CN103975851B - A kind of method of FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures and breeding - Google Patents
A kind of method of FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures and breeding Download PDFInfo
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Abstract
The invention discloses a kind of tissue cultures of FLOS CHRYSANTHEMI ALBA from Haizhou of China and the method for Vitro Quick Reproduction, make to obtain a large amount of high-quality FLOS CHRYSANTHEMI ALBA from Haizhou of China seedlings in a short time, meet the needs in market; Described method be divided into the acquisition of sterilizable material and Fiber differentiation, bud propagation and take root, strong seedling culture and transplantation of seedlings of taking root; The present invention take axillalry bud as explant, and the method for application tissue cultures carries out Fast-propagation, overcomes 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; With the plantlet in vitro that the method is produced have virus less, the advantage such as genetic stability.
Description
(1) technical field
The present invention relates to plant tissue culture technique, particularly a kind of tissue cultures of FLOS CHRYSANTHEMI ALBA from Haizhou of China 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 (ChrysanthemummorifoliumRamat) is the catananche of a kind of medicine-food two-purpose originating in China Zhejiang, is one of Zhejiang Province eight your name medicinal material " eight Zhe's ".FLOS CHRYSANTHEMI ALBA from Haizhou of China both can make medicinal material, can being made into again beverage for drinking, also can viewing and admiring 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, diuresis of refreshing oneself, can be used for treatment jaundice with damp-heat pathogen, stomachache food less, the disease such as oedema oliguria, also have sedation to nervous centralis.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 capillary resistance be strengthened, suppress the general character of capillary, play the effect that anti-inflammatory is kept fit.Studied discovery in recent years, also containing abundant nickel, zinc, ferro element in FLOS CHRYSANTHEMI ALBA from Haizhou of China, especially iron content is very high.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 in the internal metabolism and human immunity defense function of coordination zinc, calcium, magnesium.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 except 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 current FLOS CHRYSANTHEMI ALBA from Haizhou of China mainly adopts cottage propagation and division propagation, and repoductive time is long, and reproduction coefficient is low, cannot meet the production & marketing demand grown to even greater heights.Virus disease is one of important disease in FLOS CHRYSANTHEMI ALBA from Haizhou of China planting process; and FLOS CHRYSANTHEMI ALBA from Haizhou of China cottage propagation and these propagation methods of division propagation easily cause infection and the propagation of virus; 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 conservation and utilization, directly affects the seed output and quality of its flower, 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, find a kind of FLOS CHRYSANTHEMI ALBA from Haizhou of China propagation method to be rapidly and efficiently very important.
The report of the domestic and international research to FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures is at present all for explant carries out tissue cultures (Luo Zijuan, 1987 of FLOS CHRYSANTHEMI ALBA from Haizhou of China with blade, petiole, hypocotyl, stem section and petal; Zhang Yuan, 2008; Most intelligent, 2004; Niu Yanbing, 2007; Zhang Na, 2009), but these methods all effectively can not slough 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 become indefinite bud from Calli Differentiation, easily there is variation in the indefinite bud obtained by this process, have impact on the original excellent genetic character of seedling in breeding.The present invention is directly differentiation-inducing 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 obtained by this approach effectively can avoid the variation occurred in shoot proliferation process, keeps the original merit of maternal plant better.
(3) summary of the invention
The object of the invention is to provide a kind of tissue cultures of FLOS CHRYSANTHEMI ALBA from Haizhou of China and the method for Vitro Quick Reproduction, makes to obtain a large amount of high-quality FLOS CHRYSANTHEMI ALBA from Haizhou of China seedlings in a short time, meets the needs in market.
The technical solution used in the present invention is:
A kind of FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures and propagation method, described method is carried out as follows:
(1) acquisition of sterilizable material and Fiber differentiation: cut and grow fine, without the tender axillalry bud of the FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with tap water 0.5 ~ 2h, be then jolting 2 ~ 5mins in the aqueous solution of 1% liquid detergent in volumetric concentration, wash down with sterile water; Superclean bench soaks 30 ~ 60s with volumetric concentration 70 ~ 75% ethanol water, then with mixing medicining liquid dipping 10 ~ 20mins, then uses aseptic water washing 3 ~ 5 times; Suck dry moisture on the filter paper of sterilizing, the axillalry bud point of 0.3 ~ 0.6mm is stripped with scalpel, then axillalry bud point is inoculated in the blake bottle of dress inducing clumping bud medium, cover bottle cap and use ParafilmTM bottleneck, be placed in incubator, at 24 ~ 26 DEG C, cultivate 15 ~ 25 days under illumination 1500 ~ 2500lx condition; Described mixing thimerosal is the polysorbas20 of volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is add 6-BA(and 6-benzyl aminoadenine) 1.0 ~ 3.0mg/L, NAA(and methyl α-naphthyl acetate) the MS minimal medium of 0.05 ~ 0.5mg/L and glutamine 80 ~ 120mg/L;
(2) bud propagation and take root: observe step (1) described 24 ~ 26 DEG C, cultivate under illumination 1500 ~ 2500lx condition after 5 ~ 15 days, there is green projection in cutting part, Multiple Buds is there is after 20 ~ 40 days, cultivate 10 ~ 20 days again, when Multiple Buds grows to 2 ~ 4cm, Multiple Buds is cut and is transferred in Shoot propagation medium, at 24 ~ 26 DEG C, Multiplying culture is carried out under illumination 1500 ~ 2500lx condition, obtain the tufted seedling of a large amount of propagation, cultivate 6 ~ 10 days again, tufted seedling grows 2 ~ 5 root systems, one step completes Shoot propagation and step of taking root, final rooting rate is 90 ~ 95%, form whole plant, described Shoot propagation medium is the MS minimal medium adding 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 cleaned by potato, and peeling, takes potato 80 ~ 120 grams, be cut into 2 × 2cm
3fritter, put into 500ml deionized water, boil 15 ~ 25mins, after being cooled to 50 ~ 60 DEG C, with double gauze filter, filtrate is added in MS minimal medium, is and adds 80g/L ~ 120g/L potato extract in MS minimal medium, (potato extract of the present invention refers to the filtrate to boil filtration after potato is peeled in water after, and the addition of filtrate is to boil the weighing scale of front peeled potatoes),
(3) strong seedling culture: the tufted seedling of taking root (namely growing the tufted seedling of 2 ~ 5 root systems) is accessed in 1/2MS solid culture medium, at 24 ~ 26 DEG C, under illumination 1500 ~ 2500lx condition, carry out strong seedling culture, obtain that blade is unfolded, leaf look dark green, root system is sturdy, the seedling of height of seedling 3 ~ 5cm;
(4) to take root transplantation of seedlings: open bottle cap hardening after 2 ~ 4 days, agar on seedling plant and foreign material are cleaned, transplant on the seedling medium to sterilization, water permeable, cover film, keep humidity more than 80%, put ventilating and cooling place, 24 ~ 26 DEG C, cultivate 8 ~ 12d under illumination 1500 ~ 2500lx condition, 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 dries up, and regularly carries out the generation of 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, covered rearing with plastic film is opened film and is tanned by the sun matrix again 1 ~ 3 day after 3 ~ 5 days, complete the sterilization to seedling medium, or adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, covered rearing with plastic film opens film at 60 DEG C dry 2 days again after 3 ~ 5 days, complete the sterilization to seedling medium.
Further, the described inducing clumping bud medium of step (1) preferably adds the MS minimal medium of 6-BA1.5mg/L, NAA0.2mg/L and glutamine 100mg/L.
Further, the Shoot propagation medium described in step (2) preferably adds the MS minimal medium of 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 liter, agar powder 7 ~ 11 grams per liter, 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.Described macroelement composition is when configuration 1 liter (1000 milliliters) medium, 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.Described trace element composition is when configuration 1 liter (1000 milliliters) medium, adds potassium iodide (KI) 0.83 milligram, 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.Described molysite element composition is when configuration 1 liter (1000 milliliters) medium, adds disodium ethylene diamine tetraacetate (Na
2eDTA) 37.25 milligrams, ferrous sulfate (FeSO
47H
2o) 27.85 milligrams.Described organic matter composition is when configuration 1 liter (1000 milliliters) medium, adds inositol 100 milligrams, glycine 2 milligrams, 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 and the content of macroelement in MS minimal medium is reduced to the content of 1/2, and trace element, molysite, organic matter and sucrose all do not add, and namely 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 liter, solvent is water, and pH is 5.6 ~ 6.0.
Liquid detergent of the present invention is commercially available various liquid detergents, usually adds water with volume ratio 1:100 when using as cleaning agent.
Compared with prior art, beneficial effect of the present invention is mainly reflected in:
(1) take axillalry bud as explant, the method for application tissue cultures carries out Fast-propagation, overcomes 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; With the plantlet in vitro that the method is produced have virus less, the advantage such as genetic stability;
(2) improve explant disinfection method: little with toxicity and 84 thimerosals of easily degraded replace traditional nondegradable mercury chloride disinfectant, mercury chloride belongs to heavy metal sizable corrosivity, and containing hypertoxic; 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, with the addition of glutamine and resist brown agent, avoid using active carbon to reduce the shortcoming of agar solidification ability as anti-brown agent, and ascorbic acid will through the complicated process of filtration sterilization as anti-brown agent, other brown agents are expensive again.Add relatively cheap potato natural extract in the medium, the active factors in extract substantially increases FLOS CHRYSANTHEMI ALBA from Haizhou of China Bud polarization and the rate of increase;
(4) group training program is improved, the FLOS CHRYSANTHEMI ALBA from Haizhou of China differentiation adventitious buds that this method obtains and Induction Process, without callus dedifferentiation, directly with axillary bud propagation inducing sterile bud, ratio is that explant first obtains callus by blade and petal, the aseptic bud material obtained from callus is again faster, easier, and can effectively avoid the seedling caused in shoot proliferation process to make a variation, improve the stability of its proterties, ensure that quality and the quality of seedling, be conducive to Germ-plasma resources protection and Commercial cultivation, effectively can solve the supply problem of the high quality seedling of FLOS CHRYSANTHEMI ALBA from Haizhou of China.
(5) directly take root in the Shoot propagation stage, disposablely complete Shoot propagation and process of rooting culture, the special root media of configuration is not needed to carry out process of rooting culture, cellar culture takes 3 months from the aseptic bud of inoculation to taking root, about 90 days, the inventive method needed 2 first quarter moons, about 75 days, greatly save incubation time, reduce the cost of factorial praluction.
(4) accompanying drawing explanation
Fig. 1 is that FLOS CHRYSANTHEMI ALBA from Haizhou of China is not adding the upper growth of Shoot propagation 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 Shoot propagation 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: described macroelement composition is when configuration 1 liter (1000 milliliters) medium, 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.Described trace element composition is when configuration 1 liter (1000 milliliters) medium, adds potassium iodide (KI) 0.83 milligram, 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.Described molysite element composition is when configuration 1 liter (1000 milliliters) medium, adds disodium ethylene diamine tetraacetate (Na
2eDTA) 37.25 milligrams, ferrous sulfate (FeSO
47H
2o) 27.85 milligrams.Described organic matter composition is when configuration 1 liter (1000 milliliters) medium, adds inositol 100 milligrams, glycine 2 milligrams, 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 and the content of macroelement in MS minimal medium is reduced to the content of 1/2, and trace element, molysite, organic matter and sucrose all do not add, and namely 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 liter, solvent is water, and 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 cleaned by potato, peels, and takes potato 80 ~ 120 grams, be cut into 2 × 2cm
3fritter, put into 500ml deionized water, boil 15 ~ 25mins, after being cooled to 50 ~ 60 DEG C, with double gauze filter, filtrate is added in MS minimal medium, is and adds 80g/L ~ 120g/L potato extract in MS minimal medium.
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 Fiber differentiation: cut and grow fine, without the tender axillalry bud of the FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, use tap water 0.5h, be then jolting 2mins in the aqueous solution of 1% liquid detergent in volumetric concentration, wash down with sterile water; Superclean bench soaks 30s with volumetric concentration 70% ethanol water, then with mixing medicining liquid dipping 10mins, then uses aseptic water washing 3 times; Suck dry moisture on the filter paper of sterilizing, strips the axillalry bud point of 0.3mm with scalpel, be then inoculated into by axillalry bud point in the blake bottle of dress inducing clumping bud medium, cover bottle cap and use ParafilmTM bottleneck, be placed in incubator, at 24 DEG C, cultivate 15 days under illumination 1500lx condition; Described mixing thimerosal is the polysorbas20 of volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is the MS minimal medium adding 6-BA1.0mg/L, NAA0.05mg/L and glutamine 80mg/L;
(3) bud propagation and take root: observe step (1) described 24 DEG C, cultivate under illumination 1500lx condition after 5 days, there is green projection in cutting part, Multiple Buds is there is after 20 days, cultivate 10 days again, when Multiple Buds grows to 2cm, Multiple Buds is cut and is transferred in Shoot propagation medium, at 24 DEG C, under illumination 1500lx condition, carry out Multiplying culture, obtain tufted seedling, cultivate about 6 days again, tufted seedling grows 2 ~ 5 root systems, and a step completes Shoot propagation and step of taking root, rooting rate is 90%, forms whole plant; Described Shoot propagation medium is the MS minimal medium adding 6-BA0.5mg/L, NAA0.05mg/L, glutamine 80mg/L and potato extract 80g/L;
(4) strong seedling culture: the tufted seedling of taking root (growing the tufted seedling of 2 ~ 5 root systems) is accessed in 1/2MS solid culture medium, at 24 DEG C, under illumination 1500lx condition, carry out strong seedling culture, obtain that blade is unfolded, leaf look dark green, root system is sturdy, the seedling of height of seedling 3cm.
(5) to take root transplantation of seedlings: open bottle cap hardening and within 2 days, prepare afterwards to transplant, before transplanting, the agar on seedling and foreign material are cleaned, transplant on the seedling medium to sterilization, water permeable, cover film, keep humidity more than 80%, put ventilating and cooling place, 24 DEG C, cultivate 8 days under illumination 1500lx condition, 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 the wetting of matrix is suitable for as well, and too wet meeting causes rotten, and the too dry blade that there will be dries up, and regularly carries out the generation of 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, covered rearing with plastic film is opened film and is tanned by the sun matrix again 1 day after 3 days, complete the sterilization to seedling medium.
Embodiment 2 disinfectant is on the impact of FLOS CHRYSANTHEMI ALBA from Haizhou of China explant growth
Use the hydrogen peroxide (H of mass concentration 10% respectively
2o
2) mercury chloride (HgCl of the aqueous solution and mass concentration 0.1%
2) explant material (i.e. axillalry bud) of mixing thimerosal to FLOS CHRYSANTHEMI ALBA from Haizhou of China in the aqueous solution two kinds of disinfectant alternate embodiment 1 steps (2) carry out disinfection, other operation is with embodiment 1, observe the impact (namely observe axillalry bud point growth on inducing clumping bud medium) of the different disinfectant of statistics on FLOS CHRYSANTHEMI ALBA from Haizhou of China bud inducement and growth, the results are shown in Table shown in 1.Result shows, mixing thimerosal and mass concentration 0.1%HgCl
2disinfection Effect substantially identical, the disinfectant with hydrogen peroxide effect of mass concentration 10% is slightly poor, and pollution rate reaches 10%, poor growth.But mass concentration 0.1%HgCl
2sterilization has impact to the induction of bud, inductivity only 88%, and explant has and sends out phenomenon brown, and the growth of bud is also slow, may be HgCl
2the induction of Cytotoxic to bud has a certain impact.
The different disinfectant of table 1 is on the impact of FLOS CHRYSANTHEMI ALBA from Haizhou of China explant growth
Embodiment 3 different hormone combinations affects the induction of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud
Be seeded in by FLOS CHRYSANTHEMI ALBA from Haizhou of China axillalry bud in the MS minimal medium (i.e. inducing clumping bud medium) that with the addition of variable concentrations 6-BA and NAA respectively, other operation is 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 all can the formation of induced bud to some extent.Be wherein best with medium MS+6-BA1.5mg/L+NAA0.2mg/L+100mg/L glutamine, the regeneration bud induced is maximum, growth is fast, the long 2.05cm of average bud, bud induction rate reaches 100%, seedling is healthy and strong greatly, and without vitrifying seedling phenomenon, and the medium cutting part not adding glutamine all presents brownization in various degree.
Table 2 hormon concentration affects the induction of 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 Shoot propagation
Being inoculated into respectively by the Multiple Buds of FLOS CHRYSANTHEMI ALBA from Haizhou of China with the addition of in the Shoot propagation medium of variable concentrations glutamine and potato natural extract, other operation is with embodiment 1, obtain tufted seedling, glutamine and potato natural extract on FLOS CHRYSANTHEMI ALBA from Haizhou of China bud inducement and propagation take root to affect result as shown in table 3.Found that, FLOS CHRYSANTHEMI ALBA from Haizhou of China indefinite bud is in the medium not adding glutamine and potato natural extract, all present differentiation rate low, leaf brownization is serious, and adventitious bud proliferation is few, grow slow phenomenon, even if in the proliferated culture medium MS+6-BA1.0mg/L+NAA0.1mg/L optimized, when not adding glutamine and potato natural extract, although FLOS CHRYSANTHEMI ALBA from Haizhou of China indefinite bud also has propagation, but brownization is serious, and the highest 185%(that only reaches of the rate of increase is shown in Fig. 1).Independent interpolation glutamine 80 ~ 120mg/L, can improve the state of blade brownization, and adds separately potato natural extract 80 ~ 120g/L and all can promote bud Growth and Differentiation and hestening rooting.When adding glutamine 80 ~ 120mg/L simultaneously, during potato natural extract 80 ~ 120g/L, not only improve the state of blade brownization, and promote the propagation of FLOS CHRYSANTHEMI ALBA from Haizhou of China bud and take root, the interpolation concentration 100mg/L of its GLN, potato natural extract add concentration 100g/L to Browning control and Shoot propagation rooting efficiency better, the glutamine of 100mg/L is added in the Shoot propagation medium MS+6-BA1.0mg/L+NAA0.1mg/L optimized, 100g/L potato natural extract, the rate of increase can reach 280%, seedling is larger, leaf green, healthy and strong and propagation is many, well developed root system and grow vigorous (see figure 2).
Table 3 glutamine and potato natural extract are on FLOS CHRYSANTHEMI ALBA from Haizhou of China Shoot propagation and the impact of taking root
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 Fiber differentiation: cut and grow fine, without the tender axillalry bud of the FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, use tap water 1h, be then jolting 3mins in the aqueous solution of 1% liquid detergent in volumetric concentration, wash down with sterile water; Superclean bench soaks 40s with volumetric concentration 75% ethanol water, then with mixing medicining liquid dipping 15mins, then uses aseptic water washing 4 times; Suck dry moisture on the filter paper of sterilizing, strips the axillalry bud point of 0.5mm with scalpel, be then inoculated into by axillalry bud point in the blake bottle of dress inducing clumping bud medium, cover bottle cap and use ParafilmTM bottleneck, be placed in incubator, at 25 DEG C, cultivate 20 days under illumination 2000lx condition; Described mixing thimerosal is the polysorbas20 of volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is the MS minimal medium adding 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) bud propagation and take root: observe step (1) described 25 DEG C, cultivating after 10 days under illumination 2000lx condition, there is green projection in cutting part, there is Multiple Buds after 30 days, then cultivate 15 days, when Multiple Buds grows to 3cm, Multiple Buds is cut and is transferred in Shoot propagation medium, at 25 DEG C, under illumination 2000lx condition, carry out Multiplying culture, obtain tufted seedling, cultivate about 8 days again, tufted seedling grows 2 ~ 5 root systems, and a step completes Shoot propagation and step of taking root, and rooting rate is 93%; Described Shoot propagation medium is the MS minimal medium adding 6-BA1.0mg/L, NAA0.1mg/L, glutamine 100mg/L, potato extract 100g/L.
(4) strong seedling culture: the tufted seedling of taking root (growing the tufted seedling of 2 ~ 5 root systems) is accessed in 1/2MS solid culture medium, at 25 DEG C, under illumination 2000lx condition, carry out strong seedling culture, obtain that blade is unfolded, leaf look dark green, root system is healthy and strong, the seedling of height of seedling 4cm.
(5) to take root the transplanting of seedling: transplant front opening bottle cap hardening 3 days, agar on seedling and foreign material are cleaned, transplant on the seedling medium of sterilizing, water permeable, cover film, keep humidity more than 80%, put ventilating and cooling place, 25 DEG C, cultivate 10 days under illumination 2000lx condition, 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 dries up, and regularly carries out the generation of 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, covered rearing with plastic film is opened film and is tanned by the sun matrix again 2 days after 4 days, complete 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 Fiber differentiation: cut and grow fine, without the tender axillalry bud of the FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, use tap water 2h, be then jolting 5mins in the aqueous solution of 1% liquid detergent in volumetric concentration, wash down with sterile water; Superclean bench soaks 60s with volumetric concentration 75% ethanol water, then with mixing medicining liquid dipping 20mins, then uses aseptic water washing 5 times; Suck dry moisture on the filter paper of sterilizing, strips the axillalry bud point of 0.6mm with scalpel, be then inoculated into by axillalry bud point in the blake bottle of dress inducing clumping bud medium, cover bottle cap and use ParafilmTM bottleneck, be placed in incubator, at 26 DEG C, cultivate 25 days under illumination 2500lx condition; Described mixing thimerosal is the polysorbas20 of volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is the MS minimal medium adding 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) bud propagation and take root: observe step (1) described 26 DEG C, cultivating after 15 days under illumination 2500lx condition, there is green projection in cutting part, there is Multiple Buds after 40 days, then cultivate 20 days, when Multiple Buds grows to 4cm, being cut by indefinite Multiple Buds is transferred in Shoot propagation medium, at 26 DEG C, under illumination 2500lx condition, carry out Multiplying culture, obtain tufted seedling, cultivate about 10d again, tufted seedling grows 2 ~ 5 root systems, and a step completes Shoot propagation and step of taking root, and rooting rate is 95%; Described Shoot propagation medium is the MS minimal medium adding 6-BA2.0mg/L, NAA0.2mg/L, glutamine 120mg/L, potato extract 120g/L.
(4) strong seedling culture: the tufted seedling of taking root (growing the tufted seedling of 2 ~ 5 root systems) is accessed in 1/2MS solid culture medium, at 26 DEG C, under illumination 2500lx condition, carry out strong seedling culture, obtain that blade is unfolded, leaf look dark green, root system is healthy and strong, the seedling of height of seedling 5cm.
(5) to take root the transplanting of seedling: transplant front opening bottle cap hardening 4 days, agar on seedling plant and foreign material are cleaned, transplant on the seedling medium of sterilizing, water permeable, cover film, keep humidity more than 80%, put ventilating and cooling place, 26 DEG C, cultivate 12 days under illumination 2500lx condition, 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 dries up, and regularly carries out the generation of 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, covered rearing with plastic film opens film at 60 DEG C dry 2 days again after 5 days, complete the sterilization to seedling medium.
Claims (4)
1. FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures and a propagation method, is characterized in that described method is carried out as follows:
(1) acquisition of sterilizable material and Fiber differentiation: cut and grow fine, without the tender axillalry bud of the FLOS CHRYSANTHEMI ALBA from Haizhou of China children of damage by disease and insect, with tap water 0.5 ~ 2h, be then jolting 2 ~ 5mins in the aqueous solution of 1% liquid detergent in volumetric concentration, wash down with sterile water; Superclean bench soaks 30 ~ 60s with volumetric concentration 70 ~ 75% ethanol water, then with mixing medicining liquid dipping 10 ~ 20mins, then uses 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 use ParafilmTM bottleneck, be placed in incubator, at 24 ~ 26 DEG C, cultivate 15 ~ 25 days under illumination 1500 ~ 2500lx condition; Described mixing thimerosal is the polysorbas20 of volume ratio 1:200 and the mixed liquor of 84 thimerosals; Described inducing clumping bud medium is the MS minimal medium adding 6-BA1.0 ~ 3.0mg/L, NAA0.05 ~ 0.5mg/L and glutamine 80 ~ 120mg/L;
(2) bud propagation and take root: observe step (1) described 24 ~ 26 DEG C, cultivate under illumination 1500 ~ 2500lx condition after 5 ~ 15 days, there is green projection in cutting part, Multiple Buds is there is after 20 ~ 40 days, cultivate 10 ~ 20 days again, when Multiple Buds grows to 2 ~ 4cm, Multiple Buds is cut and is transferred in Shoot propagation medium, at 24 ~ 26 DEG C, under illumination 1500 ~ 2500lx condition, carry out Multiplying culture, obtain tufted seedling, cultivate 6 ~ 10 days again, tufted seedling grows 2 ~ 5 root systems, completes Shoot propagation and takes root, and forms whole plant; Described Shoot propagation medium is the MS minimal medium adding 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 cleaned by potato, and peeling, takes potato 80 ~ 120 grams, be cut into 2 × 2cm
3fritter, put into 500ml deionized water, boil 15 ~ 25mins, after being cooled to 50 ~ 60 DEG C, with double gauze filter, filtrate is added in MS minimal medium, is and adds 80g/L ~ 120g/L potato extract in MS minimal medium;
(3) strong seedling culture: the tufted seedling of taking root is accessed in 1/2MS solid culture medium, at 24 ~ 26 DEG C, carries 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; 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 liter, solvent is water, and pH is 5.6 ~ 6.0;
(4) to take root transplantation of seedlings: open bottle cap hardening after 2 ~ 4 days, agar on seedling plant and foreign material are cleaned, transplant on the seedling medium to sterilization, water permeable, cover film, keep humidity more than 80%, put ventilating and cooling place, 24 ~ 26 DEG C, cultivate 8 ~ 12d under illumination 1500 ~ 2500lx condition, 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 form according to the proportions 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, covered rearing with plastic film is opened film and is tanned by the sun matrix again 1 ~ 3 day after 3 ~ 5 days, complete the sterilization to seedling medium, or adopt volumetric concentration 0.5% formlinata aquae concentratac evenly to spray matrix, covered rearing with plastic film opens film at 60 DEG C dry 2 days again after 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 the MS minimal medium adding 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 Shoot propagation medium described in step (2) is the MS minimal medium adding 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 liter, agar powder 7 ~ 11 grams per liter, solvent is water, and pH is 5.6 ~ 6.0.
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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 |
| CN107646690A (en) * | 2017-11-07 | 2018-02-02 | 中国计量大学 | The tissue cultures and propagation method of a kind of clerodendrum bungei |
| 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 |
| 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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