CN102960246A - Tissue culturing method for effectively improving general flavone content of tartary buckwheat - Google Patents
Tissue culturing method for effectively improving general flavone content of tartary buckwheat Download PDFInfo
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- CN102960246A CN102960246A CN2012104732067A CN201210473206A CN102960246A CN 102960246 A CN102960246 A CN 102960246A CN 2012104732067 A CN2012104732067 A CN 2012104732067A CN 201210473206 A CN201210473206 A CN 201210473206A CN 102960246 A CN102960246 A CN 102960246A
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- buckwheat
- flavone content
- callus
- culturing method
- tissue culturing
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Abstract
The invention discloses a tissue culturing method for effectively improving a general flavone content of tartary buckwheat. The tissue culturing method uses a precursor feeding technology. A metabolic intermediate sodium acetate or yeast extract of a flavonoid matter is added in a buckwheat cell suspension culture, so that not only can the general flavone content in the buckwheat culture cell be obviously improved, but also the tissue culturing method has obvious acceleration effect on biomass multiplication of a tissue culture matter. According to the tissue culturing method provided by the invention, the foundation is laid for further developing a buckwheat plant secondary metabolite in a large scale, so that the rapid generation of effective components of the buckwheat plant is achieved; and meanwhile, the tissue culturing method can provide important technical support for further research on molecule conditioning of flavones secondary metabolism in the buckwheat plant.
Description
Technical field
The present invention relates to a kind of method for tissue culture of buckwheat, but particularly relate to a kind of tissue culture method of Effective Raise buckwheat total flavone content.
Background technology
Buckwheat is a kind of plant of integration of drinking and medicinal herbs, and two kinds of bitter buckwheat and sweet buckwheats are arranged.Owing to all containing a large amount of Flavonoid substances in the multiple organs such as its seed of tartarian buckwheat, root, stem, leaf and flower, thus be described as " hypoglycemic, hypotensive, reducing blood lipid " three food falls.Along with the continuous discovery of its nutritive value and medical value, the tartarian buckwheat plant resources more and more is subject to people's favor.But because the tartarian buckwheat plant has autophilous characteristics, thereby causes it to exist artificial hybridization to be difficult to successfully in the agricultural production breeding, this also is current tartarian buckwheat is difficult to obtain the high yield improved seeds aspect breeding major reason.Tissue is cultivated and can solve to a certain extent the bitter problem that wheat runs into of supporting in breeding.Have at present the report of cultivating its callus take tartarian buckwheat stem section, blade, petiole as explant.
Studies show that in a large number, in the suspension incubation of plant cell, by adding the precursor of final purpose compound, can greatly improve the Secondary Metabolite Contents of suspended culture cell.Yet there are no the report that adopts the precursor feeding technology to significantly improve general flavone content in the tartarian buckwheat group training thing.
Summary of the invention
The tissue culture method that the object of the invention is to a kind of Effective Raise tartarian buckwheat general flavone content, the method is come general flavone content in the Effective Raise buckwheat culture by add specific precursor in the liquid culture of buckwheat cell.
For achieving the above object, the solution that the present invention adopts comprises the steps:
(1) processing of explant: choose healthy growth and without the bitter buckwheat stem section of damage by disease and insect as explant, and disinfection;
(2) callus induce cultivation: the explant behind the cancellation poison, be cut into the length of 0.5cm～1.5cm after blotting the moisture on surface, then access callus inducing medium MS+6-BA 0.1～2.0mgL+2,4-D 1～6mgL+IAA 0～1mgL+ sucrose 20～60 gL
-1+ agar 5.0～7.0 gL
-1In cultivate, condition of culture is 18～28 ℃ of pH values 5.5～6.8, illumination every day 8～16 hours, intensity of illumination 1000～2000 lx, cultivation temperature;
(3) suspension of callus is cultivated: cut 15～25 days callus of growth, with 10～50 gL
-1Inoculum concentration be linked into the liquid nutrient medium MS+6-BA1.0～2.0mgL that is added with the precursor feeding thing
-1+ NAA0.1～0.5 mgL
-1+ sucrose 20～60 gL
-1+ Vc 100～300 mg L
-1In the cultivation that suspends, condition of culture is pH value 5.5～6.5,18～24 ° of C of cultivation temperature, illumination every day 8～12 hours, intensity of illumination 600～1000 lx, shaking speed is 120～140r/min, described precursor feeding thing is sodium acetate or yeast extract, the addition of sodium acetate is 1～10 mgL, and the addition of yeast extract is 0.5～5gL.
The callus cell that above-mentioned suspension was cultivated after 25 days leaches from culture fluid, wipe away the dry substance surface moisture with dry filter paper after, take by weighing weight, be fresh weight; Biomass propagation multiple=(harvest yield fresh weight-inoculum concentration fresh weight)/inoculum concentration fresh weight.
Place 55 ℃ baking oven to dry to constant weight the buckwheat callus cell of above-mentioned cultivation, and with spectrophotometric method flavones content in the buckwheat group training thing is detected, working sample is directly measured absorbance by colour developing is rear at the 500nm place, and calculates the content of flavones in the group training thing.
Disinfect described in the above-mentioned steps (1) and refer to that with bitter buckwheat stem section concentration be 0.1% mercuric chloride sterilization 3～8 minutes, use again sterile water wash 3～5 times.
Bitter buckwheat stem section in the above-mentioned steps (1) is selected the tender stem segments near tartarian buckwheat plant terminal bud end.
The callus that callus in the above-mentioned steps (3) is selected that growth is vigorous, quality is loose and produced without brown stain.
The present invention has such thinking: because the biosynthesis of flavones comprises acetoxymalonic acid approach and shikimic acid pathway, and that the A of flavones ring is acetic acid is synthetic through the acetoxymalonic acid approach, therefore, feed and raise the direct precursor that the precursor sodium acetate is synthetic flavones, thereby add the sodium acetate of suitable concentration, can improve significantly the synthetic of secondary metabolite flavones in the buckwheat suspended culture cell.And the precursor yeast extract of feeding, can by a series of key enzymes in the activation phenylpropyl alcohol alkanes approach, can impel the accumulation of flavonoids such as phenylalnine ammonialyase (PAL), cinnamic acid 4-hydroxylase (CA4H) and coumaric acid-CoA connection enzyme (4CL) etc. as a kind of biological derivant.
Therefore the present invention adopts the precursor feeding technology, by in the suspending nutrient solution of tartarian buckwheat stem section cell, but the general flavone content in the metabolic intermediate Effective Raise tartarian buckwheat cultured cell of the synthetic class material of the flavones such as interpolation sodium acetate or yeast extract, for the production of further carrying out on a large scale the plant buckwheat secondary metabolite is laid a good foundation, also provide important technical support for the molecular regulation of further studying flavones secondary metabolism in the plant buckwheat.
Embodiment
Embodiment 1
(1) processing of explant: choose healthy growth and without the 1st～2 tender stem segments of the close bitter buckwheat plant terminal bud end of damage by disease and insect as explant, be 0.1% mercuric chloride sterilization 4 minutes with concentration, use again sterile water wash 4 times;
(2) callus induce cultivation: the explant behind the cancellation poison, blot the length that is cut into 0.6cm behind the moisture on surface, then access callus inducing medium MS+6-BA 1.0mgL+2,4-D 5mgL+IAA 0.5mgL+ sucrose 35 gL
-1+ agar 6.0 gL
-1In cultivate, condition of culture is: 25 ℃ of pH values 6.0, illumination every day 12 hours, intensity of illumination 1500 lx, cultivation temperature, cultivate 15 days statistics, the inductivity of callus is 100%;
(3) suspension of callus is cultivated: the callus that cuts that 20 days growth of growth is vigorous, quality is loose and produce without brown stain, and with 30 gL
-1Inoculum concentration be linked into the liquid nutrient medium MS+6-BA1.0mgL that is added with sodium acetate
-1+ NAA0.2 mgL
-1+ sucrose 25gL
-1+ Vc 200 mg L
-1In the cultivation that suspends, condition of culture is pH value 6.0,22 ° of C of cultivation temperature, illumination every day 10 hours, intensity of illumination 800 lx, shaking speed is 130r/min, the addition of described sodium acetate is 5 mg/L, namely adds the 5mg sodium acetate in every liter of medium;
(4) calculating of culture biomass propagation multiple: suspending cultivation after 25 days, callus cell in the culture fluid is leached from culture fluid, after wiping away the dry substance surface moisture with dry filter paper, take by weighing weight, and the biomass propagation multiple that calculates the buckwheat cell is 2.71, exceeded 33.50% than the control group biomass propagation multiple 2.03 that does not add any precursor feeding thing;
(5) mensuration of general flavone content in the culture: place 55 ℃ baking oven to dry to constant weight the buckwheat histocyte of cultivating, and measure absorbance at 500 nm places with spectrophotometric method, the general flavone content that further calculates in the culture is 5.04%, has exceeded 52.73% than the control group general flavone content 3.30% that does not add any precursor feeding thing.
Embodiment 2
(1) processing of explant: choose healthy growth and without 1～2 bitter buckwheat stem section of the close bitter buckwheat plant terminal bud end of damage by disease and insect as explant, be 0.1% mercuric chloride sterilization 6 minutes with concentration, use again sterile water wash 3 times;
(2) callus induce cultivation: the explant behind the cancellation poison, blot the length that is cut into 1.0 cm behind the moisture on surface, then access callus inducing medium MS+6-BA 0.5mgL+2,4-D 2mgL+ sucrose 50 gL
-1+ agar 6.0 gL
-1In cultivate, condition of culture is: 25 ℃ of pH values 5.8, illumination every day 10 hours, intensity of illumination 1200 lx, cultivation temperature;
(3) suspension of callus is cultivated: cut 24 days callus of growth, with 40 gL
-1Inoculum concentration be linked into the liquid nutrient medium MS+6-BA1.5mgL that is added with sodium acetate
-1+ NAA0.4 mgL
-1+ sucrose 30 gL
-1+ Vc 150mg L
-1In the cultivation that suspends, condition of culture is pH value 6.0,22 ° of C of cultivation temperature, illumination every day 10 hours, intensity of illumination 800 lx, shaking speed is 130r/min, the addition of described sodium acetate is 5 mg/L, namely adds the 5mg sodium acetate in every liter of medium;
(4) calculating of culture biomass propagation multiple: suspending cultivation after 25 days, callus cell in the culture fluid is leached from culture fluid, after wiping away the dry substance surface moisture with dry filter paper, take by weighing weight, and the biomass propagation multiple that calculates the buckwheat cell is 2.51;
(5) mensuration of general flavone content in the culture: place 55 ℃ baking oven to dry to constant weight the buckwheat histocyte of cultivating, and measure absorbance at 500 nm places with spectrophotometric method, the general flavone content that further calculates in the culture is 4.84%.
Embodiment 3
Change the addition of the sodium acetate in embodiment 1 step (3) into 10 mg/L, other step is with embodiment 1, the biomass propagation multiple that records the buckwheat cell after cultivating 25 days is 2.15, has exceeded 5.91% than the control group biomass propagation multiple 2.03 that does not add any precursor feeding thing; And the general flavone content in the mensuration culture is 5.46%, has exceeded 65.45% than the control group general flavone content 3.30% that does not add any precursor feeding thing.Thus explanation, in the liquid nutrient medium of buckwheat cell, when the sodium acetate precursor feeding substrate concentration that adds is brought up to 10 mg/L, buckwheat cellular biomass propagation is not increased significantly, but the general flavone content that improves in the buckwheat cell is had significantly facilitation.
Embodiment 4
Change the precursor feeding thing in embodiment 1 step (3) into yeast extract, addition is 2 g/L, and other step is with embodiment 1.The biomass propagation multiple that records the buckwheat cell after cultivating 25 days is 2.65, has exceeded 30.54% than the control group biomass propagation multiple 2.03 that does not add any precursor feeding thing; And the general flavone content that records is 5.26%, has exceeded 59.39% than the control group general flavone content 3.30% that does not add any precursor feeding thing.
Embodiment 5
Change the addition of the yeast extract in embodiment 3 steps (3) into 5 g/L, other step is with embodiment 3.The biomass propagation multiple that records the buckwheat cell after cultivating 25 days is 2.09, has only exceeded 2.96% than the control group biomass propagation multiple 2.03 that does not add any precursor feeding thing; And the general flavone content that records is 4.96%, has exceeded 50.30% than the control group general flavone content 3.30% that does not add any precursor feeding thing, can find out that relatively biomass propagation multiple and the general flavone content of the present embodiment all is lower than embodiment 3.
Claims (4)
1. the tissue culture method of an Effective Raise tartarian buckwheat general flavone content is characterized in that comprising the steps:
(1) processing of explant: choose healthy growth and without the bitter buckwheat stem section of damage by disease and insect as explant, and disinfection;
(2) callus induce cultivation: the explant behind the cancellation poison, be cut into the length of 0.5cm～1.5cm after blotting the moisture on surface, then access callus inducing medium MS+6-BA 0.1～2.0mgL+2,4-D 1～6mgL+IAA 0～1mgL+ sucrose 20～60 gL
-1+ agar 5.0～7.0 gL
-1In cultivate, condition of culture is 18～28 ℃ of pH values 5.5～6.8, illumination every day 8～16 hours, intensity of illumination 1000～2000lx, cultivation temperature;
(3) suspension of callus is cultivated: cut 15～25 days callus of growth, with 10～50 gL
-1Inoculum concentration be linked into the liquid nutrient medium MS+6-BA1.0～2.0mgL that is added with the precursor feeding thing
-1+ NAA0.1～0.5 mgL
-1+ sucrose 20～60 gL
-1+ Vc 100～300 mg L
-1In the cultivation that suspends, condition of culture is pH value 5.5～6.5,18～24 ° of C of cultivation temperature, illumination every day 8～12 hours, intensity of illumination 600～1000 lx, shaking speed is 120～140r/min, described precursor feeding thing is sodium acetate or yeast extract, the addition of sodium acetate is 1～10 mg/L, and the addition of yeast extract is 0.5～5g/L.
2. the tissue culture method of a kind of Effective Raise buckwheat total flavone content according to claim 1, it is characterized in that: disinfect described in the step (1) and refer to that with bitter buckwheat stem section concentration be 0.1% mercuric chloride sterilization 3～8 minutes, use again sterile water wash 3～5 times.
3. the tissue culture method of a kind of Effective Raise buckwheat total flavone content according to claim 1 is characterized in that: the bitter buckwheat stem section in the step (1) is selected the tender stem segments near tartarian buckwheat plant terminal bud end.
4. the tissue culture method of a kind of Effective Raise buckwheat total flavone content according to claim 1 is characterized in that: callus described in the step (3) is vigorous for growth, quality is loose and the callus that produces without brown stain.
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| CN105580735A (en) * | 2016-02-01 | 2016-05-18 | 珀莱雅化妆品股份有限公司 | Culture solution capable of improving content of resveratrol in polygonum cuspidatum callus and culture method |
| CN107548831A (en) * | 2017-08-01 | 2018-01-09 | 六安玫瑰红茶品有限公司 | A kind of mulberry tree management method for improving flavones in mulberry leaves content |
| CN107864860A (en) * | 2017-12-06 | 2018-04-03 | 赵腾骅 | A kind of cultural method for improving sec-o-glucosylhamaudol content in windproof callus |
| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105580735A (en) * | 2016-02-01 | 2016-05-18 | 珀莱雅化妆品股份有限公司 | Culture solution capable of improving content of resveratrol in polygonum cuspidatum callus and culture method |
| CN105580735B (en) * | 2016-02-01 | 2021-10-22 | 珀莱雅化妆品股份有限公司 | Culture solution capable of improving resveratrol content in polygonum cuspidatum callus and culture method |
| CN108112474A (en) * | 2016-11-28 | 2018-06-05 | 山东农业大学 | A kind of method that cultured in vitro improves STEVIA REBAUDIANA RA contents |
| CN107548831A (en) * | 2017-08-01 | 2018-01-09 | 六安玫瑰红茶品有限公司 | A kind of mulberry tree management method for improving flavones in mulberry leaves content |
| CN107864860A (en) * | 2017-12-06 | 2018-04-03 | 赵腾骅 | A kind of cultural method for improving sec-o-glucosylhamaudol content in windproof callus |
| CN107864860B (en) * | 2017-12-06 | 2019-02-12 | 广州今成生物科技有限公司 | A kind of cultural method improving sec-o-glucosylhamaudol content in windproof callus |
| CN116574668A (en) * | 2023-06-13 | 2023-08-11 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
| CN116574668B (en) * | 2023-06-13 | 2023-12-05 | 浙江觅得优生物科技有限公司 | Method for promoting liquorice cells to release secondary metabolite liquorice total flavonoids into suspension culture medium |
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