阅读说明：本技术 一种筛选式获得高含量***二酚工业***植株的方法 (screening process of obtaining industrial hemp plant with high cannabidiol content ) 是由 司民真 罗煜 徐成东 李国树 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种筛选式获得高含量大麻二酚工业大麻植株的方法,1)标准确定,2)工业大麻处理,3)分析,4)培养,本发明不需要提取,通过工业大麻雌株植株的叶柄部位,应用拉曼光谱技术,获得叶柄部位的拉曼光谱,与大麻二酚标样的拉曼光谱的特征谱进行对比,应用比尔定律即可筛选出高大麻二酚含量的植株,在植物幼苗期就可进行筛选,有利于后期的组织培养,与其它育种技术相比,可以大大缩短培育性状优良的品种的周期,对开发利用工业大麻,提供了一种新途径。(The invention discloses a method for obtaining high-content cannabidiol industrial hemp plants in a screening mode by , which comprises the steps of 1) standard determination, 2) industrial hemp treatment, 3) analysis and 4) culture, wherein the method does not need extraction, a raman spectrum of a petiole part is obtained by applying a raman spectrum technology to the petiole part of a female strain plant of the industrial hemp without extraction, the plant with high cannabidiol content can be screened out by applying the beer law by comparing with a characteristic spectrum of the raman spectrum of a cannabidiol standard sample, the screening can be carried out at the seedling stage of the plant, the later-stage tissue culture is facilitated, compared with other breeding technologies, the period for cultivating varieties with excellent characters can be greatly shortened, and new ways are provided for developing and utilizing the industrial hemp.)

1, method for obtaining high content cannabidiol industrial hemp plant by screening, which is characterized by comprising the following steps:

1) determining a standard, namely dripping a cannabidiol standard sample on a quartz slide glass, placing the quartz slide glass on an objective table of a micro-Raman spectrometer, carrying out micro-Raman spectrum detection on cannabidiol to obtain a Raman spectrum of the cannabidiol standard sample, and determining a characteristic spectrum of the cannabidiol Raman spectrum through analysis and research;

2) processing industrial hemp, namely taking sections of leaf stalks of female industrial hemp plants when the industrial hemp reaches 5-10 cm in seedling culture in the seedling culture period of the industrial hemp, respectively processing the taken leaf stalks, placing the processed leaf stalks on an object stage of a micro-Raman spectrometer, and carrying out micro-Raman spectrum detection on the processed leaf stalks to obtain Raman spectra of the essential oil of the female industrial hemp plants;

3) analyzing, namely taking at least 10 spectral lines from the Raman spectrum of the industrial female marijuana essential oil obtained from each leaf stalk, calculating an average Raman spectrum, comparing the average Raman spectrum obtained from each leaf stalk with the characteristic spectrum of the cannabidiol Raman spectrum in the step 1), determining the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, comparing the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, and screening out the leaf stalk with the highest content of the cannabidiol through the beer law;

4) culturing, namely performing tissue culture by taking the industrial hemp plant with the petiole with high cannabidiol content screened in the step 3) as a female parent.

2. The method of screening for industrial cannabis sativa plants with high cannabidiol content as claimed in claim 1, wherein the average Raman spectrum obtained from each petiole in step 3) is compared with the characteristic spectrum of the Raman spectrum of the cannabidiol standard in step 1), and the characteristic spectrum of cannabidiol of the average Raman spectrum obtained from each petiole is determined by comparing the peak positions of the average Raman spectrum of each petiole with the characteristic spectrum of the Raman spectrum of the cannabidiol standard in step 1).

3. The method for screening industrial marijuana plants with high cannabidiol content according to claim 1, wherein the peak heights of the characteristic spectrum of cannabidiol of the average Raman spectrum of the petioles of each plant in step 3) are compared, and the petiole with the highest cannabidiol content is screened out by the beer's law.

4. The method for obtaining industrial hemp plants with high cannabidiol content through the screening methods of claim 1, wherein the Raman spectrum of the essential oil of the female industrial hemp plant obtained from the petioles in the step 3) is 10-20 spectral lines.

5. The method for obtaining industrial marijuana plants with high cannabidiol content through the screening methods of claim 1, wherein the length of leaf stalks in step 2) is 0.5-1.5 cm.

6. The screening methods for obtaining industrial hemp plants with high cannabidiol content as claimed in claim 1, wherein the power of laser is 2mw and the wavelength of laser is 785nm in the micro-raman spectrometer used in the micro-raman spectroscopy detection of step 1), and the power of laser is 10 mw-15 mw and the wavelength of laser is 785nm in the micro-raman spectrometer of step 2).

7. The method of screening for obtaining high cannabidiol content industrial hemp plants as claimed in claim 1, wherein the step 2) of treating the removed petioles separately comprises slicing the petioles with a slicer, placing the slices in a petri dish containing deionized water, picking transparent whole slices with a brush pen, placing the transparent whole slices on a quartz slide, and immersing the slices in deionized water completely.

8. The method of screening industrial hemp plants with high cannabidiol content according to claim 1, wherein the step 2) of placing the processed quartz slide on the stage of the micro-Raman spectrometer comprises placing the quartz slide with the section on the stage of the micro-Raman spectrometer, searching the oil storage organ on the section, performing Raman spectroscopy on the essential oil in the oil storage organ to obtain the Raman spectrum of the essential oil in the oil storage organ of each industrial hemp female plant, and keeping the laser power unchanged during the measurement.

9. The method for obtaining industrial hemp plants with high cannabidiol content through the screening methods of claim 1, wherein the average Raman spectrum is analyzed by using Raman spectrometer software.

Technical Field

The invention relates to the technical field of screening culture of industrial cannabis, in particular to a method for obtaining high-content cannabidiol industrial cannabis plants through screening methods.

Background

The application of industrial hemp relates to the fields of medicine, cosmetology, health care, food, textile, bioengineering materials, paper making, military requirements and other light industries, in recent years, Cannabidiol (CBD) extracted from industrial hemp flowers and leaves is developed very rapidly, the cannabidiol has multiple functions of anti-inflammation, analgesia, anxiety resistance, spasm resistance, tumor resistance and the like, and has extensive application prospects in multiple fields of medicine, cosmetology, health care and the like.

In order to meet the requirement of rapid development of CBD development, in recent years, the production scale of industrial hemp (CBD raw material) is rapidly expanded in China, but the cultivation time is long, the cultivation quality can be detected only after cultivation is completed, and the cultivation period is long.

Through literature search, the same public reports as the technical scheme of the invention are not found.

Disclosure of Invention

The invention provides methods for obtaining industrial hemp plants with high content of cannabidiol by screening.

The scheme of the invention is as follows:

method for obtaining industrial hemp plant with high content of cannabidiol by screening, comprising the following steps:

1) determining a standard, namely dripping a cannabidiol standard sample on a quartz slide glass, placing the quartz slide glass on an objective table of a micro-Raman spectrometer, carrying out micro-Raman spectrum detection on cannabidiol to obtain a Raman spectrum of the cannabidiol standard sample, and determining a characteristic spectrum of the cannabidiol Raman spectrum through analysis and research;

2) processing industrial hemp, namely taking sections of leaf stalks of female industrial hemp plants when the industrial hemp reaches 5-10 cm in seedling culture in the seedling culture period of the industrial hemp, respectively processing the taken leaf stalks, placing the processed leaf stalks on an object stage of a micro-Raman spectrometer, and carrying out micro-Raman spectrum detection on the processed leaf stalks to obtain Raman spectra of the essential oil of the female industrial hemp plants;

3) analyzing, namely taking at least 10 spectral lines from the Raman spectrum of the industrial female marijuana essential oil obtained from each leaf stalk, calculating an average Raman spectrum, comparing the average Raman spectrum obtained from each leaf stalk with the characteristic spectrum of the cannabidiol Raman spectrum in the step 1), determining the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, comparing the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, and screening out the leaf stalk with the highest content of the cannabidiol through the beer law;

4) culturing, namely performing tissue culture by taking the industrial hemp plant with the petiole with high cannabidiol content screened in the step 3) as a female parent.

Preferably, in the step 3), the average raman spectrum obtained from each petiole is compared with the characteristic spectrum of the raman spectrum of the cannabidiol standard sample in the step 1), and the characteristic spectrum of the cannabidiol of the average raman spectrum obtained from each petiole is determined by comparing the peak positions of the average raman spectrum of each petiole with the characteristic spectrum of the raman spectrum of the cannabidiol standard sample in the step 1).

Preferably, the peak heights of the characteristic spectra of cannabidiol in the average raman spectrum of each petiole in step 3) are compared, and the petiole with the highest cannabidiol content is selected by beer's law.

As a preferable technical scheme, 10-20 spectral lines are taken from the Raman spectrum of the industrial hemp female plant essential oil obtained from the petiole in the step 3).

Preferably, the length of the -segment petiole in the step 2) is 0.5-1.5 cm.

As a preferable technical scheme, in the micro-raman spectrometer used in the micro-raman spectroscopy detection in the step 1), the laser power is 2mw, and the laser wavelength is 785 nm; the laser power in the micro-Raman spectrometer of the step 2) is 10 mw-15 mw, and the laser wavelength is 785 nm.

As a preferable technical solution, the method of processing the extracted petioles in the step 2) comprises: the petioles were sliced with a microtome, the slices were placed in a petri dish containing deionized water, transparent and intact slices were picked with a writing brush and placed on a quartz slide, and the slices were completely immersed in deionized water.

As a preferred technical solution, the method of placing the processed sample on the stage of the micro-raman spectrometer in the step 2) comprises: placing the quartz glass slide with the slices on an object stage of a micro-Raman spectroscopy spectrometer, searching an oil storage organ on the slices, performing Raman spectrum measurement on essential oil in the oil storage organ to obtain Raman spectra of the essential oil in the oil storage organ of each industrial hemp female plant, wherein the laser power is kept unchanged in the measurement process.

As a preferred embodiment, the method of analyzing the average raman spectrum comprises: and (4) averaging the Raman spectrum by using self-contained software of the Raman spectrometer.

The technical scheme is adopted, screening methods for obtaining industrial hemp plants with high content of cannabidiol are adopted, 1) standard determination is carried out, cannabidiol standard samples are taken and dripped on quartz glass slides, the quartz glass slides are placed on an object stage of a micro-Raman spectrometer, the cannabidiol is subjected to micro-Raman spectrum detection to obtain Raman spectra of the cannabidiol standard samples, characteristic spectra of the cannabidiol Raman spectra are determined through analysis and research, 2) industrial hemp treatment is carried out, when the industrial hemp reaches 5-10 cm in seedling culture, leaf stalks of female marijuana plants of each industrial hemp are taken, the taken leaf stalks are respectively treated and placed on the object stage of the micro-Raman spectrometer after treatment, the micro-Raman spectrum detection is carried out on the leaf stalks to obtain Raman spectra of essential oil of the female marijuana plants of each industrial hemp, 3) analysis is carried out, at least 10 spectral lines are taken for the Raman spectra of the female marijuana essential oil of each industrial hemp obtained from each leaf stalk, average Raman spectra are obtained, the average Raman spectra obtained from each leaf stalk are compared with the spectrum obtained in the spectrum of the spectral lines of the industrial hemp female mariana essential oil of each leaf stalk, the leaf stalk is used as the average Raman spectrum of the characteristic spectra of the leaf stalks of the cannabidiol obtained from the leaf stalks, the average Raman spectra obtained by the comparison of the leaf spectra obtained by the step 3), the comparison of the leaf spectra obtained by the leaf tissue comparison of the leaf tissue of.

The invention has the advantages that:

1. the characteristic spectrum of the Raman spectrum of the petiole part is obtained by applying the Raman spectrum technology to the petiole part of the female industrial hemp plant without extraction, and the plant with high content of cannabidiol can be screened out by comparing the characteristic spectrum with the characteristic spectrum of the Raman spectrum of a cannabidiol standard sample by applying the beer law.

2. The screening can be carried out at the seedling stage of the plant, which is beneficial to the tissue culture at the later stage.

3. Compared with other breeding techniques, it can greatly shorten the period of breeding variety with good characters, and provides new approaches for developing and utilizing industrial hemp.

Detailed Description

In order to make up for the above deficiencies, the present invention provides screening methods for obtaining industrial hemp plants with high cannabidiol content to solve the above problems in the background art.

method for obtaining industrial hemp plant with high content of cannabidiol by screening, comprising the following steps:

1) determining a standard, namely dripping a cannabidiol standard sample on a quartz slide glass, placing the quartz slide glass on an objective table of a micro-Raman spectrometer, carrying out micro-Raman spectrum detection on cannabidiol to obtain a Raman spectrum of the cannabidiol standard sample, and determining a characteristic spectrum of the cannabidiol Raman spectrum through analysis and research;

2) processing industrial hemp, namely taking sections of leaf stalks of female industrial hemp plants when the industrial hemp reaches 5-10 cm in seedling culture in the seedling culture period of the industrial hemp, respectively processing the taken leaf stalks, placing the processed leaf stalks on an object stage of a micro-Raman spectrometer, and carrying out micro-Raman spectrum detection on the processed leaf stalks to obtain Raman spectra of the essential oil of the female industrial hemp plants;

3) analyzing, namely taking at least 10 spectral lines from the Raman spectrum of the industrial female marijuana essential oil obtained from each leaf stalk, calculating an average Raman spectrum, comparing the average Raman spectrum obtained from each leaf stalk with the characteristic spectrum of the cannabidiol Raman spectrum in the step 1), determining the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, comparing the characteristic spectrum of the cannabidiol in the average Raman spectrum of each leaf stalk, and screening out the leaf stalk with the highest content of the cannabidiol through the beer law;

4) culturing, namely performing tissue culture by taking the industrial hemp plant with the petiole with high cannabidiol content screened in the step 3) as a female parent.

And in the step 3), comparing the average Raman spectrum obtained by each petiole with the characteristic spectrum of the Raman spectrum of the cannabidiol standard sample in the step 1), and determining the characteristic spectrum of the cannabidiol of the average Raman spectrum obtained by each petiole through peak position comparison between the average Raman spectrum of each petiole and the characteristic spectrum of the Raman spectrum of the cannabidiol standard sample in the step 1).

Comparing the peak heights of the characteristic spectrum of the cannabidiol of the average Raman spectrum of each leaf stalk in the step 3), and screening out the leaf stalk with the highest cannabidiol content through the beer law.

And in the step 3), 10-20 spectral lines are taken from the Raman spectrum of the industrial hemp female plant essential oil obtained from the petiole.

The length of the -section petiole in the step 2) is 0.5-1.5 cm.

The laser power of the Raman micro-Raman spectrometer used in the micro-Raman spectrum detection of the step 1) is 2mw, and the laser wavelength is 785 nm; the laser power in the micro-Raman spectrometer in the step 2) is 10-15 mw, and the laser wavelength is 785 nm.

The method for respectively processing the taken out petioles in the step 2) comprises the following steps: the petioles were sliced with a microtome, the slices were placed in a petri dish containing deionized water, transparent and intact slices were picked with a writing brush and placed on a quartz slide, and the slices were completely immersed in deionized water.

The method for placing the processed sample on the object stage of the micro-Raman spectrometer in the step 2) comprises the following steps: and placing the quartz glass slide with the slices on an object stage of a micro-Raman spectrometer, searching an oil storage organ on the slices, performing Raman spectrum measurement on essential oil in the oil storage organ to obtain the Raman spectrum of the essential oil in the oil storage organ of each industrial hemp female plant, wherein the laser power is kept unchanged in the measurement process.

The method for analyzing the average Raman spectrum comprises the following steps: and (4) averaging the Raman spectrum by using self-contained software of the Raman spectrometer.

In order to make the technical means, objectives and functions of the present invention easy to understand, the present invention will be further described with reference to the following embodiments.