CN103975851A - Chrysanthemum morifolium tissue culturing and breeding method - Google Patents
Chrysanthemum morifolium tissue culturing and breeding method Download PDFInfo
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Abstract
The invention discloses a Chrysanthemum morifolium tissue culturing and breeding method. The method enables a large amount of high-quality Chrysanthemum morifolium seedlings to be obtained within a short period to be obtained in order to meet the needs of the market. The method comprises the steps of aseptic material obtaining, inductive culturing bud propagation and rooting, strong seedling culturing, and rooted seedling transplanting. Axillalry buds are used as an explants, and the tissue culturing method is used to rapidly breed, so the disadvantages of long period, low breeding coefficient, easy infection and propagation of viruses, and the like of routine cutting and division breeding of Chrysanthemum morifolium are overcome; and tissue culture seedlings produced by using the method have the advantages of few viruses, stable heredity and the like.
Description
(1) technical field
The present invention relates to plant tissue culture technique, particularly a kind of tissue of FLOS CHRYSANTHEMI ALBA from Haizhou of China is cultivated and fast seedling-breeding method, by this method, can carry out good seed large-scale production.
(2) background technology
FLOS CHRYSANTHEMI ALBA from Haizhou of China (Chrysanthemum morifolium Ramat) is the catananche of originating in a kind of medicine-food two-purpose in China Zhejiang, is one of Zhejiang Province's eight your name's medicinal materials " Zhejiang eight tastes ".FLOS CHRYSANTHEMI ALBA from Haizhou of China both can be made medicinal material, can be made into again beverage for drinking, and also can view and admire as garden.FLOS CHRYSANTHEMI ALBA from Haizhou of China contains volatile oil, flavonoids, Polyphenols, chrysanthemum glucoside, purine, choline, stachydrine, vitamin A, Cobastab and amino acid etc. in spending.The research of traditional Chinese and western medicine shows, FLOS CHRYSANTHEMI ALBA from Haizhou of China has loose wind heat-clearing, flat liver improving eyesight, the effect of detoxicating, relieving inflammation, the diuresis of refreshing oneself, can be used for treating jaundice with damp-heat pathogen, stomachache food less, the disease such as oedema oliguria, nervous centralis is also had to sedation.If take a shower with chrysanthemum soup, there is the function of itch refreshing body, cosmetology.Often drink FLOS CHRYSANTHEMI ALBA from Haizhou of China, can also strengthen capillary resistance, suppress the general character of capillary, play the effect that anti-inflammatory is kept fit.Research is in recent years found, also very high containing abundant nickel, zinc, ferro element, especially iron content in FLOS CHRYSANTHEMI ALBA from Haizhou of China.Iron is the important composition composition in cell, and in cell metabolism, the activity of many enzymes all needs iron or needs iron as co-factor, and ferro element plays an important role aspect the internal metabolism of zinc, calcium, magnesium and human immunity defense function coordinating.The extract of FLOS CHRYSANTHEMI ALBA from Haizhou of China all has inhibitory action to staphylococcus aureus, shigella dysenteriae, proteus, typhoid bacillus, comma bacillus, beta hemolysis streptococcus, Escherichia coli, Pseudomonas aeruginosa, influenza virus.
New research also finds that FLOS CHRYSANTHEMI ALBA from Haizhou of China has the effects such as treatment hypertension, antimigraine, acute conjunctivitis.Therefore, FLOS CHRYSANTHEMI ALBA from Haizhou of China, except as food and drink, health products, is also developed to novel antibacterial and drug for hypertension, is subject to paying close attention to more and more widely.
The breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China at present mainly adopts cottage propagation and division propagation, and repoductive time is long, and reproduction coefficient is low, cannot meet the production & marketing demand growing to even greater heights.Virus disease is one of important disease in FLOS CHRYSANTHEMI ALBA from Haizhou of China planting process; and these propagation methods of FLOS CHRYSANTHEMI ALBA from Haizhou of China cottage propagation and division propagation easily cause viral infection and propagation; and pass on from generation to generation; increase the weight of year by year; cause its kind serious degradation; the improved seeds resource of many FLOS CHRYSANTHEMI ALBA from Haizhou of China can not get protection and utilizes, and has directly affected output and the quality of its flower, and the economic benefit of FLOS CHRYSANTHEMI ALBA from Haizhou of China is under some influence.
The fast development of biotechnology, particularly plant tissue and cell culture technology, for the Fast-propagation breeding research of FLOS CHRYSANTHEMI ALBA from Haizhou of China provides important foundation.Therefore, finding a kind of FLOS CHRYSANTHEMI ALBA from Haizhou of China propagation method is rapidly and efficiently very important.
The research report of at present both at home and abroad FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue being cultivated is all to take blade, petiole, hypocotyl, stem section and petal to carry out tissue cultivation (Luo Zijuan, 1987 of FLOS CHRYSANTHEMI ALBA from Haizhou of China as explant; Zhang Yuan, 2008; Most intelligent, 2004; Niu Yanbing, 2007; Zhang Na, 2009), but these methods all can not effectively be sloughed the virus in FLOS CHRYSANTHEMI ALBA from Haizhou of China body, although most intelligent use stem apex is cultivated, but first produce a large amount of callus, then from Calli Differentiation, become indefinite bud, easily there is variation in the indefinite bud obtaining by this process, affected the original good genetic character of seedling in breeding.The present invention directly induces differentiation in the mode of the numerous bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud, then induces lateral bud redifferentiation indefinite bud to breed, and the indefinite bud obtaining by this approach can effectively be avoided the variation occurring in shoot proliferation process, keeps better the original merit of maternal plant.
(3) summary of the invention
The object of the invention is to provide a kind of tissue cultivation of FLOS CHRYSANTHEMI ALBA from Haizhou of China and the method for Vitro Quick Reproduction, makes to obtain in a short time a large amount of high-quality FLOS CHRYSANTHEMI ALBA from Haizhou of China seedlings, meets the needs in market.
The technical solution used in the present invention is:
FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue is cultivated and a propagation method, and described method is carried out as follows:
(1) acquisition of sterilizable material and induction are cultivated: cut grow fine, without the tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with running water, rinse 0.5～2h, in volumetric concentration, be then jolting 2～5mins in the aqueous solution of 1% liquid detergent, with sterile water, wash down; On superclean bench, with volumetric concentration 70～75% ethanol waters, soak 30～60s, then with mixing medicining liquid dipping 10～20mins, then use aseptic water washing 3～5 times; Suck dry moisture on the filter paper of sterilizing, with scalpel, strip the axillalry bud point of 0.3～0.6mm, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and seal bottleneck with sealed membrane, be placed in incubator, at 24～26 ℃, under illumination 1500～2500lx condition, cultivate 15～25 days; The polysorbas20 that described mixing thimerosal is volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is 6-benzyl aminoadenine for adding 6-BA() 1.0～3.0mg/L, NAA(be methyl α-naphthyl acetate) the MS minimal medium of 0.05～0.5mg/L and glutamine 80～120mg/L;
(2) propagation of bud and taking root: observe step (1) described 24～26 ℃, under illumination 1500～2500lx condition, cultivate after 5～15 days, there is green projection in cutting part, after 20～40 days, there is Multiple Buds, cultivate again 10～20 days, when Multiple Buds grows to 2～4cm, Multiple Buds is cut and is transferred in bud proliferated culture medium, at 24～26 ℃, under illumination 1500～2500lx condition, breed cultivation, obtain the seedling of growing thickly of a large amount of propagation, cultivate again 6～10 days, the seedling of growing thickly grows 2～5 root systems, one step completes bud propagation and the step of taking root, final rooting rate is 90～95%, form whole plant, described bud proliferated culture medium is for adding the MS minimal medium of 6-BA0.5～2.0mg/L, NAA0.05～0.2mg/L, glutamine 80～120mg/L and potato extract 80g/L～120g/L, described potato extract is that potato is cleaned, and peeling, takes 80～120 grams of potatos, is cut into 2 * 2cm
3fritter, put into 500ml deionized water, boil 15～25mins, be cooled to after 50～60 ℃, with double gauze, filter, filtrate is added in MS minimal medium, is and in MS minimal medium, adds 80g/L～120g/L potato extract, (potato extract of the present invention refers to after potato is peeled and in water, boils the filtrate after filtration, and the addition of filtrate is to boil the weighing scale of front peeling potato),
(3) strong seedling culture: by the seedling of growing thickly of taking root (growing the seedling of growing thickly of 2～5 root systems) access 1/2MS solid culture medium, at 24～26 ℃, under illumination 1500～2500lx condition, carry out strong seedling culture, acquisition blade is unfolded, leaf look dark green, root system is sturdy, the seedling of height of seedling 3～5cm;
(4) transplantation of seedlings of taking root: open bottle cap hardening after 2～4 days, agar on seedling plant and foreign material are cleaned, transplant to the seedling medium after sterilization, water permeablely, cover film, keep humidity more than 80%, put ventilating and cooling place, under 24～26 ℃, illumination 1500～2500lx condition, cultivate 8～12d, FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf look dark green, stem is normally stood upright, and realizes the breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China; Matrix is dry wet suitable, and too wet meeting causes rotten, and the too dry blade that there will be is withered, and the generation of regularly spraying the pre-preventing disease and pest of medicine; Described seedling medium is that fine sand, humus soil and rice hull ash form according to the proportional arrangement of mass ratio 5:2:2, the sterilization method of described seedling medium is: adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film after covering 3～5 days and is tanned by the sun matrix 1～3 day again, complete the sterilization to seedling medium, or adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film and at 60 ℃, is dried 2 days again after covering 3～5 days, complete the sterilization to seedling medium.
Further, the described inducing clumping bud medium optimization of step (1) is for adding the MS minimal medium of 6-BA1.5mg/L, NAA0.2mg/L and glutamine 100mg/L.
Further, the described bud proliferated culture medium of step (2) is preferably the MS minimal medium that adds 6-BA1.0mg/L, NAA0.1mg/L, glutamine 100mg/L and potato extract 100g/L.
Further, described MS minimal medium final concentration consists of: NH
4nO
31.65 grams per liters, KNO
31.9 grams per liters, CaCl
22H
2o0.44 grams per liter, MgSO
47H
2o0.37 grams per liter, KH
2pO
40.17 grams per liter, KI0.83 mg/litre, H
3bO
36.2 mg/litre, MnSO
44H
2o22.3 mg/litre, ZnSO
47H
2o8.6 mg/litre, Na
2moO
42H
2o0.25 mg/litre, CuSO
45H
2o0.025 mg/litre, CoCl
26H
2o0.025 mg/litre, Na
2eDTA37.25 mg/litre, FeSO
47H
2o27.85 mg/litre, inositol 100 mg/litre, glycine 2 mg/litre, thiamine hydrochloride 0.4 mg/litre, puridoxine hydrochloride 0.5 mg/litre, nicotinic acid 0.5 mg/litre, sucrose 20～40 grams per liters, agar powder 7～11 grams per liters, solvent is water, and pH is 5.6～6.0.
MS minimal medium of the present invention comprises macroelement, trace element, molysite, organic matter, sucrose and agar powder.It is when 1 liter of (1000 milliliters) medium of configuration that described macroelement forms, and adds ammonium nitrate (NH
4nO
3) 1.65 grams, potassium nitrate (KNO
3) 1.9 grams, calcium chloride (CaCl
22H
2o) 0.44 gram, magnesium sulfate (MgSO
47H
2o) 0.37 gram, potassium dihydrogen phosphate (KH
2pO
4) 0.17 gram.It is when 1 liter of (1000 milliliters) medium of configuration that described trace element forms, and adds 0.83 milligram of potassium iodide (KI), boric acid (H
3bO
3) 6.2 milligrams, manganese sulphate (MnSO
44H
2o) 22.3 milligrams, zinc sulphate (ZnSO
47H
2o) 8.6 milligrams, sodium molybdate (Na
2moO
42H
2o) 0.25 milligram, copper sulphate (CuSO
45H
2o) 0.025 milligram, cobalt chloride (CoCl
26H
2o) 0.025 milligram.It is when 1 liter of (1000 milliliters) medium of configuration that described molysite element forms, and adds disodium ethylene diamine tetraacetate (Na
2eDTA) 37.25 milligrams, ferrous sulfate (FeSO
47H
2o) 27.85 milligrams.It is when 1 liter of (1000 milliliters) medium of configuration that described organic matter forms, and adds 100 milligrams of inositols, 2 milligrams of glycine, thiamine hydrochloride (VB
1) 0.4 milligram, puridoxine hydrochloride (VB
6) 0.5 milligram, nicotinic acid (VB
5) 0.5 milligram.Sucrose concentration is 20～40 grams per liters, and agar powder concentration is 7～11 grams per liters.
The tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China of the present invention children is selected from FLOS CHRYSANTHEMI ALBA from Haizhou of China base, Tongxiang, Zhejiang and grows fine, without the FLOS CHRYSANTHEMI ALBA from Haizhou of China of damage by disease and insect.
1/2MS solid culture medium of the present invention refers to the content of macroelement in MS minimal medium is reduced to 1/2 content, and trace element, molysite, organic matter and sucrose all do not add, and final concentration consists of: NH
4nO
30.825 grams per liter, KNO
30.95 grams per liter, CaCl
22H
2o0.22 grams per liter, MgSO
47H
2o0.185 grams per liter, KH
2pO
40.085 grams per liter, agar powder 9 grams per liters, solvent is water, pH is 5.6～6.0.
Liquid detergent of the present invention is commercially available various liquid detergents, while using as cleaning agent, conventionally with volume ratio 1:100, adds water.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
(1) take axillalry bud as explant, the method that application tissue is cultivated is carried out Fast-propagation, has overcome FLOS CHRYSANTHEMI ALBA from Haizhou of China normal cutting propagation and the shortcoming such as the division propagation cycle is long, reproduction coefficient is low, easy infection and transmitted virus; The group training seedling of producing by the method have virus less, the advantage such as genetic stability;
(2) improve explant sterilization method: 84 thimerosals little by toxicity and easy degraded replace traditional nondegradable mercury chloride disinfectant, and mercury chloride belongs to heavy metal sizable corrosivity, and contains severe toxicity; Long Term Contact more may cause allergic or nephrotic syndrome, and entered environment also has sizable destructiveness to human foods chain, adopts 84 thimerosals to the safer environmental protection of ecotope;
(3) medium is improved, added the anti-brown agent of glutamine, avoid using active carbon as anti-brown agent, to reduce the shortcoming of agar coagulation ability, and ascorbic acid will be through the complicated process of filtration sterilization as anti-brown agent, other brown agents are expensive again.In medium, add relatively cheap potato natural extract, the active factors in extract has improved FLOS CHRYSANTHEMI ALBA from Haizhou of China bud Differentiation and proliferation rate greatly;
(4) improved group training program, FLOS CHRYSANTHEMI ALBA from Haizhou of China differentiation adventitious buds and Induction Process that this method obtains, without callus dedifferentiation, directly with the aseptic bud of axillalry bud breeding induction, ratio is that explant first obtains callus by blade and petal, the aseptic bud material obtaining from callus is again faster, easier, and can effectively avoid the seedling causing in shoot proliferation process to make a variation, improved the stability of its proterties, guarantee quality and the quality of seedling, be conducive to germ plasm resource and preserve and produce and utilize, can effectively solve the supply problem of the high quality seedling of FLOS CHRYSANTHEMI ALBA from Haizhou of China.
(5) in the bud multiplicative stage, directly take root, disposable bud propagation and the process of rooting culture of completing, do not need to configure special root media and carry out process of rooting culture, cellar culture takes 3 months from inoculating aseptic bud to taking root, approximately 90 days, the inventive method needed 2 first quarter moons, approximately 75 days, greatly save incubation time, reduced batch production production cost.
(4) accompanying drawing explanation
Fig. 1 is that FLOS CHRYSANTHEMI ALBA from Haizhou of China is not adding the upper growth of bud proliferated culture medium (MS+6-BA1.0mg/L+NAA0.1mg/L) (20 days) situation of glutamine and potato natural extract;
Fig. 2 is that FLOS CHRYSANTHEMI ALBA from Haizhou of China is adding the upper growth of bud proliferated culture medium (MS+6-BA1.0mg/L+NAA0.1mg/L) (20 days) situation of glutamine (100mg/L) and potato natural extract (100g/L).
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
MS minimal medium of the present invention comprises macroelement, trace element, molysite, organic matter, sucrose and agar powder: it is when 1 liter of (1000 milliliters) medium of configuration that described macroelement forms, and adds ammonium nitrate (NH
4nO
3) 1.65 grams, potassium nitrate (KNO
3) 1.9 grams, calcium chloride (CaCl
22H
2o) 0.44 gram, magnesium sulfate (MgSO
47H
2o) 0.37 gram, potassium dihydrogen phosphate (KH
2pO
4) 0.17 gram.It is when 1 liter of (1000 milliliters) medium of configuration that described trace element forms, and adds 0.83 milligram of potassium iodide (KI), boric acid (H
3bO
3) 6.2 milligrams, manganese sulphate (MnSO
44H
2o) 22.3 milligrams, zinc sulphate (ZnSO
47H
2o) 8.6 milligrams, sodium molybdate (Na
2moO
42H
2o) 0.25 milligram, copper sulphate (CuSO
45H
2o) 0.025 milligram, cobalt chloride (CoCl
26H
2o) 0.025 milligram.It is when 1 liter of (1000 milliliters) medium of configuration that described molysite element forms, and adds disodium ethylene diamine tetraacetate (Na
2eDTA) 37.25 milligrams, ferrous sulfate (FeSO
47H
2o) 27.85 milligrams.It is when 1 liter of (1000 milliliters) medium of configuration that described organic matter forms, and adds 100 milligrams of inositols, 2 milligrams of glycine, thiamine hydrochloride (VB
1) 0.4 milligram, puridoxine hydrochloride (VB
6) 0.5 milligram, nicotinic acid (VB
5) 0.5 milligram.Sucrose concentration is 30 grams per liters, and agar powder concentration is 9 grams per liters, and solvent is water, and pH is 5.6～6.0.
Described 1/2MS solid culture medium refers to the content of macroelement in MS minimal medium is reduced to 1/2 content, and trace element, molysite, organic matter and sucrose all do not add, and final concentration consists of: NH
4nO
30.825 grams per liter, KNO
30.95 grams per liter, CaCl
22H
2o0.22 grams per liter, MgSO
47H
2o0.185 grams per liter, KH
2pO
40.085 grams per liter, agar powder 9 grams per liters, solvent is water, pH is 5.6～6.0.
Liquid detergent of the present invention is Zhejiang development of evil in febrile disease liquid detergent.
Sealed membrane of the present invention is purchased from the biological Co., Ltd of Qingdao Prandtl.
Described potato extract is that potato is cleaned, and peels, and takes 80～120 grams of potatos, is cut into 2 * 2cm
3fritter, put into 500ml deionized water, boil 15～25mins, be cooled to after 50～60 ℃, with double gauze, filter, filtrate is added in MS minimal medium, is and in MS minimal medium, adds 80g/L～120g/L potato extract.
Embodiment 1
(1) field seed selection: select to grow fine in FLOS CHRYSANTHEMI ALBA from Haizhou of China base, Tongxiang, Zhejiang, without the FLOS CHRYSANTHEMI ALBA from Haizhou of China of damage by disease and insect.
(2) acquisition of sterilizable material and induction are cultivated: cut grow fine, without the tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with running water, rinse 0.5h, in volumetric concentration, be then jolting 2mins in the aqueous solution of 1% liquid detergent, with sterile water, wash down; On superclean bench, with volumetric concentration 70% ethanol water, soak 30s, then with mixing medicining liquid dipping 10mins, then use aseptic water washing 3 times; Suck dry moisture on the filter paper of sterilizing, the axillalry bud that strips 0.3mm with scalpel is sharp, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and seal bottleneck with sealed membrane, be placed in incubator, at 24 ℃, under illumination 1500lx condition, cultivate 15 days; The polysorbas20 that described mixing thimerosal is volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is for adding the MS minimal medium of 6-BA1.0mg/L, NAA0.05mg/L and glutamine 80mg/L;
(3) propagation of bud and taking root: observe step (1) described 24 ℃, under illumination 1500lx condition, cultivate after 5 days, there is green projection in cutting part, after 20 days, there is Multiple Buds, cultivate again 10 days, when Multiple Buds grows to 2cm, Multiple Buds is cut and is transferred in bud proliferated culture medium, at 24 ℃, under illumination 1500lx condition, breed cultivation, the acquisition seedling of growing thickly, cultivate about 6 days, the seedling of growing thickly grows 2～5 root systems again, and a step completes bud propagation and the step of taking root, rooting rate is 90%, forms whole plant; Described bud proliferated culture medium is for adding the MS minimal medium of 6-BA0.5mg/L, NAA0.05mg/L, glutamine 80mg/L and potato extract 80g/L;
(4) strong seedling culture: by the seedling of growing thickly of taking root (growing the seedling of growing thickly of 2～5 root systems) access 1/2MS solid culture medium, at 24 ℃, under illumination 1500lx condition, carry out strong seedling culture, acquisition blade is unfolded, leaf look dark green, root system is sturdy, the seedling of height of seedling 3cm.
(5) transplantation of seedlings of taking root: open bottle cap hardening and prepare afterwards for 2 days to transplant, before transplanting, the agar on seedling and foreign material are cleaned, transplant to the seedling medium after sterilization, water permeablely, cover film, keep humidity more than 80%, put ventilating and cooling place, under 24 ℃, illumination 1500lx condition, cultivate 8 days, FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf look dark green, stem is normally stood upright, and realizes the breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China; Dry wet being suitably of matrix, too wet meeting causes rotten, the too dry blade that there will be is withered, and the generation of regularly spraying the pre-preventing disease and pest of medicine.Described seedling medium is that fine sand, humus soil and rice hull ash form according to the proportional arrangement of mass ratio 5:2:2, the sterilization method of described seedling medium is: adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film after covering 3 days and is tanned by the sun matrix 1 day again, completes the sterilization to seedling medium.
The impact of embodiment 2 disinfectants on FLOS CHRYSANTHEMI ALBA from Haizhou of China explant growth
Use respectively the hydrogen peroxide (H of mass concentration 10%
2o
2) mercury chloride (HgCl of the aqueous solution and mass concentration 0.1%
2) mixing thimerosal in two kinds of disinfectant alternate embodiment 1 steps of the aqueous solution (2) carries out disinfection to the explant material of FLOS CHRYSANTHEMI ALBA from Haizhou of China (being axillalry bud), other operation is with embodiment 1, observe the impact (observe axillalry bud point growth on inducing clumping bud medium) of the different disinfectants of statistics on the induction of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud and growth, the results are shown in Table shown in 1.Result shows, mixes thimerosal and mass concentration 0.1%HgCl
2disinfection Effect basic identical, the disinfectant with hydrogen peroxide effect of mass concentration 10% is slightly poor, pollution rate reaches 10%, poor growth.But mass concentration 0.1%HgCl
2sterilization has impact to the induction of bud, inductivity only 88%, and also explant has and sends out phenomenon brown, and the growth of bud is also slow, may be HgCl
2poisoning cell has a certain impact to the induction of bud.
The impact of the different disinfectant of table 1 on FLOS CHRYSANTHEMI ALBA from Haizhou of China explant growth
The induction impact of embodiment 3 different hormone combinations on FLOS CHRYSANTHEMI ALBA from Haizhou of China bud
FLOS CHRYSANTHEMI ALBA from Haizhou of China axillalry bud is seeded in respectively in the MS minimal medium (being inducing clumping bud medium) that has added variable concentrations 6-BA and NAA, and other operates with embodiment 1, and FLOS CHRYSANTHEMI ALBA from Haizhou of China Multiple Buds growth result is shown in Table 2.Result shows, the medium of different hormone combinations is the formation of induced bud to some extent all.Wherein take medium MS+6-BA1.5mg/L+NAA0.2mg/L+100mg/L glutamine as best, the regeneration bud inducing is maximum, growth is fast, the average long 2.05cm of bud, bud induction rate reaches 100%, seedling is large and healthy and strong, and without vitrifying seedling phenomenon, and the medium cutting part that does not add glutamine all presents brownization in various degree.
The induction impact of table 2 hormon concentration on FLOS CHRYSANTHEMI ALBA from Haizhou of China bud
The impact that embodiment 4 glutamine and potato natural extract are taken root on FLOS CHRYSANTHEMI ALBA from Haizhou of China bud propagation
The Multiple Buds of FLOS CHRYSANTHEMI ALBA from Haizhou of China is inoculated into respectively in the bud proliferated culture medium that has added variable concentrations glutamine and potato natural extract, other operation is with embodiment 1, the acquisition seedling of growing thickly, glutamine and potato natural extract are taken root on the induction of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud and propagation, and to affect result as shown in table 3.Found that, FLOS CHRYSANTHEMI ALBA from Haizhou of China indefinite bud is not in adding the medium of glutamine and potato natural extract, all present differentiation rate low, brownization of leaf is serious, and adventitious bud proliferation is few, the slow phenomenon of growing, even in the proliferated culture medium MS+6-BA1.0mg/L+NAA0.1mg/L optimizing, while not adding glutamine and potato natural extract, although FLOS CHRYSANTHEMI ALBA from Haizhou of China indefinite bud also has propagation, but brownization is serious, the highest 185%(that only reaches of the rate of increase is shown in Fig. 1).Add separately glutamine 80～120mg/L, can improve the state of brownization of blade, and add separately potato natural extract 80～120g/L, all can promote bud Growth and Differentiation hestening rooting.When simultaneously, add glutamine 80～120mg/L, during potato natural extract 80～120g/L, not only improved the state of brownization of blade, and promote the propagation of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud and take root, the interpolation concentration 100mg/L of glutamine wherein, it is better to Browning control and bud propagation rooting efficiency that potato natural extract adds concentration 100g/L, in the bud proliferated culture medium MS+6-BA1.0mg/L+NAA0.1mg/L optimizing, add the glutamine of 100mg/L, 100g/L potato natural extract, the rate of increase can reach 280%, seedling is larger, leaf green, stalwartness and propagation are many, well developed root system and the vigorous (see figure 2) of growing.
The impact that table 3 glutamine and potato natural extract are bred and taken root FLOS CHRYSANTHEMI ALBA from Haizhou of China bud
Embodiment 6
(1) field seed selection, selects to grow fine in FLOS CHRYSANTHEMI ALBA from Haizhou of China base, Tongxiang, Zhejiang, without the FLOS CHRYSANTHEMI ALBA from Haizhou of China of damage by disease and insect.
(2) acquisition of sterilizable material and induction are cultivated: cut grow fine, without the tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with running water, rinse 1h, in volumetric concentration, be then jolting 3mins in the aqueous solution of 1% liquid detergent, with sterile water, wash down; On superclean bench, with volumetric concentration 75% ethanol water, soak 40s, then with mixing medicining liquid dipping 15mins, then use aseptic water washing 4 times; Suck dry moisture on the filter paper of sterilizing, the axillalry bud that strips 0.5mm with scalpel is sharp, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and seal bottleneck with sealed membrane, be placed in incubator, at 25 ℃, under illumination 2000lx condition, cultivate 20 days; The polysorbas20 that described mixing thimerosal is volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is for adding the MS minimal medium of 6-BA1.5mg/L, NAA0.2mg/L and glutamine 100mg/L, and in MS minimal medium, sucrose 30g/L, agar powder 9g/L, solvent are water, pH5.8.
(3) propagation of bud and taking root: observe step (1) described 25 ℃, cultivate after 10 days under illumination 2000lx condition, green projection appears in cutting part, after 30 days, there is Multiple Buds, then cultivate 15 days, when Multiple Buds grows to 3cm, Multiple Buds is cut and is transferred in bud proliferated culture medium, at 25 ℃, under illumination 2000lx condition, breed cultivation, obtain the seedling of growing thickly, cultivate again about 8 days, the seedling of growing thickly grows 2～5 root systems, and a step completes bud propagation and the step of taking root, and rooting rate is 93%; Described bud proliferated culture medium is for adding the MS minimal medium of 6-BA1.0mg/L, NAA0.1mg/L, glutamine 100mg/L, potato extract 100g/L.
(4) strong seedling culture: by the seedling of growing thickly of taking root (growing the seedling of growing thickly of 2～5 root systems) access 1/2MS solid culture medium, at 25 ℃, under illumination 2000lx condition, carry out strong seedling culture, acquisition blade is unfolded, leaf look dark green, root system is healthy and strong, the seedling of height of seedling 4cm.
(5) the take root transplanting of seedling: transplant front opening bottle cap hardening 3 days, agar on seedling and foreign material are cleaned, transplant to the seedling medium of sterilizing, water permeablely, cover film, keep humidity more than 80%, put ventilating and cooling place, under 25 ℃, illumination 2000lx condition, cultivate 10 days, FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf look dark green, stem is normally stood upright, and realizes the breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China; Matrix is dry wet suitable, and too wet meeting causes rotten, and the too dry blade that there will be is withered, and the generation of regularly spraying the pre-preventing disease and pest of medicine.Described seedling medium is that fine sand, humus soil and rice hull ash form according to the proportional arrangement of mass ratio 5:2:2, the sterilization method of described seedling medium is: adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film after covering 4 days and is tanned by the sun matrix 2 days again, completes the sterilization to seedling medium.
Embodiment 7
(1) field seed selection, selects to grow fine in FLOS CHRYSANTHEMI ALBA from Haizhou of China base, Tongxiang, Zhejiang, without the FLOS CHRYSANTHEMI ALBA from Haizhou of China of damage by disease and insect.
(2) acquisition of sterilizable material and induction are cultivated: cut grow fine, without the tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with running water, rinse 2h, in volumetric concentration, be then jolting 5mins in the aqueous solution of 1% liquid detergent, with sterile water, wash down; On superclean bench, with volumetric concentration 75% ethanol water, soak 60s, then with mixing medicining liquid dipping 20mins, then use aseptic water washing 5 times; Suck dry moisture on the filter paper of sterilizing, the axillalry bud that strips 0.6mm with scalpel is sharp, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and seal bottleneck with sealed membrane, be placed in incubator, at 26 ℃, under illumination 2500lx condition, cultivate 25 days; The polysorbas20 that described mixing thimerosal is volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is for adding the MS minimal medium of 6-BA3.0mg/L, NAA0.5mg/L and glutamine 120mg/L, and in MS minimal medium, sucrose 40g/L, agar powder 11g/L, solvent are water, pH5.8.
(3) propagation of bud and taking root: observe step (1) described 26 ℃, cultivate after 15 days under illumination 2500lx condition, green projection appears in cutting part, after 40 days, there is Multiple Buds, then cultivate 20 days, when Multiple Buds grows to 4cm, indefinite Multiple Buds is cut and is transferred in bud proliferated culture medium, at 26 ℃, under illumination 2500lx condition, breed cultivation, obtain the seedling of growing thickly, cultivate again 10d left and right, the seedling of growing thickly grows 2～5 root systems, and a step completes bud propagation and the step of taking root, and rooting rate is 95%; Described bud proliferated culture medium is for adding the MS minimal medium of 6-BA2.0mg/L, NAA0.2mg/L, glutamine 120mg/L, potato extract 120g/L.
(4) strong seedling culture: by the seedling of growing thickly of taking root (growing the seedling of growing thickly of 2～5 root systems) access 1/2MS solid culture medium, at 26 ℃, under illumination 2500lx condition, carry out strong seedling culture, acquisition blade is unfolded, leaf look dark green, root system is healthy and strong, the seedling of height of seedling 5cm.
(5) the take root transplanting of seedling: transplant front opening bottle cap hardening 4 days, agar on seedling plant and foreign material are cleaned, transplant to the seedling medium of sterilizing, water permeablely, cover film, keep humidity more than 80%, put ventilating and cooling place, under 26 ℃, illumination 2500lx condition, cultivate 12 days, FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf look dark green, stem is normally stood upright, and realizes the breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China; Matrix is dry wet suitable, and too wet meeting causes rotten, and the too dry blade that there will be is withered, and the generation of regularly spraying the pre-preventing disease and pest of medicine.Described seedling medium is that fine sand, humus soil and rice hull ash form according to the proportional arrangement of mass ratio 5:2:2, the sterilization method of described seedling medium is: adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film and at 60 ℃, is dried 2 days again after covering 5 days, complete the sterilization to seedling medium.
Claims (5)
1. FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue is cultivated and a propagation method, it is characterized in that described method carries out as follows:
(1) acquisition of sterilizable material and induction are cultivated: cut grow fine, without the tender axillalry bud of FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with running water, rinse 0.5～2h, in volumetric concentration, be then jolting 2～5mins in the aqueous solution of 1% liquid detergent, with sterile water, wash down; On superclean bench, with volumetric concentration 70～75% ethanol waters, soak 30～60s, then with mixing medicining liquid dipping 10～20mins, then use aseptic water washing 3～5 times; Suck dry moisture on the filter paper of sterilizing, strip the axillalry bud point of 0.3～0.6mm, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and seal bottleneck with sealed membrane, be placed in incubator, at 24～26 ℃, under illumination 1500～2500lx condition, cultivate 15～25 days; The polysorbas20 that described mixing thimerosal is volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is for adding the MS minimal medium of 6-BA1.0～3.0mg/L, NAA0.05～0.5mg/L and glutamine 80～120mg/L;
(2) propagation of bud and taking root: observe step (1) described 24～26 ℃, under illumination 1500～2500lx condition, cultivate after 5～15 days, there is green projection in cutting part, after 20～40 days, there is Multiple Buds, cultivate again 10～20 days, when Multiple Buds grows to 2～4cm, Multiple Buds is cut and is transferred in bud proliferated culture medium, at 24～26 ℃, under illumination 1500～2500lx condition, breed cultivation, obtain the seedling of growing thickly, cultivate again 6～10 days, the seedling of growing thickly grows 2～5 root systems, completes bud propagation and takes root, and forms whole plant; Described bud proliferated culture medium is for adding the MS minimal medium of 6-BA0.5～2.0mg/L, NAA0.05～0.2mg/L, glutamine 80～120mg/L and potato extract 80g/L～120g/L; Described potato extract is that potato is cleaned, and peeling, takes 80～120 grams of potatos, is cut into 2 * 2cm
3fritter, put into 500ml deionized water, boil 15～25mins, be cooled to after 50～60 ℃, with double gauze, filter, filtrate is added in MS minimal medium, is and in MS minimal medium, adds 80g/L～120g/L potato extract;
(3) strong seedling culture: by growing thickly in seedling access 1/2MS solid culture medium of taking root, at 24～26 ℃, carry out strong seedling culture under illumination 1500～2500lx condition, acquisition blade is unfolded, leaf look dark green, and root system is sturdy, the seedling of height of seedling 3～5cm;
(4) transplantation of seedlings of taking root: open bottle cap hardening after 2～4 days, agar on seedling plant and foreign material are cleaned, transplant to the seedling medium after sterilization, water permeablely, cover film, keep humidity more than 80%, put ventilating and cooling place, under 24～26 ℃, illumination 1500～2500lx condition, cultivate 8～12d, FLOS CHRYSANTHEMI ALBA from Haizhou of China leaf look dark green, stem is normally stood upright, and realizes the breeding of FLOS CHRYSANTHEMI ALBA from Haizhou of China; Described seedling medium is that fine sand, humus soil and rice hull ash are formulated according to the ratio of mass ratio 5:2:2, the sterilization method of described seedling medium is: adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film after covering 3～5 days and is tanned by the sun matrix 1～3 day again, complete the sterilization to seedling medium, or adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, plastic film is opened film and at 60 ℃, is dried 2 days again after covering 3～5 days, complete the sterilization to seedling medium.
2. FLOS CHRYSANTHEMI ALBA from Haizhou of China method for tissue culture as claimed in claim 1, is characterized in that the described inducing clumping bud medium of step (1) is for adding the MS minimal medium of 6-BA1.5mg/L, NAA0.2mg/L and glutamine 100mg/L.
3. FLOS CHRYSANTHEMI ALBA from Haizhou of China method for tissue culture as claimed in claim 1, is characterized in that the described bud proliferated culture medium of step (2) is for adding the MS minimal medium of 6-BA1.0mg/L, NAA0.1mg/L, glutamine 100mg/L and potato extract 100g/L.
4. FLOS CHRYSANTHEMI ALBA from Haizhou of China method for tissue culture as claimed in claim 1, is characterized in that described MS minimal medium final concentration consists of: NH
4nO
31.65 grams per liters, KNO
31.9 grams per liters, CaCl
22H
2o0.44 grams per liter, MgSO
47H
2o0.37 grams per liter, KH
2pO
40.17 grams per liter, KI0.83 mg/litre, H
3bO
36.2 mg/litre, MnSO
44H
2o22.3 mg/litre, ZnSO
47H
2o8.6 mg/litre, Na
2moO
42H
2o0.25 mg/litre, CuSO
45H
2o0.025 mg/litre, CoCl
26H
2o0.025 mg/litre, Na
2eDTA37.25 mg/litre, FeSO
47H
2o27.85 mg/litre, inositol 100 mg/litre, glycine 2 mg/litre, thiamine hydrochloride 0.4 mg/litre, puridoxine hydrochloride 0.5 mg/litre, nicotinic acid 0.5 mg/litre, sucrose 20～40 grams per liters, agar powder 7～11 grams per liters, solvent is water, and pH is 5.6～6.0.
5. FLOS CHRYSANTHEMI ALBA from Haizhou of China method for tissue culture as claimed in claim 1, is characterized in that described 1/2MS solid culture medium final concentration consists of: NH
4nO
30.825 grams per liter, KNO
30.95 grams per liter, CaCl
22H
2o0.22 grams per liter, MgSO
47H
2o0.185 grams per liter, KH
2pO
40.085 grams per liter, agar powder 9 grams per liters, solvent is water, pH is 5.6～6.0.
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| CN112273233A (en) * | 2020-11-06 | 2021-01-29 | 合肥戬谷生物科技有限公司 | Method for establishing efficient chrysanthemum morifolium regeneration system and application thereof |
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| CN107787839A (en) * | 2017-11-07 | 2018-03-13 | 中国计量大学 | A kind of method for tissue culture of Edgeworthia chrysantha |
| CN111837960A (en) * | 2020-08-05 | 2020-10-30 | 贵州茗香茶业发展有限公司 | Breeding method suitable for karst landform golden-silk yellow chrysanthemum |
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