CN108056021B - Method for inducing purslane callus and producing flavonoid - Google Patents
Method for inducing purslane callus and producing flavonoid Download PDFInfo
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Abstract
The invention provides a purslane callus induction and flavonoid production method, belonging to the callus induction field, wherein a seedling hypocotyl after purslane seed germination for 7 days is taken as an explant, the explant is inoculated in MB5+ 1.0 mg/L6-BA +0.5 mg/L2, 4-D + AgNO34.0 mg/L culture medium, and after the culture is carried out for 30 days under blue light, the proliferation coefficient and the flavonoid content are highest. The method is simple, and provides a faster and effective way for the industrial production of the flavonoid by the purslane.
Description
Technical Field
The invention belongs to the field of callus induction, and particularly relates to a method for inducing purslane callus and producing flavonoid.
Background
Purslane (purslane)Portulaca oleraceaL), also called longevity vegetable, longevity grass, etc., is a fleshy wild herb plant that can complete the whole growth cycle in the same year, and is also called Wuxing grass in Ben Cao Tu Jing because of the different colors of its root (white), stem (red), leaf (green), flower (yellow) and seed (black). The purslane has strong adaptability in south and north of great rivers in China, has the same protection characteristic as other common wild plants, and has obvious heat resistance and drought resistance. The purslane is popular with consumers and scholars because it not only contains various chemical components with certain medicinal value, such as mineral substances, amino acids, polysaccharides, fatty acids, vitamins, alkaloids, acids, flavonoids and the like, but also has the functions of resisting bacteria, stopping bleeding and dispersingThe health food has the functions of detumescence, disinsection and sterilization, inflammation diminishing and diuresis, heat clearing and detoxification, diuresis inducing, immunity enhancing, oxidation resistance, aging resistance, tumor resistance and the like, and is one of health foods certified by the national ministry of health. In recent years, purslane has been studied more in the aspects of chemical components, clinical application, and health food processing and cultivation technology, and because purslane can be eaten and used as a medicine, but wild resources are deficient, scholars also strengthen the application of purslane in tissue culture, so as to meet the demand of seed resources and cultivate high-yield and high-quality purslane varieties. The first report of domestic research on tissue culture of purslane plants appears in 2003. The study on tissue culture and rapid propagation of the purslane, namely Qinninghong and Li 28952is to establish a plant regeneration system, and 0.5-2.0 mg/L6-BA and 0.5 mg/L2, 4-D are added in an MS culture medium, so that the induction of the callus is favorably carried out by taking cotyledon and hypocotyl as explants. Later researchers also achieved the same results.
The light is one of the most important environmental factors in plant growth and development. It is an energy source that can control photosynthesis, but also an induction signal that can affect tissue growth. Different photoreceptors in the tissue culture seedlings sense optical signals, so that the growth and development, the photosynthetic property and the property expression of the tissue culture seedlings are influenced. For the application of light quality in the aspects of plant callus induction and growth, researches on various plants in the aspects have been reported, such as jellyfish saussurea involucrata researched by Zhao De Xie and the like, grapes researched by Zhang Zhen and the like, garlic researched by Malin and the like, No. 47 Huai Yam researched by Guojunli and the like, and the research results show that the plant callus induction or growth is greatly influenced by the light quality, and the appropriate light quality is favorable for the pair of the callus induction and growth. Therefore, the influence of the light quality on the induction and growth of the purslane callus has certain research significance, and the invention is expected to provide a faster and effective way for the industrial production of flavonoids in the purslane callus.
Disclosure of Invention
The invention aims to provide a method for inducing purslane callus and producing flavonoid.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for inducing callus of purslane and producing flavonoid comprises the steps of taking seedling hypocotyls of purslane seeds after sprouting for 7 days as explants, and inoculating the explants to MB5+ 1.0 mg/L6-BA +0.5 mg/L2, 4-D + AgNO34.0mg/L medium, cultured under blue light for 30 days. The culture conditions were 12h light each day at 25 ℃.
The seedling culture method comprises the following steps: wrapping herba Portulacae seed with filter paper, soaking in 50-55 deg.C warm water for 10-15min, soaking in warm water for sterilization, and washing with running water for 20 min; then sterilizing the seeds wrapped by the filter paper in 75% alcohol for 60 s; then at 0.1% HgCl2Sterilizing for 8min while stirring; then washing with sterile water for 4-5 times; and finally inoculating the seedlings into an MS basic culture medium subjected to high-pressure sterilization treatment, and culturing the MS basic culture medium under the conditions of illumination intensity of 2000-3000 lx, illumination time of 12h/d and temperature of 25 +/-2 ℃ to obtain sterile seedlings.
The invention has the advantages that:
1. establishes a rapid and efficient purslane callus induction system, saves the culture time
2. The culture condition for improving the flavonoid content in the purslane callus is established, annual production of the purslane flavonoid can be realized, and resources are saved.
Detailed Description
Example 1
1 materials and methods
1.1 test materials
The test uses wild purslane seeds produced in Sichuan as test materials.
1.2 test methods
1.2.1 obtaining of sterile seedlings
Wrapping herba Portulacae seed with filter paper, soaking in 50-55 deg.C warm water for 10-15min, soaking in warm water for sterilization, washing with running water for 20 min, and sterilizing and inoculating in a clean bench. Firstly, disinfecting seeds wrapped by filter paper for 60s in 75% alcohol; then at 0.1% HgCl2Sterilizing for 8min while stirring; washing with sterile water for 4-5 times(ii) a And finally inoculating the seeds into an MS basic culture medium which is subjected to high-pressure sterilization treatment, inoculating 10 seeds into each bottle, inoculating 40 bottles, and culturing under the conditions that the illumination intensity is 2000-3000 lx, the illumination time is 12h/d and the temperature is 25 +/-2 ℃ to obtain aseptic seedlings.
1.2.2 Effect of different seedling ages and tissue parts on the induction of purslane callus
Sterile seedlings of 4d, 7d, 10 d, 13 d and 16 d after purslane seed germination are taken as materials, cotyledons and hypocotyls are respectively inoculated into callus induction culture media MB5+6-BA1.0mg/L +2,4-D0.5mg/L for induction, and the influence of different seedling ages and tissue parts on purslane callus is compared.
1.2.3 Effect of different hormones on callus induction of Portulaca oleracea
(1) Influence of different concentrations of 6-BA on callus induction of purslane
Inoculating 7D seedling-aged cotyledons to callus induction culture medium MB5+ 0.5 mg/L2, 4-D + AgNO34.0mg/L and 6-BA (0.5 mg/L, 1.0mg/L, 1.5 mg/L, 2.0 mg/L and 3.0 mg/L) in different concentrations are added for induction, and the influence of the 6-BA in different concentrations on the induction of the purslane callus is compared. Statistical callus induction rates were observed at 30d of culture. Each treatment was inoculated with 30 explants, 3 replicates.
(2) Influence of IAA with different concentrations on callus induction of purslane
Inoculating 7d seedling-aged cotyledons to callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and was induced with different concentrations of IAA (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L).
(3) Different concentrations of 2,4-D
Inoculating 7d seedling-aged cotyledons to callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and 2,4-D (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L) was added at different concentrations for induction.
(4) Different concentrations of NAA
Inoculating 7d seedling-aged cotyledons to callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and adding NAA (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L) at different concentrations for induction.
1.2.4 Effect of different photophobic substances on callus induction of purslane
Selecting tissue culture strong seedlings with good and consistent growth vigor as test materials, cutting leaves into 0.5cm2The young stem of the left and right squares is cut into stem segments of about 1-2cm, inoculated on the optimum culture medium MS +6-BA1.0mg/L +2,4-D0.5mg/L for inducing the callus of the purslane, the back of the leaf is contacted with the culture medium substrate during inoculation, 18 bottles of inoculation are processed, 5 bottles are processed, and 3 times of repetition are carried out. Inoculating, culturing in 6 light-treated culture racks of red light, blue light, white light, yellow light, green light and dark at 25 deg.C for 12 h/d. Then, the growth status of the callus was observed 1 time every 1d or 2d, the appearance time and morphology of each photo-treated callus was recorded, and the number of the callus induced was counted after 40 d.
1.2.5 Effect of different photophobic substances on the proliferation of purslane callus
Firstly, weighing the purslane callus cultured in advance under aseptic condition, then cutting the purslane callus into small stem segments, inoculating the stem segments to a proliferation culture medium MS +6-BA1.0mg/L + IAA1.0mg/L, respectively placing the stem segments on 6 light treatment culture racks of red light, blue light, white light, yellow light, green light and darkness for subculture, inoculating 30 bottles of the callus, 3 bottles of the callus, and repeating for 3 times. The culture conditions were 12h light each day at 25 ℃. The observed calli were weighed after 30 days.
1.2.6 Effect of different photophobs on flavonoid content in purslane callus
Inoculating the purslane callus into the optimal proliferation culture medium, and then respectively placing the purslane callus on 6 light treatment culture shelves of red light, blue light, white light, yellow light, green light and dark for culture, wherein 30 bottles are inoculated for each treatment, and 3 bottles are inoculated for each treatment, and the process is repeated for 3 times. The culture conditions were 12h light each day at 25 ℃. After 30 days, flavonoid content was measured. Drying callus, weighing 0.1g, placing in 30mL 85% ethanol water bath at constant temperature of 70 deg.C for 2 hr, sucking supernatant 4mL into test tube, sucking into cuvette, and placing at 510nm wavelengthDetecting by light splitting, measuring the value of the light splitting degree, and obtaining the value of the light splitting degree through a regression equation α =1.2C +4.8x10 of a standard curve formula-3The linear range is 0-0.5 mg, the correlation coefficient is r =0.9955, and the flavonoid content is calculated.
1.3 data processing and analysis
Callus induction rate (%) = number of callus explants induced/total number of explants inoculated × 100%;
callus growth rate (%) = callus growth weight/callus inoculation weight × 100%.
Statistical software such as Excel and SPSS 19.0 is adopted to process and analyze the test data.
2 results and analysis
2.1 obtaining and strengthening aseptic seedlings
And (3) taking purslane seeds as test materials, and obtaining the sterile seedlings after 7d, wherein the plant height is 4-5 cm, and cotyledons and hypocotyls are purple red.
2.2 Effect of different seedling ages and tissue parts on the induction of purslane callus
Taking aseptic seedlings of 4D, 7D, 10D, 13D and 16D after purslane seed germination as materials, respectively inoculating leaves and hypocotyls to callus induction culture medium MB5+6-BA1.0mg/L +0.5 mg/L2, 4-D + AgNO34.0mg/L, and comparing the influence of different seedling ages and tissue parts on the purslane callus.
As can be seen from the table, the callus induction of purslane is facilitated at the seedling age of 7d, the induction rate of hypocotyl as an explant is slightly higher than that of cotyledon, and the two materials can be used as explants for callus induction.
2.3 Effect of different hormones on callus induction of Portulaca oleracea
(1) Influence of different concentrations of 6-BA on callus induction of purslane
Inoculating 7D seedling-old embryonic axis to callus induction culture medium MB5+ 0.5 mg/L2, 4-D + AgNO34.0mg/L, and adding 6-B with different concentrationA (0, 0.5mg/L, 1.0mg/L, 1.5 mg/L, 2.0 mg/L).
It is proved that the 6-BA concentration of 0.5mg/L is beneficial to the induction of the callus.
(2) Influence of IAA with different concentrations on callus induction of purslane
Inoculating 7d seedling-age embryonic axis into callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and was induced with different concentrations of IAA (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L). As can be seen from the table, IAA is not suitable for purslane callus induction. The highest induction rate is only 62.3%.
(3) Different concentrations of 2,4-D
Inoculating 7d seedling-aged cotyledons to callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and 2,4-D (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L) was added at different concentrations for induction. As can be seen from the table, the 2,4-D concentration of 0.5 or 1.0mg/L is beneficial to callus induction of purslane.
(4) Different concentrations of NAA
Inoculating 7d seedling-aged cotyledons to callus induction culture medium MB5+ 1.0 mg/L6-BA + AgNO34.0mg/L, and adding NAA (0 mg/L, 0.2 mg/L, 0.5mg/L, 0.8 mg/L, 1.0 mg/L) at different concentrations for induction. As can be seen from the table, NAA is also not beneficial to purslane callus induction.
2.4 Effect of different light qualities on purslane-induced callus
2.4.1 Effect of different photophobs on callus induced by purslane hypocotyls
Inoculating the oleracea hypocotyls to a callus induction culture medium, culturing in 6 light treatments of red light, blue light, white light, yellow light, green light and darkness, and studying the influence of different light qualities on the earliest emergence time and induction rate of the oleracea stem induced callus. The results are shown in the table, the photoplasm has certain influence on the induction of the purslane hypocotyl callus, and various photoplasmic hypocotyls can induce the callus, wherein the time for inducing the callus by red light is shortest, the callus is generated after 6 days, the induction rate is the highest and reaches 100%, although the induction rate of blue light is also 100%, compared with the induction rate of red light, the induction time of the callus is delayed by 2 days. The induction rate of the stem callus under each light quality is red light = blue light > white light > dark light > yellow light > green light in the order from high to low.
Influence of light on the earliest emergence time and induction rate of purslane hypocotyl callus
Capital letters in the table indicate significance of 0.01 level differences.
2.4.2 Effect of different light qualities on quality of purslane callus
Different lights also have certain influence on the quality of the purslane callus, and the chart II shows that the texture of the callus generated by the leaves of the purslane under red light is very compact, the texture of the callus generated under the light yellow and blue lights is compact, the texture of the callus generated under the white light is light yellow green and is compact, the texture of the callus generated under the light green and dark lights is loose, the texture of the callus generated under the light yellow and yellow lights is loose, the texture of the callus generated under the yellow green and green lights is compact, and the color of the callus generated under the green and yellow lights is green and white. The stem section has loose texture and slightly brownish color compared with the leaf section when the stem section is treated by different light qualities.
From the above experimental data, comparing the effect of light quality on the induction of purslane stem and leaf callus results in: the induction rates of the stem sections and the leaf callus under the light treatment are consistent, the healing time of the stem sections under the light treatment is obviously earlier than that of the leaf, and the induction rates are also obviously higher than that of the leaf, so that the earliest time for inducing the callus by the stem sections is shorter than that of the leaf, and the growth speed of the callus is higher than that of the leaf. The callus quality induced by the leaves under various light qualities is better than that of the stem section, the browning occurrence probability is lower than that of the stem section, and the materials are easily obtained, so that a large amount of operations can be carried out. Different explants can therefore be used for callus induction depending on the particular requirements of the experiment.
2.5 Effect of different light qualities on the proliferation of purslane callus
Inoculating the purslane callus onto a proliferation culture medium, and then carrying out subculture in 6 light treatments of red light, blue light, white light, yellow light, green light and darkness, and researching the influence of different lights on the growth of the purslane leaf callus. The results are shown in the table, each light quality can promote the growth of the purslane callus, wherein the fresh increment of blue light is the largest and is 9.71g, and the growth rate is the highest and reaches 359%. The difference between the blue light and other light treatments is obvious, and the growth influence of the blue light on the purslane callus is obviously higher than that of other light qualities. The red light was significantly different from the other light treatments, and the white, yellow and dark light were not significantly different at the 5% significant level. The photoplasm has a certain positive significance in the callus induction of the purslane.
Effect of epilucite on growth of purslane callus
Lower case letters in the table indicate 0.05 level difference significance.
2.6 Effect of photoplasm on flavonoid content in purslane callus
As can be seen from the table, blue light is beneficial to the accumulation of flavonoids in the purslane callus, and the content is the highest.
Finally, the best culture medium and culture conditions for the induction and proliferation and flavonoid content accumulation of the purslane callus are determined by the study: after purslane seeds germinate for 7 days, seedling hypocotyls are used as explants and inoculated in MB5+ 1.0 mg/L6-BA +0.5 mg/L2, 4-D + AgNO34.0 mg/L culture medium, and after the purslane seeds are cultured for 30 days under blue light, the proliferation coefficient and the flavonoid content are the highest.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. A method for inducing purslane callus and producing flavonoid is characterized in that: the method comprises the steps of taking the embryonic axis of the seedling after purslane seed germination for 7 days as an explant, and inoculating the explant to MB5+ 1.0 mg/L6-BA +0.5 mg/L2, 4-D + AgNO3Culturing in 4.0mg/L culture medium under blue light for 30 days;
the seedling culture method comprises the following steps: wrapping herba Portulacae seed with filter paper, soaking in 50-55 deg.C warm water for 10-15min, disinfecting by soaking in warm soup, and washing with running water for 20 min; then sterilizing the seeds wrapped by the filter paper in 75% alcohol for 60 s; then at 0.1% HgCl2Sterilizing for 8min while stirring; then washing with sterile water for 4-5 times; and finally inoculating the seedlings into an MS basic culture medium subjected to high-pressure sterilization treatment, and culturing the MS basic culture medium under the conditions of illumination intensity of 2000-3000 lx, illumination time of 12h/d and temperature of 25 +/-2 ℃ to obtain sterile seedlings.
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