CN102405833B - Method for improving adventitious bud differentiation rate of tartary buck wheat through suspension culture - Google Patents
Method for improving adventitious bud differentiation rate of tartary buck wheat through suspension culture Download PDFInfo
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- CN102405833B CN102405833B CN201110238818A CN201110238818A CN102405833B CN 102405833 B CN102405833 B CN 102405833B CN 201110238818 A CN201110238818 A CN 201110238818A CN 201110238818 A CN201110238818 A CN 201110238818A CN 102405833 B CN102405833 B CN 102405833B
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Abstract
The invention discloses a method for improving adventitious bud differentiation rate of tartary buck wheat through suspension culture, comprising the following steps: building a solid-liquid-solid culture system of the tartary buck wheat under the in-vitro culture condition, wherein the frequency of inducting the callus tissue is 100%, the adventitious bud differentiation rate is 90.7%, and the rooting rate is 100%; and obtaining the regeneration plants after 8-10 weeks through manually controlling. The method of the invention is simple and easy to implement, reduces the browning degree of the callus tissues of the tartary buck wheat, promotes the differentiation of the adventitious buds, reduces the culture time, and provides the technical base for improving the establishment of the in-vitro high-frequency regeneration system of the tartary buck wheat.
Description
Technical field
The invention belongs to field of plant tissue culture technique, be specifically related to a kind of method that improves the hull buckwheat differentiation adventitious buds through suspension culture.
Background technology
Buckwheat is polygonaceae (Polygonaceae), the dicotyledonous cereal crop of Fagopyrum (Fagopyrum Mill); Main cultispecies has sweet buckwheat (F.esculentum Moench; Also claim general buck wheat) and bitter buckwheat (F.tartaricum (L.) Gaerth also claims hull buckwheat).Buckwheat is the only cereal crops that contain rutin; Found to utilize research gradually; Nutritious; To the elderly's fat-reducing, promote longevity and increase children ' s intelligence development and can play good effect, and diseases such as tuberculosis, hepatopathy, diabetes, dysentery are had the obvious treatment effect, and contain multivitamin and mineral matter.China working people recognizes the medical value of hull buckwheat very early, just just has in the far away monumental work Prescriptions Worth Thousand Gold for Emergencies of famous physician Sun Si in Tang: the record of " acid of buckwheat flavor, be slightly cold, nontoxic ".Effect to hull buckwheat in the medical science masterpiece " draft detailed outline " of China has following record: " the tartarian buckwheat nature and flavor are bitter, flat, cold, useful strength, slow spirit, sharp knowledge, sending down abnormally ascending, wide intestines, the effect of being good for the stomach ".The scientist of countries in the world carries out each side research to the plant of this unique integration of drinking and medicinal herbs from different perspectives.
Rutin is a citrin, and the major physiological function is to strengthen blood vessel, increases the capillary wall permeability, reduces fragility of blood vessels, promotes hyperplasia and prevents condensing of haemocyte.Rutin can reduce blood fat and cholesterol; Hypertension and angiocardiopathy there are prevention and therapeutic action preferably; And control is arranged and treat diabetes, prevent canceration, the effect of aspects such as anti-infective, antiallergy, diuresis, antibechic, level and smooth relaxed muscle, purposes is widely arranged in food and medicine industry.The rutin content of hull buckwheat is higher 13.5 times than sweet buckwheat, and vitamin B2 is high 3.16 times.So the research for hull buckwheat seems particularly important.
Because hull buckwheat has significant researching value, research emphasis is mainly at plant genetic operation, aspects such as genetic transformation at present; But the tissue culture technique of hull buckwheat is stagnation always but; Factors such as difficult mainly due to the hull buckwheat differentiation, callus is prone to brownization, and incubation time is long cause.These unfavorable factors make that hull buckwheat research is serious to lag behind, so, through promoting the differentiation rate of indefinite bud, thereby set up stable plant high frequency regenerating system, have positive role for the further investigation of hull buckwheat.At present; The existing a small amount of report of hull buckwheat (F.tartaricum (L.) Gaerth) tissue culture; People such as Li Zhanqi and Wu Chongming has obtained regeneration plant (northwest Botany Gazette, 2006 of hull buckwheat (F.tartaricum (L.) Gaerth) through hypocotyl and cotyledon; Use and the environmental organism journal 2009), and promote the research of hull buckwheat differentiation adventitious buds that report is not arranged both at home and abroad through callus being carried out suspension culture.
Summary of the invention
The object of the invention is, a kind of method through suspension culture raising hull buckwheat differentiation adventitious buds rate is provided, and research lays the foundation with genetic manipulation for the hull buckwheat scale is fast numerous.This method is an explant with hull buckwheat aseptic seedling hypocotyl in 7 day age; Set up solid callus induction-liquid and induce the system of differentiation-solid hestening rooting; Regulate the intensity of illumination of inducing each stage simultaneously, can prevent brownization of hull buckwheat callus, and promote the differentiation of bud greatly.
In order to realize above-mentioned task, the present invention takes following technical solution to be achieved:
A kind of method through suspension culture raising hull buckwheat differentiation adventitious buds rate is characterized in that, carries out according to the following steps:
(1) acquisition of explant
The hull buckwheat seed of maturation is soaked peeling after 10 minutes in water, be surface sterilization 1min in 70% the alcohol with the seed that strips skin at mass fraction, and the 15min that in the mercuric chloride of concentration 0.1%, sterilizes again uses rinsed with sterile water four times at last;
Seed after the sterilization is inoculated on sucrose free 1/2MS (Murashige-Skoog) solid culture medium, and being 25 ℃ ± 2 ℃ in temperature is to sprout under the 2000lx condition with light intensity.
(2) callus induces
The bitter buckwheat aseptic seedling hypocotyl that sprouts 7 day age being cut the long segment into 0.5cm, be seeded on the evoked callus medium, is that 25 ℃ ± 2 ℃, illumination cultivation condition are to cultivate 15 days under the condition of 500-2000Lx in temperature; Consisting of of described evoked callus medium: the adding mass concentration is 0.8% agar in the MS of routine medium, and mass concentration is 3% sucrose, 2 of 1-2mg/L, 4-D, the 6-BA of 0.1-1mg/L, the AgNO of 10mg/L
3
(3) indefinite bud induces
The callus of inducing is transferred on the liquid MS differentiation culture liquid, and 20rpm is that 25 ℃ ± 2 ℃, intensity of illumination are under the 1000-2500Lx condition in temperature, and illumination every day 16h induces the differentiation of seedling; Consisting of of described liquid MS differentiation culture liquid: in the MS of routine medium, add the NAA of 0.5-2mg/L, the 6-BA of 1-2mg/L, the TDZ of 0.1-1mg/L, the AgNO of 5mg/L
3, the caseinhydrolysate of 0.1-0.3mg/L, inoculum density are 40g/L, the 250ml triangular flask contains 60ml liquid MS differentiation culture liquid (will have only the middle and lower part of callus to be dipped in the liquid MS differentiation culture liquid); After treating that 2-3 week, bud point appearred in callus, the callus that will have bud point takes out from liquid MS differential medium, changes over to continue in the solid MS differential medium to cultivate; (MS solid differentiation medium component is identical with MS liquid differentiation culture based component, and the agar mass concentration is 0.8%);
(4) seedling takes root
Treat that indefinite bud grows to 2-3cm; Cut inducing the indefinite bud that obtains; Insert in the root induction medium and cultivate; Consisting of of described root induction medium: in the MS of routine medium, add the NAA of 1mg/L, cultivation temperature is that 25 ℃ ± 2 ℃, light intensity are 2000lx, illumination every day 16h, cultivates 2-3 and can obtain a large amount of tissue cultivating seedling after week.
Adopt method of the present invention, hull buckwheat differentiation adventitious buds rate can be up to 90.7%.Cultivate with conventional solid and to compare, solid through utilize solid-liquid-the growth system with regulate intensity of illumination, suppressed brownization of callus greatly, promote callus growth and improve the differentiation adventitious buds rate.
Embodiment
The embodiment that provides below in conjunction with the inventor does further detailed description to the present invention.
In following examples; The MS medium of described routine is to use the most general medium at present; Those skilled in the art all can obtain, and it has higher inorganic salt concentration, can guarantee that the required mineral nutrition of tissue growth can also quicken callus Growth.Its main component is:
Macroelement: NH
4NO
3, KNO
3, CaC
122H
2O, MgSO
47H
2O, KH
2PO
4
Trace element: KI, H
3BO
3, MnSO
44H2
0, ZnSO
47H
2O, Na2MoO
42H
2O, CuSO
45H
2O, CoCl
26H
2O;
Molysite: FeSO
47H2
0Na
2-EDTA2H
2O;
Organic principle: inositol, nicotinic acid, puridoxine hydrochloride (vitamin B6), thiamine hydrochloride (vitamin B1), glycine.
Embodiment 1:
(1) acquisition of explant
The hull buckwheat seed of maturation is soaked peeling after 10 minutes in water, be surface sterilization 1min in 70% the alcohol with the seed that strips skin at mass fraction, and the 15min that in mass concentration is 0.1% mercuric chloride, sterilizes again uses rinsed with sterile water four times at last.
Seed after the sterilization is inoculated on sucrose free 1/2MS (Murashige-Skoog) solid culture medium, and being 25 ℃ ± 2 ℃ in temperature is to sprout under the 2000Lx condition with light intensity.
(2) callus induces
The bitter buckwheat aseptic seedling hypocotyl that sprouts 7 day age is cut the long segment into 0.5cm; Be seeded on the evoked callus medium the consisting of of evoked callus medium: in the MS of routine medium, add mass concentration and be 0.8% agar, mass concentration is 3% sucrose; 2 of 2mg/L; 4-D, the 6-BA of 0.5mg/L, the AgNO of 10mg/L
3In temperature is 25 ℃ ± 2 ℃, 500Lx, cultivates 15 days.Expanding rapidly in the hypocotyl two ends, induces callus, and inductivity reaches 100%, and the callus of being induced is most to be open-textured faint yellow or pale red callus.
(3) indefinite bud induces
The callus of inducing is transferred on the liquid MS differentiation culture liquid; Inoculum density is 40g/L; The 250ml triangular flask contains the liquid MS differentiation culture liquid (having only the middle and lower part of callus to be dipped in the liquid MS differentiation culture liquid) of 60ml; 20rpm, temperature is 25 ℃ ± 2 ℃, 2500Lx, induces the differentiation of seedling under the condition of illumination every day 16h.Consisting of of liquid MS differentiation culture liquid: in the liquid MS of routine culture fluid, add the NAA of 0.5mg/L, the 6-BA of 2mg/L, the TDZ of 0.5mg/L, the AgNO of 5mg/L
3, the caseinhydrolysate of 0.3mg/L.
2-3 is after week; Green bud point appears in callus; And grow up rapidly, will have the grow thickly callus of bud of 0.5cm size and from liquid MS differential medium, take out, change over to and continue in the solid MS differential medium to cultivate; Solid MS differential medium main component is identical with liquid MS differentiation culture liquid composition, agar 0.8%.The differentiation rate of bud can reach 90.7%.
(4) seedling takes root
Treat that indefinite bud grows to 2-3cm, with inducing the indefinite bud that obtains to cut, insert in the root induction medium of MS+NAA 1.0mg/L and cultivate that temperature is that 25 ℃ ± 2 ℃, light intensity are 2000lx, illumination every day 16h condition.Rooting rate is 100%.Cultivate 2-3 and can obtain a large amount of tissue cultivating seedling after week.
Embodiment 2:
(1) acquisition of explant
The hull buckwheat seed of maturation is soaked peeling after 10 minutes in water, with the seed that strips skin surface sterilization 1min in the alcohol of mass fraction 70%, the 15min that in the mercuric chloride of concentration 0.1%, sterilizes again uses rinsed with sterile water four times at last.
Seed after the sterilization is inoculated on 1/2MS (Murashige-Skoog) solid culture medium of no sucrose, and being 25 ℃ ± 2 ℃ in temperature is to sprout under the 2000Lx condition with light intensity.
(2) callus induces
The bitter buckwheat aseptic seedling hypocotyl that sprouts 7 day age is cut the long segment into 0.5cm; Be seeded on the evoked callus medium the consisting of of evoked callus medium: in the MS of routine medium, add mass concentration and be 0.8% agar, mass concentration is 3% sucrose; 2 of 1mg/L; 4-D, the 6-BA of 0.5mg/L, the AgNO of 10mg/L
3In temperature is 25 ℃ ± 2 ℃, 500Lx, cultivates 15 days.The hypocotyl two ends expand into dumb-bell shape, induce callus, and inductivity reaches 89%, and the callus great majority of being induced are open-textured yellow or red callus.
(3) indefinite bud induces
The callus of inducing transferred in the liquid MS differentiation culture liquid (have only the middle and lower part of callus to be dipped in the liquid MS differentiation culture liquid); 20rpm is 25 ℃ ± 2 ℃, 2500Lx in temperature, induces the differentiation of seedling under the illumination every day 16h condition; Consisting of of liquid MS differentiation culture liquid: the NAA that in the liquid MS medium of routine, adds 0.5mg/L; The 6-BA of 1mg/L, the TDZ of 0.1mg/L, the AgNO of 5mg/L
3, the caseinhydrolysate of 0.1mg/L.2-3 is after week, and green bud point appears in the part callus, a small amount of adventive root occurs on the callus simultaneously.To have the grow thickly callus of bud of 0.5cm size and from liquid nutrient medium, take out, and change over to and continue to cultivate (main component of solid MS differential medium is identical with the composition of liquid MS differentiation culture liquid) in the solid differential medium.The differentiation rate of bud reaches 55.7%.
(4) seedling takes root
Treat that indefinite bud grows to 2-3cm, with inducing the indefinite bud that obtains to cut, insert in the root induction medium of MS+NAA 1mg/L and cultivate that temperature is that 25 ℃ ± 2 ℃, light intensity are 2000lx, illumination every day 16h condition.Rooting rate is 100%.Cultivate 2-3 and can obtain tissue cultivating seedling after week.
Embodiment 3:
(1) acquisition of explant
The hull buckwheat seed of maturation is soaked peeling after 10 minutes in water, with the seed that strips skin surface sterilization 1min in the alcohol of mass fraction 70%, the 15min that in the mercuric chloride of concentration 0.1%, sterilizes again uses rinsed with sterile water four times at last.
Seed after the sterilization is inoculated on 1/2MS (Murashige-Skoog) solid culture medium of no sucrose, and being 25 ℃ ± 2 ℃ in temperature is to sprout under the 2000Lx condition with light intensity.
(2) callus induces
The bitter buckwheat aseptic seedling hypocotyl that sprouts 7 day age is cut the length into 0.5cm, be seeded in (composition of evoked callus medium is identical with embodiment 1) on the evoked callus medium.In temperature is 25 ℃ ± 2 ℃, 2000Lx, cultivates 15 days.The hypocotyl two ends expand into dumb-bell shape, induce callus, and inductivity reaches 94.5%, and the callus of being induced is most to be open-textured yellow or red callus, and minority is the yellow closely or red callus of quality.
(3) indefinite bud induces
The callus of inducing transferred to (composition of liquid MS differentiation culture liquid is identical with embodiment 1 on the liquid MS differentiation culture liquid; Have only the middle and lower part of callus to be dipped in liquid MS differentiation culture liquid); 20rpm; Temperature is 25 ℃ ± 2 ℃, 1000Lx, induces the differentiation of seedling under the illumination every day 16h condition.2-3 is after week, and green bud point appears in the part callus, a small amount of adventive root occurs on the callus simultaneously.To have the grow thickly callus of bud of 0.5cm size and from liquid nutrient medium, take out, and change over to and continue in the MS solid differential medium to cultivate (main component of MS solid differential medium is identical with the composition of liquid MS differentiation culture liquid), the differentiation rate of bud reaches 69.6%.
(4) seedling takes root
Treat that indefinite bud grows to 2-3cm, with inducing the indefinite bud that obtains to cut, insert in the root induction medium of MS+NAA 1mg/L and cultivate that temperature is that 25 ℃ ± 2 ℃, light intensity are 2000lx, illumination every day 16h condition.Rooting rate is 100%.Cultivate 2-3 and can obtain a large amount of tissue cultivating seedling after week.
Claims (1)
1. the method through suspension culture raising hull buckwheat differentiation adventitious buds rate is characterized in that, carries out according to the following steps:
(1) acquisition of explant
The hull buckwheat seed of maturation is soaked peeling after 10 minutes in water, be surface sterilization 1min in 70% the alcohol with the seed that strips skin at mass fraction, and the 15min that in 0.1% mercuric chloride, sterilizes again uses rinsed with sterile water four times at last;
Seed after the sterilization is inoculated on the sucrose free 1/2MS solid culture medium, and being 25 ℃ ± 2 ℃ in temperature is to sprout under the 2000lx condition with light intensity;
(2) callus induces
The bitter buckwheat aseptic seedling hypocotyl that sprouts 7 day age being cut the long segment into 0.5cm, be seeded on the evoked callus medium, is that 25 ℃ ± 2 ℃, illumination cultivation condition are to cultivate 15 days under the condition of 500-2000Lx in temperature; Consisting of of described evoked callus medium: the adding mass concentration is 0.8% agar in the MS of routine medium, and mass concentration is 3% sucrose, 2 of 1-2mg/L, 4-D, the 6-BA of 0.1-1mg/L, the AgNO of 10mg/L
3
(3) indefinite bud induces
The callus of inducing is transferred on the liquid MS differentiation culture liquid; The 250ml triangular flask contains 60ml liquid MS differentiation culture liquid; Have only the middle and lower part of callus to be dipped in the liquid MS differentiation culture liquid, 20rpm is that 25 ℃ ± 2 ℃, intensity of illumination are under the 1000-2500Lx condition in temperature; Illumination every day 16h induces the differentiation of seedling; Consisting of of described liquid MS differentiation culture liquid: in liquid MS medium, add the NAA of 0.5-2mg/L, the 6-BA of 1-2mg/L, the TDZ of 0.1-1mg/L, the AgNO of 5mg/L
3, the caseinhydrolysate of 0.1-0.3mg/L, inoculum density are 40g/L; After treating that 2-3 week, bud point appearred in callus; The callus that will have bud point takes out from liquid MS differential medium, changes over to continue in the solid MS differential medium to cultivate, wherein; MS solid differentiation medium component is identical with MS liquid differentiation culture based component, and the agar mass concentration is 0.8%;
(4) seedling takes root
Treat that indefinite bud grows to 2-3cm; Cut inducing the indefinite bud that obtains; Insert in the root induction medium and cultivate, the consisting of of described root induction medium: in the MS of routine solid culture medium, add the NAA of 1mg/L, agar 0.8%; Cultivation temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 2000lx, illumination every day 16h, cultivates 2-3 and can obtain a large amount of tissue cultivating seedling after week.
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| CN102960246B (en) * | 2012-11-20 | 2015-03-04 | 成都大学 | Tissue culturing method for effectively improving general flavone content of tartary buckwheat |
| CN103651131B (en) * | 2013-12-05 | 2015-07-29 | 西北大学 | A kind of method being applicable to the efficient evoking adventive bud of Damask Rose suspension cultivation |
| CN103695365B (en) * | 2013-12-24 | 2015-12-02 | 成都大学 | A kind of buckwheat cell culture processes improving buckwheat cell synchronization |
| CN109349108A (en) * | 2018-11-05 | 2019-02-19 | 长江大学 | A kind of sweet tea buckwheat somatic embryo occurs and plant regeneration method |
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| JP2008228609A (en) * | 2007-03-19 | 2008-10-02 | Akita Prefectural Univ | Tissue culture method of buckwheat, and plant body of buckwheat |
| CN101869073B (en) * | 2009-04-22 | 2012-07-04 | 中国科学院成都生物研究所 | Tartary buckwheat isolated regeneration culture method |
| CN101946698A (en) * | 2010-05-11 | 2011-01-19 | 石国荣 | Fast propagation technology for wild buckwheat rhizome |
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Non-Patent Citations (6)
| Title |
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