CN101595843A - Kuh-seng tissue culture and method for quickly breeding - Google Patents
Kuh-seng tissue culture and method for quickly breeding Download PDFInfo
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- CN101595843A CN101595843A CNA2009100946900A CN200910094690A CN101595843A CN 101595843 A CN101595843 A CN 101595843A CN A2009100946900 A CNA2009100946900 A CN A2009100946900A CN 200910094690 A CN200910094690 A CN 200910094690A CN 101595843 A CN101595843 A CN 101595843A
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Abstract
Kuh-seng tissue culture and method for quickly breeding are provided, and are that explant carries out tissue culture with kuh-seng stem apex, terrestrial stem and subterranean stem, successfully induce test-tube plantlet.Explant is seeded in MS+2, and on the 4-D0.2mg/L+6-BA 0.8mg/L medium, inductivity has a large amount of callus to produce up to 100%, and color is a light green, and growth is very fast; Induce from propagation and the differentiation phase of sprouting with MS+IBA0.1mg/L+6-BA1.5mg/L, growth coefficient can reach 7.3, and the bud coefficient of differentiation reaches 8.3 times; Carry out culture of rootage with 1/2MS+IBA1.5mg/L or 1/2MS+NAA1.0mg/L, rooting rate is up to 95%, and mean elements is cultivated more than 6 at a certain temperature and humidity conditions, and transplanting survival rate can reach more than 87%.
Description
Technical field:
The invention belongs to the biotechnology field of tissue culture, particularly, relate to the tissue culture and the method for quickly breeding of kuh-seng.
Technical background:
Kuh-seng (Sophora flavescens Ait.) is a pulse family Sophora machaka, and China has 12 kinds approximately, concentrates to be distributed in northern desert area.English name Lighiyellow Sophora Root claim again bitter bone, bitter Chinese scholartree, water Chinese scholartree, white, the mound youth of Chinese scholartree, wild Chinese scholartree, white stem, tiger fiber crops, high post, river ginseng etc.Root is cylindrical, the crust yellow.The cane green has sparse fine, soft fur.The leaf alternate is imparipinnate leaf, and leaflet is to life, oblong, and full edge tip point or blunt, the blade face green, back side pale asphyxia, raceme, armpit is given birth to or give birth on the top, and the flower butterfly is faint yellow.Fruit is a pod, includes 2～6 pieces in black seed, and the seed oil-containing is 14.7%.The pod elongated cylindrical, tip is long, and ripe back pitchy does not ftracture.1～5 in seed, filbert, elongated cylindrical, 5～June of florescence, really 7～September of phase.
Kuh-seng is China's traditional Chinese medicine, and is both on the books in the Compendium of Material Medica: its medicinal part is a root, has the effect of heat-clearing, clearing damp, desinsection, diuresis.What research was comparatively ripe in the active ingredient of kuh-seng is alkaloid and flavonoids, and alkaloid (matrine) has effects such as antitumor, antibiotic, antiviral; Chromocor extract has effects such as heat-clearing, desinsection, diuresis, clearing damp.Be developed to various kinds of drug at present, be used for treating multiple diseases such as cancer, hepatitis, acute inflammation; On agricultural, also have been widely used, various pests and germ are had inhibitory action; Kuh-seng can be controlled various livestock diseases, also is the first-selected additive of green feed during livestock breeding is produced.In addition, kuh-seng is again good anti-blown sand, saline alkali tolerant plant.
The kuh-seng wild resource is fewer and feweri, and the market demand is increasing, and artificial planting is based on seed propagation, but percentage of seedgermination is low and the genetic variation and genetic differentiation problem is serious.Utilize tissue culture technique, not only can keep the merit of original plant, and fine individual plant is bred into clone fast, can promote aborning.So far, do not see the report that kuh-seng tissue culture aspect is arranged in the prior art.
Summary of the invention:
The present invention be intended to screen and definite kuh-seng tissue-culturing rapid propagation process in the principal element that influences callus, proliferation and differentiation and take root, ripe kuh-seng method for tissue culture reliably is provided, provide a feasible approach for solving shortage of resources.
Above-mentioned purpose of the present invention is to realize by following technical scheme:
The method for tissue culture of kuh-seng comprises that explant is sterilized, induced, subculture, culture of rootage, the transfer step, get kuh-seng stem apex or terrestrial stem or subterranean stem, clean, 75% alcohol is handled 30s, and 0.1% mercuric chloride soaks 8min, use sterile water wash again 4～5 times, dry, be cut into every section 0.5cm and be with 1～2 bud, insert MS+2, carry out callus induction among the 4-D 0.2mg/L+6-BA0.8mg/L, callus is divided into 3～5mm
2Segment, insert in the MS+IBA0.1mg/L+6-BA1.5mg/L medium, carry out successive transfer culture with every 25d one-period, carry out from propagation and the differentiation of sprouting, then the regeneration bud of high 3cm bunch is downcut from bud, insert among root media 1/2MS+IBA1.5mg/L or the 1/2MS+NAA1.0mg/L, in humus soil+vermiculite of 1: 1 to the greenhouse of the plantlet of transplant after taking root; Sucrose mass concentration in the above-mentioned medium except that culture of rootage is 15g/L, is 30g/L, and the agar mass concentration is 8g/L, medium pH 5.8～6.2, the prepared culture medium 20min that sterilizes under 121 ℃ of conditions of 1.05MPa; Condition of culture is 22～28 ℃ of temperature, every day light application time 10～12h, intensity of illumination 1500～3000Lx; Inducing adventitious root under the low light level 500～1000Lx condition.
Wherein earlier at 121 ℃ of following autoclaving 2h, preceding 7 days of transfer is watered the next day of with 1/2MS macroelement and clear water before use for transfer greenhouse humus soil and vermiculite, and transfer was used clear water instead and watered after 7 days, transplants after 4 weeks Routine Management under field conditions (factors).
Embodiment:
Further specify essentiality content of the present invention with embodiments of the invention below, but do not limit the present invention with this.
Embodiment 1:
The tissue culture of kuh-seng:
1 materials and methods:
1.1 material:
1.1.1 examination material:
During elementary cultivation, selecting kuh-seng stem apex, terrestrial stem and subterranean stem for use is explant, and research medium and hormone are during to the influencing of kuh-seng sprout propagation and differentiation, and explant is the stem apex or the stem section of aseptic seedling.
1.1.2 reagent is formed:
Each composition of MS medium, methyl (NAA), 6-benzyl purine (6-BA), indolebutyric acid (IBA), 2,4 dichlorophenoxyacetic acid (2,4-D), 2%NaClO, 0.1%HgCl etc.
1.2 method:
1.2.1 the sterilization of explant:
Under running water, wash 30s, be placed in the beaker that fills suitable quantity of water, dip in soap and stirring, repeat 2 times with writing brush, clean with flushing with clean water then.Pick out tenderer stem apex, terrestrial stem and subterranean stem, sterilize with three kinds of modes on aseptic superclean bench respectively: (1) 2%NaClO soaks 8min; (2) 0.1%HgCl soaks 8min; (3) 75% alcohol is handled 30s, and 0.1%HgCl soaks 8min.The every interval of above disinfecting process 30s jog once after sterilization is finished, uses sterile water wash 4～5 times, is put on the aseptic filter paper and dries, and cuts about 0.5cm (being with 1～2 bud), is used for initial culture for every section.
1.2.2 callus induction:
Employing is easy to the MS medium of evoked callus, explores the influence of the different proportionings with the basic element of cell division of growth hormone to callus induction.This paper selects basic element of cell division 6-BA, and the amount of getting is respectively 0.5,1.0,2.0,3.0mg/L; Growth hormone 2,4-D, NAA and IBA, the amount of getting is 0.2mg/L.Contrast MS is set
0, filter out the combination of the suitable growth hormone and the basic element of cell division, at the combination consumption proportion of further testing out the suitableeest kuh-seng explant callus induction.After cultivating 30d, observe the callus growth situation and add up inductivity.
1.2.3 breed and differentiation from sprouting:
To grow preferably, callus is divided into 3～5mm
2Segment, forward inserts in the new bud inducing culture, carries out successive transfer culture with every 25d one-period.Adopt MS medium+6-BA+IBA,, done the 3 horizontal quadrature experimental designs of 3 factors, handle (table 2) totally 9 times for exploring the influence of hormone concentration to bud propagation and differentiation.After cultivating 25d, observe the growing state of explant and add up growth coefficient and the bud differentiation rate.This test subculture 3 times, the each commentaries on classics for the time is to cultivate back 25 days.
1.2.4 rooting of vitro seedling and transplanting:
Regeneration bud about high 3cm is downcut from bud bunch, insert root media (1) 1/2MS+IBA, IBA gets 0,1.0,1.5,2.0mg/L, and (2) 1/2MS+NAA, NAA get 1.0,1.5,2.0mg/L, the mean elements of the statistics rooting rate and the bud of taking root behind the 30d; Complete plantlet after taking root is transplanted on greenhouse humus soil+vermiculite (1: 1).
1.2.5 medium and condition of culture:
Sucrose mass concentration in the medium (except that culture of rootage 15g/L) is 30g/L, the agar mass concentration is 8g/L, medium pH 5.8～6.2, the prepared culture medium 20min that sterilizes under 1.05MPa (121 ℃) condition, culture medium after sterilization lies against on the workbench, cools off standby.The culture materials of inoculation is placed on the culturing rack of culturing room cultivates.Condition of culture is 22～28 ℃ of temperature, every day light application time 10～12h, intensity of illumination 1500～3000Lx; At the low light level (inducing adventitious root under 500～1000Lx) conditions.All processing all are that every processing repeats for 4 times in the test, repeat 4 bottles at every turn, and 5 every bottle, promptly each processing number is 20.
1.2.6 data analysis:
More than the single-factor comparative trial sterilization of explant (remove) is all adopted in test, cultivates behind certain fate statistics and calculates desired data.Adopt analyses such as data such as DPS software returns, the significance of difference.
2, result and analysis:
2.1 the influence of the external implant body pollution rate of different sterilization methods:
The influence of different disinfectants and the external implant body pollution rate of sterilization method is very big, pollution rate 6.1%～16.7%, and the pollution rate of processing 1～3 descends gradually.As seen from Table 1, the pollution rate of the 3rd kind of disinfection way is minimum, is 6.1%, and it is better than 2%NaClO Disinfection Effect to draw the 0.1%HgCl Disinfection Effect, and multiple disinfectant is used in combination, and can reduce pollution rate better.
The influence of the external implant body pollution rate of the different disinfection way of table 1
Tab?1.Different?sterilization?methods?on?the?impact?of?explantscontamination?rate
In 3 kinds of sterilization methods, terrestrial stem and rhizomatous pollution rate are lower, and the pollution rate of terrestrial stem is 3.8%, and rhizomatous pollution rate is 3.2%; The pollution rate of stem apex is 6.7% (table 2).
Table 2 different explants is to the influence of pollution rate
Tab?2.Explants?different?from?the?impact?of?the?rate?of?pollution
In sum, select the flushing of running water and soap, 75% alcohol is handled 30s, and the sterilization method of 0.1% mercuric chloride immersion 8min can be controlled the pollution rate of kuh-seng explant preferably.
2.2 different hormone combination medium are to the effect of inducing of callus:
The proportioning of the basic element of cell division and growth hormone is induced the very big influence of existence to callus, and the MS medium that does not contain hormone can not induce callus, IBA and 2,4-D all can induce callus, NAA but can not induce callus, and 2, the callus that 4-D induces is faster than IBA.Low hormone concentration (IBA0.2mg/L, though 6-BA0.5mg/L) have down callus to produce, along with the prolongation of time, callus Growth stops gradually, colour-darkening; The hormone-content height (2,4-D0.2mg/L, the callus that induces in the time of 6-BA2.0mg/L) is tight, and poor growth, color are dark deeply, and as 6-BA content 3mg/L, no callus produces; Hormone-content quite (2, have a large amount of callus to produce when 4-D0.2mg/L, 6-BA0.5～1.0mg/L), color is a light green, growth is very fast.Medium is MS+2,4-D0.2mg/L+6-BA0.5mg/L, MS+2,4-D0.2mg/L+6-BA 1.0mg/L, MS+IBA0.2mg/L+6-BA1.0mg/L, MS+NAA 0.2mg/L+6-BA 1.0mg/L etc., MS
0It is the MS medium that does not add hormone.
(after the combination of 6-BA0.5～1.0mg/L), for selecting the combination consumption proportion of the suitableeest kuh-seng explant callus induction, ad hoc meter orthogonal experiment is as table 3 to filter out the suitable growth hormone (2,4-D 0.2mg/L) and the basic element of cell division.
The different hormone combination medium of table 3 are to the effect of inducing of callus
Tab?3.Effect?of?callus?induction?on?different?hormone?combinations
Annotate: good: forms a large amount of, green, callus closely; Generally: form a spot of, loose callus; Do not have: do not form callus; Different letter representatives are remarkable at 0.05 level difference, down together.
Note：Good：a?lot?of?green，compact?calli?growth；General：a?small?amountof?loose?calli?growth；None：no?callus?growth；Different?letters?within?columnsindicate?significant?different?by?Duncan’s?multiple?rangtest?at?5％level，thesame?below.
As can be seen from Table 3, the hormone-content that kuh-seng explant callus induction requires is lower, and when 2,4-D is 0.1～0.5mg/L, all has callus to produce when 6-BA is 0.5～1.0mg/L; The processing of medium numbering 5 and other are handled significant difference, so the suitableeest medium is MS+2, and 4-D0.2mg/L+6-BA 0.8mg/L, and through twice with growth behind the concentration medium successive transfer culture better.
2.3 different hormone combination medium are to propagation and differentiation effect from sprouting:
Find by the selection test that growth hormone is influenced the kuh-seng proliferation and differentiation: best aspect the quality and quantity that IBA is sprouted at induced bundle; NAA is better than IAA on the quantity of induced bud, is better than NAA at IAA aspect the quality of induced bud.When 6-BA induced proliferation and differentiation, the concentration of 6-BA can not be too high or too low.When too low, the bud number of growing thickly of inducing is less, only produces simple bud or 4～5 buds, can not form bud bunch; When too high, again the vitrifying seedling can appear.From the otherness between handling as can be seen, the otherness of handling between the growth coefficient of medium numbering 5, bud differentiation rate all significantly handles in other, determined that by hormone combination kuh-seng is MS+IBA0.1mg/L+6-BA1.5mg/L (table 4) from the optimal medium of the proliferation and differentiation of sprouting.
The different hormone combination medium of table 4 are to propagation and differentiation effect from sprouting
Tab?4.Effect?of?bud?proliferation?and?differentiation?on?different?hormonecombinations
2.4 the rooting efficiency of different hormone combination medium and transplanting:
The root of self-sow is normally underground, thereby the generation of the plant roots of some cultured in vitro is very sensitive to intensity of illumination.In the cultured in vitro of part plant, adopt the low light level according to the generation of inducing root, can guarantee certain photosynthetic carrying out, for plant provides necessary nutrient, the inhibition that can avoid high light that root is taken place again.(it is best under 500～1000Lx) conditions kuh-seng being taken root at the low light level.The present invention has tested 2 kinds of influences that hormone is taken root to kuh-seng, the results are shown in Table 5.When medium was 1/2MS+IBA1.5mg/L and 1/2MS+NAA1.0mg/L, rooting rate all reached 95%, and root system is flourishing, growing way also is best, what rooting rate was minimum is that 1/2MS is 35%, and the processing otherness of numbering 2～7 is significantly in numbering 1, and it is obvious to the kuh-seng rooting efficiency to add hormone.It is remarkable that the mean elements of bud of taking root is respectively handled differences, and the processing of numbering 3 and 6 has obvious effect to the mean elements of the bud of taking root.To sum up, the optimum kuh-seng medium of taking root is: 1/2MS+IBA1.5mg/L and 1/2MS+NAA1.0mg/L.
After treating that test-tube plantlet is cultivated 30d, with stalwartness, the plantlet of well developed root system takes out from blake bottle, carefully wash the medium of adhesion off, be transplanted in greenhouse humus soil+vermiculite (1: 1), soil and vermiculite are all in advance at 121 ℃ of following autoclaving 2h, preceding 7 days, water the next day of with 1/2MS macroelement and clear water, after 7 days, use the clear water pouring instead.After transplanting for 4 weeks, tissue cultivating seedling begins the strong point young leaves, sends new root, and this is a normal management under field conditions (factors), and survival rate is at (table 5) more than 87%.
The rooting efficiency of the different hormone combination medium of table 5
Tab?5.Effect?of?root?induction?on?different?hormone?combinations
Annotate: "+" expression small and weak and vein jaundice of seedling of taking root, illustrate that growing way is poor, "+" is many more, and the expression seedling of taking root is healthy and strong more, and color is a green, illustrates that growing way is good more.
Note：“+”weak?root?and?that?the?Health?and?yellow?veins?that?poor?growth，“+”more，said?Health?rooted?more?robust，color?is?green，that?the?bettergrowth.
3 conclusions:
3.1 the sterilization of explant:
From extraneous or the indoor explant of choosing, all have various microorganisms to some extent at first, in a single day these pollution source are brought in the blake bottle, just can cause medium to pollute, and all have to abandon usually.Therefore, adopt the vegetable material that is used to cultivate, all must handle through careful surface sterilizing.Can find out exploring the best disinfectant kind of kuh-seng different parts explant surface-treated and 3 kinds of set disinfection way of time from the present invention: different plants, position, the degree of carrying disease germs is different, and it is also different to the responsive fitness of the disinfectant of variety classes and concentration, for this reason, the explant of different parts should carry out disinfection respectively, and multiple disinfectant is used in combination and can reduces pollution rate.The pollution rate height of terrestrial stem may be capped certain fine hair because of its epidermis, has influenced sterilization effect.
3.2 callus induction:
The callus that induces vigorous growth is the important foundation of next step successive transfer culture, and hormone is to influence the key factor that callus produces.It is generally acknowledged leguminous plant difficulty induce callus, in the present invention test, can not evoked callus when hormone-content is too low or the callus color of inducing browning gradually; When hormone-content was too high, the callus of inducing was tight, poor growth; The kind of hormone also influences the generation of callus: IBA and 2, and 4-D all can induce callus, and NAA but can not induce callus.As seen, it is very high to induce required hormone kind of kuh-seng callus and hormone combination to require.
3.3 adventitious buds proliferation and induction:
Seek out the high efficiency and the high-quality thereof of test-tube plantlet breeding, carry out the basic element of cell division and the growth hormone proportioning is a primary factor.The used basic element of cell division 6-BA of the present invention induces the effect of sprout propagation slightly to be better than ZT, is suitable for use in the batch process tissue cultivating seedling, and promptly economy is reliable again.6-BA is the most frequently used a kind of basic element of cell division in Plant Tissue Breeding.6-BA and growth hormone are to inducing from the influence test of sprouting and growing, and growth hormone IBA effect is better.
3.4 take root and transplant:
The bud of growing thickly stops propagation and differentiation after transferring to root media, takes root rapidly, and seedling has also grown tall simultaneously.It is generally acknowledged that mineral element concentration helps developing cauline leaf when higher, and help taking root when low, so adopt the MS medium of 1/2 or 1/4 amount more, all remove or only use the very low basic element of cell division, and add an amount of growth hormone, use NAA the most generally, with the floristics difference, general 2～4 weeks can take root, can the bottle outlet plantation when growing pure white normal short root.What employing was maximum in test is taken root in Plant Tissue Breeding is the MS medium of 1/2～1/8 amount, according to the otherness of each plant, explores the medium of the suitableeest amount.
The best-of-breed technology scheme that 4 the present invention select is:
(1), the explant disinfection way is: running water and soap wash, and 75% alcohol is handled 30s, and 0.1% mercuric chloride soaks 8min, and this disinfection way pollution rate is minimum, is 6.1%.
(2), the evoked callus medium is: MS+2,4-D 0.2mg/L+6-BA 0.8mg/L, inductivity are up to 100%, and the callus color is a light green, and growth is very fast.
(3), from sprout propagation and differential medium be: MS+IBA0.1mg/L+6-BA1.5mg/L, growth coefficient can reach 7.3, the bud differentiation rate reaches 830%.
(4), root media is: 1/2MS+IBA1.5mg/L and 1/2MS+NAA1.0mg/L, rooting rate reach 95%, and root system is flourishing, and growing way is also fine.Be transplanted in greenhouse humus soil+vermiculite (1: 1), survival rate is more than 87%.
Claims (2)
1, the method for tissue culture of kuh-seng comprises that explant is sterilized, induced, subculture, culture of rootage, the transfer step, get kuh-seng stem apex or terrestrial stem or subterranean stem, clean, 75% alcohol is handled 30s, and 0.1% mercuric chloride soaks 8min, use sterile water wash again 4～5 times, dry, be cut into every section 0.5cm and be with 1～2 bud, insert MS+2, carry out callus induction among the 4-D 0.2mg/L+6-BA 0.8mg/L, callus is divided into 3～5mm
2Segment, insert in the MS+IBA0.1mg/L+6-BA1.5mg/L medium, carry out successive transfer culture with every 25d one-period, carry out from propagation and the differentiation of sprouting, then the regeneration bud of high 3cm bunch is downcut from bud, insert among root media 1/2MS+IBA1.5mg/L or the 1/2MS+NAA1.0mg/L, in humus soil+vermiculite of 1: 1 to the greenhouse of the plantlet of transplant after taking root; Sucrose mass concentration in the above-mentioned medium except that culture of rootage is 15g/L, is 30g/L, and the agar mass concentration is 8g/L, medium pH 5.8～6.2, the prepared culture medium 20min that sterilizes under 1.05MPa121 ℃ of condition; Condition of culture is 22～28 ℃ of temperature, every day light application time 10～12h, intensity of illumination 1500～3000Lx; Inducing adventitious root under the low light level 500～1000Lx condition.
2, the method for tissue culture of kuh-seng as claimed in claim 1, wherein the greenhouse humus soil of transfer and vermiculite are before use earlier at 121 ℃ of following autoclaving 2h, preceding 7 days of transfer, water the next day of with 1/2MS macroelement and clear water, after the transfer 7 days, use clear water pouring instead, transplant after 4 weeks Routine Management under field conditions (factors).
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106069763A (en) * | 2016-06-25 | 2016-11-09 | 杨文平 | The pierre method of Radix Scrophulariae |
| CN106105620A (en) * | 2016-06-25 | 2016-11-16 | 杨文平 | The asexual reproduction method of Radix Sophorae Flavescentis |
| CN106106151A (en) * | 2016-06-25 | 2016-11-16 | 杨文平 | The prevalent variety cultivation method of Radix Sophorae Flavescentis |
| CN107593460A (en) * | 2017-11-13 | 2018-01-19 | 陈培党 | A kind of establishing techniques of kuh-seng regenerating system |
-
2009
- 2009-07-03 CN CNA2009100946900A patent/CN101595843A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106069763A (en) * | 2016-06-25 | 2016-11-09 | 杨文平 | The pierre method of Radix Scrophulariae |
| CN106105620A (en) * | 2016-06-25 | 2016-11-16 | 杨文平 | The asexual reproduction method of Radix Sophorae Flavescentis |
| CN106106151A (en) * | 2016-06-25 | 2016-11-16 | 杨文平 | The prevalent variety cultivation method of Radix Sophorae Flavescentis |
| CN106069763B (en) * | 2016-06-25 | 2018-04-24 | 杨文平 | The pierre method of radix scrophulariae |
| CN107593460A (en) * | 2017-11-13 | 2018-01-19 | 陈培党 | A kind of establishing techniques of kuh-seng regenerating system |
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Application publication date: 20091209 |