CN114885635A - Method for accumulating carotenoids in seedling stage of orange Chinese cabbage - Google Patents
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Abstract
The invention discloses a method for accumulating carotenoid in seedling stage of orange Chinese cabbage, which comprises the following steps: preparing a Chinese cabbage material, selecting orange Chinese cabbage seeds with plump seeds; preparing a tool before the Chinese cabbage shading treatment: black cloth bag, iron wire or bamboo stick, ruler, binding rope; and (3) shading treatment of the Chinese cabbages in the seedling stage: in the period from five leaves to eight leaves to one heart of orange Chinese cabbage seedlings, the color of the inner leaves is changed after the Chinese cabbage in the seedling stage is induced for 1 hour by monochromatic blue light; measuring the content of carotene in the Chinese cabbage by using an HPLC high performance liquid chromatography technology. The black cloth shading technology is used for simulating the inner leaf environment of orange Chinese cabbage in the mature period, so that the carotenoid in the seedling period can be quickly accumulated.
Description
Technical Field
The invention relates to the technical field of Chinese cabbage planting, in particular to a method for accumulating carotenoid in seedling stage of orange Chinese cabbage.
Background
Chinese cabbage is one of the most important vegetables in Asian regions, and white, yellow and orange are the most popular leaf ball inner leaf colors in Chinese cabbage. The orange Chinese cabbage has unique color, rich nutrition value and delicious taste, is deeply favored by consumers, and in addition, the content of carotenoid of the orange Chinese cabbage is higher than that of carotenoid of normal white Chinese cabbage, more importantly, the orange Chinese cabbage contains lycopene, and the material also provides favorable conditions for researching the metabolic pathway of carotenoid in inner leaves of the Chinese cabbage of horticultural crops. The research on the positioning cloning of carotenoid isomerase in the inner leaves of orange Chinese cabbages is carried out in the early stage of a Chinese cabbage subject group of horticulture academy of northwest agriculture and forestry science and technology university, and the research on the influence of various exogenous monochromatic light qualities on the accumulation of carotenoid in the inner leaves of orange Chinese cabbages in an in-vitro maturation period is carried out, but the growth period of the orange Chinese cabbages is long, the acquisition of leaf materials in the maturation period needs 4-5 months, the test period is long, and the operation is troublesome. In addition, in the researches, in-vitro leaves of the inner leaves of the Chinese cabbage in the mature period are taken as research materials, the in-vitro leaves cannot completely represent the response process of living plants to the environment, and the accurate research on the color generation mechanism of the inner leaves of the living orange Chinese cabbage under the environmental conditions is limited, so that a method for quickly realizing the carotenoid accumulation in the seedling period of the orange Chinese cabbage is needed to be established, and the method has important significance for researching the color generation mechanism of the inner leaves of the orange Chinese cabbage and provides technical support for producing high-quality orange Chinese cabbage seedling. At present, no method for realizing the accumulation of carotenoid in the seedling stage of orange Chinese cabbages exists.
Disclosure of Invention
The invention aims to provide a method for accumulating carotenoids in the seedling stage of orange Chinese cabbages, which simulates the inner leaf environment of orange Chinese cabbages in the mature stage by using a black cloth shading technology so as to quickly realize the quick accumulation of the carotenoids in the seedling stage.
In order to realize the aim, the invention provides a method for accumulating carotenoid in seedling stage of orange Chinese cabbage, which comprises the following steps:
s1, Chinese cabbage material preparation:
selecting full-grain orange Chinese cabbage seeds, soaking the seeds in warm soup at 55 ℃ for 20min, soaking the seeds in clear water for 5h, accelerating germination in a dark-black artificial climate box at 25 ℃, exposing the seeds to white for 4d, directly sowing the exposed seeds in a plastic nutrition pot of 10 multiplied by 10cm, transferring the plastic nutrition pot to a light incubator for normal growth, and culturing for 30d until the Chinese cabbage of five-leaf one-heart to eight-leaf one-heart seedling age is used for subsequent shading treatment;
s2, preparing a tool before the Chinese cabbage shading treatment:
storing a black cloth bag, an iron wire or bamboo stick, a ruler and a binding rope;
s3, shading treatment of the Chinese cabbage in the seedling stage:
selecting a black cloth bag made of black cloth to shade the Chinese cabbages with the age of five-leaf one-heart to eight-leaf one-heart seedlings, and then transferring the Chinese cabbages into a light incubator to be cultured, wherein the color of the inner leaves of the seedlings is changed into orange yellow after shading treatment for 25 days, the color state of the inner leaves of the mature period of the orange Chinese cabbages is achieved, and the growth period is shortened by two months compared with the orange Chinese cabbages grown in a field to the mature period;
s4, inducing the color change of the Chinese cabbage inner leaves by blue light:
based on the process that the color of inner leaves of the orange Chinese cabbage is changed from yellow to orange under the induction of natural light, the inner leaves of the orange Chinese cabbage are treated by LED lamps with different colors, the Chinese cabbage in the seedling stage is induced by monochromatic blue light (lambda is 460nm) for 1h, then the color of the inner leaves is changed, if the color is changed from yellow to orange, the Chinese cabbage is an orange Chinese cabbage, otherwise, if the color of the inner leaves is not changed, the Chinese cabbage is a common Chinese cabbage;
s5, measuring the content of the carotene in the Chinese cabbage by using an HPLC high performance liquid chromatography technology.
Preferably, the test material in the step S1 is one of common chinese cabbage 14S23, orange chinese cabbage 14S837, 15S1094, 20S530, and 27-2.
Preferably, the seeds are directly sown in the plastic nutrition bowls in an exposed white manner in the step S1, and are transferred to a light incubator for normal growth, wherein the cultivation conditions in the process are illumination for 16h at 25 ℃ or illumination for 8h at 20 ℃, the relative humidity is 65%, and the illumination intensity is 20000 lx.
Preferably, the cultivation conditions of the light incubator of step S3 are illumination at 25 ℃ for 16h or 20 ℃ for 8h, relative humidity is 45%, and light intensity is 20000 lx.
Preferably, in the step S5, the content of carotene in the Chinese cabbage is determined by using an HPLC high performance liquid chromatography technology, specifically, 2g of the soft leaf part of the test material is stored in a centrifuge tube, freeze-dried in a vacuum freeze dryer, and stored at-80 ℃; then grinding the freeze-dried cabbage leaves, adding 5mL of carotenoid extracting solution, fully and uniformly mixing, extracting for 70min, taking out, centrifuging for 10min at 12000rmp, repeating the operation twice, and combining supernate; placing the supernatant in a round-bottomed flask, evaporating to dryness at 35 ℃ under reduced pressure, dissolving with 1mL of ethyl acetate, filtering through a 0.22-micron organic filter head after dissolution to a 1.5mL brown sample injection bottle for detection on the machine, and analyzing and determining the carotenoid by adopting an Agilent1260InfinityII high performance liquid chromatography system (lee, 2000).
Preferably, the carotenoid extracting solution is a mixture of acetone and ethanol, wherein the mass ratio of the acetone to the ethanol is 1: 1.
Preferably, the analysis and determination conditions of the Agilent1260InfinityII high performance liquid chromatography system are C30 column (4.6mm multiplied by 250mm, 5 μm), the detection wavelength is 450nm, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample introduction amount is 20 μ L;
the mobile phase comprises a phase A and a phase B, wherein the phase A: acetonitrile: methanol 3: 1 (containing 0.01% BHT and 0.05% TEA); phase B: 100% MTBE (containing O.01% BHT) at a flow rate of 1 ml/min;
the elution conditions were as follows:
0min：A-B(95：5)；
0-10min：A-B(95：5)；
10—19min：A-B(86：14)；
19-29min：A-B(75：25)；
29-54min：A-B(50：50)；
54-66min：A-B(26：74)；
67min：A-B(95：5)。
therefore, the method for accumulating the carotenoid in the seedling stage of the orange Chinese cabbage is adopted, and the black cloth shading technology is used for simulating the inner leaf environment of the orange Chinese cabbage in the mature stage, so that the carotenoid in the seedling stage is quickly accumulated.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a flow chart of an embodiment of the method for accumulating carotenoids in the seedling stage of orange Chinese cabbage of the present invention;
FIG. 2 is a schematic view showing the measurement of the height of Chinese cabbage and the length of leaf crown before shading treatment;
FIG. 3 is a schematic view of a preparation tool before shading orange Chinese cabbage;
FIG. 4 is a schematic view of orange Chinese cabbage 15S1094 in the sun-shading treatment of five leaves in one heart phase, wherein A is the phenotype of Chinese cabbage before sun-shading; b, preparing a black cloth bag for shading treatment; c, sheathing the base part, and slightly tying the base part to avoid damaging the inner leaves of the Chinese cabbage; d is the phenotype after shading treatment; e, inserting bamboo sticks around the Chinese cabbage, and binding ropes to prevent the inner leaves from being damaged by the falling of the shading black cloth bag; f, shading the celery cabbage, and then normally culturing for 25 days in an illumination incubator (the culture temperature is 16h under illumination of 25 ℃ or 8h under illumination of 20 ℃, the relative humidity is 45%, and the illumination intensity is 20000 lx);
FIG. 5 shows the phenotype of inner leaves of five-leaf one-heart common Chinese cabbage 14S23 after shading treatment, 25 days without shading treatment, and 90 days after normal field growth;
FIG. 6 is a graph of the inner leaf phenotype of a five-leaf, one-heart orange Chinese cabbage 15S1094 after shading treatment, 25 days without shading treatment, and 90 days after normal field growth;
FIG. 7 shows the phenotype of 20S530, 27-2, 15S1094, and 14S23 after blue-light treatment of four light-shielded seedlings and Chinese cabbage for 1 h;
FIG. 8 shows the change in the major carotenoid content in inner leaves after 25 days of shading treatment (Shade) and non-shading treatment (WT) in different materials.
Detailed Description
In the embodiment, the black cloth shading treatment technology is used for simulating the conditions of growth of the inner leaves of the Chinese cabbage to accumulate the carotenoid in the inner leaves of the orange Chinese cabbage, and meanwhile, an effective means is provided for screening new germplasm resources of the orange Chinese cabbage, so that convenient conditions are further provided for researching the color development mechanism of the inner leaves.
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
Examples
The invention provides a method for accumulating carotenoid in seedling stage of orange Chinese cabbage, which comprises the following steps:
s1, Chinese cabbage material preparation:
the test material is one of common Chinese cabbage 14S23, orange Chinese cabbage 14S837, 15S1094, 20S530, and 27-2. Selecting full-seed Chinese cabbage seeds, soaking the seeds in warm soup at 55 deg.C for 20min, soaking in clear water for 5h, and performing germination acceleration in a dark-black artificial climate box at 25 deg.C; exposing the seeds for 4 days, directly sowing the seeds in a 10 multiplied by 10cm plastic nutrition pot, transferring the seeds to an illumination incubator for normal growth under the conditions that the illumination is carried out for 16 hours at 25 ℃ or 8 hours at 20 ℃, the relative humidity is 65 percent, the illumination intensity is 20000lx, and the seeds are cultured for about 30 days until the seedlings of five-leaf one-heart to eight-leaf one-heart Chinese cabbages are used for subsequent shading treatment.
(2) Shading treatment of seedling Chinese cabbage
In order to simulate the outer leaf environment of orange Chinese cabbage in the mature period, black cloth bags made of black cloth are selected to shade the Chinese cabbage of five-leaf one-heart to eight-leaf one-heart seedling age, as shown in fig. 4, and then the Chinese cabbage is transferred to an illumination incubator for cultivation, wherein the cultivation temperature is 16h under illumination of 25 ℃ or 8h under illumination of 20 ℃, the relative humidity is 45%, and the illumination intensity is 20000 lx. After shading treatment for 25 days, the color of the inner leaves becomes orange yellow, the color state of the inner leaves of the mature period of the orange Chinese cabbage is achieved, and the growth period is shortened by two months compared with that of the orange Chinese cabbage growing to the mature period in the field.
(3) Blue light induction of Chinese cabbage inner leaf color change
Based on the process that the color of the inner leaves of the orange Chinese cabbage is changed from yellow to orange under the induction of natural light, the inner leaves of the orange Chinese cabbage are further treated by different LED lamps, the fact that sunlight promotes the increase of the total carotenoid content of the orange Chinese cabbage is found, the increase of lutein is also induced, red light inhibits the accumulation of the carotenoid in the Chinese cabbage leaves, the accumulation of zeaxanthin is probably promoted through red light and blue light treatment, and in addition, the fact that the lycopene content in the 14 hetero 1 orange Chinese cabbage leaves is increased through blue light treatment is found through other researches. After being treated by red light, yellow light and green light, the contents of alpha-carotene, beta-carotene and lycopene in the 14 za 1 and 13 za 99 orange Chinese cabbage leaves are reduced in different degrees, but the common Chinese cabbage does not have the phenomenon, so that the color of the inner leaves of the Chinese cabbage is changed after the Chinese cabbage in the seedling stage is induced for 1 hour by monochromatic blue light (lambda is 460nm), if the color is changed from yellow to orange, the Chinese cabbage is orange, otherwise, if the color of the inner leaves is not changed, the Chinese cabbage is the common Chinese cabbage, and the technology can also be used for rapidly and efficiently screening and identifying the orange Chinese cabbage strain.
In the embodiment, the inner leaves of the orange Chinese cabbage in the seedling stage are changed into orange yellow, the common Chinese cabbage is yellow or white, and after the blue light treatment is carried out for 1 hour, the inner leaves of the orange Chinese cabbage are changed into orange, and the inner leaves of the common Chinese cabbage are unchanged in color.
(4) And (3) determining carotenoid components of the Chinese cabbage in the seedling stage, the Chinese cabbage not subjected to shading treatment and the Chinese cabbage in the mature stage by using an HPLC high performance liquid chromatography technology.
In order to further clarify the change of carotenoid components in the leaf during the seedling stage after the light-shielding treatment, the carotenoid content in the leaf of Chinese cabbage was measured by HPLC. The test material soft leaf part 2g is stored in a centrifuge tube, freeze-dried in a vacuum freeze dryer and stored at-80 ℃. Used for extracting carotenoid and analyzing components. Grinding the freeze-dried cabbage leaves, adding mL carotenoid extracting solution (acetone: ethanol is 1:1, the mass ratio of the two is equal), fully mixing, extracting for 70min, taking out, centrifuging at 12000rmp for 10min, repeating the operation twice, and combining the supernate. The supernatant was evaporated to dryness under reduced pressure at 35 ℃ in a round-bottomed flask, and dissolved in 1mL of ethyl acetate, and then filtered through a 0.22 μm organic filter head into a 1.5mL brown sample bottle for on-machine detection (the whole process should be protected from light).
Carotenoid assay reference (lee, 2000) using an Agilent1260Infinity II high performance liquid chromatography system, C30 column (4.6 mm. times.250 mm, 5 μm); the detection wavelength is 450nm, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 20 mu L; the mobile phase is, A phase: acetonitrile: methanol 3: 1 (containing 0.01% BHT and 0.05% TEA); phase B: 100% MTBE (containing 0.01% BHT). The flow rate was 1 ml/min. The elution conditions are as follows, 0min: A-B (95: 5); 0-10 min: A-B (95: 5); 10-19 min: A-B (86: 14); 19-29 min: A-B (75: 25); 29-54 min: A-B (50: 50); 54-66 min: A-B (26: 74); 67 min: A-B (95: 5). The influence of black cloth bag shading treatment on the accumulation of inner leaf carotenoids is further determined by comparing the components and the content of the inner leaf carotenoids of the Chinese cabbage.
The results show that: the shading treatment does not influence the change of the main carotenoid components of the orange Chinese cabbage; compared with the non-shading material, the shading treatment reduces the contents of lutein, zeaxanthin, alpha-carotenoid and beta-carotenoid in the orange Chinese cabbage, and the shading treatment increases the content of lycopene in different orange Chinese cabbage materials, thereby providing favorable conditions for researching the metabolism pathway of the carotenoid of the orange Chinese cabbage. Compared with materials in the mature period, the shading treatment in the seedling period is beneficial to the accumulation of bioactive substances such as beta-carotene, lutein, zeaxanthin and the like. The treatment also provides certain help for the production of high-quality Chinese cabbage diet products.
Therefore, the method for accumulating the carotenoid in the seedling stage of the orange Chinese cabbage is adopted, and the black cloth shading technology is used for simulating the inner leaf environment of the orange Chinese cabbage in the mature stage, so that the carotenoid in the seedling stage is quickly accumulated.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Claims (7)
1. A method for accumulating carotenoid in the seedling stage of orange Chinese cabbage is characterized by comprising the following steps:
s1, Chinese cabbage material preparation:
selecting full-grain orange Chinese cabbage seeds, soaking the seeds in warm soup at 55 ℃ for 20min, soaking the seeds in clear water for 5h, accelerating germination in a dark-black artificial climate box at 25 ℃, exposing the seeds to white for 4d, directly sowing the exposed seeds in a plastic nutrition pot of 10 multiplied by 10cm, transferring the plastic nutrition pot to a light incubator for normal growth, and culturing for 30d until the Chinese cabbage of five-leaf one-heart to eight-leaf one-heart seedling age is used for subsequent shading treatment;
s2, preparing a tool before the Chinese cabbage shading treatment:
storing a black cloth bag, an iron wire or bamboo stick, a ruler and a binding rope;
s3, shading treatment of the Chinese cabbage in the seedling stage:
selecting a black cloth bag made of black cloth to shade the Chinese cabbages with the age of five-leaf one-heart to eight-leaf one-heart seedlings, and then transferring the Chinese cabbages into a light incubator to be cultured, wherein the color of the inner leaves of the seedlings is changed into orange yellow after shading treatment for 25 days, the color state of the inner leaves of the mature period of the orange Chinese cabbages is achieved, and the growth period is shortened by two months compared with the orange Chinese cabbages grown in a field to the mature period;
s4, inducing the color change of the Chinese cabbage inner leaves by blue light:
based on the process that the color of inner leaves of the orange Chinese cabbage is changed from yellow to orange under the induction of natural light, the inner leaves of the orange Chinese cabbage are treated by LED lamps with different colors, the Chinese cabbage in the seedling stage is induced by monochromatic blue light (lambda is 460nm) for 1h, then the color of the inner leaves is changed, if the color is changed from yellow to orange, the Chinese cabbage is an orange Chinese cabbage, otherwise, if the color of the inner leaves is not changed, the Chinese cabbage is a common Chinese cabbage;
s5, measuring the content of the carotene in the Chinese cabbage by using an HPLC high performance liquid chromatography technology.
2. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 1, wherein: the tested material in the step S1 is one of common Chinese cabbage 14S23 and orange Chinese cabbage 14S837, 15S1094, 20S530 and 27-2.
3. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 1, wherein: in the step S1, the seeds are exposed and directly sown in a plastic nutrition pot and transferred to an illumination incubator for normal growth, wherein the culture conditions in the process are illumination for 16h at 25 ℃ or 8h at 20 ℃, the relative humidity is 65%, and the illumination intensity is 20000 lx.
4. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 1, wherein: the culture conditions of the illumination incubator of the step S3 are illumination for 16h at 25 ℃ or illumination for 8h at 20 ℃, the relative humidity is 45%, and the illumination intensity is 20000 lx.
5. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 1, wherein: in the step S5, the HPLC high performance liquid chromatography is utilized to measure the carotene content in the Chinese cabbage, specifically, 2g of soft leaf part of a test material is stored in a centrifuge tube, freeze-dried in a vacuum freeze-drying instrument and stored at the temperature of minus 80 ℃; then grinding the freeze-dried cabbage leaves, adding 5mL of carotenoid extracting solution, fully and uniformly mixing, extracting for 70min, taking out, centrifuging for 10min at 12000rmp, repeating the operation twice, and combining supernate; placing the supernatant into a round-bottomed flask, evaporating to dryness at 35 ℃ under reduced pressure, dissolving with 1mL of ethyl acetate, filtering by a 0.22-micron organic filter head after dissolution into a 1.5mL brown sample injection bottle for detection by an upper machine, and analyzing and determining carotenoid by adopting an Agilent1260InfinityII high performance liquid chromatography system according to a reference lee, 2000.
6. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 5, wherein: the carotenoid extracting solution is a mixture of acetone and ethanol, wherein the mass ratio of the acetone to the ethanol is 1: 1.
7. The method for accumulating carotenoids in the seedling stage of orange Chinese cabbage according to claim 5, wherein: the analysis and determination conditions of an Agilent1260InfinityII high performance liquid chromatography system are C30 column (4.6mm multiplied by 250mm, 5 mu m), the detection wavelength is 450nm, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample introduction amount is 20 mu L;
the mobile phase comprises a phase A and a phase B, wherein the phase A: acetonitrile: methanol 3: 1 (containing 0.01% BHT and 0.05% TEA); phase B: 100% MTBE (containing O.01% BHT) at a flow rate of 1 ml/min;
the elution conditions were as follows:
0min：A-B(95：5)；
0-10min：A-B(95：5)；
10—19min：A-B(86：14)；
19-29min：A-B(75：25)；
29-54min：A-B(50：50)；
54-66min：A-B(26：74)；
67min：A-B(95：5)。
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