CN103749298A - Lolium multiflorum tissue culture and rapid propagation method - Google Patents

Lolium multiflorum tissue culture and rapid propagation method Download PDF

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CN103749298A
CN103749298A CN201410005547.0A CN201410005547A CN103749298A CN 103749298 A CN103749298 A CN 103749298A CN 201410005547 A CN201410005547 A CN 201410005547A CN 103749298 A CN103749298 A CN 103749298A
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mother liquor
reagent
aseptic
settled
tissue culture
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CN103749298B (en
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王晓凌
施江
李晓倩
赵威
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Henan University of Science and Technology
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Henan University of Science and Technology
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Abstract

The invention belongs to the field of plant biology and in particular relates to a Lolium multiflorum tissue culture and rapid propagation method. The method comprises the steps of preparation of aseptic seedlings, preparation of aseptic no-root tillered cluster buds, and preparation and transplanting of aseptic seedlings with root systems; the required instruments are simple, the cost is low and the economic benefit is improved; the constructed Lolium multiflorum tissue culture and rapid propagation system including seed germination culture, tillered cluster bud culture, rooting culture and seedling transplanting is adopted, the seedling time is 2-3 months, the survival rate is improved, and the method is suitable for scale production; a stem is cultured, the survival rate of the tissue culture seedling is greatly improved, and the culture period is shortened. According to the method, the stem is used for culture, the material drawing range of the tissue culture of the Lolium multiflorum can be greatly expanded, the development of the tissue culture of the Lolium multiflorum is promoted, and therefore, the method can be widely applied to scale production.

Description

A kind of Annual Ryegrass tissue culture and rapid propagation method
Technical field
The invention belongs to phytobiology field, be specifically related to a kind of Annual Ryegrass tissue culture and rapid propagation method.
Background technology
Annual Ryegrass is important gramineous forage grass, in recent years, becomes herbage and turfgrass kind that China widelys popularize, and the effect in China's animal husbandry development and town and country are beautified is increasing.Perennial ryegrass tillers many, and output is high, quality softness, and the green phase is long, is good green feed.In addition, its well developed root system, suitable natural disposition is strong, and resistance to trample, has Salt And Alkali Tolerance potentiality, can increase the soil organic matter, improves soil structure, prevents erosion, and is good turfgrass.For adapting to the needs of perennial ryegrass breeding work, preserve and Fast-propagation high-quality starting material, and for the physiological ecological research about perennial ryegrass gene expression provides reliable research material in good time, the cultured in vitro of perennial ryegrass and quick propagating technology are subject to increasing attention.About the tissue of perennial ryegrass, cultivate and only limit to Seed inducement callus and plant regeneration at present, and with protoplast, young fringe, stem apex etc., do the formation of sugarcane explants through callus induction or Multiple Buds, but induction time is long, growth rate is slow, the seedling cycle is long, and group culturation rapid propagating technology is also immature.
Summary of the invention
The present invention is directed to the deficiency of the problems referred to above, a kind of Annual Ryegrass tissue culture and rapid propagation method is provided, its cultural method is simple and easy to do, and cultivation cycle is short.
The present invention for addressing the above problem adopted technical scheme is: a kind of Annual Ryegrass tissue culture and rapid propagation method, comprises the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation that has the aseptic seedling of root system and transplanting;
The described preparation method without the aseptic Multiple Buds of tillering of root system is: in superclean bench, choose healthy and strong aseptic seedling, and be placed in sterile petri dish, under aseptic condition, first cut off the root system of aseptic seedling, the then upwards position of the 1cm cutting in rhizome junction, obtains stem foot, the stem foot obtaining is put in to the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprout in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in to temperature is 23～27 ℃ again, light application time is 16h/d, light intensity is to cultivate after 20 ~ 35 days in the illumination box of 1500 ~ 2000Lx, single stem foot sprouts high 6 ~ 10cm, quantity is the Multiple Buds of tillering of 8 ~ 20, the Multiple Buds of must tillering, then therefrom chooses quantity and is the Multiple Buds of tillering of 18 ~ 48 as without the aseptic Multiple Buds of tillering of root system;
The described preparation method who has the aseptic seedling of root system is: in superclean bench, taking-up make without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the medical surgical knife of sterilizing, excise the unnecessary blade on Multiple Buds top, leave the Multiple Buds of long 2 ~ 3cm, then at tillering node place, longitudinally cut, be divided into complete simple bud, then simple bud root is put down gently to the culture of rootage primary surface in tissue culture bottle downwards, light pressure submerged in medium its root, after having inoculated, cover tightly bottle cap, it is 23～27 ℃ that postvaccinal tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is to cultivate after 15 ~ 25 days in the illumination box of 1500 ~ 2000Lx, seedling base portion grows 6 ~ 10 coring roots, obtain aseptic seedling.
The preparation method of described aseptic seedling chooses the ryegrass seed of complete maturation, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, the alcohol that is first 75% with percent by volume mark is by ryegrass seed sterilization 30 ~ 60s, use again aseptic water washing 2 ~ 3 times, put into afterwards mass percent concentration and be mercuric chloride soaking disinfection 6 ~ 8min of 0.1%, with after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtaining is put into sterile petri dish, under aseptic condition, put down gently in aseptic minimal medium surface, then gently press, seed is submerged in medium, after having inoculated, cover tightly tissue culture bottle lid, it is 23～27 ℃ that tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is in the illumination box of 1000 ~ 2000Lx, cultivate after 20 ~ 35 days, obtaining is highly the aseptic seedling of 5 ~ 8cm.
The method of described transplanting is that vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, standby;
From medium take out preparation the aseptic seedling that has root system, with clear water, wash the medium of root remnants off, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23～27 ℃ in temperature, and light application time is 16h/d, intensity of illumination is to cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in again temperature and be the glass greenhouse of 23～27 ℃, under natural lighting, cultivate after 6～7 days, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 2014100055470100002DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 34019DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 256446DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 329444DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 297400DEST_PATH_IMAGE002
eDTA
Figure 206581DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 980502DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 845690DEST_PATH_IMAGE002
eDTA
Figure 296132DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 941877DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 457172DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 989916DEST_PATH_IMAGE002
7H2O, Na 2moO 4 2H 2o, CuSO 4
Figure 432715DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 800636DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4 4H 2o 22.3 mg/L, ZnSO 4
Figure 183393DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 554462DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 208298DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 214169DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add afterwards 15g sucrose and 3.2g agar powder, continue heating and constantly stir, the shape until be translucent, the minimal medium solution that obtains not sterilizing, standby,
Step 4: the not minimal medium solution of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid minimal medium of transparence.
The compound method of the described medium of sprouting, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 193626DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 973363DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4 7H 2o 370 mg/L, CaCl 2
Figure 995994DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 880773DEST_PATH_IMAGE002
eDTA
Figure 474040DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 672940DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 404135DEST_PATH_IMAGE002
eDTA
Figure 210549DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 964878DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 764207DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 287592DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 29021DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 270646DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 608087DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 64607DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4 7H 2o 8.6 mg/L, Na 2moO 4
Figure 191012DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 269826DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 82318DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, the shape until be translucent, then add the 6-BA of 0.9mg and the NAA of 0.09mg, and stir evenly, the culture medium solution of sprouting that obtains not sterilizing, standby.
Step 4: the not culture medium solution of sprouting of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid medium of sprouting.
The collocation method of described root media, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4 7H 2o and CaCl 2
Figure 804603DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4 7H 2o 370 mg/L, CaCl 2 2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 44458DEST_PATH_IMAGE002
eDTA
Figure 295441DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 716058DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 250945DEST_PATH_IMAGE002
eDTA
Figure 364395DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 289625DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 825518DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 887015DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 171365DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 646209DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 674208DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4 4H 2o 22.3 mg/L, ZnSO 4
Figure 858513DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 758336DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 324446DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 688431DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add again 15g sucrose and 3.1 ~ 3.25g agar powder, continue heating and constantly stir, the shape until be translucent, then add the IBA of 0.7mg and the NAA of 0.09mg, and stir, the culture of rootage based sols that obtains not sterilizing, standby,
Step 4: the not culture of rootage based sols of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain sterile solid root media.
beneficial effect
1, instrument required for the present invention is simple, with low cost, has improved economic benefit.
2, the constructed seed sprouting of the present invention cultivate, Multiple Buds cultivation, the culture of rootage of tillering and become transplantation of seedlings perennial ryegrass tissue culture sprout quick traditional font system, can be between 2 months to 3 months seedling, and improved survival rate, be applicable to the production of scale.
3, the present invention, by adopting stem foot to cultivate, has improved the survival rate of group training seedling greatly, has shortened cultivation cycle.And the present invention has greatly expanded the tissue of Annual Ryegrass and has cultivated scope of selecting material by adopting stem foot to cultivate, promoted the development that Annual Ryegrass tissue is cultivated, and makes it widely for large-scale production.
Embodiment
A kind of Annual Ryegrass tissue culture and rapid propagation method, comprises the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation that has the aseptic seedling of root system and transplanting;
The described preparation method without the aseptic Multiple Buds of tillering of root system is: in superclean bench, choose healthy and strong aseptic seedling, and be placed in sterile petri dish, under aseptic condition, first cut off the root system of aseptic seedling, the then upwards position of the 1cm cutting in rhizome junction, obtains stem foot, the stem foot obtaining is put in to the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprout in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in to temperature is 23～27 ℃ again, light application time is 16h/d, light intensity is to cultivate after 20 ~ 35 days in the illumination box of 1500 ~ 2000Lx, single stem foot sprouts high 6 ~ 10cm, quantity is the Multiple Buds of tillering of 8 ~ 20, the Multiple Buds of must tillering, then therefrom chooses quantity and is the Multiple Buds of tillering of 18 ~ 48 as without the aseptic Multiple Buds of tillering of root system;
The described preparation method who has the aseptic seedling of root system is: in superclean bench, taking-up make without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the medical surgical knife of sterilizing, excise the unnecessary blade on Multiple Buds top, leave the Multiple Buds of long 2 ~ 3cm, then at tillering node place, longitudinally cut, be divided into complete simple bud, then simple bud root is put down gently to the culture of rootage primary surface in tissue culture bottle downwards, light pressure submerged in medium its root, after having inoculated, cover tightly bottle cap, it is 23～27 ℃ that postvaccinal tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is to cultivate after 15 ~ 25 days in the illumination box of 1500 ~ 2000Lx, seedling base portion grows 6 ~ 10 coring roots, obtain aseptic seedling.。
The preparation method of described aseptic seedling chooses the ryegrass seed of complete maturation, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, the alcohol that is first 75% with percent by volume mark is by ryegrass seed sterilization 30 ~ 60s, use again aseptic water washing 2 ~ 3 times, put into afterwards mass percent concentration and be mercuric chloride soaking disinfection 6 ~ 8min of 0.1%, with after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtaining is put into sterile petri dish, under aseptic condition, put down gently in aseptic minimal medium surface, then gently press, seed is submerged in medium, after having inoculated, cover tightly tissue culture bottle lid, it is 23～27 ℃ that tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is in the illumination box of 1000 ~ 2000Lx, cultivate after 20 ~ 35 days, obtaining is highly the aseptic seedling of 5 ~ 8cm.
The method of described transplanting is that vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, standby;
From medium take out preparation the aseptic seedling that has root system, with clear water, wash the medium of root remnants off, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23～27 ℃ in temperature, and light application time is 16h/d, intensity of illumination is to cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in again temperature and be the glass greenhouse of 23～27 ℃, under natural lighting, cultivate after 6～7 days, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 49006DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 750639DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 120440DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 11036DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 870407DEST_PATH_IMAGE002
eDTA
Figure 479243DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 652736DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 476466DEST_PATH_IMAGE002
eDTA
Figure 444422DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 540554DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4
Figure 318520DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 213533DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 463249DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 448522DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 820598DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 891322DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4 5H 2o 0.025mg/L, CoCl 2
Figure 597558DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add afterwards 15g sucrose and 3.2g agar powder, continue heating and constantly stir, the shape until be translucent, the minimal medium solution that obtains not sterilizing, standby,
Step 4: the not minimal medium solution of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid minimal medium of transparence.
The compound method of the described medium of sprouting, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 78218DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 432976DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 290073DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 984360DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 950435DEST_PATH_IMAGE002
eDTA
Figure 995752DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 390961DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 2071DEST_PATH_IMAGE002
eDTA
Figure 90113DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4 7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 369095DEST_PATH_IMAGE002
4H 2o, ZnSO 4 7H2O, Na 2moO 4
Figure 31338DEST_PATH_IMAGE002
2H 2o, CuSO 4 5H 2o, CoCl 2
Figure 584996DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 419966DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4 7H 2o 8.6 mg/L, Na 2moO 4
Figure 825856DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4 5H 2o 0.025mg/L, CoCl 2
Figure 72347DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, the shape until be translucent, then add the 6-BA of 0.9mg and the NAA of 0.09mg, and stir evenly, the culture medium solution of sprouting that obtains not sterilizing, standby.
Step 4: the not culture medium solution of sprouting of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid medium of sprouting.
The collocation method of described root media, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 407513DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 949484DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 28299DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2 2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 94661DEST_PATH_IMAGE002
eDTA
Figure 310878DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 193384DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 848663DEST_PATH_IMAGE002
eDTA
Figure 791211DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 229146DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 712080DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 184649DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 845569DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 770800DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 995108DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 384501DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 668851DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 81378DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 155382DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 336965DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add again 15g sucrose and 3.1 ~ 3.25g agar powder, continue heating and constantly stir, the shape until be translucent, then add the IBA of 0.7mg and the NAA of 0.09mg, and stir, the culture of rootage based sols that obtains not sterilizing, standby,
Step 4: the not culture of rootage based sols of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain sterile solid root media.
Embodiment 1
A kind of Annual Ryegrass tissue culture and rapid propagation method, comprises the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation that has the aseptic seedling of root system and transplanting;
The described preparation method without the aseptic Multiple Buds of tillering of root system is: in superclean bench, choose healthy and strong aseptic seedling, and be placed in sterile petri dish, under aseptic condition, first cut off the root system of aseptic seedling, the then upwards position of the 1cm cutting in rhizome junction, obtains stem foot, the stem foot obtaining is put in to the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprout in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in to temperature is 23 ℃ again, light application time is 16h/d, light intensity is to cultivate after 20 days in the illumination box of 1500Lx, single stem foot sprouts high 6cm, quantity is the Multiple Buds of tillering of 8, the Multiple Buds of must tillering, then therefrom chooses quantity and is the Multiple Buds of tillering of 18 ~ 48 as without the aseptic Multiple Buds of tillering of root system;
The described preparation method who has the aseptic seedling of root system is: in superclean bench, taking-up make without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the medical surgical knife of sterilizing, excise the unnecessary blade on Multiple Buds top, leave the Multiple Buds of long 2cm, then at tillering node place, longitudinally cut, be divided into complete simple bud, then simple bud root is put down gently to the culture of rootage primary surface in tissue culture bottle downwards, light pressure submerged in medium its root, after having inoculated, cover tightly bottle cap, it is 23 ℃ that postvaccinal tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is to cultivate after 15 days in the illumination box of 1500Lx, seedling base portion grows 6 ~ 10 coring roots, obtain aseptic seedling.
The preparation method of described aseptic seedling chooses the ryegrass seed of complete maturation, put into running water and soak 0.5h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, the alcohol that is first 75% with percent by volume mark is by the ryegrass seed 30s that sterilizes, use again aseptic water washing 2 times, put into afterwards mass percent concentration and be 0.1% mercuric chloride soaking disinfection 6min, with after aseptic water washing 4 times, obtain aseptic seed, in superclean bench, the aseptic seed obtaining is put into sterile petri dish, under aseptic condition, put down gently in aseptic minimal medium surface, then gently press, seed is submerged in medium, after having inoculated, cover tightly tissue culture bottle lid, it is 23 ℃ that tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is in the illumination box of 1000Lx, cultivate after 20 days, obtain the highly aseptic seedling for 5cm.
The method of described transplanting is that vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, standby;
From medium take out preparation the aseptic seedling that has root system, with clear water, wash the medium of root remnants off, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23 ℃ in temperature, and light application time is 16h/d, intensity of illumination is to cultivate after 7 days in the illumination box of 1500Lx, be placed in again temperature and be the glass greenhouse of 23 ℃, under natural lighting, cultivate after 6 days, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 792217DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 754357DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 320467DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 356557DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2 eDTA
Figure 182878DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 349417DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 505592DEST_PATH_IMAGE002
eDTA
Figure 302647DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 225997DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 399489DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 410171DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 440444DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 536576DEST_PATH_IMAGE002
5H 2o, CoCl 2 6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 926417DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 65274DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 711019DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 960735DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 946008DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add afterwards 15g sucrose and 3.2g agar powder, continue heating and constantly stir, the shape until be translucent, the minimal medium solution that obtains not sterilizing, standby,
Step 4: the not minimal medium solution of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid minimal medium of transparence.
The compound method of the described medium of sprouting, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 567351DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 638076DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 691482DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 593579DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 74239DEST_PATH_IMAGE002
eDTA 2H 2o and FeSO 4
Figure 36827DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 731114DEST_PATH_IMAGE002
eDTA
Figure 382675DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 47H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 490308DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 885517DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 748825DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 836866DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 103900DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 99537DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 768416DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4 2H 2o 0.25 mg/L, CuSO 4
Figure 329159DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 331750DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, the shape until be translucent, then add the 6-BA of 0.9mg and the NAA of 0.09mg, and stir evenly, the culture medium solution of sprouting that obtains not sterilizing, standby.
Step 4: the not culture medium solution of sprouting of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid medium of sprouting.
The collocation method of described root media, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 855135DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 81717DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 323342DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 864045DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 553521DEST_PATH_IMAGE002
eDTA
Figure 154267DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 679926DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2 eDTA
Figure 522297DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 841414DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 57632DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 940137DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 354938DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 31907DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 469842DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4 4H 2o 22.3 mg/L, ZnSO 4
Figure 677543DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4 2H 2o 0.25 mg/L, CuSO 4 5H 2o 0.025mg/L, CoCl 2
Figure 2848DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add again 15g sucrose and 3.1 ~ 3.25g agar powder, continue heating and constantly stir, the shape until be translucent, then add the IBA of 0.7mg and the NAA of 0.09mg, and stir, the culture of rootage based sols that obtains not sterilizing, standby,
Step 4: the not culture of rootage based sols of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain sterile solid root media.
Result shows, adopt this method to get final product seedling with 70 days, and survival rate is 94%.
Embodiment 2
A kind of Annual Ryegrass tissue culture and rapid propagation method, comprises the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation that has the aseptic seedling of root system and transplanting;
The described preparation method without the aseptic Multiple Buds of tillering of root system is: in superclean bench, choose healthy and strong aseptic seedling, and be placed in sterile petri dish, under aseptic condition, first cut off the root system of aseptic seedling, the then upwards position of the 1cm cutting in rhizome junction, obtains stem foot, the stem foot obtaining is put in to the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprout in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in to temperature is 27 ℃ again, light application time is 16h/d, light intensity is to cultivate after 35 days in the illumination box of 2000Lx, single stem foot sprouts high 10cm, quantity is the Multiple Buds of tillering of 20, the Multiple Buds of must tillering, then therefrom chooses quantity and is the Multiple Buds of tillering of 48 as without the aseptic Multiple Buds of tillering of root system;
The described preparation method who has the aseptic seedling of root system is: in superclean bench, taking-up make without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the medical surgical knife of sterilizing, excise the unnecessary blade on Multiple Buds top, leave the Multiple Buds of long 3cm, then at tillering node place, longitudinally cut, be divided into complete simple bud, then simple bud root is put down gently to the culture of rootage primary surface in tissue culture bottle downwards, light pressure submerged in medium its root, after having inoculated, cover tightly bottle cap, it is 27 ℃ that postvaccinal tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is to cultivate after 25 days in the illumination box of 2000Lx, seedling base portion grows 6 ~ 10 coring roots, obtain aseptic seedling.
The preparation method of described aseptic seedling chooses the ryegrass seed of complete maturation, put into running water and soak 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, the alcohol that is first 75% with percent by volume mark is by ryegrass seed sterilization 30 ~ 60s, use again aseptic water washing 3 times, put into afterwards mass percent concentration and be mercuric chloride soaking disinfection 6 ~ 8min of 0.1%, with after aseptic water washing 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtaining is put into sterile petri dish, under aseptic condition, put down gently in aseptic minimal medium surface, then gently press, seed is submerged in medium, after having inoculated, cover tightly tissue culture bottle lid, it is 27 ℃ that tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is in the illumination box of 2000Lx, cultivate after 35 days, obtain the highly aseptic seedling for 8cm.
The method of described transplanting is that vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, standby;
From medium take out preparation the aseptic seedling that has root system, with clear water, wash the medium of root remnants off, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 27 ℃ in temperature, and light application time is 16h/d, intensity of illumination is to cultivate after 7 days in the illumination box of 2000Lx, be placed in again temperature and be the glass greenhouse of 27 ℃, under natural lighting, cultivate after 7 days, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4 7H 2o and CaCl 2
Figure 427324DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 574272DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 602270DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 846170DEST_PATH_IMAGE002
eDTA
Figure 301422DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 512829DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 78940DEST_PATH_IMAGE002
eDTA
Figure 115029DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 803499DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 925039DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 294841DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 264065DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 61119DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 669955DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 640185DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4 7H 2o 8.6 mg/L, Na 2moO 4
Figure 945056DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 41188DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 752792DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add afterwards 15g sucrose and 3.2g agar powder, continue heating and constantly stir, the shape until be translucent, the minimal medium solution that obtains not sterilizing, standby,
Step 4: the not minimal medium solution of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid minimal medium of transparence.
The compound method of the described medium of sprouting, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 945876DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 84733DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4 7H 2o 370 mg/L, CaCl 2
Figure 730926DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4
Figure 893420DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 946827DEST_PATH_IMAGE002
eDTA
Figure 786607DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 578851DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 136871DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 728390DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 750572DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 402134DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 994920DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 390129DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 204502DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4 2H 2o 0.25 mg/L, CuSO 4
Figure 356314DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 555214DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, the shape until be translucent, then add the 6-BA of 0.9mg and the NAA of 0.09mg, and stir evenly, the culture medium solution of sprouting that obtains not sterilizing, standby.
Step 4: the not culture medium solution of sprouting of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid medium of sprouting.
The collocation method of described root media, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4 7H 2o and CaCl 2
Figure 531971DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 286300DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 351208DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 874594DEST_PATH_IMAGE002
eDTA
Figure 38859DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 93534DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 634236DEST_PATH_IMAGE002
eDTA
Figure 74445DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4 7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 138533DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 794511DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 26909DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 798556DEST_PATH_IMAGE002
5H 2o, CoCl 2 6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 694017DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 46501DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 802098DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 240033DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 926229DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add again 15g sucrose and 3.25g agar powder, continue heating and constantly stir, the shape until be translucent, then add the IBA of 0.7mg and the NAA of 0.09mg, and stir, the culture of rootage based sols that obtains not sterilizing, standby,
Step 4: the not culture of rootage based sols of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain sterile solid root media.
Result shows, adopt this method to get final product seedling with 75 days, and survival rate is 92%.

Claims (6)

1. an Annual Ryegrass tissue culture and rapid propagation method, comprises the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation that has the aseptic seedling of root system and transplanting, it is characterized in that:
The described preparation method without the aseptic Multiple Buds of tillering of root system is: in superclean bench, choose healthy and strong aseptic seedling, and be placed in sterile petri dish, under aseptic condition, first cut off the root system of aseptic seedling, the then upwards position of the 1cm cutting in rhizome junction, obtains stem foot, the stem foot obtaining is put in to the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprout in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in to temperature is 23～27 ℃ again, light application time is 16h/d, light intensity is to cultivate after 20 ~ 35 days in the illumination box of 1500 ~ 2000Lx, single stem foot sprouts high 6 ~ 10cm, quantity is the Multiple Buds of tillering of 8 ~ 20, the Multiple Buds of must tillering, then therefrom chooses quantity and is the Multiple Buds of tillering of 18 ~ 48 as without the aseptic Multiple Buds of tillering of root system;
The described preparation method who has the aseptic seedling of root system is: in superclean bench, taking-up make without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the medical surgical knife of sterilizing, excise the unnecessary blade on Multiple Buds top, leave the Multiple Buds of long 2 ~ 3cm, then at tillering node place, longitudinally cut, be divided into complete simple bud, then simple bud root is put down gently to the culture of rootage primary surface in tissue culture bottle downwards, light pressure submerged in medium its root, after having inoculated, cover tightly bottle cap, it is 23～27 ℃ that postvaccinal tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is to cultivate after 15 ~ 25 days in the illumination box of 1500 ~ 2000Lx, seedling base portion grows 6 ~ 10 coring roots, obtain aseptic seedling.
2. a kind of Annual Ryegrass tissue culture and rapid propagation method according to claim 1, it is characterized in that: the preparation method of described aseptic seedling chooses the ryegrass seed of complete maturation, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, the alcohol that is first 75% with percent by volume mark is by ryegrass seed sterilization 30 ~ 60s, use again aseptic water washing 2 ~ 3 times, put into afterwards mass percent concentration and be mercuric chloride soaking disinfection 6 ~ 8min of 0.1%, with after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtaining is put into sterile petri dish, under aseptic condition, put down gently in aseptic minimal medium surface, then gently press, seed is submerged in medium, after having inoculated, cover tightly tissue culture bottle lid, it is 23～27 ℃ that tissue culture bottle is placed in to temperature, light application time is 16h/d, intensity of illumination is in the illumination box of 1000 ~ 2000Lx, cultivate after 20 ~ 35 days, obtaining is highly the aseptic seedling of 5 ~ 8cm.
3. a kind of Annual Ryegrass tissue culture and rapid propagation method according to claim 1, is characterized in that: the method for described transplanting is that vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, standby;
From medium take out preparation the aseptic seedling that has root system, with clear water, wash the medium of root remnants off, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23～27 ℃ in temperature, and light application time is 16h/d, intensity of illumination is to cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in again temperature and be the glass greenhouse of 23～27 ℃, under natural lighting, cultivate after 6～7 days, get final product seedling.
4. a kind of Annual Ryegrass tissue culture and rapid propagation method according to claim 1, is characterized in that, the compound method of described aseptic minimal medium, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 2014100055470100001DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 447654DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4
Figure 330160DEST_PATH_IMAGE002
7H 2o 370 mg/L, CaCl 2
Figure 682644DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 562875DEST_PATH_IMAGE002
eDTA
Figure 809DEST_PATH_IMAGE002
2H 2o and FeSO 4
Figure 421427DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2 eDTA
Figure 434875DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4 7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 522097DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 583594DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 805627DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 218154DEST_PATH_IMAGE002
5H 2o, CoCl 2 6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 365419DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 555092DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 891133DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 457243DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 758912DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add afterwards 15g sucrose and 3.2g agar powder, continue heating and constantly stir, the shape until be translucent, the minimal medium solution that obtains not sterilizing, standby,
Step 4: the not minimal medium solution of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid minimal medium of transparence.
5. a kind of Annual Ryegrass tissue culture and rapid propagation method according to claim 1, is characterized in that, described in the sprout compound method of medium, comprise the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4
Figure 57169DEST_PATH_IMAGE002
7H 2o and CaCl 2
Figure 444288DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4 7H 2o 370 mg/L, CaCl 2 2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 439423DEST_PATH_IMAGE002
eDTA 2H 2o and FeSO 4
Figure 159434DEST_PATH_IMAGE002
7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 170116DEST_PATH_IMAGE002
eDTA 2H 2o 37.3 mg/L, FeSO 4
Figure 673352DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 322639DEST_PATH_IMAGE002
4H 2o, ZnSO 4
Figure 187827DEST_PATH_IMAGE002
7H2O, Na 2moO 4
Figure 326684DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 847795DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 97511DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 82784DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4
Figure 330226DEST_PATH_IMAGE002
7H 2o 8.6 mg/L, Na 2moO 4
Figure 400950DEST_PATH_IMAGE002
2H 2o 0.25 mg/L, CuSO 4
Figure 454357DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 730355DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, the shape until be translucent, then add the 6-BA of 0.9mg and the NAA of 0.09mg, and stir evenly, the culture medium solution of sprouting that obtains not sterilizing, standby,
Step 4: the not culture medium solution of sprouting of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain the sterile solid medium of sprouting.
6. a kind of Annual Ryegrass tissue culture and rapid propagation method according to claim 1, is characterized in that, the collocation method of described root media, comprises the following steps:
Step 1: the configuration of mother liquor:
Mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 4 7H 2o and CaCl 2
Figure 441139DEST_PATH_IMAGE002
2H 2o composition, wherein the mass concentration of each component is KNO 31900 mg/L, NH 4nO 31650 mg/L, KH 2pO 4170 mg/L, MgSO 4 7H 2o 370 mg/L, CaCl 2
Figure 992523DEST_PATH_IMAGE002
2H 2o 440 mg/L;
Mother liquor B is by Na 2
Figure 581768DEST_PATH_IMAGE002
eDTA 2H 2o and FeSO 4 7H 2o composition, wherein the mass concentration of each component is Na 2
Figure 22293DEST_PATH_IMAGE002
eDTA
Figure 508769DEST_PATH_IMAGE002
2H 2o 37.3 mg/L, FeSO 4
Figure 596811DEST_PATH_IMAGE002
7H 2o 27.8 mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4
Figure 302992DEST_PATH_IMAGE002
4H 2o, ZnSO 4 7H2O, Na 2moO 4
Figure 905192DEST_PATH_IMAGE002
2H 2o, CuSO 4
Figure 101818DEST_PATH_IMAGE002
5H 2o, CoCl 2
Figure 856148DEST_PATH_IMAGE002
6H 2o composition, wherein the mass concentration of each component is respectively KI 0.83mg/L, H 3bO 36.2 mg/L, MnSO 4
Figure 593159DEST_PATH_IMAGE002
4H 2o 22.3 mg/L, ZnSO 4 7H 2o 8.6 mg/L, Na 2moO 4 2H 2o 0.25 mg/L, CuSO 4
Figure 397801DEST_PATH_IMAGE002
5H 2o 0.025mg/L, CoCl 2
Figure 938504DEST_PATH_IMAGE002
6H 2o 0.025 mg/L;
Mother liquor D is comprised of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2 mg/L, thiamine hydrochloride VB1 0.1 mg/L, puridoxine hydrochloride VB6 0.5 mg/L, nicotinic acid 0.5 mg/L;
Mother liquor E is comprised of inositol, and the mass concentration of inositol is 100 mg/L;
Step 2: the each reagent quality concentration of mother liquor A in step 1 is expanded to 20 times, and constant volume is to 1L; The each reagent quality concentration of mother liquor B expands 100 times, and adjust pH is 5.5, is settled to 1L, is stored in Brown Glass Brown glass bottles and jars only; Each reagent quality concentration in mother solution C expands 100 times, and is settled to 0.5L; Each reagent quality concentration in mother liquor D expands 200 times, and is settled to 1L; Each reagent quality concentration in mother liquor E expands 50 times, and is settled to 0.5L, standby;
Step 3: get the each reagent 25ml respectively in the mother liquor A that step 2 makes, each reagent in the mother liquor B that step 2 makes 5ml respectively, each reagent in the mother solution C that step 2 makes 2.5ml respectively, each reagent in the mother liquor D that step 2 makes 2.5ml respectively, each reagent in the mother liquor E that step 2 makes 5ml respectively, after being mixed, the above composition taking is settled to 0.5L with distilled water, and put into beaker heating and boil, add again 15g sucrose and 3.1 ~ 3.25g agar powder, continue heating and constantly stir, the shape until be translucent, then add the IBA of 0.7mg and the NAA of 0.09mg, and stir, the culture of rootage based sols that obtains not sterilizing, standby,
Step 4: the not culture of rootage based sols of sterilization that step 3 is made is loaded in blake bottle while hot, covers tightly bottle cap and is placed in high-pressure sterilizing pot and carries out autoclaving, takes out standingly and cooling after 15min, obtain sterile solid root media.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105684895A (en) * 2014-11-28 2016-06-22 上海市农业科学院 A method for constructing a haploid population of a cereal crop
CN107926675A (en) * 2017-12-13 2018-04-20 佛山推启农业研究院（普通合伙） A kind of implantation methods of selenium-rich water spinach

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1597933A (en) * 2004-08-13 2005-03-23 山东大学 Process for construvting transgene teceptor system of rye grass and its application

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1597933A (en) * 2004-08-13 2005-03-23 山东大学 Process for construvting transgene teceptor system of rye grass and its application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
刘珍等: "多年生黑麦草高频丛生芽增殖培养体系的研究", 《草地学报》 *
郑若男等: "原种黑麦草组培再生体系建立初探", 《安徽农业科学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105684895A (en) * 2014-11-28 2016-06-22 上海市农业科学院 A method for constructing a haploid population of a cereal crop
CN107926675A (en) * 2017-12-13 2018-04-20 佛山推启农业研究院（普通合伙） A kind of implantation methods of selenium-rich water spinach

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