CN108450333A - A kind of abductive approach of Lilium lancifo1ium Thunb callus - Google Patents
A kind of abductive approach of Lilium lancifo1ium Thunb callus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a kind of abductive approach of Lilium lancifo1ium Thunb callus, include the following steps：By after pretreatment and sterilizing Lilium lancifo1ium Thunb bulbil or scale be inoculated in inducing culture, intensity of illumination is 700 lx, 900 lx, it cultivates 20 30 days, obtain the callus of Lilium lancifo1ium Thunb, the inducing culture is 6 BA+0.15 mg/L NAA+0.20 mg/L KT+0.05 ~ 0.1 mg/L 2 of MS+1.00 mg/L, 4 D, Medium's PH Value are 5.8 ~ 6.2.By 6 BA, NAA, KT and 2, the collaboration compatibility effect between 4 D component contents and certain illumination, the common plant endogenous hormones that adjust are horizontal, and to induce Lilium lancifo1ium Thunb to generate callus, inductivity reaches 71%.It can effectively induce the callus of Lilium lancifo1ium Thunb to be formed within only 20 days using the method for the present invention, overcome the problem of its callus is difficult to induce, lay the foundation for vegetative propagation, breeding for disease resistance and the molecular biology research etc. of Lilium lancifo1ium Thunb.
Description
Technical field
The invention belongs to lily tissue culture technology fields, and in particular to a kind of induction side of Lilium lancifo1ium Thunb callus
Method.
Background technology
Lilium lancifo1ium Thunb (Lilium lancifolium Thunb.) is Liliaceae (Liliaceae) lilium (Lilium)
Cultigen the most ancient, although area of origin is unknown, distributed pole is wide.Wild Lilium lancifo1ium Thunb is distributed in east Asia, Japan more
And Korea.In China, distributed pole is wide, there is its trace, main distributed area from the Changbaishan area in Xi Nan the Yunnan-Guizhou Plateau to northeast
For Tibet, Qinghai, Gansu, Shaanxi, Jiangsu, Zhejiang, Anhui, Hebei, Shandong and Jilin and other places.Lilium lancifo1ium Thunb is a kind of collection medicine
With, the edible and ornamental good plant being integrated.Lilium lancifo1ium Thunb medical value is embodied in removing heat from the lung to relieve cough, clearing away the heart fire and tranquillizing and other effects,
In the middle and lower reach of Yangtze River, Lilium lancifo1ium Thunb is widely cultivated, and is the main source of lily medicinal material.Lilium lancifo1ium Thunb (Yixing lily) is the same as Lanzhou hundred
It closes, the referred to as Chinese three big edible lily kinds of Lilium brownii var viridulum (lily), the necessary amino acid content of 8 kinds of human bodies is than blue in Lilium lancifo1ium Thunb
State lily is high by 7.05%, and total amino acid content is higher by 11.24% than lanzhou lily；In mineral element, outside dephosphorization, potassium, tiger lily hundred
Calcium, iron, copper, manganese, zinc, magnesium, Se content are above lanzhou lily in conjunction；In basic nutrition ingredient, except starch, pectin, reduced sugar
Outside, albumen, fat, crude fibre, Vc, total phospholipids content are above lanzhou lily in Lilium lancifo1ium Thunb.Therefore, Lilium lancifo1ium Thunb is in low fat
Fat, high nutrition and the potential value of good healthcare function are worth exploitation.Lilium lancifo1ium Thunb spends big, various colors, low temperature resistant and arid,
Its bulbil seed propagation facilitates equal merits, may be directly applied to urban landscaping, family with can making in gardens
Courtyard and potting etc. are commonly used one of the flowering bulbs in gardens.Therefore, Lilium lancifo1ium Thunb has good market value.
Lilium lancifo1ium Thunb disease resistance is poor, and wherein Common Diseases such as gray mold (Botrytis elliptica), blade tip are withered
Disease (Phoma lilii) and anthracnose (Colletotrichum lilii) etc., common virus has cucumber mosaic virus
(CMV), more than the 20 kinds of virus such as lily asymptomatic virus (LSV), lily mottle virus (LMoV).Disease can lead to the plant of Lilium lancifo1ium Thunb
Strain is withered, and ornamental value declines, and bulb quality is affected, and medical value substantially reduces.Improving problem above can pass through
The method of breeding solves.Common Resistant breeding means have systematic breeding, distant hybridization and biotechnology breeding etc., knot
It closes the disease-resistant external selection of biotechnology and effective means that the somaclonal variation person that is research in recent years begins attempt to, and takes
It must be in progress.The method that somaclonal variation carries out breeding for disease resistance is without external selection, directly to being regenerated through tissue cultures
Plant or transplanted seedling carry out disease-resistant detection, greatly shorten the breeding time limit compared with screening disease-resistant plant in big Tanaka.Wherein,
Dedifferentiation and breaking up again is easy to generate variation, thus somaclonal variation is frequently with callus tissue culture, callus
(Callus) refer to plant carried out after by wound self-regeneration having wound place grow new cell or group
It knits.Starting period, three periods of division stage and maturity period are undergone from explant to callus is formed.Different plant callus
The ability of induction and forming part difference；The ability of same plant different parts callus induction is also different.Related tiger lily hundred
There are reports for research in terms of conjunction tissue cultures, such as Guo Haibin and Lei Jiajun (2006), Ma Suxian (2012) and poplar
Gentle Song Xiao of profit macro (2013) etc.；But it is all studies have shown that the callus induction of Lilium lancifo1ium Thunb is extremely inefficient, it is particularly difficult to
Induction generates a large amount of callus (block).In addition, using the common kind such as existing rattlebush, lanzhou lily inducing culture simultaneously
Lilium lancifo1ium Thunb is not suitable for it, reason may be that Lilium lancifo1ium Thunb is triploid variety, can not effectively induce and be generated on its explant
Callus, majority can only generate adventitious bud.Therefore, very necessary to the post-directed training of Lilium lancifo1ium Thunb callus, there is weight
Want scientific meaning.
Invention content
For deficiencies of the prior art, the purpose of the present invention is to provide a kind of Lilium lancifo1ium Thunb callus
It is high with pollution rate mainly to solve the problems, such as that Lilium lancifo1ium Thunb explant callus is difficult to induce for abductive approach.
To achieve the above object, the present invention adopts the following technical scheme that：A kind of abductive approach of Lilium lancifo1ium Thunb callus,
Include the following steps：
By after pretreatment and sterilizing Lilium lancifo1ium Thunb scale or bulbil be inoculated in inducing culture and carry out Initial culture, light
It is 700lx-900lx according to intensity, Fiber differentiation 20-30 days is to get to the callus of Lilium lancifo1ium Thunb and sterile adventitious bud；It is described
Inducing culture is MS+1.00mg/L 6-BA+0.15mg/L NAA+0.20mg/L KT+0.05~0.1mg/L 2-4, D, training
It is 5.8~6.2 to support base pH value.
Preferably, the scale is middle level or the outer scale of bulb.
Preferably, sterile adventitious bud base portion enlargement (the calling sterile clove in the following text) switching can also be lured in described
It leads and continues to cultivate in culture medium, intensity of illumination 700lx-900lx, Fiber differentiation 20-30 days is to get to the callus of Lilium lancifo1ium Thunb
Tissue.
Preferably, it is 25 ± 2 DEG C that the condition of the Fiber differentiation, which is in cultivation temperature, and humidity is 60%~70%, illumination
Time is 12~14h/d.
Preferably, lily bulbil or scale detergent solution is impregnate 5-10min by the pretreatment, then with flowing
Tap water rinses 2h or more；Pretreated Lilium lancifo1ium Thunb bulbil or scale is are placed on superclean bench by the sterilizing, first
The ethanol water for being 70~75% with volumetric concentration handles 20~30s, then with sterile water wash at least 2 times, then uses quality
A concentration of 0.1~0.2% mercuric chloride solution 10~25min of sterilization treatment, finally with sterile water washing at least 4 times.
Preferably, the detergent is soap, washing powder or sapindust saponin.
Compared with prior art, the present invention has the advantages that：
1, the present invention uses MS culture mediums as minimal medium, and is aided with exogenous plant hormones, growth regulating substance,
Energy is provided for the generation of Lilium lancifo1ium Thunb callus, the growth for accelerating Lilium lancifo1ium Thunb callus can be promoted, to meet volume
Red lily is nutritionally and physiologically to the requirement of cell dedifferentiation, especially by 6-BA, NAA, KT and 2,4-D component contents
And the collaboration compatibility effect between certain dim light, the common Endogenous Hormones for adjusting Lilium lancifo1ium Thunb, to induce tiger lily hundred
It closes itself and generates callus, be obtained within 20 days a large amount of callus, inductivity solves Lilium lancifo1ium Thunb group up to 71%
Knit the technical issues of being difficult to generate callus in incubation, the present invention be the vegetative propagation of Lilium lancifo1ium Thunb, breeding for disease resistance with
And molecular biology research lays the foundation.
2, the explant that uses of the present invention for bulbil, bulb middle outer scale or Initial culture after the sterile small squama that generates
Stem, have the characteristics that material be easy to get, be easy to operate, breed it is quick, wherein the sterile Bulblet induction culture through Initial culture
Pollution rate is far below other explants in journey, and only 2.5%, and callus induction rate solves Lilium lancifo1ium Thunb group up to 35%
The technical issues of knitting pollution rate height in incubation and being difficult to generate callus.
Description of the drawings
Fig. 1 is the metamorphosis for inducing Lilium lancifo1ium Thunb bulbil；
Scheme the bulbil scale that A is 2-3 days, B is the bulbil scale after 10 days, and C is the bulbil scale after 2 weeks, D 20-30
The callus that its bulbil scale generates, E are that the callus that bulbil scale generates after 30 days starts death；
Fig. 2 is the callus for inducing sterile clove to generate under different illumination conditions；
Figure A is the callus that intensity of illumination is induced synthesis under the conditions of 0-300lx；It is 700- that B, which is in intensity of illumination,
The callus of induced synthesis under the conditions of 900lx；C is the callus of induced synthesis under the conditions of illumination condition is 1500-2000lx
Tissue.
Specific implementation mode
With reference to specific embodiments and the drawings, invention is further described in detail.
1, instrument and equipment：
Steam pressure sterilization pot, filtration sterilization device, spraying sterilizer, ultraviolet lamp, electronic balance, surpasses dry heat sterilization cabinet
Net workbench, refrigerator, balance, electromagnetic oven, culture bottle, stainless steel inoculation disk, operation inoculation is cut, straight forceps, gun shaped are inoculated with tweezer, operation
Handle of a knife etc..
2, preparation of reagents：
(1) 75% alcohol：95% alcohol of 75ml adds water to be settled to 95ml.
The mercuric chloride solution of (2) 0.1% (0.2%)：Weigh 1 gram of (2 grams) HgCl2, add distilled water to 1000ml.
(3) 1mol/LHCL solution：The concentrated hydrochloric acid for taking 8.25ml, adds distilled water to be settled to 100ml.
(4) 1mol/LNaOH solution：Claim 8gNaOH, 200ml is settled to distilled water.
3, culture medium is prepared：
The minimal medium (MS) of tissue cultures：
Every liter of culture medium contains NH4NO31650mg, KNO31900mg, CaCl2·2H2O 440mg, MgSO4·7H2O
370mg, KH2PO4170mg, KI 0.83mg, H2BO36.2mg, MnSO4·4H20 22.3mg, ZnSO4·7H2O 8.6mg,
Na2MoO4·5H2O 0.25mg, CuSO4·5H2O 0.025mg, CoCl2·6H2O 0.025mg, FeSO4·7H2O 27.8mg,
Na2-EDTA·2H2O 37.3mg, inositol 100mg, niacin 0.5mg, glycine 2mg, puridoxine hydrochloride (vitamin B6) 0.5mg,
Thiamine hydrochloride (vitamin B1) 0.4mg and 30g sucrose.
PH is adjusted：HC1 the or NaOH aqueous solutions that 1mol is added dropwise adjust Medium's PH Value to 5.8-6.2.
The culture medium configured is dispensed to culture bottle, then by the culture medium steam pressure sterilization pot dispensed 121
After sterilizing under DEG C high temperature, be placed in gnotobasis store it is for use.
4, auxin：
Auxins：Methyl α-naphthyl acetate (1-Naphthylacetic acid, NAA), 2,4- dichlorphenoxyacetic acids (2,4-
Dichlorophenoxyacetic acid, 2,4-D)；
Cytokinin：6- benzyls aminoadenine (6-Benzylaminopurine, 6-BA), 6- glycosyl adenine phosphates
(6-Furfurylaminopurine, KT).
5, observation statistics is carried out to callus：
Material is observed every 2-3 days, sees its growth and development situation, such as the variation of the color, size of material, and
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.
Embodiment 1
1) pretreatment of Lilium lancifo1ium Thunb bulbil
Dirt on originally underwater cleaning Lilium lancifo1ium Thunb bulbil surface, choose surface have mechanical damage, pest and disease damage bulbil,
By the bulbil of health, 7-8min is impregnated with clear suds, then 2h or more is rinsed with the tap water of flowing.
2) sterilizing of Lilium lancifo1ium Thunb bulbil
Bulbil is pushed aside with dissecting needle on aseptic operating platform and is divided into several uniform bulbil scales, first with 75% second
Cleaning and sterilizing 30s is cleaned 2-3 times after outwelling with high-temperature sterilization water and is added 0.20% mercuric chloride solution (mercuric chloride solution will flood by alcohol
Do not have bulbil scale), covered container lid, which is placed on shaking table, rocks 10-15min, and allowing mercuric chloride solution to be come into full contact with bulbil scale makes
Disinfection is more thorough, then again outwells mercuric chloride solution on aseptic operating platform, and high-temperature sterilization water is added and cleans 4-6 times.
3) induction of Lilium lancifo1ium Thunb bulbil
The bulbil scale after preprocessed and sterilizing is inoculated in containing various concentration 2 respectively, 4-D (0.01,0.05,
0.1,0.5mg/L) inducing culture in, 5 bulbil scales are put on every bottle of culture medium, each concentration gradient respectively puts 10 bottles of trainings
Support base.It is 25 ± 2 DEG C that the condition of Fiber differentiation, which is in cultivation temperature, is cultivated 20 days, and humidity is 60%~70%, and illumination condition is
1500lx-2000lx, 12~14h/d.
Fiber differentiation based component：MS+6-BA 1.00mg/L+NAA 0.15mg/L+KT 0.20mg/L+2-4,D(0.01、
0.05,0.1 or 0.5mg/L).
Material is observed every 2-3 days, sees its growth and development situation, such as the variation of the color, size of material.
As shown in Figure 1, after inoculation 2-3 days, the appearance of bulbil scale becomes brown (figure A).There is bulbil scale after 1 week expands situation and goes out
It is existing, it can see the cell mass of protrusion after 10 days around bulbil scale gradually or (figure B) occur in bud point, it can be apparent after 2 weeks
See the appearance (figure C) of callus and adventitious bud, 20-30 days are that callus occurs at most (figure D), but callus group after 30 days
It is slowly dead to knit beginning, the amount of adventitious bud then starts to increase, and root also begins to occur, and illustrates that callus starts differentiation (figure E).
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.As shown in table 1.
Table 1 induces Lilium lancifo1ium Thunb bulbil scale (Initial culture) callus culture situation
Embodiment 2
1) pretreatment of Lilium lancifo1ium Thunb scale
The soil and impurity that Lilium lancifo1ium Thunb kind ball surface is washed under tap water are peelled off bulb periphery and are encroached on by pest and disease damage
Or the scale for thering are other to damage, take middle level to have no mechanical damage, the scale of no disease and pests harm, health, with certain density soapberry soap
Plain solution impregnates 5min, then spare with the tap water flushing 2h or more of flowing.
2) sterilizing of Lilium lancifo1ium Thunb scale
First 30s will be sterilized at the beginning of the scale cleaned up with 70% ethyl alcohol on aseptic operating platform, it is clear with high-temperature sterilization water afterwards
It washes 2-3 times, adds the immersion of 0.10% mercuric chloride solution and cover container lid and be placed on shaking table and rock 20-24min, make scale and rise
Mercury solution comes into full contact with, and will outwell in mercuric chloride solution on aseptic operating platform after taking-up, is cleaned 4-6 times with high-temperature sterilization water.
3) induction of Lilium lancifo1ium Thunb scale
Scale after preprocessed and sterilizing is crosscutting at 1cm2The fritter of left and right, is inoculated in respectively containing various concentration 2,4-
D (0.01,0.05,0.1,0.5mg/L) inducing culture on, 5 pieces of scales, each concentration cultures are put on every bottle of culture medium
Respectively put 10 bottles of culture mediums.It is 25 ± 2 DEG C that the condition of Fiber differentiation, which is in cultivation temperature, is cultivated 20 days, and humidity is 60%~70%,
Illumination condition is 1500lx-2000lx, 12~14h/d.
Fiber differentiation based component：MS+6-BA 1.00mg/L+NAA 0.15mg/L+KT 0.20mg/L+2-4,D(0.01、
0.05,0.1 or 0.5mg/L).
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.As shown in table 2.
Table 2 induces Lilium lancifo1ium Thunb scale (Initial culture) callus culture situation
Embodiment 3
1) pretreatment of Lilium lancifo1ium Thunb plateau
Plateau is cut from bulb base portion with pocket knife after washing the soil and impurity of tiger lily kind ball surface under tap water
Take, take no disease and pests harm, mechanical damage, healthy basal disc, impregnate 10min with aqueous soup solution, after cleaned with clear water after
It is allowed under water under clean circulating water and cleans 3h or more.
2) sterilizing of Lilium lancifo1ium Thunb plateau
The plateau cleaned up is first sterilized into 1min with 75% ethyl alcohol on aseptic operating platform, 2- is washed with high-temperature sterilization
3 times, then plateau 10-15min is impregnated with 0.20% mercuric chloride solution, it is during which placed on shaking table and it is allowed not stop to shake, purpose exists
Keep disinfection more thorough in allowing bulb basal disc adequately to invade bubble by mercuric chloride solution, high-temperature sterilization water is added after finally outwelling thimerosal
Cleaning 4-6 times.
3) induction of Lilium lancifo1ium Thunb plateau
Plateau after preprocessed and sterilizing is crosscutting at 1cm2The fritter of left and right, is inoculated in respectively containing various concentration
2,4-D (0.01,0.05,0.1,0.5mg/L) inducing culture in, 5 pieces of bulb basal discs, Mei Genong are put on every bottle of culture medium
Degree gradient puts 10 bottles of culture mediums.The condition of Fiber differentiation be in cultivation temperature be 25 ± 2 DEG C, cultivate 20 days, humidity is 60%~
70%, illumination condition 1500lx-2000lx, 12~14h/d.
Fiber differentiation based component：MS+6-BA 1.00mg/L+NAA 0.15mg/L+KT 0.20mg/L+2-4,D(0.01、
0.05,0.1 or 0.5mg/L).
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.As shown in table 3.
Table 3 induces Bulb of Lilium lancifolium disk callus culture situation
Embodiment 4
It randomly selects and generates after being cultivated 20 days in the inducing culture of every group of identical 2,4-D concentration in embodiment 1 or 2
Sterile clove 200, is inoculated in corresponding same concentrations 2 respectively, on the inducing culture of 4-D, inducing culturing condition：Culture temperature
Degree is 25 ± 2 DEG C, and humidity is 60%~70%, illumination condition 1500lx-2000lx, 12~14h/d Fiber differentiation 20-30
It.
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.As shown in table 4.
Table 4 induces the sterile clove callus culture situation of tiger lily
Pollution rate highest when explant is Bulb of Lilium lancifolium disk is can be seen that from table 1-4, pollution rate has reached 80% or more.
When explant is sterile clove, pollution rate is far below other explants, and only 2.5%, and callus induction rate and explant
Body is that bulbil or scale phase difference are little, can effectively solve the problems, such as that evoked callus pollution rate is high.When in inducing culture
Bud ratio highest when 2,4-D a concentration of 0.1mg/L, bulbil can reach 86%, and sterile clove can reach 87.5%.When luring
Callus rate highest when leading a concentration of 0.05mg/L of 2,4-D in culture medium, bulbil can reach 38%, but its pollution rate 14%；Nothing
Bacterium clove can reach 35%, and pollution rate is only 5%.Therefore, inducing culture is MS+6-BA 1.00mg/L+NAA
The induction for being conducive to Lilium lancifo1ium Thunb callus when 0.15mg/L+KT 0.20mg/L+2-4, D 0.05mg/L, with existing callus
Tissue lily inducing culture in plant hormone differ greatly, reason may be different cultivars lily Endogenous Hormones not
Together, therefore in cultured in vitro required exogeneous growth conditioning agent also differs.Because exogenous hormone is mainly by influencing to plant
Object endogenous hormones play a role, the function and effect of exogenous hormone and the type of explant and callus itself endogenous hormones and
Level has substantial connection.In addition, after sterile adventitious bud being transferred in fresh inducing culture and carrying out continuing culture, can limit
It differentiates adventitious bud, and induced synthesis callus (block).
Embodiment 5
Experimental procedure is respectively 0-300lx, 700-900lx and 1500-2000lx, light with embodiment 4, wherein intensity of illumination
It is 12~14h/d according to the time, inducing culture is MS+6-BA 1.00mg/L+NAA 0.15mg/L+KT 0.20mg/L+2-4, D
0.05mg/L。
Sterile clove is transferred in fresh inducing culture and carries out continuing to cultivate, material is seen at 20 days
It examines, after being transferred to new culture medium, can induce and form callus, and limit it and differentiate adventitious bud.As shown in Fig. 2, in light
The callus size formed under the conditions of being 0-300lx according to condition is unequal, near-white transparence (figure A).It is in illumination condition
Under the conditions of 700-900lx, callus lines are easily formed, the callus pigment content of generation is low, is transparence (figure B)；And
The callus pigment content that illumination condition is formed under the conditions of being 1500-2000lx is higher, opaque (figure C).
Pollution rate, bud ratio, the Callus induction rate of statistical material at 20 days.As shown in table 5.
Under 5 different illumination intensity of table the case where Lilium lancifo1ium Thunb callus
Such as table 5 as can be seen that when intensity of illumination is 700lx-900lx, callus rate is highest, and sterile clove is cured within 20 days
Hinder inductivity and reach 71%, it is extremely inefficient to solve the problems, such as that existing inducing culture induction Lilium lancifo1ium Thunb generates callus.
Improve 2.38 times compared with dark condition callus rate, 1.22 times improved compared with intense light conditions callus rate, this with generally believe it is dark right
Lily callus has facilitation not to be consistent.This is because certain illumination can adjust it is certain endogenous in Lilium lancifo1ium Thunb body
Hormonal readiness, to induce generation callus, and the Endogenous Hormones of different cultivars lily are different, so to intensity of illumination
Demand it is different.
Finally illustrate, the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although with reference to compared with
Good embodiment describes the invention in detail, it will be understood by those of ordinary skill in the art that, it can be to the skill of the present invention
Art scheme is modified or replaced equivalently, and without departing from the objective and range of technical solution of the present invention, should all be covered at this
In the right of invention.
Claims (7)
1. a kind of abductive approach of Lilium lancifo1ium Thunb callus, which is characterized in that include the following steps：
By after pretreatment and sterilizing Lilium lancifo1ium Thunb scale or bulbil be inoculated in inducing culture and carry out Initial culture, illumination is strong
Degree is 700 lx-900 lx, and Fiber differentiation 20-30 days is to get to the callus of Lilium lancifo1ium Thunb and sterile adventitious bud；It is described to lure
It is MS+1.00 mg/L 6-BA+0.15 mg/L NAA+0.20 mg/L KT+0.05 ~ 0.1 mg/L 2-4, D, training to lead culture medium
It is 5.8 ~ 6.2 to support base pH value.
2. the abductive approach of Lilium lancifo1ium Thunb callus according to claim 1, which is characterized in that the scale is bulb
Middle level or outer scale.
3. the abductive approach of Lilium lancifo1ium Thunb callus according to claim 1, which is characterized in that further including will be described sterile
The base portion enlargement switching of adventitious bud continues to cultivate in the inducing culture, and intensity of illumination is 700 lx-900 lx, is lured
Culture is led 20-30 days to get to the callus of Lilium lancifo1ium Thunb.
4. the abductive approach of Lilium lancifo1ium Thunb callus according to claim 1, which is characterized in that the inducing culture is
MS+1.00 mg/L 6-BA+0.15 mg/L NAA+0.20 mg/L KT+0.05 mg/L 2-4, D, Medium's PH Value is 5.8 ~
6.2。
5. according to the abductive approach of the Lilium lancifo1ium Thunb callus of claim 1 or 3, which is characterized in that the Fiber differentiation
Condition be cultivation temperature be 25 ± 2 DEG C, humidity is 60% ~ 70%, and light application time is 12 ~ 14 h/d.
6. the abductive approach of Lilium lancifo1ium Thunb callus according to claim 1, which is characterized in that the pretreatment is by hundred
It closes bulbil or scale detergent solution and impregnates 5-10 min, then 2 h or more are rinsed with the tap water of flowing；The sterilizing is will
Pretreated Lilium lancifo1ium Thunb bulbil or scale are placed on superclean bench, and it is 70 ~ 75% ethanol water first to use volumetric concentration
Handle 20 ~ 30 s, then with sterile water wash at least 2 times, then with mass concentration for 0.1 ~ 0.2% mercuric chloride solution sterilization treatment
10 ~ 25 min, finally with sterile water washing at least 4 times.
7. the abductive approach of Lilium lancifo1ium Thunb callus according to claim 6, which is characterized in that the detergent is fertilizer
Soap, washing powder or sapindust saponin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810343821.3A CN108450333B (en) | 2018-04-17 | 2018-04-17 | Induction method of lilium tigrinum callus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810343821.3A CN108450333B (en) | 2018-04-17 | 2018-04-17 | Induction method of lilium tigrinum callus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108450333A true CN108450333A (en) | 2018-08-28 |
| CN108450333B CN108450333B (en) | 2020-02-07 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113475399A (en) * | 2021-07-30 | 2021-10-08 | 重庆临空都市农业开发建设有限公司 | Quick breeding method for lily non-toxic seedling |
Citations (2)
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| JPH04287623A (en) * | 1991-03-15 | 1992-10-13 | Mitsui Petrochem Ind Ltd | Method for proliferating bulbs by plant tissue culture |
| CN104396742B (en) * | 2014-10-30 | 2017-03-15 | 毕节市中药研究所 | The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04287623A (en) * | 1991-03-15 | 1992-10-13 | Mitsui Petrochem Ind Ltd | Method for proliferating bulbs by plant tissue culture |
| CN104396742B (en) * | 2014-10-30 | 2017-03-15 | 毕节市中药研究所 | The method that aseptic seedling is differentiated again with five-step approach induction Lilium sulphureum Baker bulbil calluss |
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| 文涛等: "不同光照强度对虎杖愈伤组织的影响", 《中国中药杂志》 * |
| 谢杰等: "宜兴百合通过愈伤组织诱导再生鳞茎的研究", 《上海农业学报》 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113475399A (en) * | 2021-07-30 | 2021-10-08 | 重庆临空都市农业开发建设有限公司 | Quick breeding method for lily non-toxic seedling |
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