CN101498703B - Cell line selection method for tea plant containing different catechin content and components - Google Patents
Cell line selection method for tea plant containing different catechin content and components Download PDFInfo
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- CN101498703B CN101498703B CN2009101161955A CN200910116195A CN101498703B CN 101498703 B CN101498703 B CN 101498703B CN 2009101161955 A CN2009101161955 A CN 2009101161955A CN 200910116195 A CN200910116195 A CN 200910116195A CN 101498703 B CN101498703 B CN 101498703B
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Abstract
The invention relates to a screening method for the tea plant clone containing different catechuic acid contents and components, which solves the problems that the detection for the catechuic acid contents and the components in the callus or clone of tea plants needs long time and the operation is complex in the screening technology. The screening method includes four operating steps of inducing and successively transferring the clone, screening the callus containing different catechuic acid contents, screening the clone containing different catechuic acid contents, and screening the clone containing different catechuic acid components. The invention can screen the clone containing different catechuic acid contents and components and display the difference of the catechuic acid contents of the callus within the short time of 5-10 min, thereby having the great advantages that the catechuic acid detection can be performed during the successive transfer operation process; a plurality of samples can be analyzed once by the thin layer chromatography and identification of the catechuic acid components of the clone of the tea plant, in addition, a new component can be also found by the thin layer chromatography and identification, thereby the screening of the clone containing different catechuic acid contents and components is accelerated.
Description
Technical field
The present invention relates to the screening technique of plant cell, the cell line selection method of tea plant containing different catechin content and component.
Background technology
The synthetic influence that is subjected to the external environment factor of plant flavonoids, factors such as illumination, temperature, nutrition all can have influence on the synthetic of aldehydes matter in the tea tree.Because utilize the biosynthetic metabolism of the individual research of complete tea tree aldehydes matter that many limiting factors are arranged, tea callus and cell suspension cultures system have become the good platform of research tea tree secondary metabolism undoubtedly.Filtering out the tea clone that contains different catechin content and component is the final prerequisite of setting up of this good platform.
The common screening means that contain particular matter clone have multiple, are the bases that the screening system is set up and seek a kind of effectively easily special quality testing survey means of row.For coloured special thing, adopt direct visual method screening high yield clone usually
[1], as containing the callus or the clone of materials such as anthocyanidin, naphthoquinones, carotenoid, chlorophyll, the depth of its color has shown the variation of inclusion content.Yamamotos etc. have in high yield and the stable yields anthocyanidin clone process in screening, utilizing visual method to select the darker callus subculture of color cultivates, like this repeated screening nearly 30 generations, the mean value of the pigment content of cell lump keeps stable after the 23rd generation, and higher 7 times than archaeocyte strain content
[2]Wu Yunqi etc. utilize dichotomy in conjunction with efficient liquid phase chromatographic analysis (HPLC), and the taxol high yield clone in the taxus callus is screened, and have obtained a taxol high yield clone Ts-19, and its content of taxol reaches 4.0% of dry cell weight
[3]
But tree plant catechin is colourless, can't directly carry out the identification screening of tea clone by naked eyes, can only take round-about way.The content of catechin can be by the spectrophotometry of 1% vanillic aldehyde hydrochloric acid reagent colour developing, the component of catechin can detect by high performance liquid chromatography (HPLC) method, but its mensuration process complexity, required time are longer, at least need 30 minutes as extracting spectrophotometry from the catechin of sample, need at least 1 hour and extract from the catechin of sample that efficient liquid phase chromatographic analysis finishes, can't reach the purpose of fast detecting, thereby limit the screening of the tea clone that contains different catechin content and component.
Summary of the invention
Longer in order to solve in tea tree callus or the clone detection required time of catechin content and component, complicated operation, can't reach the problem of fast detecting, the invention provides a kind of easy and simple to handle, can realize the tea plant containing different catechin content of rapid screening and the cell line selection method of component.
Concrete technical scheme is as follows:
The cell line selection method of tea plant containing different catechin content and component comprises tea callus induction and subculture, contain different catechin content the screening of tea callus, contain different catechin content the tea cell line selection, contain the tea cell line selection 4 road operation stepss of different catechin component, concrete operation method is as follows:
(1) tea callus induction and subculture
A, induce
Choosing the tender stem of tea tree children is explant, with the explant sterilization, and is inoculated on the solid medium and induces inductive condition: 25 ± 1 ℃ of temperature, the dark cultivation; Per 21 days switching subcultures are once transferred 1-3 time, and stem section two ends can grow lurid tea callus;
B, subculture
Light yellow tea callus is cut into the 0.5cm*0.5cm size, continues subculture cultivation on solid medium, per 21 days switching subcultures are once transferred the subculture condition of culture 5 times at least: 25 ± 1 ℃ of temperature, the dark cultivation; Constantly discard the bad tea callus of brownization and growing way in the subculture process, keep the tea callus of fast growth;
(2) the tea callus that contains different catechin content screens
Get tea callus subculture on solid medium and cultivate, 21 days switching subcultures once; Subculture condition of culture: 25 ± 1 ℃ of temperature, the dark cultivation; When the 21st day switching subculture, take visual method that the tea callus is screened, the tea callus of promptly selecting different colours, differing texture on superclean bench carries out subculture respectively and cultivates; Switching subculture half a year to one year, obtain the tea callus of high-catechin content and low catechin content;
The tea callus of described high-catechin content, color are that yellow, fast growth, quality are loose;
The tea callus of described low catechin content, color are that white, fast growth, quality are loose;
(3) contain the tea cell line selection of different catechin content
The tea callus of high-catechin content and low catechin content is inoculated into respectively on the fluid nutrient medium cultivated 21 days, then be inoculated on the solid medium cultivation more respectively 21 days; Solid culture condition: 25 ± 1 ℃ of temperature, the dark cultivation; Liquid Culture condition: shaking speed 100rpm/min, culture flask are triangular pyramidal bottle, liquid amount 30ml, the inoculum concentration 1g of 150ml, 25 ± 1 ℃ of temperature, the dark cultivation; When the alternately subinoculation of liquid and solid culture, on superclean bench the tea callus of high-catechin content and low catechin content being numbered the back one by one is divided into two with scalpel, half of getting wherein put into the test tube that 5ml1% vanillic aldehyde hydrochloric acid reagent is housed in advance, developed the color 10 minutes, and second half of correspondence carried out subculture respectively cultivate; Circulation so repeatedly, at least 4 circulations, the tea clone of acquisition high-catechin content and low catechin content;
The tea clone of described high-catechin content presents peony behind the 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing; The catechin content of tea clone reaches every gram dry weight more than 7.7689 milligrams;
The tea clone of described low catechin content presents light red behind the 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing; The catechin content of tea clone reaches every gram dry weight below 0.1272 milligram;
(4) contain the tea cell line selection of different catechin component
The tea callus of high-catechin content and low catechin content is inoculated into respectively on the fluid nutrient medium cultivated 21 days, then be inoculated on the solid medium cultivation more respectively 21 days; Solid culture condition: 25 ± 1 ℃ of temperature, the dark cultivation; Liquid Culture condition: shaking speed 100rpm/min, culture flask are triangular pyramidal bottle, liquid amount 30ml, the inoculum concentration 1g of 150ml, 25 ± 1 ℃ of temperature, the dark cultivation; When the alternately subinoculation of liquid and solid culture, the thin-layer chromatography that half of the tea callus of high-catechin content and low catechin content is carried out catechin composition respectively detects, and second half of correspondence is carried out subculture respectively cultivate; Circulation so repeatedly, at least 4 circulations; Obtain the two or more tea clones that contain six kinds of catechin compositions and catechin composition below six kinds;
Thin-layer chromatography detecting operation step is as follows:
A. prepare tea clone catechin extract
The two or more tea clones that contain six kinds of catechin compositions and catechin composition below six kinds are prepared tea clone catechin extract respectively;
Get the tea clone 3g that contains six kinds of catechin compositions and catechin composition below six kinds, add a little silica sand, liquid nitrogen grinding is to pulverizing, add 3ml 95% ethanol and grind, 4000g is centrifugal, draws supernatant, precipitation adds 3ml 95% ethanol, centrifugal once more, this method triplicate, last 95% ethanol is settled to 10ml; With the above-mentioned 10ml ethanolic solution that obtains, evaporate to dryness is removed ethanol, and reusable heat is water-soluble to be separated, and uses the equivalent ethyl acetate extraction again 3 times, gets the organic phase concentrate drying, uses the 1ml methanol constant volume; Methanol constant volume solution is 0.45 μ m organic membrane filter with the aperture, gets two or more catechin extracts;
B. thin-layer chromatography detects
Respectively two or more catechin extracts being carried out thin-layer chromatography detects;
With catechin extract point sample on 2.5*10 silica G F-254 plate, open up layer a 2～3h down for 25 ℃, developping agent takes out silica gel plate near back, silica gel plate upper end and dries, and silica gel plate is sprayed through 1% (g/v) vanillic aldehyde hydrochloric acid solution and developed the color, after hair-dryer dries up, observe at once and take pictures;
The described tea clone that contains six kinds of catechin compositions, its catechin extract on the silica gel plate behind the exhibition layer, through 1% (g/v) vanillic aldehyde hydrochloric acid solution spraying colour developing, presents four red stripes; High performance liquid chromatography detects (public knowledge method) and contains Epigallo-catechin gallate (EGCG) ((-)-epigallocatechin-3-gallate, EGCG), L-Epicatechin gallate ((-)-epicatechin-3-gallate, ECG), epigallocatechin ((-)-epigallocatechin, EGC), nutgall catechin ((+)-gallocatechin, GC), catechin ((+)-catechin, C) and epicatechin ((-)-epicatechin, EC) six kinds of catechins;
The described tea clone that contains catechin composition below six kinds, its catechin extract on the silica gel plate behind the exhibition layer, through 1% (g/v) vanillic aldehyde hydrochloric acid solution spraying colour developing, presents four following red stripes; High performance liquid chromatography detects (public knowledge method) and contains Epigallo-catechin gallate (EGCG) ((-)-epigallocatechin-3-gallate, EGCG), L-Epicatechin gallate ((-)-epicatechin-3-gallate, ECG), epigallocatechin ((-)-epigallocatechin, EGC), nutgall catechin (㈩-gallocatechin, GC), catechin ((+)-catechin, C) and epicatechin ((-)-epicatechin, EC) in six kinds of catechins below five kinds;
Described solid medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg, agar 8g and sucrose 30g; PH is 5.5;
Described fluid nutrient medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg and sucrose 30g; PH is 5.5.
The explanation of invention technical characterstic:
1. the selection of nutrient culture media
Different nutrient culture media can have influence on tea callus Growth characteristic and catechin synthesis capability, table 2, Fig. 7 found that, tea callus in the H of pH5.5 nutrient culture media (the H nutrient culture media is: 1L MS nutrient culture media contains the solid medium of 10mg indolebutyric acid (IBA), 2mg kinetin (KT), 8g gram agar and 30g sucrose) growth, not only the catechin synthesis capability is limited, catechin content is low, and tissue is tightly tied, the speed of growth is slow; (the S solid medium is: 1L MS nutrient culture media contains 2mg2 at the S of pH5.5 solid medium, 4-dichlorphenoxyacetic acid (2,4-D), 0.5mg kinetin (KT), 8g agar and 30g sucrose) last tea callus of growing, though catechin content height, but tissue is tightly tied, the speed of growth is slow, and these two kinds of nutrient culture media all are not suitable for carrying out inducing and screening of homogeneous tea clone; Only (the B solid culture is: 1L B in the B of pH5.5 solid culture
5Nutrient culture media contains 0.5mg/L2, the tea callus of inducing on the 4-dichlorphenoxyacetic acid (2,4-D), 0.1mg kinetin (KT), 8g gram agar and 30g sucrose), and fast growth, tissue looseness is fit to induce the tea clone of screening homogeneous.
In addition, in the B solid medium, hormone concentration also can influence the growth and the catechin accumulation of tea clone.By table 3 as seen, under the situation that kinetin (KT) remains unchanged, suitably increase by 2, (2, concentration 4-D) can promote the growth of tea clone to the 4-dichlorphenoxyacetic acid, but the accumulation of catechin is not had too much influence; Under the situation that kinetin (KT) remains unchanged, increase methyl (NAA) concentration, not only help the tea cell line growth, it is synthetic also to help catechin; 2, and the 4-dichlorphenoxyacetic acid (2, under the situation about 4-D) remaining unchanged, increase kinetin (KT) concentration, be unfavorable for the tea cell line growth, the accumulation of also not obvious raising tea clone catechin; When improving 2 simultaneously, and the 4-dichlorphenoxyacetic acid (2,4-D), when keeping its constant rate, all be unfavorable for the growth of tea clone and the accumulation of catechin with the concentration of kinetin (KT).
2. contain the tea callus screening of different catechin content
Early stage tea callus is uneven, from appearance, yellow, white is arranged, and light green color on quality, has hard, loose difference.Fig. 8-shown in Figure 13, after the colour developing of 1% (w/v) vanillic aldehyde hydrochloric acid solution, the color of yellow tea callus is darker, white and light green color tea callus then develop the color more shallow, explanation can tentatively be distinguished catechin content in the tea callus body in appearance from the tea callus, and the catechin content of promptly yellow tea callus is greater than white, jade-green.But will distinguish in test the tea callus color and luster flavescence that causes owing to old and feeble, the catechin content in the tea callus of flavescence aging is very low.
3. the tea cell line selection that contains different catechin content
Because the difference of condition of culture, the tea cell mass under the Liquid Culture condition is rounded, and cell is more even, and the tea cell mass of solid culture is scrambling, tea clone homogeneity is relatively poor, so Liquid Culture helps obtaining the tea clone (Figure 14, Figure 15, Figure 16, Figure 17) of homogeneous.
But the Liquid Culture mode also is not suitable for the operation of the screening of tea clone, therefore in the tea cell line selection, select liquid and solid to replace training method, again in conjunction with dichotomy, 1% vanillic aldehyde concentrated hydrochloric acid reagent live body development process, screening has the tea clone of obvious catechin content difference, for example, adopt this mode in the embodiment of the invention, from cloud stem 63 callus, screening obtains the tea clone that two kinds of catechin contents differ greatly, " cloud stem 63XA " and " cloud stem 63YA "; From the appearance, " cloud stem 63XA " tea cell color turns white, loose, suspension is better, and the jaundice of " cloud stem 63YA " tea cell color, slightly tight, suspension relatively poor (Fig. 2); High performance liquid chromatography (HPLC) the analysis showed that (table 1, Fig. 3), the catechin content of cloud stem 63Y tea cell is far above cloud stem 63X tea cell.
4. the tea cell line selection that contains the different catechin component
(HPLC) compares with high performance liquid chromatography, utilizes thin-layer chromatography to have characteristics quick, that method simply, does not need expensive instrument at the catechin composition of short time fast detecting tea delivery clone.For example, adopt this mode in the embodiment of the invention, from cloud stem 63 tea callus, screening obtains the less clone cloud stem 63H of a kind of catechin composition, from the appearance, " cloud stem 63H " is similar to " cloud stem 63Y ", but " cloud stem 63H " cell only contains simple catechin GC, EGC, C, EC, do not contain ester catechin ECG, EGCG (Fig. 5, Fig. 6).
The present invention compared with prior art has following useful technique effect:
1. present, the method that acquisition contains the tea callus of different catechin content or component mainly reaches by changing condition of culture, as increasing illumination, changes medium component etc.But illumination and nutrient culture media all may change cell characteristics significantly, can't obtain valuable, uniform tea clone.
Still there is not at present to set up the triage techniques of the tea clone that contains different catechin content or component.
2. though tree plant catechin is colourless.But of the present invention discovering, the appearance color and the catechin content of tea callus have correlativity, and therefore directly visual method is to be used for screening the tea clone that contains different catechin content.
3. compare with the spectrophotometric method of 1% vanillic aldehyde hydrochloric acid reagent colour developing, the vanillic aldehyde hydrochloric acid reagent live body developing technology of tea tree callus, though can not accurately measure catechin content in the callus, have simple to operate, characteristics rapidly develop the color.Owing to can in 5-10 minute short time, show the difference of the catechin content of callus, therefore its great advantage is can carry out catechin simultaneously in the operating process of switching subculture to detect, thereby has accelerated to contain the breakneck acceleration of the tea clone of different catechin content.
5. compare with high-efficient liquid phase chromatogram technology, the thin-layer chromatography of tea tree clone catechin composition is identified, can once analyze a plurality of samples, therefore has the characteristics of Rapid identification.In addition, thin-layer chromatography identifies and can also find new composition, thereby accelerated to contain the breakneck acceleration of the tea clone of different catechin component.
Description of drawings
Fig. 1 is that dichotomy screens tea clone synoptic diagram in conjunction with 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing method,
Fig. 2 is cloud stem 63YB (going up a left side, bottom left) and cloud stem 63XB (going up the right side, bottom right) tea clone synoptic diagram,
Fig. 3 is the comparison diagram of the catechin composition of " cloud stem 63XB " and " cloud stem 63YB " tea clone,
Fig. 4 is the tea cell line selection program synoptic diagram that contains the different catechin component,
Fig. 5 is the thin-layer chromatogram of different tea clone catechin compositions,
Fig. 6 is the high-efficient liquid phase chromatogram of different tea clone catechin compositions,
Fig. 7 is the high-efficient liquid phase chromatogram of the tea callus catechin composition of different medium culture,
Fig. 8 is constitutional diagram before the green tea callus dyeing,
Fig. 9 is green tea callus dyeing back constitutional diagram,
Figure 10 is constitutional diagram before the white tea callus dyeing,
Figure 11 is white tea callus dyeing back constitutional diagram,
Figure 12 is constitutional diagram before the yellow tea callus dyeing,
Figure 13 is yellow tea callus dyeing back constitutional diagram,
Figure 14 is the preceding constitutional diagram of Liquid Culture screening system dyeing,
Figure 15 is the dyeing back constitutional diagram of Liquid Culture screening system,
Figure 16 is the preceding constitutional diagram of solid culture screening system dyeing,
Figure 17 is the dyeing back constitutional diagram of solid culture screening system,
Figure 18 is " cloud stem 63XB " and " cloud stem 63YB " catechin content, sees Table 1,
The characteristic of the callus that Figure 19 induces for different nutrient culture media sees Table 2,
Figure 20 influence that to be hormone synthesize the growth of tea tree cell and catechin sees Table 3.
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described by embodiment.
Embodiment:
Major equipment
1. scalpel; 2. high-pressure steam sterilizing pan; 3.2.5*10 silica G F-254 plate; 4.9cm double dish; 5. hydro-extractor; 6.pH meter; 7. superclean bench; 8. high performance liquid chromatography (HPLC)
Material and reagent
1. material:
Tea tree big-leaf species in yunnan (Camellia sinensis (L.) O.Kuntze.var.sinensis cultivar Yun Nan Da YeZhong) tender stem segments.
2. main agents:
(1) 70% ethanol: absolute ethyl alcohol by volume: water=preparation in 750: 282,
(2) 0.1% mercuric chloride (HgCl
2): get 0.1g mercuric chloride (HgCl
2) be dissolved in the 100ml pure water,
(3) solid medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg, agar 8g and sucrose 30g; PH is 5.5,
(4) fluid nutrient medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg and sucrose 30g; PH is 5.5,
(5) 1% vanillic aldehyde concentrated hydrochloric acid solutions: take by weighing the 1.0g vanillic aldehyde, be dissolved in the 100ml concentrated hydrochloric acid solution,
(6) thin-layer chromatography developping agent: normal butyl alcohol: glacial acetic acid: water=4: 1: 5, the developping agent after the preparation is placed after 24 hours through after the jolting repeatedly, discards after the lower floor stand-by.
Method of operating
The cell line selection method of tea plant containing different catechin content and component comprises tea callus induction and subculture, contain different catechin content the screening of tea callus, contain different catechin content the tea cell line selection, contain the tea cell line selection 4 road operation stepss of different catechin component, concrete operation method is as follows:
1. tea callus induction and subculture
A: tea callus induction
Choosing the tender stem of tea tree children is explant.It is clean with flushing with clean water to adopt the explant that comes, and on superclean bench, earlier with 70% alcohol immersion 30 seconds, through aseptic water washing 3～5 times, puts into 0.1%HgCl then
2The middle immersion 5～7 minutes used aseptic water washing 3～5 times again, gets aseptic explant.Immediately aseptic explant is inoculated in and induces the tea callus on the solid medium.Tea callus induction condition is 25 ± 1 ℃ of temperature; The dark cultivation.Per 21 days of explant switching subculture is once transferred after 1-3 time, and stem section two ends can grow lurid tea callus.
B: tea callus subculture
Light yellow tea callus is cut into the 0.5cm*0.5cm size, is seeded in subculture cultivation on the solid medium.In the 21st day transfers the subculture process, on superclean bench, discard the bad tea callus of brownization and growing way, the tea callus that keeps fast growth continues to cultivate.The subculture condition of culture is 25 ± 1 ℃ of temperature; The dark cultivation.Per 21 days later on switching subcultures once.Subculture 4 times obtains tea callus comparatively uniformly, called after cloud stem 63 callus.
2. contain the tea callus screening of different catechin content
Cloud stem 63 tea callus are seeded on the solid medium, in the 21st day transfers the subculture process, on superclean bench, take visual method that callus is screened, the callus of promptly selecting different colours, differing texture carries out subculture respectively and cultivates.The subculture condition of culture is 25 ± 1 ℃ of temperature; The dark cultivation.Switching was 1 time in per 21 days later on, adopted visual method that callus is proceeded screening simultaneously.Subculture is cultured to not a half year, can tentatively obtain the tea callus of high-catechin content and low catechin content, respectively called after cloud stem 63YA and cloud stem 63XA callus.
The tea callus of high-catechin content (cloud stem 63YA): color yellow, fast growth, quality are loosened.
The tea callus (cloud stem 63XA) of low catechin content: colours white, fast growth, quality are loose.
3. the tea cell line selection that contains different catechin content
The tea callus of high-catechin content and low catechin content is carried out liquid and alternately cultivation of solid, be about to the tea callus and be seeded in and carry out liquid suspension in the fluid nutrient medium and cultivate, at the 21st day the tea callus inoculated and carry out solid culture on the solid medium; Solid culture condition: 25 ± 1 ℃ of temperature; The dark cultivation; The Liquid Culture condition: culture flask is the triangular pyramidal bottle of 150ml; Liquid amount 30ml; Inoculum concentration 1g; Shaking speed 100rpm/min; Cultivation cycle 21 days; (25 ± 1) ℃; The dark cultivation; In the alternation procedure of liquid and solid culture, on superclean bench, utilize dichotomy, in conjunction with 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing method (Fig. 1), screen the tea clone of high-catechin content and low catechin content respectively, promptly on superclean bench cell mass is numbered the back one by one and be divided into two with scalpel, half cell mass of getting is wherein put into the test tube that 5ml 1% vanillic aldehyde hydrochloric acid reagent is housed in advance, develops the color 10 minutes.Second half tea cell mass of tea callus correspondence of the different depths of colour developing is inoculated respectively, continued to cultivate.Alternately cultivating 1 time of per 21 days later on liquid and solid culture carried out dichotomy simultaneously, screens respectively in conjunction with 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing method.At least alternately cultivate 4 cycles, obtained the tea clone of high-catechin content and low catechin content, respectively called after cloud stem 63YB clone and cloud stem 63XB clone (Fig. 2, Fig. 3, table 1).
The tea clone of high-catechin content (cloud stem 63YB) is (Fig. 2): color yellow, fast growth, quality is loose, cell granulations is big, suspension is poor; Present peony behind the 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing.
The tea clone (cloud stem 63XB) of low catechin content is (Fig. 2): colours white, fast growth, quality is loose, cell granulations is little, suspension is good; Present light red behind the 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing.
4. the tea cell line selection that contains the different catechin component
The tea callus of high-catechin content and low catechin content is carried out liquid and alternately cultivation of solid, be about to the tea callus and be seeded in and carry out liquid suspension in the fluid nutrient medium and cultivate, at the 21st day the tea callus inoculated and carry out solid culture on the solid medium.In the alternation procedure of liquid and solid culture, on superclean bench, utilize dichotomy, in conjunction with thin-layer chromatography and 1% vanillic aldehyde concentrated hydrochloric acid solution spraying development process, screening contains more than the catechin composition and contains the few tea clone (Fig. 4) of catechin composition respectively.Alternately cultivating 1 time of per 21 days later on liquid and solid culture carried out dichotomy simultaneously, screens respectively in conjunction with 1% vanillic aldehyde hydrochloric acid reagent live body quick colour-developing method.At least alternately cultivate 4 cycles, it is many and contain the few tea clone of catechin composition to have obtained to contain catechin composition, called after cloud stem 63YC respectively, cloud stem 63H clone (Fig. 5, Fig. 6).
Clone catechin thin-layer chromatography detection method is as follows:
A. screen the preparation of clone catechin extract and get screening clone 3g, add a little silica sand, liquid nitrogen grinding adds 3ml 95% ethanol and grinds to pulverizing, and 4000g is centrifugal, draws supernatant, and precipitation adds 3ml 95% ethanol, and is centrifugal once more.This method triplicate, last 95% ethanol is settled to 10ml.With the above-mentioned 10ml ethanolic solution that obtains, evaporate to dryness is removed ethanol, and reusable heat is water-soluble to be separated, and uses the equivalent ethyl acetate extraction again 3 times, gets the organic phase concentrate drying, uses the 1ml methanol constant volume.The sample that obtains is 0.45 μ m organic membrane filter with the aperture, gets the catechin extract.
B. thin-layer chromatography detects
Catechin extract point sample is opened up layer a 2～3h down for 25 ℃ on 2.5*10 silica G F-254 plate, developping agent takes out silica gel plate near back, silica gel plate upper end and dries, and 1% (g/v) vanillic aldehyde hydrochloric acid solution is sprayed and developed the color, and after hair-dryer dries up, observes at once and takes pictures.
Thin-layer chromatography detects (Fig. 5) and shows that six kinds of catechins in the tea tree can present four red stripes at silica gel plate, and the mobility of each band is also inequality.The band number of clone that contains the different catechin component is variant, cloud stem 63YC contains four red stripes, and cloud stem 63H has only catechin ((+)-catechin, C) and epicatechin ((-)-epicatechin, EC) and epigallocatechin ((-)-epigallocatechin, EGC) red stripes, but cloud stem 63H has unknown band.
High performance liquid chromatography (HPLC) further confirms (Fig. 6), contain the many clone (cloud stem 63YC) of catechin composition and contain Epigallo-catechin gallate (EGCG) ((-)-epigallocatechin-3-gallate, EGCG), L-Epicatechin gallate ((-)-epicatechin-3-gallate, ECG), epigallocatechin ((-)-epigallocatechin, EGC), nutgall catechin ((+)-gallocatechin, GC), catechin ((+)-catechin, C) and epicatechin ((-)-epicatechin, EC), has only epigallocatechin ((-)-epigallocatechin and contain the few clone of catechin composition (cloud stem 63H), EGC), nutgall catechin ((+)-gallocatechin, GC), catechin ((+)-catechin, C) and epicatechin ((-)-epicatechin, EC).
5. screen tea clone Detection of Stability
Utilize high performance liquid chromatography (HPLC) method, regularly detect the catechin content and the component of the tea clone of cultivating, to determine the stability of tea clone catechin synthesis capability.
Tea clone catechin high performance liquid chromatography (HPLC) detection method is as follows:
A. screen the preparation (with the thin-layer chromatography detection method) of tea clone catechin extract,
B. high performance liquid chromatography (HPLC) detection method
The catechin extract is diluted to suitable concentration with chromatogram methyl alcohol, and HPLC detects.
HPLC chromatographic condition: chromatogram pump: water these 600 (Waters 600); Controller: water these 600 (Waters 600); Chromatographic column: the dark 250*4.6mm of the holy and pure blue 4u spoke of F door (Phenomenex Synergi 4u Fusion 250*4.6mm); Detecting device: this (Waters) 2487 of UV water; Sensitivity: 0.01; Detect wavelength: 280nm; Flow velocity: 1.2ml/ minute; Sample size: 5 μ l; Before each sample sample introduction, with moving phase balance 15 minutes.Adopt condition of gradient elution A phase: 1% acetate, the B phase: trifluoroacetic acid aqueous solution, gradient are that A is by 90% to 87% in preceding 20 minutes, and B is by 10% to 13%; 20 minutes to 40 minutes A are by 87% to 70%, and B is by 13% to 30%.External standard method is quantitative.
Whether the synthetic ability of catechin that purpose is to detect the tea clone that has screened remains unchanged, if change, tea clone must be preserved by nitrogen ultra low temperature.
List of references:
[1] Su Guoqing, Wang Li, Liu Yujun. the high yield cell line selection [J] in the culture plant cell. Hainan Teachers College's journal (natural science edition), 2005,18 (4): 364-368.
[2] Y. Yamamoto, the R. mouth of a river. the strategy [J] of high-load cyanine plain sheet Malus spectabilis cell culture is stablized in screening. gene theory and application, 1982,61:113-116
(Y.Yamamoto，R.Mizuguchi.Selection?of?a?high?and?stable?pigment-producingstrain?in?cultured?euphorbia?millii?Cells[J].Theor?Appl?Genet，1982，61：113-116.)
[3] Wu Yunqi, Zhu Weihua, Lu Jian, Hu Qiu, Li Xinlan. the selection of taxol high yield clone and cultivation [J] thereof in the taxus callus. Chinese medicine and natural drug, 1998,33 (1): 15-18.
Claims (1)
1. the cell line selection method of tea plant containing different catechin content and component, it is characterized in that: comprise tea callus induction and subculture, contain different catechin content tea callus screening, contain different catechin content the tea cell line selection, contain the tea cell line selection 4 road operation stepss of different catechin component, concrete operation method is as follows:
(1) tea callus induction and subculture
A, induce
Choosing the tender stem of tea tree children is explant, with the explant sterilization, and is inoculated on the solid medium and induces inductive condition: 25 ± 1 ℃ of temperature, the dark cultivation; Per 21 days switching subcultures are once transferred 1-3 time, and stem section two ends can grow lurid tea callus;
B, subculture
Light yellow tea callus is cut into the 0.5cm*0.5cm size, continues subculture cultivation on solid medium, per 21 days switching subcultures are once transferred the subculture condition of culture 5 times at least: 25 ± 1 ℃ of temperature, the dark cultivation; Constantly discard the bad tea callus of brownization and growing way in the subculture process, keep the tea callus of fast growth;
(2) the tea callus that contains different catechin content screens
Get tea callus subculture on solid medium and cultivate, 21 days switching subcultures once; Subculture condition of culture: 25 ± 1 ℃ of temperature, the dark cultivation; When the 21st day switching subculture, take visual method that the tea callus is screened, the tea callus of promptly selecting different colours, differing texture on superclean bench carries out subculture respectively and cultivates; Switching subculture half a year to one year, obtain the tea callus of high-catechin content and low catechin content;
The tea callus of described high-catechin content, color are that yellow, fast growth, quality are loose;
The tea callus of described low catechin content, color are that white, fast growth, quality are loose;
(3) contain the tea cell line selection of different catechin content
The tea callus of high-catechin content and low catechin content is inoculated into respectively on the fluid nutrient medium cultivated 21 days, then be inoculated on the solid medium cultivation more respectively 21 days; Solid culture condition: 25 ± 1 ℃ of temperature, the dark cultivation; Liquid Culture condition: shaking speed 100rpm, culture flask are triangular pyramidal bottle, liquid amount 30ml, the inoculum concentration 1g of 150ml, 25 ± 1 ℃ of temperature, the dark cultivation; When the alternately subinoculation of liquid and solid culture, on superclean bench the tea callus of high-catechin content and low catechin content being numbered the back one by one is divided into two with scalpel, half of getting wherein put into the test tube that 5ml1%g/v vanillic aldehyde hydrochloric acid reagent is housed in advance, developed the color 10 minutes, and second half of correspondence carried out subculture respectively cultivate; Circulation so repeatedly, at least 4 circulations, the tea clone of acquisition high-catechin content and low catechin content;
The tea clone of described high-catechin content presents peony behind the 1%g/v vanillic aldehyde hydrochloric acid reagent live body quick colour-developing; The catechin content of tea clone reaches every gram dry weight more than 7.7689 milligrams;
The tea clone of described low catechin content presents light red behind the 1%g/v vanillic aldehyde hydrochloric acid reagent live body quick colour-developing; The catechin content of tea clone reaches every gram dry weight below 0.1272 milligram;
(4) contain the tea cell line selection of different catechin component
The tea callus of high-catechin content and low catechin content is inoculated into respectively on the fluid nutrient medium cultivated 21 days, then be inoculated on the solid medium cultivation more respectively 21 days; Solid culture condition: 25 ± 1 ℃ of temperature, the dark cultivation; Liquid Culture condition: shaking speed 100rpm/min, culture flask are triangular pyramidal bottle, liquid amount 30ml, the inoculum concentration 1g of 150ml, 25 ± 1 ℃ of temperature, the dark cultivation; When the alternately subinoculation of liquid and solid culture, the thin-layer chromatography that half of the tea callus of the low catechin content of one half-sum of the tea callus of high-catechin content is carried out catechin composition respectively detects, and second half of correspondence is carried out subculture respectively cultivate; Circulation so repeatedly, at least 4 circulations; Obtain the two or more tea clones that contain six kinds of catechin compositions and catechin composition below six kinds;
Thin-layer chromatography detecting operation step is as follows:
A. prepare tea clone catechin extract
The two or more tea clones that contain six kinds of catechin compositions and catechin composition below six kinds are prepared tea clone catechin extract respectively;
Get the tea clone 3g that contains six kinds of catechin compositions and catechin composition below six kinds, add a little silica sand, liquid nitrogen grinding is to pulverizing, add 3ml 95% ethanol and grind, 4000g is centrifugal, draws supernatant, precipitation adds 3ml 95% ethanol, centrifugal once more, repeated centrifugation three times, last 95% ethanol is settled to 10ml; With the above-mentioned 10ml ethanolic solution that obtains, evaporate to dryness is removed ethanol, and reusable heat is water-soluble to be separated, and uses and the ethyl acetate extraction of hot water equivalent 3 times again, gets the organic phase concentrate drying, uses the 1ml methanol constant volume; Methanol constant volume solution is 0.45 μ m organic membrane filter with the aperture, gets two or more catechin extracts;
B. thin-layer chromatography detects
Respectively two or more catechin extracts being carried out thin-layer chromatography detects;
Thin-layer chromatography developping agent: normal butyl alcohol: glacial acetic acid: water=4: 1: 5, the developping agent after the preparation is placed after 24 hours through after the jolting repeatedly, discards after the lower floor stand-by;
With catechin extract point sample on 2.5*10 silica G F-254 plate, open up layer 2～3h down for 25 ℃, developping agent takes out silica gel plate near back, silica gel plate upper end and dries, silica gel plate process 1%g/v vanillic aldehyde hydrochloric acid solution spraying colour developing, after hair-dryer dries up, observe at once and take pictures;
The described tea clone that contains six kinds of catechin compositions, its catechin extract on the silica gel plate behind the exhibition layer, through 1%g/v vanillic aldehyde hydrochloric acid solution spraying colour developing, presents four red stripes; High performance liquid chromatography detects and contains Epigallo-catechin gallate (EGCG), L-Epicatechin gallate, epigallocatechin, nutgall catechin, catechin and six kinds of catechins of epicatechin;
The described tea clone that contains catechin composition below six kinds, its catechin extract on the silica gel plate behind the exhibition layer, through 1%g/v vanillic aldehyde hydrochloric acid solution spraying colour developing, presents four following red stripes; High performance liquid chromatography detect contain in Epigallo-catechin gallate (EGCG), L-Epicatechin gallate, epigallocatechin, nutgall catechin, catechin and six kinds of catechins of epicatechin below five kinds;
Described solid medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg, agar 8g and sucrose 30g; PH is 5.5;
Described fluid nutrient medium is: at 1L B
5Add 2 in the nutrient culture media, 4-dichlorphenoxyacetic acid 0.5mg, kinetin 0.1mg and sucrose 30g; PH is 5.5.
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| CN108051522B (en) * | 2017-12-29 | 2020-09-25 | 福建省农业科学院茶叶研究所 | Method for quickly identifying tea plant germplasm suitable for making flower and fruit flavor type tea |
| CN110879269B (en) * | 2019-12-10 | 2022-04-08 | 浙江尖峰健康科技有限公司 | Combined identification method of cranberry extract |
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