CN103749298B - A kind of Annual Ryegrass tissue culture and rapid propagation method - Google Patents

A kind of Annual Ryegrass tissue culture and rapid propagation method Download PDF

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CN103749298B
CN103749298B CN201410005547.0A CN201410005547A CN103749298B CN 103749298 B CN103749298 B CN 103749298B CN 201410005547 A CN201410005547 A CN 201410005547A CN 103749298 B CN103749298 B CN 103749298B
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mother liquor
reagent
aseptic
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CN103749298A (en
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王晓凌
侯小改
施江
李晓倩
戴攀峰
张有福
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 phytobiology field, be specifically related to a kind of Annual Ryegrass tissue culture and rapid propagation method.Comprise the preparation of aseptic seedling, the preparation without the aseptic Multiple Buds of tillering of root system, the preparation having the aseptic seedling of root system and transplanting, instrument required for the present invention is simple, with low cost, improves economic benefit.Seed sprouting cultivation constructed by the present invention, adventitious shoots culture of tillering, culture of rootage and become transplantation of seedlings perennial ryegrass plantlet in vitro rapid propagation system, can between 2 months to 3 months seedling, and improve survival rate, be applicable to the production of scale.The present invention cultivates by adopting stem foot, substantially increases the survival rate of plantlet in vitro, shortens cultivation cycle.And the present invention carries out cultivating by adopting stem foot the tissue cultures scope of selecting material greatly extending Annual Ryegrass, facilitates the development of Annual Ryegrass tissue cultures, makes it widely for large-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, and in recent years, become herbage and turfgrass kind that China widelys popularize, the effect in China's animal husbandry development and town and country beautify is increasing.Perennial ryegrass tillers many, and output is high, and quality is soft, 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, improve soil structure, prevent erosion, and is good turfgrass.For adapting to the needs of perennial ryegrass breeding work, preserve and Fast-propagation high-quality starting material, and providing reliable research material for the physiological ecological research about perennial ryegrass gene expression in good time, the cultured in vitro of perennial ryegrass and quick propagating technology are subject to increasing attention.At present about the tissue cultures of perennial ryegrass is only limitted to Seed inducement callus and plant regeneration, and the formation of sugarcane explants through callus induction or Multiple Buds is done with protoplast, young fringe, stem apex etc., 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, provide a kind of Annual Ryegrass tissue culture and rapid propagation method, its cultural method is simple and easy to do, and cultivation cycle is short.
The present invention for the adopted technical scheme that solves the problem 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 having 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, aseptically first cut off the root system of aseptic seedling, then in rhizome junction, upwards the position of 1cm is cut, and obtains stem foot, the stem foot obtained is put in the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprouts in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in temperature is 23 ~ 27 DEG C again, light application time is 16h/d, light intensity is 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, must tiller Multiple Buds, then therefrom choosing quantity is that the Multiple Buds of tillering of 18 ~ 48 is as without the aseptic Multiple Buds of tillering of root system;
Described have the preparation method of the aseptic seedling of root system to be: in superclean bench, take out obtained without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the unnecessary blade on the medical surgical knife excision Multiple Buds top of sterilizing, leave the Multiple Buds of long 2 ~ 3cm, then the longitudinally cutting at tillering node place, be divided into complete simple bud, then simple bud root is put down gently culture of rootage primary surface in tissue culture bottle downwards, light pressure makes its root submerge in medium, bottle cap is covered tightly after having inoculated, postvaccinal tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, light application time is 16h/d, intensity of illumination is 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 mature, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, be first that the alcohol of 75% is by ryegrass seed sterilization 30 ~ 60s with percent by volume mark, use aseptic water washing again 2 ~ 3 times, put into mercuric chloride soaking disinfection 6 ~ 8min that mass percent concentration is 0.1% afterwards, after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtained is put into sterile petri dish, aseptically put down gently in aseptic minimal medium surface, then gently press, seed is made to submerge in medium, tissue culture bottle lid is covered tightly after having inoculated, tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, 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 vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, for subsequent use;
From medium take out preparation the aseptic seedling having root system, the medium of root remnants is washed off with clear water, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23 ~ 27 DEG C in temperature, and light application time is 16h/d, intensity of illumination is cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in the glass greenhouse that temperature is 23 ~ 27 DEG C again, cultivate after 6 ~ 7 days under natural lighting, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.2g agar powder afterwards, continue heating and constantly stir, the shape until be translucent, the minimal medium solution of not sterilized, for subsequent use,
Step 4: the minimal medium solution of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain the sterile solid minimal medium of transparence after 15min.
The compound method of described medium of sprouting, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, the shape until be translucent, then the NAA of 6-BA and 0.09mg of 0.9mg is added, and stir evenly, the culture medium solution of sprouting of not sterilized, for subsequent use.
Step 4: the culture medium solution of sprouting of not sterilizing step 3 obtained 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 and leave standstill and cool after 15min, obtains sterile solid and to sprout medium.
The collocation method of described root media, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.1 ~ 3.25g agar powder again, continue heating and constantly stir, the shape until be translucent, then the NAA of IBA and 0.09mg of 0.7mg is added, and stir, the culture of rootage based sols of not sterilized, for subsequent use,
Step 4: the culture of rootage based sols of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain sterile solid root media after 15min.
beneficial effect
1, instrument required for the present invention is simple, with low cost, improves economic benefit.
2, the seed sprouting cultivation constructed by the present invention, adventitious shoots culture of tillering, culture of rootage and become transplantation of seedlings perennial ryegrass plantlet in vitro rapid propagation system, can between 2 months to 3 months seedling, and improve survival rate, be applicable to the production of scale.
3, the present invention cultivates by adopting stem foot, substantially increases the survival rate of plantlet in vitro, shortens cultivation cycle.And the present invention carries out cultivating by adopting stem foot the tissue cultures scope of selecting material greatly extending Annual Ryegrass, facilitates the development of Annual Ryegrass tissue cultures, 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 having 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, aseptically first cut off the root system of aseptic seedling, then in rhizome junction, upwards the position of 1cm is cut, and obtains stem foot, the stem foot obtained is put in the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprouts in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in temperature is 23 ~ 27 DEG C again, light application time is 16h/d, light intensity is 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, must tiller Multiple Buds, then therefrom choosing quantity is that the Multiple Buds of tillering of 18 ~ 48 is as without the aseptic Multiple Buds of tillering of root system;
Described have the preparation method of the aseptic seedling of root system to be: in superclean bench, take out obtained without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the unnecessary blade on the medical surgical knife excision Multiple Buds top of sterilizing, leave the Multiple Buds of long 2 ~ 3cm, then the longitudinally cutting at tillering node place, be divided into complete simple bud, then simple bud root is put down gently culture of rootage primary surface in tissue culture bottle downwards, light pressure makes its root submerge in medium, bottle cap is covered tightly after having inoculated, postvaccinal tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, light application time is 16h/d, intensity of illumination is 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 mature, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, be first that the alcohol of 75% is by ryegrass seed sterilization 30 ~ 60s with percent by volume mark, use aseptic water washing again 2 ~ 3 times, put into mercuric chloride soaking disinfection 6 ~ 8min that mass percent concentration is 0.1% afterwards, after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtained is put into sterile petri dish, aseptically put down gently in aseptic minimal medium surface, then gently press, seed is made to submerge in medium, tissue culture bottle lid is covered tightly after having inoculated, tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, 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 vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, for subsequent use;
From medium take out preparation the aseptic seedling having root system, the medium of root remnants is washed off with clear water, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23 ~ 27 DEG C in temperature, and light application time is 16h/d, intensity of illumination is cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in the glass greenhouse that temperature is 23 ~ 27 DEG C again, cultivate after 6 ~ 7 days under natural lighting, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.2g agar powder afterwards, continue heating and constantly stir, the shape until be translucent, the minimal medium solution of not sterilized, for subsequent use,
Step 4: the minimal medium solution of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain the sterile solid minimal medium of transparence after 15min.
The compound method of described medium of sprouting, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, the shape until be translucent, then the NAA of 6-BA and 0.09mg of 0.9mg is added, and stir evenly, the culture medium solution of sprouting of not sterilized, for subsequent use.
Step 4: the culture medium solution of sprouting of not sterilizing step 3 obtained 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 and leave standstill and cool after 15min, obtains sterile solid and to sprout medium.
The collocation method of described root media, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.1 ~ 3.25g agar powder again, continue heating and constantly stir, the shape until be translucent, then the NAA of IBA and 0.09mg of 0.7mg is added, and stir, the culture of rootage based sols of not sterilized, for subsequent use,
Step 4: the culture of rootage based sols of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain sterile solid root media after 15min.
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 having 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, aseptically first cut off the root system of aseptic seedling, then in rhizome junction, upwards the position of 1cm is cut, and obtains stem foot, the stem foot obtained is put in the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprouts in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in temperature is 23 DEG C again, light application time is 16h/d, light intensity is 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, must tiller Multiple Buds, then therefrom choosing quantity is that the Multiple Buds of tillering of 18 ~ 48 is as without the aseptic Multiple Buds of tillering of root system;
Described have the preparation method of the aseptic seedling of root system to be: in superclean bench, take out obtained without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the unnecessary blade on the medical surgical knife excision Multiple Buds top of sterilizing, leave the Multiple Buds of long 2cm, then the longitudinally cutting at tillering node place, be divided into complete simple bud, then simple bud root is put down gently culture of rootage primary surface in tissue culture bottle downwards, light pressure makes its root submerge in medium, bottle cap is covered tightly after having inoculated, postvaccinal tissue culture bottle being placed in temperature is 23 DEG C, light application time is 16h/d, intensity of illumination is 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 mature, put into running water and soak 0.5h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, with percent by volume mark be first 75% alcohol ryegrass seed to be sterilized 30s, use aseptic water washing again 2 times, put into the mercuric chloride soaking disinfection 6min that mass percent concentration is 0.1% afterwards, after aseptic water washing 4 times, obtain aseptic seed, in superclean bench, the aseptic seed obtained is put into sterile petri dish, aseptically put down gently in aseptic minimal medium surface, then gently press, seed is made to submerge in medium, tissue culture bottle lid is covered tightly after having inoculated, tissue culture bottle being placed in temperature is 23 DEG C, light application time is 16h/d, intensity of illumination is in the illumination box of 1000Lx, cultivate after 20 days, obtain being highly the aseptic seedling of 5cm.
The method of described transplanting is vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, for subsequent use;
From medium take out preparation the aseptic seedling having root system, the medium of root remnants is washed off with clear water, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23 DEG C in temperature, and light application time is 16h/d, intensity of illumination is cultivate after 7 days in the illumination box of 1500Lx, be placed in the glass greenhouse that temperature is 23 DEG C again, cultivate after 6 days under natural lighting, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.2g agar powder afterwards, continue heating and constantly stir, the shape until be translucent, the minimal medium solution of not sterilized, for subsequent use,
Step 4: the minimal medium solution of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain the sterile solid minimal medium of transparence after 15min.
The compound method of described medium of sprouting, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 47H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, the shape until be translucent, then the NAA of 6-BA and 0.09mg of 0.9mg is added, and stir evenly, the culture medium solution of sprouting of not sterilized, for subsequent use.
Step 4: the culture medium solution of sprouting of not sterilizing step 3 obtained 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 and leave standstill and cool after 15min, obtains sterile solid and to sprout medium.
The collocation method of described root media, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.1 ~ 3.25g agar powder again, continue heating and constantly stir, the shape until be translucent, then the NAA of IBA and 0.09mg of 0.7mg is added, and stir, the culture of rootage based sols of not sterilized, for subsequent use,
Step 4: the culture of rootage based sols of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain sterile solid root media after 15min.
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 having 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, aseptically first cut off the root system of aseptic seedling, then in rhizome junction, upwards the position of 1cm is cut, and obtains stem foot, the stem foot obtained is put in the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprouts in medium, then cover tightly tissue culture bottle lid; Tissue culture bottle being placed in temperature is 27 DEG C again, light application time is 16h/d, light intensity is 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, must tiller Multiple Buds, then therefrom choosing quantity is that the Multiple Buds of tillering of 48 is as without the aseptic Multiple Buds of tillering of root system;
Described have the preparation method of the aseptic seedling of root system to be: in superclean bench, take out obtained without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the unnecessary blade on the medical surgical knife excision Multiple Buds top of sterilizing, leave the Multiple Buds of long 3cm, then the longitudinally cutting at tillering node place, be divided into complete simple bud, then simple bud root is put down gently culture of rootage primary surface in tissue culture bottle downwards, light pressure makes its root submerge in medium, bottle cap is covered tightly after having inoculated, postvaccinal tissue culture bottle being placed in temperature is 27 DEG C, light application time is 16h/d, intensity of illumination is 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 mature, put into running water and soak 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, be first that the alcohol of 75% is by ryegrass seed sterilization 30 ~ 60s with percent by volume mark, use aseptic water washing again 3 times, put into mercuric chloride soaking disinfection 6 ~ 8min that mass percent concentration is 0.1% afterwards, after aseptic water washing 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtained is put into sterile petri dish, aseptically put down gently in aseptic minimal medium surface, then gently press, seed is made to submerge in medium, tissue culture bottle lid is covered tightly after having inoculated, tissue culture bottle being placed in temperature is 27 DEG C, light application time is 16h/d, intensity of illumination is in the illumination box of 2000Lx, cultivate after 35 days, obtain being highly the aseptic seedling of 8cm.
The method of described transplanting is vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, for subsequent use;
From medium take out preparation the aseptic seedling having root system, the medium of root remnants is washed off with clear water, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 27 DEG C in temperature, and light application time is 16h/d, intensity of illumination is cultivate after 7 days in the illumination box of 2000Lx, be placed in the glass greenhouse that temperature is 27 DEG C again, cultivate after 7 days under natural lighting, get final product seedling.
The compound method of described aseptic minimal medium, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.2g agar powder afterwards, continue heating and constantly stir, the shape until be translucent, the minimal medium solution of not sterilized, for subsequent use,
Step 4: the minimal medium solution of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain the sterile solid minimal medium of transparence after 15min.
The compound method of described medium of sprouting, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, the shape until be translucent, then the NAA of 6-BA and 0.09mg of 0.9mg is added, and stir evenly, the culture medium solution of sprouting of not sterilized, for subsequent use.
Step 4: the culture medium solution of sprouting of not sterilizing step 3 obtained 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 and leave standstill and cool after 15min, obtains sterile solid and to sprout medium.
The collocation method of described root media, comprises the following steps:
Step one: 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 2H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 4 7H 2o370mg/L, CaCl 2 2H 2o440mg/L;
Mother liquor B is by Na 2 eDTA 2H 2o and FeSO 4 7H 2o forms, and wherein the mass concentration of each component is Na 2 eDTA 2H 2o37.3mg/L, FeSO 4 7H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 4 4H 2o, ZnSO 4 7H2O, Na 2moO 4 2H 2o, CuSO 4 5H 2o, CoCl 2 6H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 4 4H 2o22.3mg/L, ZnSO 4 7H 2o8.6mg/L, Na 2moO 4 2H 2o0.25mg/L, CuSO 4 5H 2o0.025mg/L, CoCl 2 6H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.25g agar powder again, continue heating and constantly stir, the shape until be translucent, then the NAA of IBA and 0.09mg of 0.7mg is added, and stir, the culture of rootage based sols of not sterilized, for subsequent use,
Step 4: the culture of rootage based sols of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain sterile solid root media after 15min.
Result shows, adopt this method to get final product seedling with 75 days, and survival rate is 92%.

Claims (1)

1. an Annual Ryegrass tissue culture and rapid propagation method, comprise the preparation of aseptic seedling, without the preparation of the aseptic Multiple Buds of tillering of root system, there are preparation and the transplanting of the aseptic seedling of root system, 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, aseptically first cut off the root system of aseptic seedling, then in rhizome junction, upwards the position of 1cm is cut, obtain stem foot, the stem foot obtained is put in the media surface of sprouting in tissue culture bottle, then gently press, root is submerged and sprouts in medium, then tissue culture bottle lid is covered tightly, tissue culture bottle being placed in temperature is 23 ~ 27 DEG C again, light application time is 16h/d, light intensity is 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, and then therefrom choosing quantity is that the Multiple Buds of tillering of 18 ~ 48 is as without the aseptic Multiple Buds of tillering of root system, described have the preparation method of the aseptic seedling of root system to be: in superclean bench, take out obtained without the aseptic Multiple Buds of tillering of root system, and be placed in sterile petri dish, with the unnecessary blade on the medical surgical knife excision Multiple Buds top of sterilizing, leave the Multiple Buds of long 2 ~ 3cm, then the longitudinally cutting at tillering node place, be divided into complete simple bud, then simple bud root is put down gently culture of rootage primary surface in tissue culture bottle downwards, light pressure makes its root submerge in medium, bottle cap is covered tightly after having inoculated, postvaccinal tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, light application time is 16h/d, intensity of illumination is cultivate after 15 ~ 25 days in the illumination box of 1500 ~ 2000Lx, seedling base portion grows 6 ~ 10 coring roots, the aseptic seedling of root system must be had,
The preparation method of described aseptic seedling chooses the ryegrass seed of complete mature, put into running water and soak 0.5 ~ 1h, then in superclean bench, ryegrass seed is transferred in aseptic bottle, be first that the alcohol of 75% is by ryegrass seed sterilization 30 ~ 60s with percent by volume mark, use aseptic water washing again 2 ~ 3 times, put into mercuric chloride soaking disinfection 6 ~ 8min that mass percent concentration is 0.1% afterwards, after aseptic water washing 4 ~ 6 times, obtain aseptic seed, in superclean bench, the aseptic seed obtained is put into sterile petri dish, aseptically put down gently in aseptic minimal medium surface, then gently press, seed is made to submerge in medium, tissue culture bottle lid is covered tightly after having inoculated, tissue culture bottle being placed in temperature is 23 ~ 27 DEG C, light application time is 16h/d, intensity of illumination is in the illumination box of 1000 ~ 2000Lx, cultivate after 20 ~ 35 days, obtain the aseptic seedling of height 5 ~ 8cm,
The method of described transplanting is vermiculite and turfy soil are mixed by the weight ratio of 3:1, obtains mixed-matrix, after stirring, for subsequent use; The aseptic seedling having root system of preparation is taken out from medium, the medium of root remnants is washed off with clear water, and be placed in mixed-matrix, root system is submerged in mixed-matrix completely, is 23 ~ 27 DEG C in temperature, and light application time is 16h/d, intensity of illumination is cultivate after 7 days in the illumination box of 1500 ~ 2000Lx, be placed in the glass greenhouse that temperature is 23 ~ 27 DEG C again, cultivate after 6 ~ 7 days under natural lighting, get final product seedling;
The compound method of described aseptic minimal medium, comprises the following steps:
Step one: the configuration of mother liquor: mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 47H 2o and CaCl 22H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 47H 2o370mg/L, CaCl 22H 2o440mg/L; Mother liquor B is by Na 2eDTA2H 2o and FeSO 47H 2o forms, and wherein the mass concentration of each component is Na 2eDTA2H 2o37.3mg/L, FeSO 47H 2o27.8mg/L; Mother solution C is by KI, H 3bO 3, MnSO 44H 2o, ZnSO 47H 2o, Na 2moO 42H 2o, CuSO 45H 2o, CoCl 26H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 44H 2o22.3mg/L, ZnSO 47H 2o8.6mg/L, Na 2moO 42H 2o0.25mg/L, CuSO 45H 2o0.025mg/L, CoCl 26H 2o0.025mg/L; Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L; Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.2g agar powder afterwards, continue heating and constantly stir, the shape until be translucent, the minimal medium solution of not sterilized, for subsequent use,
Step 4: the minimal medium solution of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain the sterile solid minimal medium of transparence after 15min;
The compound method of described medium of sprouting, comprises the following steps:
Step one: the configuration of mother liquor: mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 47H 2o and CaCl 22H 2o forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 47H 2o370mg/L, CaCl 22H 2o440mg/L;
Mother liquor B is by Na 2eDTA2H 2o and FeSO 47H 2o forms, and wherein the mass concentration of each component is Na 2eDTA2H 2o37.3mg/L, FeSO 47H 2o27.8mg/L;
Mother solution C is by KI, H 3bO 3, MnSO 44H 2o, ZnSO 47H 2o, Na 2moO 42H 2o, CuSO 45H2O, CoCl 26H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 44H 2o22.3mg/L, ZnSO 47H 2o8.6mg/L, Na 2moO 42H 2o0.25mg/L, CuSO 45H 2o0.025mg/L, CoCl 26H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, the shape until be translucent, then the NAA of 6-BA and 0.09mg of 0.9mg is added, and stir evenly, the culture medium solution of sprouting of not sterilized, for subsequent use,
Step 4: the culture medium solution of sprouting of not sterilizing step 3 obtained 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 and leave standstill and cool after 15min, obtains sterile solid and to sprout medium;
The collocation method of described root media, comprises the following steps:
Step one: the configuration of mother liquor: mother liquor A is by KNO 3, NH 4nO 3, KH 2pO 4, MgSO 47H 2o and CaCl 22H2O forms, and wherein the mass concentration of each component is KNO 31900mg/L, NH 4nO 31650mg/L, KH 2pO 4170mg/L, MgSO 47H 2o370mg/L, CaCl 22H 2o440mg/L;
Mother liquor B is by Na 2eDTA2H 2o and FeSO 47H 2o forms, and wherein the mass concentration of each component is Na 2eDTA2H 2o37.3mg/L, FeSO 47H 2o27.8mg/L; Mother solution C is by KI, H 3bO 3, MnSO 44H 2o, ZnSO 47H 2o, Na 2moO 42H 2o, CuSO 45H 2o, CoCl 26H 2o forms, and wherein the mass concentration of each component is respectively KI0.83mg/L, H 3bO 36.2mg/L, MnSO 44H 2o22.3mg/L, ZnSO 47H 2o8.6mg/L, Na 2moO 42H 2o0.25mg/L, CuSO 45H 2o0.025mg/L, CoCl 26H 2o0.025mg/L;
Mother liquor D is made up of glycine, thiamine hydrochloride VB1, puridoxine hydrochloride VB6 and nicotinic acid, and wherein the mass concentration of each component is respectively glycine 2mg/L, thiamine hydrochloride VB10.1mg/L, puridoxine hydrochloride VB60.5mg/L, nicotinic acid 0.5mg/L;
Mother liquor E is made up of inositol, and the mass concentration of inositol is 100mg/L;
Step 2: each reagent quality concentration of mother liquor A in step one is expanded 20 times, and constant volume is to 1L; 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, for subsequent use;
Step 3: get each reagent 25ml respectively in the mother liquor A that step 2 obtains, each reagent in the mother liquor B that step 2 obtains 5ml respectively, each reagent in the mother solution C that step 2 obtains 2.5ml respectively, each reagent in the mother liquor D that step 2 obtains 2.5ml respectively, each reagent in the mother liquor E that step 2 obtains 5ml respectively, 0.5L is settled to distilled water by after the above composition mixing taken, and put into beaker heating boil, add 15g sucrose and 3.1 ~ 3.25g agar powder again, continue heating and constantly stir, the shape until be translucent, then the NAA of IBA and 0.09mg of 0.7mg is added, and stir, the culture of rootage based sols of not sterilized, for subsequent use,
Step 4: the culture of rootage based sols of not sterilizing step 3 obtained 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 and leaves standstill and cool, obtain sterile solid root media after 15min.
CN201410005547.0A 2014-01-07 2014-01-07 A kind of Annual Ryegrass tissue culture and rapid propagation method Expired - Fee Related CN103749298B (en)

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