedlings with bacteria into the sterile culture soil prepared in the step S1050The planting depth is 2-3 cm. Culturing for 12-16 days under the condition of avoiding direct irradiation of strong light, and irrigating the germ-carrying seedlings with spore concentration not lower than 10 every other day in order to further increase the symbiotic relationship between the penicillium citrinum and the houttuynia cordata²The CFU/ml Penicillium citrinum spore suspension ensures that the Penicillium citrinum fungal spores inoculated on average to each bacterial-bearing seedling are not lower than 20000CFU, so that the obtained houttuynia cordata planting seedlings have better properties.

Experimental example 1

Culturing Penicillium citrinum with potato glucose agar medium with diameter of 7 cm, scraping off mycelium with sterile scraper after the mycelium grows out, mixing the mycelium with pure water, filtering to obtain Penicillium citrinum spore suspension, and adjusting the Penicillium citrinum spore suspension to concentration of 10²CFU/ml。