CN105309316A - Culture medium for tissue culture seedlings of savia miltiorrhiza and rapid propagation method for savia miltiorrhiza tissue culture - Google Patents
Culture medium for tissue culture seedlings of savia miltiorrhiza and rapid propagation method for savia miltiorrhiza tissue culture Download PDFInfo
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Abstract
The invention discloses a culture medium for tissue culture seedlings of savia miltiorrhiza and a rapid propagation method for savia miltiorrhiza tissue culture. Savia miltiorrhiza stems with axillary bud seve as the materials, savia miltiorrhiza stem tissue culture and propagation are conducted, and the large number of high quality savia miltiorrhiza seedlings can be obtained through explant disinfection treatment, induction and differentiation culture, rooting culture and acclimatization and transplant. The culture medium and the rapid propagation method have the advantages that the growing cycle is short, the propagation rate is high, variation is little, the culture condition can be controlled artificially, operation is simple, output is large, a theoretical basis and a practice basis are provided for achieving the goal of producing the savia miltiorrhiza seedlings on a large scale and in an industrialized mode within a short period, and the foundation is laid for genetic transformation, breed improvement and the like of savia miltiorrhiza.
Description
Technical field
The invention belongs to field of plant tissue culture technique, particularly relate to a kind of tissue culture technique of the red sage root.
Background technology
The red sage root has another name called red ginseng, by crow, strongly fragrant cicada is careless, galloping horse is careless, and be the dry root and rhizome of the labiate red sage root (SalviamiltiorrhizaBunge), bitter, is slightly cold.The thoughts of returning home, Liver Channel, has promoting blood circulation and removing obstruction in channels, silt pain relieving of dispelling, cool blood disappears carbuncle, clear away heart-fire the effects such as relieving restlessness, to cardiovascular and hematological system effect is remarkable.Be used for clinically treat chest impediment and cardialgia, gastral cavity abdomen hypochondriac pain ， lumps in the chest and abdomen, hot numbness pain, dysphoria and insomnia, irregular menstruation, dysmenorrhoea through closing, sore, carbuncle and painful swelling etc.Along with going deep into its main chemical compositions and pharmacological research, increase the development and utilization of red sage root resource, red sage root market demand is constantly expanded.But red sage root wild resource is relatively less, and traditional cultivation mode, be faced with again growth cycle long, the relative problem such as excessively high of quality deterioration, production cost.
Plant Tissue Breeding is said in a broad sense and is referred under artificial manipulation, plant corpus organ, a tissue, individual cells or protoplast are to be placed on after plant corpus takes out glass container and under supply abundant nutrition composition and suitable extraneous condition of culture, make them be survived or develop, the final a kind of cultural method forming whole plant.Tissue cultures has more advantages, as: growth cycle is short, and reproduction rate is high, and variation is few, and condition of culture can manual control, simple to operate, is beneficial to factorial praluction and Automated condtrol, etc.
Summary of the invention:
The object of this invention is to provide a kind of method of red sage root plantlet in vitro inducing culture, root media and red sage root tissue-culturing rapid propagation.
For solving the problem, the invention provides following technical scheme:
The invention discloses a kind of red sage root plantlet in vitro medium, comprise inducing culture and root media, described inducing culture is: MS+6-BA0.5-2.0mg/L+NAA0.1-0.5mg/L+ sucrose 30g/L+ agar 7g/L, and pH value is between 6.08-6.12; Described root media is: 1/2MS+IBA1.0mg/L+KT1.0mg/L+ sucrose 15g/L+ agar 7g/L+ active carbon 0.05-0.15%, pH value is between 6.08-6.12.
Preferably, the 6-BA in described inducing culture is 1.0mg/L, NAA is 0.5mg/L, and the active carbon in described root media is 0.05%.
Wherein, the macroelement of 1/2MS and MS basic components reduces by half, and other is constant.
Described inducing culture and root media: 1L culture fluid is divided in the bottle of 15 200ml, the culture fluid of every bottle of packing about 60ml.
The present invention discloses a kind of red sage root tissue culture and rapid propagation method in addition, and with the red sage root stem with axillalry bud for material, carry out red sage root tissue culture propagating, its step is as follows:
(1) explant is disinfected;
(2) differentiation-inducing cultivation: the explant through disinfecting is inoculated in described inducing culture and carries out inducing clumping bud cultivation;
(3) step (2) is cultivated the segment that the stem obtaining red sage root seedling is cut into band joint, and proceed on described root media and carry out squamous subculture;
(4) acclimatization and transplants.
Preferably, described step (1) explant is disinfected: the tender stem section taking green tape axillalry bud is then explant, cut off leaf, stay petiole base ， Cheongju water constantly to rock and wash away surface attachments, in superclean bench, with the solution (add 2-3 and drip tween) of 1.5% clorox, sterilize 15-20min, with aseptic water washing 2-3 time, use 75% ethanol disinfection 2 times again, each 30-60s, with aseptic washing 2-3 time.
Preferably, the condition of described step (2) differentiation-inducing cultivation is: light application time is 14 hours every days, and intensity of illumination is 2000lx, and being placed in cultivation temperature is cultivate under the condition of 24-26 DEG C until form Multiple Buds.
Preferably, the condition of described step (3) culture of rootage is: light application time is 12 hours every days, and intensity of illumination is 2000lx, and cultivation temperature is 22 DEG C;
Preferably, described step (4) acclimatization and transplants: by the half-open hardening 2-3 days of cultivation bottle cap of the rooting tube plantlet of height about 5-6cm, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5-7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by 1:1 proportioning by vermiculite and perlite and to be colonizated in large Tanaka.
Preferably, described inducing culture and root media all need sterilizing before use, and put in sterilizer by the medium prepared, sterilising temp is 120 DEG C, and sterilization time is 30 minutes, after sterilizing is complete, is stored into desinfection chamber rapidly.
Preferably, in Fiber differentiation and culture of rootage, all inoculate 1 explant, with anti-cross-contamination for every bottle.
Preferably, in transplanting, seeding row spacing of planting is about 10 × 15cm, and spray water in time after transplanting, finally cost Small plastic shed, water permeable, cover plastic film, use shading screen shading, temperature controls at 20-25 DEG C, with growth after surviving, increases intensity of illumination as early as possible.
The present invention discloses a kind of method of red sage root test-tube plantlet acclimatization and transplants in addition, by the half-open hardening 2-3 days of cultivation bottle cap of the rooting tube plantlet of height about 5-6cm, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5-7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by 1:1 proportioning by vermiculite and perlite and to be colonizated in large Tanaka.Seeding row spacing of planting is about 10 × 15cm, and spray water in time after transplanting, finally cost Small plastic shed, water permeable, cover plastic film, use shading screen shading, temperature controls at 20-25 DEG C, with growth after surviving, increases intensity of illumination as early as possible.
Beneficial effect of the present invention is: the present invention is directed to traditional red sage root cultivation mode, to there is growth cycle long, quality deterioration, the relative problem such as excessively high of production cost.The present invention is to be with the red sage root stem of axillalry bud for material, red sage root tissue culture quick breeding technical system is established through steps such as explant sterilization process, Induction and differentiation cultivation, culture of rootage and acclimatization and transplantses, have found best red sage root tissue-culturing rapid propagation condition, can be used for producing red sage root high quality seedling in enormous quantities.Its beneficial effect is that growth cycle is short, reproduction rate is high, variation is few, condition of culture can manual control, simple to operate, output is large, provides theoretical foundation and practical basis for realizing large-scale industrialized production red sage root seedling in a short time, and lays the foundation for the genetic transformation of the red sage root and breed improvement etc.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Embodiment
Below in conjunction with instantiation, the present invention is described in detail.
Embodiment 1
Red sage root tissue culture and rapid propagation method, comprises the steps:
Step one, the preparation of several MS medium mother liquor.
The preparation of MS100 times of macroelement mother liquor, take potassium nitrate 190g respectively, ammonium nitrate 165g, potassium dihydrogen phosphate 17g, magnesium sulfate 37g, calcium chloride 44g, add respectively in 5 1000mL beakers, add appropriate distilled water, stirring and dissolving, constant volume becomes 1000mL, and 5 kinds of macroelement mother liquor placement refrigerators are for subsequent use.
The preparation of MS1000 doubly micro-mother liquor, takes potassium iodide 0.83g, boric acid 6.2g respectively, manganese sulphate 16.9g, zinc sulphate 8.6g, sodium molybdate 0.25g, copper sulphate 0.025g, cobalt chloride 0.025g, to in 1000mL beaker, add appropriate distilled water, stirring and dissolving, constant volume becomes 1000mL, places refrigerator for subsequent use.
The preparation of MS100 doubly organic mother liquor, takes inositol 10g, glycine 0.2g, thiamine hydrochloride 0.01g, puridoxine hydrochloride 0.05g, nicotinic acid 0.05g.Dissolve successively in distilled water, constant volume becomes 1000mL, places refrigerator for subsequent use.
The preparation of MS100 times of molysite, takes disodium ethylene diamine tetraacetate 3.73g respectively, ferrous sulfate 2.78g, and in 1000mL beaker, add appropriate distilled water, stirring and dissolving, constant volume becomes 1000mL, places refrigerator for subsequent use.
Step 2, the preparation of inducing culture, get MS1000 doubly micro-mother liquor 1mL, MS is a large amount of, MS is organic, MS molysite three kinds of each 10mL of mother liquor, adds appropriate distilled water, add 30g sucrose and 1000 μ L1.0mg/mL6-BA to 1000mL beaker, 1000 μ L0.5mg/mLNAA, stirring and dissolving, regulate PH to be about 6.10, constant volume becomes 1000mL mixed liquor.Take agar powder 7g, add 1000mL mixed liquor, heating is boiled, and divides and is filled in 15 200mL blake bottles.
Step 3, the preparation of root media, gets MS1000 doubly micro-mother liquor 1mL, MS100 times of macroelement mother liquor 5mL respectively, MS is organic, each 10mL of MS mother liquid of iron salt, to 1000mL beaker, add appropriate distilled water, add 15g sucrose and 1000 μ L1.0mg/mLIBA, 1000 μ L1mg/mLKT (6-furan feeds aminopurine), stirring and dissolving, regulate PH to be about 6.10, add 0.5g active carbon, constant volume becomes 1000mL mixed liquor.Take agar powder 7g, add 1000mL mixed liquor, heating is boiled, and divides and is filled in 15 200mL blake bottles.
Step 4, the sterilizing of medium, puts in autoclave by point medium installed, and setting sterilising temp is 120 DEG C, and sterilization time is 30 minutes, after sterilizing is complete, is stored into rapidly in desinfection chamber.
Step 5, the process of red sage root explant and Fiber differentiation, the tender stem of green tape axillalry bud is then taked to be explant, cut off leaf, stay petiole base, be cut into the segment that length is the band axillalry bud of about 2cm, surface attachments is removed in Cheongju washing, in superclean bench, with liquor natrii hypochloritis's (add 2-3 and drip tween) of 1.5% (mass fraction), sterilize 15-20min, with aseptic water washing 2-3 time, after use 75% (volume fraction) ethanol disinfection 2 times, each 20-30s, with aseptic washing 2-3 time, then be inoculated in inducing culture after sucking moisture with aseptic filter paper and carry out inducing clumping bud cultivation.Condition of culture is 14 hours every days of light application time, intensity of illumination 2000lx, cultivation temperature 24-26 DEG C.Within 25 days, bud ratio can reach 95%.
Step 6, culture of rootage, the red sage root edible tender branch obtained by Fiber differentiation, to cut and merogenesis proceeds to root media and carries out squamous subculture from base portion.Condition of culture is 12 hours every days of light application time, intensity of illumination 2000lx, cultivation temperature 24-26 DEG C.Within 20 days, rooting rate can reach 100%.
Step 7, acclimatization and transplants, by the half-open hardening 2-3 days of cultivation bottle cap of the rooting tube plantlet of height about 5-6cm, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5-7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by 1:1 quality proportioning by vermiculite and river sand and to be colonizated in large Tanaka.Seeding row spacing of planting is about 10 × 15cm, and spray water in time after transplanting, finally cost Small plastic shed, water permeable, cover plastic film, use shading screen shading, temperature controls at 20-25 DEG C, with growth after surviving, increases intensity of illumination as early as possible.
Embodiment 2
This patent has investigated different plant growth regulator and concentration affects medium composition, cultivation temperature to the impact of taking root (except investigation factor, other factors, condition equivalent integers 1) to red sage root plantlet in vitro bud inducement.Determine the best culture scheme of red sage root plantlet in vitro.Now the results list will be investigated as follows.
The different plant growth regulator of table 1 and concentration are on the impact of red sage root plantlet in vitro bud inducement
As can be seen from Table 1, when adding 6-BA1mg and NAA0.5mg in often liter of medium, best to the inducing action of Multiple Buds.
The impact that the different plant growth regulator of table 2 and concentration are taken root on red sage root plantlet in vitro
As can be seen from Table 2, when adding IBA1mg and KT1mg in often liter of medium, be conducive to taking root of red sage root plantlet in vitro.
After having groped best plant growth conditioning agent and concentration, on this basis, study the impact of taking root the composition of medium, namely often group is all added with IBA1mg/L and KT1mg/L. result as table 3 in processing
Table 3 different culture media forms the impact of taking root on red sage root plantlet in vitro
Wherein, the macroelement in 1/2MS and MS basic components reduces by half, and namely macroelement is the proportional concentration in embodiment 1, and other is constant.1/2 sucrose refers to that relative to the concentration of sucrose in inducing culture be 1/2, i.e. 15g/L.
As can be seen from Table 3, MS and sucrose all reduce by half, in medium add 0.05% active carbon be conducive to taking root of red sage root plantlet in vitro.
The impact that the different cultivation temperature of table 4 is taken root on red sage root plantlet in vitro
As can be seen from Table 4, cultivation temperature is conducive to taking root of red sage root plantlet in vitro most 22 DEG C time.
Although illustrate and describe examples and comparative examples of the present invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (10)
1. a red sage root plantlet in vitro medium, comprises inducing culture and root media, it is characterized in that,
Described inducing culture is: MS+6-BA0.5-2.0mg/L+NAA0.1-0.5mg/L+ sucrose 30g/L+ agar 7g/L, and pH value is between 6.08-6.12;
Described root media is: 1/2MS+IBA1.0mg/L+KT1.0mg/L+ sucrose 15g/L+ agar 7g/L+ active carbon 0.05-0.15%, pH value is between 6.08-6.12.
2. red sage root plantlet in vitro medium according to claim 1, is characterized in that, the 6-BA in described inducing culture is 1.0mg/L, NAA is 0.5mg/L, and the active carbon in described root media is 0.05%.
3. a red sage root tissue culture and rapid propagation method, is characterized in that, with the red sage root stem with axillalry bud for material, carry out red sage root tissue culture propagating, its step is as follows:
(1) explant is disinfected;
(2) differentiation-inducing cultivation: the explant through disinfecting is inoculated in the inducing culture described in inducing culture or claim 1 or 2 and carries out inducing clumping bud cultivation;
(3) step (2) is cultivated the segment that the stem obtaining red sage root seedling is cut into band joint, and proceed on the root media described in root media or claim 1 or 2 and carry out squamous subculture;
(4) acclimatization and transplants.
4. red sage root tissue culture and rapid propagation method according to claim 3, it is characterized in that, described step (1) explant is disinfected: the tender stem section taking green tape axillalry bud is then explant, cuts off leaf, stays petiole base Cheongju water constantly to rock and washes away surface attachments, in superclean bench, with the solution (add 2-3 and drip tween) of 1.5% clorox, sterilize 15-20min, with aseptic water washing 2-3 time, then uses 75% ethanol disinfection 2 times, each 30-60s, with aseptic washing 2-3 time.
5. red sage root tissue culture and rapid propagation method according to claim 3, it is characterized in that, the condition of described step (2) differentiation-inducing cultivation is: light application time is 14 hours every days, and intensity of illumination is 2000lx, and being placed in cultivation temperature is cultivate under the condition of 24-26 DEG C until form Multiple Buds.
6. red sage root tissue culture and rapid propagation method according to claim 3, is characterized in that, the condition of described step (3) culture of rootage is: light application time is 12 hours every days, and intensity of illumination is 2000lx, and cultivation temperature is 22 DEG C.
7. red sage root tissue culture and rapid propagation method according to claim 3, it is characterized in that, described step (4) acclimatization and transplants: by the half-open hardening 2-3 days of cultivation bottle cap of the rooting tube plantlet of height about 5-6cm, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5-7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by 1:1 proportioning by vermiculite and perlite and to be colonizated in large Tanaka.
8. red sage root tissue culture propagation method according to claim 3, it is characterized in that, described inducing culture and root media all need sterilizing before use, the medium prepared is put in sterilizer, sterilising temp is 120 DEG C, sterilization time is 30 minutes, after sterilizing is complete, is stored into desinfection chamber rapidly.
9. red sage root tissue culture propagation method according to claim 7, it is characterized in that, in transplanting, seeding row spacing of planting is about 10 × 15cm, spray water in time after transplanting, finally cost Small plastic shed, water permeable, cover plastic film, use shading screen shading, temperature controls at 20-25 DEG C, with growth after surviving, increases intensity of illumination as early as possible.
10. the method for a red sage root test-tube plantlet acclimatization and transplants, it is characterized in that, by the half-open hardening 2-3 days of cultivation bottle cap of the rooting tube plantlet of height about 5-6cm, standard-sized sheet bottle cap adds appropriate running water and is placed in natural lighting lower refining seedling 5-7 days again, test-tube plantlet is taken out from blake bottle, wash root medium off, to plant in the matrix be mixed into by 1:1 proportioning by vermiculite and perlite and to be colonizated in large Tanaka; Seeding row spacing of planting is about 10 × 15cm, and spray water in time after transplanting, finally cost Small plastic shed, water permeable, cover plastic film, use shading screen shading, temperature controls at 20-25 DEG C, with growth after surviving, increases intensity of illumination as early as possible.
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| CN110178728A (en) * | 2019-05-16 | 2019-08-30 | 四川农业大学 | A kind of method of middle river Radix Salviae Miltiorrhizae tissue-culturing rapid propagation |
| CN112167064A (en) * | 2020-11-11 | 2021-01-05 | 山东农业大学 | Tissue culture and rapid propagation method for salvia miltiorrhiza |
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