CN104472359A - Ginseng adventitious root induced proliferation method - Google Patents
Ginseng adventitious root induced proliferation method Download PDFInfo
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Abstract
The invention relates to a ginseng adventitious root induced proliferation method, which comprises the following steps: cutting ginseng tissue culture seedlings into small tissue blocks and then inoculating into a solid induced culture medium for inducing to form adventitious roots, cutting the adventitious roots into small adventitious root blocks and then inoculating into a liquid proliferation culture medium for performing proliferation culture of the adventitious roots, wherein the solid induced culture medium and the liquid proliferation culture medium take 1/2 MS(-N) culture medium as basic culture mediums and contain 1-10mg/L indolebutyric acid. By utilizing the method provided by the invention, the quantity of the adventitious roots obtained through induction can be obviously increased, the adventitious roots obtained through induction are subjected to proliferation culture, and compared with the method for performing adventitious root induction by utilizing callus tissues, the proliferation rate of the adventitious roots is higher, and the proliferation effect is better.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to a kind of ginseng adventitious root proliferative induction method.
Background technology
Ginseng (Panax ginseng C.A.Meyer) is the perennial dicotyledon of Araliaceae Panax, is ancient and famous and precious traditional Chinese medicine, has the multiple effects such as adjusting immunity of human body, anticancer and anti-diabetic.Ginsenoside is its main active substance.In recent years, both at home and abroad the demand of ginseng is increased fast.But owing to excessively excavating for a long time, ginseng wild resource is substantially exhausted, and the culture of ginseng cycle is long, the impact of easy climate, damage by disease and insect and cultivation condition, is difficult to meet the increasing market demand.The ginseng tissue culture technique cycle is short, is not subject to seasonal restrictions, and easily carries out large-scale industrial production, has very large development prospect.
The hairy root growth utilizing agrobacterium rhizogenes to induce ginseng to produce is rapid, saponin(e kind is identical with the ginseng of field cultivation, content is than common cell chulture object height, and utilize the system of bio-reactor large-scale culture ginseng poultry also to set up [Jeong GT, Park DH, Hwang B, etal.Comparison of growth characteristics of Panax ginseng hairy roots invarious bioreactors [J] .Appl Biochem Biotechnol, 2003,107:493].But because in induction of hairy roots process, the importing of foreign gene has potential safety issue, therefore this technology is not used for commercialization.
In recent years, the research of ginseng adventitious root is directly induced to achieve greater advance without agrobacterium rhizogenes conversion.But adventive root is generally induced by ginseng callus and is produced [SivakumarG, Yu KW, Paek KY.Production of biomass and ginsenosides fromadventitious roots of Panax ginseng in bioreactor cultures [J] .Eng LifeSci, 2005,4:333].The defect of said method is: need first evoked callus, causes experimental period long.
Therefore, being necessary to provide a kind of easy directly utilizes ginseng plantlet in vitro as the root induction enrichment procedure of explant.
Summary of the invention
The object of the present invention is to provide a kind of method utilizing the generation of the root of ginseng plantlet in vitro, stem section or blade inducing adventitious root and adventive root is carried out to Multiplying culture.
For reaching above object, the invention provides a kind of root induction enrichment procedure, comprising the following steps:
Ginseng plantlet in vitro is cut into after organizing fritter and be inoculated in induced synthesis adventive root in solid inducing culture, after described adventive root is cut into adventive root fritter, be inoculated in the Multiplying culture carrying out adventive root in liquid proliferated culture medium;
Wherein, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid (IBA) that minimal medium contains that concentration is 1-10mg/L with 1/2MS (-N) medium.
Preferred in order to obtain better ginseng adventitious root proliferative induction effect, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid that minimal medium contains that concentration is 2-5mg/L with 1/2MS (-N) medium.
Optionally, described solid inducing culture and liquid proliferated culture medium are also the sucrose of 1.8-2.2 quality % containing concentration.
Optionally, described proliferative induction method comprises the following steps:
1) ginseng plantlet in vitro is cut into organize fritter, utilize solid inducing culture Fiber differentiation 3-6 week under the dark condition of cultivation temperature for 23-25 DEG C to obtain adventive root;
2) by step 1) adventive root that obtains cuts into adventive root fritter, utilizes liquid proliferated culture medium to cultivate 3-9 week with the concussion of the speed of 50-80rpm under the dark condition of cultivation temperature for 23-25 DEG C.
Optionally, described fritter of organizing is carry out cutting the ginseng plantlet in vitro root fritter obtained, the ginseng plantlet in vitro stem fritter carrying out cutting acquisition to ginseng tissue culture seeding stem segment or carrying out ginseng plantlet in vitro blade cutting the ginseng plantlet in vitro leaf fritter obtained to ginseng plantlet in vitro root.
Optionally, described ginseng plantlet in vitro is the plantlet in vitro obtained through seed germination or explant cultured in vitro, and the age in days of plantlet in vitro is 28-32 days.
Optionally, the kind of described ginseng plantlet in vitro is Radix Ginseng or Da-maya ginseng.
Utilize method provided by the present invention, the adventive root quantity that induction obtains can be significantly improved, and, after the adventive root that induction obtains is carried out Multiplying culture, compared with the method for carrying out root induction with utilizing callus, the adventive root rate of increase is higher, and cultivation effect is better.In addition method provided by the present invention can shorten the time obtaining ginseng adventitious root greatly, by direct to ginseng plantlet in vitro carry out root induction propagation avoid plantlet in vitro is trained whole plant after obtain the step that explant carries out induction of callus, shorten experimental period.Produce ginsenoside for utilizing ginseng adventitious root to have laid a good foundation, the basic research also for utilizing ginseng adventitious root to carry out metabolic pathway provides important material.
Accompanying drawing explanation
Fig. 1 is the adventive root that ginseng plantlet in vitro different tissues proliferative induction produces;
In figure, (A) situation for utilizing ginseng plantlet in vitro root induction to cultivate adventive root; (B) for utilizing the situation of ginseng tissue culture seeding stem segment Fiber differentiation adventive root; (C) for utilizing the situation of ginseng plantlet in vitro blade Fiber differentiation adventive root.
Fig. 2 is the proliferative conditions that the induction of ginseng plantlet in vitro different tissues produces adventive root;
In figure, (A) is plantlet in vitro root; (B) be tissue culture seeding stem segment; (C) be plantlet in vitro blade; 1/2MS (-N)+5mg/L IBA and 1/2MS (-N)+2mg/L IBA represents type of culture medium; 1st represents first time subculture, and 2nd represents second time subculture, and 3rd represents third time subculture.
Fig. 3 is the Multiplying culture of the root-derived callus induction generation adventive root of 5 years stranger's ginsengs;
1/2MS (-N)+5mg/L IBA and 1/2MS (-N)+2mg/L IBA represents type of culture medium; 1st represents first time subculture, and 2nd represents second time subculture, and 3rd represents third time subculture.
Embodiment
Below will the present invention is described in detail by embodiment.
The invention provides a kind of ginseng adventitious root proliferative induction method, comprise the following steps:
Ginseng plantlet in vitro is cut into after organizing fritter and be inoculated in induced synthesis adventive root in solid inducing culture, after described adventive root is cut into adventive root fritter, be inoculated in the Multiplying culture carrying out adventive root in liquid proliferated culture medium;
Wherein, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid that minimal medium contains that concentration is 1-10mg/L with 1/2MS (-N) medium.
In method provided by the present invention, in order to improve the effect of induction and propagation, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid that minimal medium contains that concentration is 2-5mg/L with 1/2MS (-N) medium.
In the present invention, described ginseng plantlet in vitro is the plantlet in vitro obtained through seed germination or the regeneration plant plantlet in vitro obtained through explant cultured in vitro.In preferred situation, when utilizing method provided by the invention to carry out proliferative induction to the ginseng plantlet in vitro obtained through cultured in vitro, excellent root induction cultivation effect can be obtained.
Wherein, method provided by the present invention has no particular limits for the preparation method of the plantlet in vitro obtained through seed germination, can be obtain according to the processing method of this area routine, such as, in one embodiment of the invention, the method obtaining plantlet in vitro through seed germination can be: the ginseng breach seed after Stratificated treatment of learning from else's experience, peel off shell, through 70% alcohol immersion after 1 minute, sterilize 20 minutes in 1% clorox, after sterile water wash 3-4 time, complete embryo is stripped out in super-clean bench, be inoculated in 1/2MS medium, 24 ± 2 DEG C of dark culturing after 3 days, namely the induction of adventive root is can be used for after cultivating 4 weeks under proceeding to light.
Wherein, method provided by the present invention is had no particular limits for the method being obtained regeneration plant plantlet in vitro by explant culture in vitro system, can be obtain according to the processing method of this area routine, such as, in one embodiment of the invention, the method being obtained regeneration plant plantlet in vitro by explant culture in vitro system can be: after being shelled by ginseng seeds, with 70% ethanol surface sterilization after 1 minute, proceed to 1% liquor natrii hypochloritis to sterilize 20 minutes, sterile water wash 3-4 time, then carefully embryo is taken out at superclean bench sterile scalpel, be seeded to 1/2MS medium (containing 2% sucrose and 7g/L agar, pH value is 5.8) in 24 ± 2 DEG C of dark culturing after 3 days, cut cotyledon access MS+1mg/L2, the medium of 4-dichlorphenoxyacetic acid is (containing 3% sucrose and 7g/L agar, pH value is 5.8), in 24 ± 2 DEG C, cultivate under 16 h light/8 h dark conditions.Get the good embryo callus of growth conditions and proceed to the induction that the MS medium (containing 5% sucrose and 10g/L agar, pH value is 5.8) not containing hormone carries out somatic embryo.About after one month, the 1/2MS medium (containing 2% sucrose and 2.5g/L plant gel, pH value is 5.8) proceeded to by the somatic embryo induced containing 5mg/L gibberellin carries out the sprouting of plant, is then proceeded to by regeneration plant containing NH
4nO
31/2MS medium (containing 2% sucrose and 2.5g/L plant gel, pH value is 5.8) carry out culture of rootage, within about about 3 weeks, can take root, obtain regeneration plant plantlet in vitro.
In method provided by the present invention, described fritter of organizing is ginseng plantlet in vitro root fritter, ginseng plantlet in vitro stem fritter or ginseng plantlet in vitro leaf fritter.
One of the present invention preferred embodiment in, the described size of fritter of organizing can be about 5mm × 5mm × 3mm.Apparatus sterilizing should be noted in the process that described plantlet in vitro and adventive root are cut and keep gnotobasis to avoid polluting.
In method provided by the present invention, 1/2MS (-N) medium is that macroelement reduces by half the MS (-N) medium of other components unchanged, and wherein, MS (-N) medium is the MS medium not containing ammonium nitrate.
Wherein, the formula of MS (-N) medium is: potassium nitrate 1900mg/L, potassium dihydrogen phosphate 170mg/L, magnesium sulfate 370mg/L, calcium chloride 440mg/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulphate 22.3mg/L, zinc sulphate 8.6mg/L, sodium molybdate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L.
In one embodiment of the invention, in order to make the adventive root organizing fritter and induction to obtain of ginseng plantlet in vitro obtain nutrition more fully, the agar also containing concentration to be the sucrose of 1.8-2.2 quality % and concentration be 0.65-0.75 quality % in described solid inducing culture.Be the sucrose of 1.8-2.2 quality % containing concentration in described liquid proliferated culture medium.
In one embodiment of the invention, the pH of described solid inducing culture, liquid proliferated culture medium is 5.8-6.0.
Concrete, the step of described induction and propagation comprises:
1) ginseng plantlet in vitro is cut into organize fritter, utilize solid inducing culture Fiber differentiation 3-6 week under the dark condition of cultivation temperature for 23-25 DEG C to obtain adventive root;
2) by step 1) adventive root that obtains cuts into adventive root fritter, utilizes liquid proliferated culture medium to cultivate 3-9 week with the concussion of the speed of 50-80rpm under the dark condition of cultivation temperature for 23-25 DEG C.
Wherein in step 1) in, start to grow adventive root after organizing fritter to cultivate 14-28 days in solid inducing culture.General, cultivate in solid inducing culture after terminating and induce the number of the adventive root of acquisition can reach 4-13; Concrete, utilize ginseng plantlet in vitro root fritter generally can induce and obtain 10-13 adventive root; Utilize ginseng plantlet in vitro stem fritter generally can induce and obtain 8-12 adventive root; Utilize ginseng plantlet in vitro leaf fritter generally can induce and obtain 4-8 adventive root.
In the process of described Fiber differentiation and Multiplying culture, generally need every 3 time-of-weeks to change a solid inducing culture or liquid proliferated culture medium carries out squamous subculture, and when each subculture the induction of statistical observation adventive root and proliferative conditions.
Below by embodiment, the preferred embodiment of the present invention is described in detail.It will be appreciated that providing of following examples is only object in order to play explanation, being not used to limit scope of the present invention.Those skilled in the art, when not deviating from aim of the present invention and spirit, can carry out various amendment and replacement to the present invention.
In following examples, adopt the Radix Ginseng kind in Kuandian county.In following examples, ginseng plantlet in vitro is obtained by the mode of seed germination, be specially: the Radix Ginseng breach seed after Stratificated treatment of learning from else's experience, peel off shell, through 70% alcohol immersion after 1 minute, sterilize 20 minutes in 1% clorox, after sterile water wash 3-4 time, strip out complete embryo in super-clean bench, be inoculated in 1/2MS medium, 24 ± 2 DEG C of dark culturing are after 3 days, cultivate under proceeding to light, obtain the plantlet in vitro that age in days is 30 days.
Root induction rate=(producing the explant number/inoculation explant sum of adventive root) × 100%.
The 3rd week adventive root rate of increase=(breeding the 3rd week results adventive root fresh weight-inoculation adventive root fresh weight)/inoculation adventive root fresh weight × 100%;
The 6th week adventive root rate of increase=(breeding the 6th week results adventive root fresh weight-propagation the 3rd week results adventive root fresh weight)/propagation the 3rd week results adventive root fresh weight × 100%;
The 9th week adventive root rate of increase=(breeding the 9th week results adventive root fresh weight-propagation the 6th week results adventive root fresh weight)/propagation the 6th week results adventive root fresh weight × 100%;
Embodiment 1
The present embodiment is for illustration of utilizing ginseng plantlet in vitro root induction adventive root.
Get the root of ginseng plantlet in vitro, with the segment of sterilizing cutter cutting into about 5mm × 5mm × 3mm, proceed on the solid inducing culture containing 1/2MS (-N)+2mg/L IBA (containing 2% sucrose in solid inducing culture, 7g/L agar, pH value is 5.8), in 24 ± 2 DEG C, the generation of Fiber differentiation adventive root under dark condition.30 explants inoculated by each culture dish, and in Fiber differentiation process, every three weeks subcultures once, the indefinite radical that statistics root induction rate and each explant produce, and test repetition 3 times, averages, the results are shown in table 1.
Result shows, after three weeks cultivate, all roots can induce adventive root, and every bar root all only induces 1 adventive root, but after six weeks cultivate (situation of Fiber differentiation adventive root is shown in Figure 1A), the indefinite radical that average every bar root produces is 11.33 (see table 1).
Embodiment 2
The Multiplying culture of the adventive root that the present embodiment produces for illustration of ginseng plantlet in vitro root induction.
The adventive root that the ginseng plantlet in vitro root induction obtained after six weeks by Fiber differentiation in embodiment 1 produces takes out, be cut into the fritter of 1cm size, by in the blake bottle of the liquid nutrient medium of fritter access containing 100ml 1/2MS (-N)+2mg/L IBA+20g/L sucrose, be placed in shaking table to shake at a slow speed (60rpm) in 24 ± 2 DEG C of dark conditions, carry out the Multiplying culture of adventive root.Every three weeks subcultures once, and add up the fresh weight of adventive root, and test repetition 3 times, averages.Take out cultivating the adventive root obtained, the culture fluid of sterilizing filter paper adsorption surface, is placed in balance and weighs, be fresh weight.Calculate the rate of increase of adventive root, result display (see Fig. 2 A), cultivate three weeks in the 1/2MS (-N) culture fluid containing 2mg/LIBA and 20g/L sucrose after, the rate of increase is 17.73%; After cultivating six weeks, the rate of increase significantly improves, and is 27.45%; When after nine weeks cultivate, the rate of increase slightly declines, and is 25.88%.
Embodiment 3
The Multiplying culture of the adventive root that the present embodiment produces for illustration of ginseng plantlet in vitro root induction.
According to the method identical with embodiment 2, Multiplying culture is carried out to the adventive root that ginseng plantlet in vitro root induction produces.Difference is only, the liquid proliferated culture medium used is 1/2MS (-N) the liquid proliferated culture medium containing 5mg/L IBA and 20g/L sucrose.Calculate the rate of increase of adventive root, result display (Fig. 2 A): Multiplying culture is after three weeks, and the rate of increase is 24.4%; Multiplying culture is after six weeks, and the rate of increase is 32.27%, and when after nine weeks cultivate, the rate of increase is 22.25%.
Embodiment 4
The present embodiment is for illustration of the method utilizing ginseng plantlet in vitro stem inducing adventitious root.
Get the stem section of ginseng plantlet in vitro, with the segment of sterilizing cutter cutting into about 5mm × 5mm × 3mm, proceed on the solid culture medium containing 1/2MS (-N)+2mg/L IBA (containing 2% sucrose in solid inducing culture, 7g/L agar, pH value is 5.8), in 24 ± 2 DEG C, under dark condition, cultivate the generation of inducing adventitious root.30 explants inoculated by each culture dish, and every three weeks subcultures once, and add up the indefinite radical of root induction rate and the generation of each explant, and test repetition 3 times, averages.Result shows, after three weeks cultivate, there is the outer planting physical efficiency of 27.94% to induce and can produce adventive root, and every bar root all only induces 1 adventive root, but after six weeks cultivate, all explants all create adventive root (situation of Fiber differentiation adventive root is shown in Figure 1B), and the indefinite radical that average every bar root produces is 10.33 (see table 1).
Embodiment 5
The Multiplying culture method of the adventive root that the present embodiment produces for illustration of the induction of ginseng plantlet in vitro stem.
The adventive root that the ginseng stem induction obtained after cultivating six weeks in embodiment 4 produces is taken out, be cut into 1cm size, in the blake bottle of the liquid proliferated culture medium of access containing 100ml 1/2MS (-N)+2mg/L IBA+20g/L sucrose, be placed in shaking table, 24 ± 2 DEG C, dark condition shakes at a slow speed (60rpm), carries out the Multiplying culture of adventive root.Every three weeks subcultures once, and add up the fresh weight of adventive root, and test repetition 3 times, averages.Take out cultivating the adventive root obtained, the culture fluid of sterilizing filter paper adsorption surface, is placed in balance and weighs, be fresh weight.Calculate the rate of increase of adventive root, result display (see Fig. 2 B), cultivate three weeks in the 1/2MS (-N) culture fluid containing 2mg/L IBA and 20g/L sucrose after, the rate of increase is 270%; After cultivating six weeks, the rate of increase is 188%; When after nine weeks cultivate, the rate of increase is 142%.
Embodiment 6
The Multiplying culture of the adventive root that the present embodiment produces for illustration of the induction of ginseng plantlet in vitro stem.
The adventive root produced is induced to carry out Multiplying culture according to the method identical with embodiment 5 to ginseng plantlet in vitro stem.Difference is only, the medium used is 1/2MS (-N) the liquid proliferated culture medium containing 5mg/L IBA and 20g/L sucrose.Calculate the rate of increase of adventive root, result display (see Fig. 2 B), Multiplying culture is after three weeks, and the rate of increase is 295%; After cultivating six weeks, the fresh weight rate of increase is 169%, and when after nine weeks cultivate, the rate of increase is 126%.
Embodiment 7
The present embodiment is for illustration of the generation of ginseng plantlet in vitro leaf inducing adventitious root.
Get the blade of ginseng plantlet in vitro, with the segment of sterilizing cutter cutting into about 5mm × 5mm × 3mm, proceed on the solid culture medium containing 1/2MS (-N)+2mg/L IBA (containing 2% sucrose in solid inducing culture, 7g/L agar, pH value is 5.8), in 24 ± 2 DEG C, under dark condition, cultivate the generation of inducing adventitious root.30 explants inoculated by each culture dish, and every three weeks subcultures once, and add up the indefinite radical of root induction rate and the generation of each explant, and test repetition 3 times, averages.Result shows, after three weeks cultivate, all blades all do not have the generation of adventive root, but after six weeks cultivate (situation of Fiber differentiation adventive root is shown in Fig. 1 C), have the explant induction of 87.78% to create adventive root, the indefinite radical that average every bar root produces is 6.33 (see table 1).
Embodiment 8
The Multiplying culture of the adventive root that the present embodiment produces for illustration of the induction of ginseng plantlet in vitro blade.
The adventive root that the leaves of panax ginseng induction obtained after cultivating six weeks in embodiment 7 produces is taken out, in the blake bottle of the liquid proliferated culture medium of access containing 100ml 1/2MS (-N)+2mg/L IBA+20g/L sucrose, be placed in shaking table, 24 ± 2 DEG C, dark condition shakes at a slow speed (60rpm), carries out the Multiplying culture of adventive root.Every three weeks subcultures once, and add up the fresh weight of adventive root, and test repetition 3 times, averages.Take out cultivating the adventive root obtained, the culture fluid of sterilizing filter paper adsorption surface, is placed in balance and weighs, be fresh weight.Calculate the rate of increase of adventive root, result display (see Fig. 2 C), in 1/2MS (-N) the liquid proliferated culture medium containing 2mg/L IBA and 20g/L sucrose, Multiplying culture is after three weeks, and the rate of increase is 713%; After cultivating six weeks, the rate of increase is 331%; When after nine weeks cultivate, the rate of increase is 77.42%.
Embodiment 9
The Multiplying culture of the adventive root that the present embodiment produces for illustration of the induction of ginseng plantlet in vitro leaf.
The adventive root produced is induced to carry out Multiplying culture according to the method identical with embodiment 8 to ginseng plantlet in vitro leaf.Difference is only, the medium used is the 1/2MS (-N) culture fluid containing 5mg/L IBA and 20g/L sucrose, calculates the rate of increase of adventive root, result display (see Fig. 2 C), and Multiplying culture is after three weeks, and the rate of increase is 638%; After cultivating six weeks, the rate of increase is 216%, and when after nine weeks cultivate, the rate of increase drops to 83.44%.
Table 1
In table 1, the indefinite radical variance analysis of root induction rate and average every block callus adopts T inspection.Different letter represents significant difference.During Fiber differentiation 6 weeks, compared with leaf, root and the average indefinite radical significant difference of stem, p<0.05.Other p<0.01.
In fig. 2, different letter represents significant difference.Compared with Multiplying culture in 1/2MS (-N)+2mg/L IBA 3 weeks, adventive root rate of increase significant difference after 9 weeks Multiplying culture of ginseng plantlet in vitro root induction, p<0.05.Compared with Multiplying culture in 1/2MS (-N)+5mg/L IBA 6 weeks, adventive root rate of increase significant difference after 9 weeks Multiplying culture of panax ginseng stem segment induction, p<0.05.During the adventive root Multiplying culture 6 weeks of leaves of panax ginseng induction, with compared with 1/2MS (-N)+2mg/L IBA culture fluid, inductivity significant difference in 1/2MS (-N)+5mg/L IBA culture fluid, p<0.05.Other p<0.01.
Comparative example 1
This comparative example is for contrasting the induction of ginseng callus adventive root.
Get the root of 5 years raw Radix Ginsengs, clean through clear water, oven for drying and 75% alcohol surface sterilization be after 1 minute, with the otch of sterile scalpel at standardized the 1-2cm in root table face in superclean bench, and cut from both sides, fritter interior tissue being cut into 5mm × 5mm × 5mm is inoculated into MS+1mg/L 2, on 4-dichlorphenoxyacetic acid medium, carry out the Fiber differentiation of callus.Condition of culture is 24 ± 2 DEG C and lucifuge.All add 30g/L sucrose and 7g/L agar in medium, pH value is 5.8.Every three weeks subcultures once.The good callus of growth conditions can be obtained after six weeks cultivate.
By the callus that above-mentioned ginseng is induced, be seeded on the solid culture medium containing 1/2MS (-N)+2mg/LIBA (containing 2% sucrose in solid inducing culture, concentration is the agar of 0.7 quality %, pH value is 5.8), in 24 ± 2 DEG C, under dark condition, cultivate the generation of inducing adventitious root.30 explants inoculated by each culture dish, and every three weeks subcultures once, and add up the indefinite radical of root induction rate and the generation of each explant, and test repetition 3 times, averages.Result shows, after three weeks cultivate, the ginseng callus of 30% is had to create adventive root, the indefinite radical that average every bar root produces is 1.67, after six weeks cultivate, all explants are all induced and are created adventive root, and the indefinite radical that average every bar root produces is 3.67 (see table 1).
Comparative example 2
This comparative example is for contrasting the propagation of ginseng callus adventive root.
The adventive root that the ginseng callus induction obtained after cultivating six weeks in comparative example 1 produces is taken out, in the blake bottle of the liquid nutrient medium of access containing 100ml 1/2MS (-N)+2mg/L IBA+20g/L sucrose, be placed in shaking table, 24 ± 2 DEG C, dark condition shakes at a slow speed (60rpm), carries out the Multiplying culture of adventive root.Every three weeks subcultures once, and add up the fresh weight of adventive root, and test repetition 3 times, averages.Take out cultivating the adventive root obtained, the culture fluid of sterilizing filter paper adsorption surface, is placed in balance and weighs, be fresh weight.Result display (see Fig. 3), cultivate three weeks in the 1/2MS (-N) culture fluid containing 2mg/L IBA and 20g/L sucrose after, the rate of increase only has an appointment 1%; After cultivating six weeks, the rate of increase declines to some extent, occurs negative propagation.
Comparative example 3
The present embodiment is for illustration of the propagation utilizing ginseng callus to carry out adventive root.
According to the method identical with comparative example 2, Multiplying culture is carried out to the adventive root that ginseng callus induction produces.Difference is only, the medium used is the 1/2MS (-N) culture fluid containing 5mg/L IBA and 20g/L sucrose, fresh weight proliferation times result display (see Fig. 3), and Multiplying culture is after three weeks, and the rate of increase is 3%; After cultivating six weeks, the rate of increase 105%; When after nine weeks cultivate, the rate of increase is 386.5%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. a ginseng adventitious root proliferative induction method, is characterized in that, comprises the following steps:
Ginseng plantlet in vitro is cut into after organizing fritter and be inoculated in induced synthesis adventive root in solid inducing culture, after described adventive root is cut into adventive root fritter, be inoculated in the Multiplying culture carrying out adventive root in liquid proliferated culture medium; Wherein, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid that minimal medium contains that concentration is 1-10mg/L with 1/2MS (-N) medium.
2. root induction enrichment procedure according to claim 1, is characterized in that, described solid inducing culture and liquid proliferated culture medium are be the indolebutyric acid that minimal medium contains that concentration is 2-5mg/L with 1/2MS (-N) medium.
3. root induction enrichment procedure according to claim 1 and 2, is characterized in that, comprise the following steps:
1) ginseng plantlet in vitro is cut into organize fritter, utilize solid inducing culture Fiber differentiation 3-6 week under the dark condition of cultivation temperature for 23-25 DEG C to obtain adventive root;
2) by step 1) adventive root that obtains cuts into adventive root fritter, utilizes liquid proliferated culture medium to cultivate 3-9 week with the concussion of the speed of 50-80rpm under the dark condition of cultivation temperature for 23-25 DEG C.
4. root induction enrichment procedure according to claim 3, is characterized in that, described fritter of organizing is ginseng plantlet in vitro root fritter, ginseng plantlet in vitro stem fritter or ginseng plantlet in vitro leaf fritter.
5. root induction enrichment procedure according to claim 4, is characterized in that, also contains the sucrose that concentration is 1.8-2.2 quality % in described solid inducing culture and liquid proliferated culture medium.
6. root induction enrichment procedure according to claim 5, is characterized in that, described ginseng plantlet in vitro is the plantlet in vitro obtained through seed germination or explant cultured in vitro, and the age in days of plantlet in vitro is 28-32 days.
7. according to the root induction enrichment procedure in claim 1-2,4-6 described in any one, it is characterized in that, the kind of described ginseng plantlet in vitro is Radix Ginseng or Da-maya ginseng.
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| CN105638470A (en) * | 2016-01-09 | 2016-06-08 | 佛山市金蓝领教育科技有限公司 | Ginseng seed germination culture medium |
| CN107343443A (en) * | 2017-08-14 | 2017-11-14 | 高健强 | A kind of Talinum crassifolium large-scale method for producing |
| CN108739377A (en) * | 2018-04-28 | 2018-11-06 | 刘汉石 | A kind of ginseng adventitious root inducing culture and its application |
| CN109729974A (en) * | 2019-02-12 | 2019-05-10 | 天津大学 | A kind of method for tissue culture of Low- temperature culture ginseng adventitious root |
| CN111937748A (en) * | 2020-08-24 | 2020-11-17 | 延边大学 | Culture method for improving content of ginsenoside Rg1 and Re in ginseng adventitious roots |
| CN112813018A (en) * | 2021-04-02 | 2021-05-18 | 山东安然纳米实业发展有限公司 | Culture method of ginseng stem cells |
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| CN113025554A (en) * | 2021-04-20 | 2021-06-25 | 山东安然纳米实业发展有限公司 | Method for culturing ginseng stem cells by using biological reaction device |
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