CN106973791A - A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant - Google Patents

Abstract

A kind of method that ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi produces mutant, it is therefore an objective to which breed improvement and New idioplasm resource initiative for day lily provide EMS Vitro Mutation methods, and obtain target variant；The present invention is using the hemerocailis middendorffi bud for normal 1 2cm that grows as material, and the callus access differential medium induced by explant of ovary differentiates green budlet；Band bud callus differentiation culture uses concentration to be 0.75-1.0% after 10 days（w/v）EMS half lethal doses handle the callus lines；Callus after processing is inoculated in differential medium and carries out differentiation culture, obtains regeneration plant；Plant length to be regenerated to 1 2cm it is high when, switching is cut from callus and enters subculture medium squamous subculture 15 days, using concentration as 40%（v/v）Tawny daylily leaf spoting bacteria toxin half lethal dose for M8003 line pressure carry out stress screening, obtain resistant mutant plant.

Description

A kind of method that ethylmethane sulfonate Vitro Mutation hemerocailis middendorffi produces mutant

Technical field

The present invention relates to a kind of flowers and trees --- and the method that hemerocailis middendorffi in-vitro inducing mutant occurs, is specially a kind of utilize Chemical mutagen ethylmethane sulfonate (EMS) carries out mutagenic treatment to hemerocailis middendorffi callus, and induction produces the skill of mutant Art method.

Background technology

Tawny daylily has thousands of years culture history in China, tawny daylily also known as forgets grass, and it is exactly the meaning forgotten to forget, and also referred to as forgets sorrow grass.Tawny daylily Careless (Hemerocallis hybrida) is Liliaceae hemerocallis perennial herb.Its leaf is like blue, flower such as lily.Have light Several patterns in yellow, golden yellow, light yellow, bright red, pink, secondary color etc. ten, it is gorgeous in riotous profusion；Flower pattern is colourful, has single-lobe, polyphyll, petal anti- Volume, it is graceful, it is worth with high ornamental plantation.Hemerocailis middendorffi dries cold district in the north and shows fabulous cultivation Petter：It has short root-like stock and fleshy hypertrophy fusiform root tuber, and drought tolerance, cold resistance are extremely strong, are planted with being largely used as Thing, part substitutes lawn；Its extensive management, maintenance cost is the 1/5 of turfgrass, is outstanding Garden Greenland flowers.It is complete at present State is being widely applied the new varieties " golden doll " mainly introduced from America and Europe of plantation, and cover plant has been accounted for using area More than 40%.Its plant is short and small, tiller fast, flower amount is big, and the florescence is long, is particularly suitable for the northern area of China plantation.Polyploid " good luck " is the new varieties with great market development potentiality.

The breeding research 19th-century of tawny daylily is just in America and Europe's rise.So far, garden-variety up to more than 60,000 is planted.Last century The fifties to the eighties, people cultivate the tawny daylily kind of multi-color, gauffer and polyphyll by the method for crossbreeding.Then, Breeding direction starts to turn to polyploid breeding.With the cultivation success of tetraploid tawny daylily, tawny daylily kind has obtained great rich Richness, as one of kind most abundant Perennial Flowers.

Domestic tawny daylily breeding research is concentrated mainly on the exploitation side of cross compatibility, reproduction isolation and wild resource Face.In terms of breeding objective focuses on pattern, flower pattern, the fragrance of a flower and florescence change.More than breeding mode using crossbreeding and Ploidy breeding method.With the constantly improve of plant tissue culture technique, combined using tawny daylily tissue or Organ culture method The method of colchicine-induced polyploid greatly improves breeding efficiency.

Chemical mutation is a kind of hereditary variation of artificial utilization chemical mutagen inducing plant, can effectively be overcome Cross incompatibility and the phenomenon of reproduction isolation, obtain the mutant material for having value, further according to breeding in a short time Directed screening is used for production application after obtaining new varieties and stable heredity the need for target.It is quick with cell engineering Development, mutagenesis and excised cotyledon technology are combined, and with not carried out by the crop field ambient influnence anniversary, shorten breeding Time, the advantage for expanding spectrum of variation and raising aberration rate.Ethylmethane sulfonate (Ethyl methane sulfonate, EMS) is made It is by inducing point mutation with the directly reaction of the phosphoric acid in nucleotides, pyrimidine and purine, in many plants for chemical mutagen On all obtain different types of mutant, applied in the improvement of many crops and ornamental plant.Luo Jing^【1】Using 0.2%EMS immersion treatment 1hrs three kinds of callus lines of strawberry, wherein with the callus induction best results directly produced, Produce botrytis resistant mutant.Yang Mei^【2】Using banana callus and Bud Differentiation as acceptor, 0.5%EMS processing 40min are obtained " half lethal dose effect ", the callus group that selection obtains resisting banana vascular wilt verticillium toxin is coerced by banana blight bacteria gradient Knit block and Bud Differentiation.Cai Haiyan etc.^【3】Chrysanthemum stem apex is handled using EMS mutagenesis, the phenotypic variation rate of mutagenized populations reaches To 6.31%, Mutagenic Effect is fairly obvious.EMS Vitro Mutations acceptor material produces the research of mutant in crops and gardening The ratio carried out in plant is wide, is rarely reported in terms of gardening ornamental plant breeding, in terms of hemerocailis middendorffi mutation breeding Research has no report.

The content of the invention

The present invention seeks to overcome the shortcomings of above-mentioned prior art there is provided one kind using chemical mutagen ethylmethane sulfonate The method that Vitro Mutation hemerocailis middendorffi produces mutant.The present invention can be breed improvement and the New idioplasm resource of day lily Initiative provides the method for EMS Vitro Mutations, and obtains target variant.

The method that ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi of the present invention produces mutant, is to grow just Normal 1-2cm hemerocailis middendorffi bud is material, the callus access differential medium induced by explant of ovary, point Dissolve green budlet.The EMS semilethal agent that concentration is 0.75-1.0% (w/v) is used after breaking up culture 10 days with bud callus Amount handles the callus lines.Callus after processing is inoculated in differential medium and carries out differentiation culture, obtains regeneration and plants Strain.Plant length to be regenerated to 1-2cm it is high when, switching is cut from callus and enters subculture medium squamous subculture 15 days, with The tawny daylily leaf spoting bacteria toxin half lethal dose that concentration is 40% (v/v) is that M8003 line pressure carries out stress screening, is obtained disease-resistant Mutant plants.

Using the normal 1-2cm hemerocailis middendorffis bud that grows as material, disappeared under aseptic condition with the mercuric chloride of 0.1% concentration Poison sterilizing, aseptic water washing 3-4 times.Petal is peeled off under aseptic condition, exposes ovary, crosscutting flakiness is inoculated in callus Inducing culture；25 DEG C, 26-28 days under the conditions of light culture, evoked callus is produced；Callus inducing medium：MS is complete Amount+2mg/L2,4-D+1mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders, pH5.8.

The callus that ovary is induced is inoculated on differential medium, 25 DEG C, illumination cultivation, intensity of illumination 2000Lux, treats that callus differentiates green budlet.It is differentiated go out green bud point callus be cut into 0.3-0.5cm³ Size, continue break up culture 10 days with acceptor material of the bud callus as EMS mutagenesis.

Callus differential medium：MS full dose+0.5mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders； pH5.8.The EMS half lethal doses of 0.75-1.0% (w/v) concentration are configured to 0.05M pH7.0 phosphate buffer EMS concentration is 0.75-1.0% (w/v) EMS half lethal dose treatment fluids.

Under aseptic condition, by 0.3-0.5cm³It is 0.75-1.0% (w/v's) to be transferred with bud callus lines into concentration In EMS half lethal dose treatment fluids, with 28 DEG C, 150 revs/min, shaken cultivation 1 hour is EMS mutagenic treatment methods.At mutagenesis Band bud callus after reason is aseptically rinsed 3-4 times with sterile distilled water, and filter paper suck dry moisture accesses callus group Knit differential medium, 25 DEG C, illumination differentiation culture.Dead callus is eliminated, the callus survived continues to break up training Support, obtain mutant regeneration plant.

When mutant regeneration plant length is high to 1-2cm, is cut from callus and 15 are cultivated on subculture medium My god.Switching enters the selection pressure screening and culturing medium for the thick tawny daylily leaf spoting bacteria toxin that with the addition of 40% (v/v) concentration, is selected Select pressure stress screening.Death plant is eliminated, plant is survived and is transferred to root media, culture of rootage turns into complete disease-resistant mutation Body material.

Subculture medium：MS full dose+1.0mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders, pH5.8；

Selection pressure screening and culturing medium：The thick verticillium toxin liquid of subculture medium+40% (400ml/L) tawny daylily leaf blight, pH5.8；

Root media：1/2MS+0.1mg/LNAA+20g/L sucrose+8g/L agar powders, pH5.8.

It is day lily the method have the advantages that carrying out Vitro Mutation research on hemerocailis middendorffi using EMS induced-mutation techniques Breed improvement and New idioplasm resource initiative the method for EMS Vitro Mutations is provided, and obtain target variant, improve breeding skill Art level.Using band bud callus as first choice in the acceptor material selection of EMS Vitro Mutation hemerocailis middendorffis, it can effectively improve and lure The seedling efficiency of mutant after change.Half lethal dose of the EMS mutagenesis with bud callus is at 0.75-1.0%, 25 DEG C of vibrations Manage 1hrs.

The present invention with most widely used " golden doll " or " good luck " be material, " using ovary as explant callus induction group Knit " it is in-vitro culture method；Using agitated submerged culture as EMS mutagenic treatment methods；To cut into 0.3-0.4cm³, preculture The callus lines of 10d hemerocailis middendorffi tool Bud Differentiation are acceptor material, using 0.75-1.0% EMS concentration as semilethal agent Amount, 150r/min, 25 DEG C of vibration mutagenic treatment 1hrs, induces DNA mutation on a molecular scale；Using germ crude venom as selection Pressure orientation stress screening hemerocailis middendorffi anti-leaf blight mutant is detection method, verify the EMS mutagenesis systems validity and can Row.Take 0.75-1.0% scope actual compared with single dose, more scientific in the determination of Induced dosage.In acceptor material The planting percent after mutagenesis is improved in the selection of material, efficiency of inducing mutation is significantly improved.EMS induced-mutation techniques answering on hemerocailis middendorffi Report is had no with research.This technology is not only applicable to breeding for disease resistance, is also applied for hemerocailis middendorffi in terms of biological character The mutant type that the characters such as flower pattern pattern, blade profile leaf color and florescence change is produced, there is higher ornamental value for screening New varieties there is very universal meaning.

Brief description of the drawings

Fig. 1 is the broken line of " influence of various concentrations, different time EMS mutagenic treatments to I class callus survival rates " Figure；

Fig. 2 is the line chart of " influence of same concentration, different time EMS mutagenic treatments to II class callus survival rates "；

Fig. 3 is the line chart of " various concentrations EMS mutagenic treatment I classes, the influence of II class callus planting percents "；

Fig. 4 is the line chart of " lethal effect of the various concentrations dead leaf virusin to hemerocailis middendorffi regeneration plant ".

Embodiment

1. the acquisition of hemerocailis middendorffi ovary callus

1.1 experimental method：

1.1.1 material

Experiment derives from Dry Farm Agricultural Research Centre, Shanxi Academy of Agriculture Dongyang Demonstration Base experimental plot with tawny daylily.2- 3 years raw hemerocailis middendorffis " golden doll " and " good luck ".

1.1.2 method

Use the patented technology " the hemerocailis middendorffi tissue culture technique method by explant of ovary " of our unit【It is Chinese special Profit number 2011100940526】Carry out explant inoculation evoked callus.

Full-bloom stage takes the normotrophic bud in crop field, rinses the dust of outside well with running water plus detergent.With 75% (v/v) ethanol immersion 3-5min, distilled water flushing 1-2 times, then with 0.1% (w/v) HgCl₂Sterilize 8-10min, sterilized water Immersion, flushing 4-5 times, then strip external petal, exposes ovary, and crosscutting slabbing is seeded on inducing culture.Per ware 30 More than block (stripping and slicing is too small, bad statistics), 25 ± 1 DEG C, light culture.The callus of induction 26-28 days is transferred to differential medium Carry out 25 ± 1 DEG C of optical culture 15-26d.Callus with green bud point is cut into about 0.2-0.3cm³Fritter continue Differential medium is inoculated in, preculture for a period of time, carries out EMS processing.

Inducing culture：MS_{Full dose}+ 6BA2mg/L+IBA0.5mg/L+2.4-D1mg/L+ sucrose 30g/L+ agar powders 8.6g/ L (Ph5.8),

Differential medium：MS_{Full dose}+ 6BA0.5mg/L+IBA0.2mg/L+ sucrose 30g/L+ agar powders 8.6g/L (Ph5.8)

1.1.2 result and analysis

This method is patented technology " the hemerocailis middendorffi tissue culture technique method by explant of ovary for using our unit 【China Patent No. 2011100940526】" carry out explant inoculation and evoked callus.

The hemerocailis middendorffi bud of length 1-2cm normal developments is selected, 1-3min, 0.1% are soaked with 70% ethanol HgCl₂High intensity sterilizing bud 10-15min, sterile stripping ovary is explant, then cuts into slices and is seeded in Fiber differentiation respectively On base, 25 ± 1 DEG C, dark culturing 26-28 days, induction produce callus；Callus is transferred into subculture differential medium to enter 25 ± 1 DEG C of row, illumination cultivation 15 days, callus differentiates green bud point, is denoted as I class callus；Differentiation culture 20-26 Its callus produces Bud Differentiation, is denoted as II class callus.

2. the callus EMS half lethal dose parameters of different differentiation states

2.1 test method

2.1.1 material

Be divided into according to the different growth conditions of callus have just enter into the idiophase occur green bud point callus (note Make：I classes callus) and it is differentiated go out green budlet callus (be denoted as：II classes callus) 2 kinds, size is about 0.3-0.5cm³。

2.1.2 method

2.1.2.1 the half lethal dose of EMS mutagenesis is determined

Mutagenic treatment uses agitated submerged culture method.EMS treatment dosages are set as 0.25% (W/V), 0.5%, 0.75% With 1.0%, prepared with pH7.0,0.05M phosphate buffer, be not added with EMS for CK.150 revs/min of shaken cultivation, 28 DEG C, Time is set as 30min, 60min and 90min.Callus after processing aseptic water washing 3 times, suck dry moisture is inoculated in Subculture medium, illumination cultivation 26d, count survival number, calculate survival rate, it is every can on subculture medium continued growth or The I class callus that new callus is grown in browning tissue is designated as surviving.It is every can the continued growth on subculture medium Or browning tissue Bud Differentiation can continue survival II classes callus be all designated as surviving.This experiment to I classes callus, Two kinds of callus of II classes callus are carried out respectively, each 3 repetitions of processing, each to repeat 20 block of material.

Survival rate (%)=(survival number/inoculation number) × 100%

Subculture medium：MS full dose+6BA1.0mg/L+IBA0.2mg/L+ agar powder 8g/L+ sucrose 30g/L pH 5.8

By the calculating of survival rate, EMS half lethal doses (50%) are determined.

2.1.2.2EMS the planting percent of mutagenesis different type callus

Two kinds of callus after EMS mutagenic treatments count survival rate after differentiation culture in 26 days.By the callus group survived Knit subculture to continue to cultivate 26 days to differential medium, cut the effective regeneration seedling that length is 1-2cm, be inoculated in root media, Carry out culture of rootage.Statistics survives number, calculated seeding rate.

Planting percent (%)=effective seedling number of differentiation/callus number × 100% survived

Root media：1/2MS+NAA0.2mg/L+ agar powder 0.8g/L+ sucrose 20g/L pH 5.8

EMS optimization process effect systems are determined by finally counting differentiation seedling number.

2.2. result and analysis

2.2.1 the preculture of callus

Two class callus are cutting into 0.2-0.3cm³Fritter after the pre- of 10d will be carried out on subculture medium Culture, could then carry out EMS processing.Purpose is the wound tissue after healing cutting, impaired thin to avoid EMS from directly encroaching on Born of the same parents, cause the damage of wound face, and then trigger browning, the death of callus, cause the inaccurate of statistical result.

2.2.2 EMS half lethal dose effects

The EMS solution of 4 kinds of concentration (0.25%, 0.50%, 0.75% and 1.0%) is can be seen that to big from table 1 and Fig. 1 Flower tawny daylily callus has different degrees of injury effect.With the increase and the extension of processing time for the treatment of fluid concentration, I There is the trend gradually reduced in the survival rate of class callus.When reaching 30min between when treated, EMS dosage is 1.0% When, I class callus survival rates still reach 60.0%, and the tolerance to EMS reaches more than 1.0%.Reached between when treated During 60min, I classes callus is reduced to 0.50~0.75% to EMS tolerable concentration, survival rate 51.6%~ 47.5%.When extending to 90min between when treated, water stain sample pellucidity occurs in part callus surface, either high Concentration or low concentration, can all cause I class callus mortalities, survival rate is all below 40%.

It can also be seen that the II classes callus with Bud Differentiation is carrying out the process of EMS mutagenic treatments from table 1, Fig. 2 In show the variation tendency similar with I class callus.The difference is that II classes callus is when processing time is 60min, Tolerable concentration to EMS is 0.75~1.00%.It is same it has been observed that when extending to 90min between when treated, partial differentiation Water stain sample wet face state also occurs in the blade of bud, and this partial blade can soften yellow quickly.

This experiment filters out different EMS half lethal doses for I classes callus and II class callus.I class callus The EMS mutagenesis system of tissue is that 0.50-0.75%EMS handles 60min；The EMS mutagenesis system of II class callus is 0.75- 1.00%EMS handles 60min.

The survival rate of two class callus of the EMS mutagenic treatments of table 1

Influence of the 2.2 EMS mutagenic treatments to two class callus planting percents

Whether what EMS half lethal doses were investigated is the survival rate after acceptor material processing certain time, and can be with after surviving It is the key factor for being related to efficiency of inducing mutation to regenerate effective plant.Therefore, studied on the premise of half lethal dose rate at EMS The planting percent problem for managing different acceptor materials seems particularly significant.

It can be seen that from table 2, Fig. 3：II classes callus is with the increase of EMS mutagenesis concentration, and survival rate is in becoming for declining Gesture.The differentiation culture of the callus survived again Jing Guo a generation, even part callus appearance are light brown to tend to be dead The state died, but differentiation seedling thereon still survives and regenerates seedling.Planting percent reaches more than 250%, different EMS processing Influence of the dosage to planting percent is little, between 254.2-280.9%.That is II classes callus late growing stage by EMS long lasting effect is smaller.And the callus that I classes callus is survived in the follow-up generation of EMS half lethal doses processing Continue to be inoculated in differential medium, most of material continues to death, the green bud points of only a small number of parcels in the tissue into It is living, and subculture differentiates regeneration plant.The sustained response to EMS sensitiveness is shown, differentiation capability substantially weakens, highest is only There is 138.5% during 0.25% dosage, effective seedling number of differentiation significantly reduces 150% compared with II class callus.

The EMS of table 2 handles the planting percent of the class callus of hemerocailis middendorffi two

3. resistant mutant selection pressure screening and acquisition

3.1 test method

Hemerocailis middendorffi antitoxin mutant is screened using a step back-and-forth method

3.1.1 for examination hemerocailis middendorffi leaf blight bacterial strain

Hemerocailis middendorffi leaf spoting bacteria (Kabatiella microsticta) is taught by agricultural college of Jilin Agriculture University Bai Qingrong Separation, preservation, and friendship is awarded to give.

3.1.2 the preparation of hemerocailis middendorffi leaf spoting bacteria Raw toxin

3 pieces that the bacterium colony 1*1cm being incubated in PDA culture medium is cut under aseptic condition are inoculated in sterile 500mlPS trainings In nutrient solution, 25 DEG C are placed in, under the conditions of light culture, 150r/min shaken cultivations 20-28 days.With spectrophotometer 600nm's OD values are determined under wavelength between 1.40-1.60.Bacterium solution is centrifuged with 8 layers of filtered through gauze mycelium, filtrate through 3500r/min 30min, takes supernatant to produce pathogen crude venom.

PS culture mediums：30min, 4 layers of filtered through gauze are boiled in 100g peeled potatoes strippings and slicings.Filtrate adds 10g sucrose, constant volume It is standby after autoclaving to 500ml.

3.1.3 disease-resistant selection pressure half lethal dose screening

Acceptor type is：Effective seedling high 1-2cm that differentiates of II class callus after the processing of EMS half lethal doses.

Stress screening uses a step plate method.The leaf of 20%, 40% and 60% concentration is added in subculture medium Rot verticillium toxin liquid.It is control without toxin solution.8 plants every bottle, 8 bottles are often handled, in triplicate.25 ± 2 DEG C of illumination Illumination cultivation in incubator, illumination condition is 16hrs light/8hrs dark, intensity of illumination 2800lx, observes the life by stress material Long situation, 26d statistics survival rates.

3.2 results and analysis

Carrying out disease-resistant detection to a large amount of regeneration plants after EMS processing needs very big plantation space, when spending very long Between and long time.Disease-resistant variant is screened using in vitro stress, can in limited room and time and stably Environmental condition completes fast and efficiently to screen to bulk materials, obtains disease-resistant strain.

There is blade tip the 5th day since the regrowth illumination cultivation being inoculated on the subculture medium containing different Raw toxin liquid Turn yellow, extend gradually downward with the extension yellow of incubation time, and engender multiple-blade while occurring Disease symptoms, Until plant part somatic death.As can be seen from Figure 4：With the rise of pathogenic toxin concentration, the survival rate of regeneration plant is in Downward trend.The seedling growth of 20% toxin processing is normal, and yellow leaf defect phenomenon occur in indivedual blades of indivedual plant, Survival rate 89.3%；The indivedual plant strain growths of material of 40% toxin stress are more healthy and stronger, and plant part growth is basic to be stopped, greatly There are disease conditions in partial blade, and survival rate drops to 48.4%.The material of 60% toxin processing can clearly be seen that whole plants Strain stops growing and shows as yellow leaf, the serious plant disease stress of whole strain morbidity, and survival rate only has 39.1%.Equally, from table 3 statistics it is also seen that：40% toxin dose, when cultivating 18 days, survival rate to 48.4%, close to 50%, institute To determine that the addition of 40% toxin coerces close rate for semilethal.

Lethal effect of the dead leaf virusin of table 3 to hemerocailis middendorffi regeneration plant

4. EMS mutagenic treatments and the selection pressure screening of pathogenic toxin half lethal dose obtain anti-leaf blight mutant plants

4.1 test method

4.1.1 EMS method of mutagenesis：The result of the test drawn using this experiment 2.Test material is II class callus. EMS mutagenesis system is that 0.75%EMS half lethal doses handle 60min.Mutagenic treatment use agitated submerged culture method, 150 turns/ Point, 25 DEG C.

4.1.2 screening technique is pressed in the sick Raw toxin selection of dead leaf：The test method drawn using this experiment 3.Test material For：Effective seedling high 1-2 that differentiates of II class callus after the processing of EMS half lethal doses.

Stress screening uses a step plate method.The leaf spoting bacteria toxin of 40% concentration is added in subculture medium Liquid.Illumination cultivation 26d in 25 ± 2 DEG C of illumination box, illumination condition is 16hrs light/8hrs dark, intensity of illumination 2800lx. The positive plant survived is transferred to root media and carries out culture of rootage, and rooted seedling passes through transition domesticating and cultivating intermediate house, then Into grown in field, disease-resistant rank identification is further carried out.

4.2 results and analysis

The experiment is that we obtain the orientation stress screening process of target gene, is also the checking to EMS method of mutagenesis.

We are total to mutagenic treatment " good luck (H) " 7 batches in aforementioned manners, and totally 1939 pieces of callus, survive 935 Block callus, average survival 49.0%.806 plants of regeneration plant.760 plants of 40% crude venom sieve stress Screening Treatment regeneration plant, Into 391 plants of live seedling, average survival 52.7%.The positive plant of intermediate house has 297 plants after transiting cultivation.Primarily determine that for Anti- hemerocailis middendorffi leaf blight mutant material, disease-resistant rank needs further classification identification (being shown in Table 4).

The acquisition of the anti-leaf blight mutant material of the hemerocailis middendorffi of table 4 " good luck "

We are total to mutagenic treatment " golden doll (J) " 5 batches, and totally 1446 pieces of callus, survive 530 pieces of callus, put down Equal survival rate 46.8%, 543 plants of regeneration plant.491 plants of 40% crude venom stress Screening Treatment regeneration plant, into live seedling 184 Strain, average survival 50.7%.The positive plant of intermediate house has 204 plants after transiting cultivation.Primarily determine that as anti-hemerocailis middendorffi Leaf blight mutant material, disease-resistant rank needs further classification identification (being shown in Table 5).

The acquisition of the hemerocailis middendorffi of table 5 " golden doll " anti-leaf blight mutant material

In addition it has also been found that the variant of part biological character, such as blade streak occur and blade profile shortens the class broadened Type.

5th, discuss：

5.1 find by multiple repetition test, and the acceptor material for receiving EMS mutagenic treatments is biological tissue or organ, It is an effective dosage range to the tolerable half lethal doses of EMS, rather than a dose point.To callus semilethal Dosage is 0.50-0.75%, is then 0.75-1.0% to the callus with bud.Fatal rate is protected substantially in the range of this Hold 50% or so.Such dosage range is more scientific.

5.2 due to EMS Vitro Mutations be biological tissue, and growth conditions have very big difference between individual, between batch It is different.It is difficult to accomplish complete unified although in test selecting consistent material as far as possible.Survival rate after per batch processed Change in the presence of certain height.But in terms of multiple batches of whole result, average survival illustrates the technical side 50% or so The accuracy and feasibility of method.

5.3, from this result of study, the efficient of seedling are had more with bud callus as acceptor material than callus Property.Death, and differentiation are still may proceed in the material later stage incubation even survived after EMS infringement callus Rate is very low, shows as EMS continued damage effect.And the callus with bud also can browning, death by EMS infringement.Parcel Bud Differentiation thereon remains to the nutrient growth provided using perienchyma's cell.Although yellow leaf, cut down after It can quickly be restore normal growth on culture medium, grow young leaves.So, using with bud callus as EMS mutagenesis acceptor material Material can effectively improve efficiency of inducing mutation.

Type in research for the callus of mutagenesis acceptor material has carried out 6 repetition experiments altogether, tests every time not With Callus Types 180-200 block of material, and Germicidal efficacy and data statistics and analysis are carried out, drawn with band bud callus It is organized as suitable acceptor material.

It is more accurate for data due to the otherness of Physiology and biochemistry state between acceptor material individual and different batches Really, we have carried out 10 multiplicating experiments altogether for the treatment fluid concentration in EMS half lethal dose and processing time, often Secondary each about 100-120 pieces of callus of processing (5-6 culture dish, per about 20 pieces of ware).Through experimental observation, data statistics and Analysis show that EMS is to half lethal dose rate of the hemerocailis middendorffi with bud callus under conventional sense.

Mutagenic treatment " good luck (H) " 7 batches are total in aforementioned manners, totally 1939 pieces of callus, survive 935 pieces more Wound, average survival 49.0%.806 plants of regeneration plant.760 plants of 40% crude venom sieve stress Screening Treatment regeneration plant, is survived 391 plants of seedling, average survival 52.7%.The positive plant of intermediate house has 297 plants after transiting cultivation.

Mutagenic treatment " golden doll (J) " 5 batches are total in aforementioned manners, and totally 1446 pieces of callus, survive 530 pieces Callus, average survival 46.8%, 543 plants of regeneration plant.491 plants of 40% crude venom sieve stress Screening Treatment regeneration plant, into 184 plants of live seedling, average survival 50.7%.The positive plant of intermediate house has 204 plants after transiting cultivation.

It is anti-hemerocailis middendorffi leaf blight mutant material to primarily determine which part material.Disease resistance, disease-resistant rank needs Further identification.In addition it has also been found that 3 plants of the variant of part biological character, streak occurs in such as blade and blade profile shortens Wide type.

Outside this research, the research of half lethal dose also has been carried out to the seed of hemerocailis middendorffi " golden doll " with EMS. Mutagenesis obtains 42% survival rate (relative to control), the material of part Albino Seedling and part leaf color and Change of leaf form occurs.

Claims (6)

1. a kind of method that ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi produces mutant, it is characterized in that to grow Normal 1-2cm hemerocailis middendorffi bud is material, the callus access differential medium induced by explant of ovary, point Dissolve green budlet；Band bud callus differentiation culture uses concentration to be 0.75-1.0% after 10 days（w/v）EMS half lethal doses Handle the callus lines；Callus after processing is inoculated in differential medium and carries out differentiation culture, obtains regeneration plant；Treat Regeneration plant length to 1-2cm it is high when, switching is cut from callus and enters subculture medium squamous subculture 15 days, using concentration as 40%（v/v）Tawny daylily leaf spoting bacteria toxin half lethal dose for M8003 line pressure carry out stress screening, obtain resistant mutant plant Strain.

2. ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi as claimed in claim 1 produces the method for mutant, its feature It is：Using the normal 1-2cm hemerocailis middendorffis bud that grows as material, gone out under aseptic condition with the mercuric chloride sterilization of 0.1% concentration Bacterium, aseptic water washing 3-4 times；Petal is peeled off under aseptic condition, exposes ovary, crosscutting flakiness is inoculated in callus induction Culture medium；25 DEG C, 26-28 days under the conditions of light culture, evoked callus is produced；Callus inducing medium is：MS full doses+ 2mg/L2,4-D+1mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders；pH5.8.

3. the method that ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi as claimed in claim 1 or 2 produces mutant, its It is characterized in：The callus that ovary is induced is inoculated on differential medium, 25 DEG C, illumination cultivation, intensity of illumination 2000Lux, treats that callus differentiates green budlet；It is differentiated go out green bud point callus be cut into 0.3-0.5cm³Greatly It is small, continue break up culture 10 days with acceptor material of the bud callus as EMS mutagenesis；Callus differential medium is： MS full dose+0.5mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders；pH5.8.

4. ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi as claimed in claim 1 produces the method for mutant, its feature It is 0.75-1.0%（w/v）The EMS half lethal doses of concentration are to be configured to EMS concentration with 0.05M pH7.0 phosphate buffer For 0.75-1.0%（w/v）EMS half lethal dose treatment fluids.

5. the method that the ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi as described in claim 1,3 or 4 produces mutant, It is characterized in that：Under aseptic condition, by 0.3-0.5cm³It is 0.75-1.0% that band bud callus lines, which are transferred into concentration,（w/v）'s In EMS half lethal dose treatment fluids, with 28 DEG C, 150 revs/min, shaken cultivation 1 hour is EMS mutagenic treatment methods；Mutagenic treatment Band bud callus afterwards is aseptically rinsed 3-4 times with sterile distilled water, filter paper suck dry moisture, access callus point Change culture medium, 25 DEG C, illumination differentiation culture；Dead callus is eliminated, the callus survived continues to break up culture, obtains Mutant regeneration plant.

6. ethylmethane sulfonate EMS Vitro Mutations hemerocailis middendorffi as claimed in claim 1 produces the method for mutant, its feature It is：When mutant regeneration plant length is high to 1-2cm, cuts and cultivated 15 days on subculture medium from callus；Switching Into with the addition of 40%（v/v）The selection pressure screening and culturing medium of the thick tawny daylily leaf spoting bacteria toxin of concentration, carries out selection pressure stress sieve Choosing；Death plant is eliminated, plant is survived and is transferred to root media, culture of rootage turns into complete resistant mutant material；

Subculture medium：MS full dose+1.0mg/L6BA+0.2mg/LIBA+30g/L sucrose+8g/L agar powders, pH5.8；

Selection pressure screening and culturing medium：Subculture medium+40%（400ml/L）The thick verticillium toxin liquid of tawny daylily leaf blight, pH5.8；

Root media：1/2MS+0.1mg/LNAA+20g/L sucrose+8g/L agar powders, pH5.8.