CN110476817A - A kind of iris radioinduction dose screening method - Google Patents
A kind of iris radioinduction dose screening method Download PDFInfo
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- CN110476817A CN110476817A CN201910933472.5A CN201910933472A CN110476817A CN 110476817 A CN110476817 A CN 110476817A CN 201910933472 A CN201910933472 A CN 201910933472A CN 110476817 A CN110476817 A CN 110476817A
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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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- 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
Abstract
The invention discloses a kind of iris radioinduction dose screening methods, by constructing iris clonal tissue culture seedling, and by iris clonal tissue culture seedling, radioinduction processing is carried out with various dose, continue to cultivate after radiation treatment, tissue-cultured seedling when choosing mutagenic treatment 40 days measures malonaldehyde (MDA) content, ultra-oxygen anion free radical (O2) generate rate, H2O2Content, antioxidase (SOD, CAT) activity, MDA and H when by 40 days after measurement mutagenic treatment2O2Content, O2The suitable Induced dosage of rate, SOD and CAT activity judgment is generated, index measured value is bigger, and respective material survival rate is lower, and corresponding radioinduction dosage is more improper, and measured value is smaller, and survival rate is higher, and corresponding radioinduction dosage is more suitable.The present invention first constructs iris clonal tissue culture seedling as mutant materials, overcome filial generation seed be mutant materials when itself there are problems that certain trait segregation, after radioinduction, the correlation analysis of iris tissue-cultured seedling physiological and biochemical index and survival rate, it filters out evaluation mutagenesis and is tied to form the relevant physiological and biochemical index of motility rate, be suitable for Induced dosage for screening iris.
Description
Technical field
The present invention relates to radiaction mutation fields, and in particular to a kind of iris radioinduction dose screening method.
Background technique
Iris is Iridaceae (Iridaceae) Jris (Iris L.) herbaceos perennial, the usual corolla of iris compared with
Greatly, pattern is more gorgeous, flower pattern is peculiar, has higher ornamental value.
Currently, the breeding of iris mainly has conventional cross-breeding, somatic hybridization, ploidy breeding, spoke both at home and abroad
Penetrate breeding, transgenic technology etc..Wherein common technology of the crossbreeding as plant breeding, but a large amount of result of study shows kite
Tail category kind is mostly between sibling species, interbreed generates, and remote edge interspecific hybridization is less, however remote edge interspecific hybridization can be ornamental
Property, adaptability etc. bigger potentiality are provided.However, Jris chromosome number changes greatly, lead to remote edge interspecific hybridization not
It is easily solid, limit the process of Jris distant hybridization breeding.
In recent years, mutation breeding is because of its simplicity, safety, economy and the high advantage of mutation rate, and the variation generated stablize compared with
Fastly, new varieties can be brought out in a short time, and are paid attention to by breeder.Common method for mutation breeding is lured including chemistry
Change, radioinduction, aviation mutagenesis etc., wherein radiaction mutation is because its mutation rate is generally up to some thousandths of or even percent
It is several, it is 1,100 times higher than natural mutation rate, and phytomorph structure can be caused and and phytomorph structure and Physiology and biochemistry can be caused
Various variations, since the color of ornamental plant, size, type are readily selected, many materials are genetically height heterozygosis
, the high frequency of mutation is easily obtained, more suitable for people when cultivating ornamental plant the characteristics of seeking new thoughts.Therefore, radiation lures
It is turned into a kind of very important ways and means (field outstanding person etc., 2012) in current ornamental plant breed of variety and improvement, and passes through
The development of decades is crossed, only China is with regard to carrying out radioactive breeding and improvement to more than 40 kinds of flowers, and has successfully obtained more than 60 and dashed forward
Mutation, including the famous flower in China, such as chrysanthemum, orchid, lily, Chinese rose, ornamental value Malus spectabilis, narcissus and iris (Zhao Li
Female and Qiang Jiye, 2013).
There are commonly gamma-rays, neutron, X-ray, β ray and Non-ionizing radiations for radiation source used in radioinduction
Ultraviolet light and laser.60Co- gamma-rays is most common radioinduction source on flowers and crops, between different plant species, same species
Different cultivars and different tissues, it is most suitable60There are larger differences for Co- gamma-rays dosage, and radioinduction dosage is excessively high, causes
Lethality is excessively high, and Induced dosage is too low, may fail to obtain enough new germ plasms.
Simple repeated sequence (Simple Sequence Repeats, SSR), also referred to as microsatellite DNA, SSR principle are roots
According to microsatellite sequence both ends complementary series design primer, the product Ago-Gel of PCR amplification is carried out to template DNA or is gathered
Acrylamide gel electrophoresis detection determines genotype according to the size of amplified production and calculates gene frequency.With other points
Sub- labelling technique is compared, and since microsatellite is in codominant inheritance, can identify heterozygote and homozygote；In addition also there is DNA
The advantages that dosage is few, and DNA quality requirement is not high, fast and convenient, reproducible.Currently, SSR marker is successfully used for oyster mushroom, wolf
The mutagenesis system of the different plant species such as tail grass category herbage, Festuca Arundinacea, alfalfa identifies.
With the maturation of group culturation rapid propagating technology, iris germ plasm resource clone is constructed using group culturation rapid propagating technology, both may be used
For subsequent mutation breeding, and it is conducive to the phenotypic evaluation of later period mutagenesis system.To the material of mutagenesis, by the measurement of Physiology and biochemistry,
Suitable radioinduction dosage is allowed rapid screening out, work can be mitigated in terms of the breeding of iris new varieties for Scientific Research Workers
Amount shortens breeding time, accelerates the breeding process of the new quality product kind of iris.
Summary of the invention
In order to overcome problems of the prior art, the purpose of the present invention is to provide a kind of iris radioinduction dosage
Screening technique.
Technical solution: the present invention is achieved through the following technical solutions:
One aspect of the present invention provides a kind of iris radioinduction dose screening method, comprising the following steps:
S1 constructs iris clonal tissue culture seedling；
S2 handles iris clonal tissue culture seedling in S1 with various dose radioinduction, continues to cultivate after radiation treatment；
S3, tissue-cultured seedling when mutagenic treatment 40 days in selecting step 2, measurement malonaldehyde (MDA) content, superoxide anion from
By base (O2) generate rate, H2O2Content, antioxidase (SOD, CAT) activity；
S4, Induced dosage screening, is screened according to the measurement result of the step S3, passes through after measurement mutagenic treatment 40
It when MDA and H2O2Content, O2The suitable Induced dosage of generating rate, SOD and CAT activity judgment, index measured value are got over
Greatly, respective material survival rate is lower, and corresponding radioinduction dosage is more improper, and measured value is smaller, and survival rate is higher, corresponding
Radioinduction dosage is more suitable.
Preferably, using the iris current year new stem apex for sprouting young shoot as explant, using 0.1%HgCl2 pairs in step S1
Explant sterilizes 7min, and Initial culture based formulas is MS+6-BA 1.5mg/L+IAA 0.2mg/L, and squamous subculture based formulas is MS
+6-BA 2.0mg/L+IAA 0.5mg/L.Cultivation temperature is 25 DEG C ± 2 DEG C, humidity 60%-70%, intensity of illumination 2000lx, light
According to time 12h/d.
Preferably, in step S2, the mutation source of the radioinduction is60Co- gamma-rays.
Preferably, the dosage of the radioinduction is 1-40Gy, dosage rate 1Gymin in step S2-1。
Preferably, the invention also includes following steps,
S5: mutant materials of the screening for SSR marker identification select MDA, H2O2、O2, 5 indexs of SOD and CAT survey
Definite value is respectively in 2.77~3.24nmolg-1, 0.524~0.564 μm of ol/g, 0.558~0.736nmol/ (g FWmin),
Mutagenesis seedling within the scope of 234.44~320.40U/gFW and 132.33~134.17U/gFW is used for subsequent identification；
S6: the mutagenesis system based on SSR marker identifies, the mutagenesis seedling of indication range is met in selecting step S5, passes through screening
Analysis of genetic diversity is carried out with verifying SSR marker, the genetic distance of mutant materials and parent, Cong Zhongjian are identified by dendrogram
Make with the consistent material of parent's genetic background, filter out the mutant materials with parent there are hereditary difference, the later period is to this part
Material expansion is numerous to take root, plants after hardening.
Beneficial effect
The present invention uses tissue culture rapid propagation system, can expand to mutagenesis system in a short time numerous, is conducive to new excellent iris kind
The expansion of matter is numerous and promotes.The present invention first constructs iris clonal tissue culture seedling as mutant materials, and overcoming filial generation seed is
There are problems that certain trait segregation when mutant materials itself, it is easier to observe the variation of its phenotype (leaf color, growing way), and can survey
Its fixed Leaf Physiology biochemical indicator evaluates influence of the various dose to iris by overall target；After radioinduction, iris
The correlation analysis of tissue-cultured seedling physiological and biochemical index and survival rate filters out evaluation mutagenesis and is tied to form the relevant Physiology and biochemistry of motility rate and refers to
Mark is suitable for Induced dosage for instructing screening iris, and by SSR marker for identifying mutagenesis system and the genetic diversity of parent
Property, further diminution mutagenesis system range rapidly and efficiently, mitigates breeding work amount.
Detailed description of the invention
Fig. 1；Influence of the different dose of radiations to iris tissue-cultured seedling MOD content.
Fig. 2；Different dose of radiations are to iris tissue-cultured seedling H2O2The influence of content.
Fig. 3；Different dose of radiations are to iris tissue-cultured seedling O2Generating rate influences.
Fig. 4；Different dose of radiations are on the active influence of iris tissue-cultured seedling SOD.
Fig. 5: different dose of radiations are on the active influence of iris tissue-cultured seedling CAT.
Fig. 6: different dose of radiations are on the active influence of iris tissue-cultured seedling POD.
Fig. 7: the iris clustering based on 10 pairs of SSR markers.
Specific embodiment
Below with reference to embodiment, the present invention will be further described, but not as a limitation of the invention.The following example
Middle test method without specific conditions, usually according to the known approaches of this field.
1, the clonal building of iris
With Louisiana iris kind ' Heather Stream ' for test material, test material is planted in double-layer plastic
In greenhouse, its tissue-cultured seedling clone is constructed.Concrete operations are as follows: using the current year new stem apex for sprouting young shoot as explant, use 0.1%
HgCl2 sterilizes 7min to explant, and Initial culture based formulas is MS+6-BA 1.5mg/L+IAA 0.2mg/L, subculture medium
Formula is MS+6-BA 2.0mg/L+IAA 0.5mg/L.Cultivation temperature is 25 DEG C ± 2 DEG C, humidity 60%-70%, intensity of illumination
2000lx, light application time 12h/d.
2, radioinduction is handled
Select growing way is consistent, plant height be 2-3cm ' Heather Stream ' tissue-cultured seedling is handled for radioinduction.Benefit
With60Co- gamma-rays carries out radioinduction processing, sets 2.5,5,10,20 and 40Gy totally 5 dosage, dosage rate 1Gymin- altogether
1.After radioinduction processing, condition of culture (culture medium, intensity of illumination and period, temperature and humidity) is consistent with clone building period,
Every 30d is forwarded to the subculture medium newly configured.
3, mutagenesis system Leaf Physiology biochemical measurement and survival rate statistics
Iris tissue culture seedling leaf is taken when 0,5,10,15,20,30 and 40d after mutagenic treatment, according to every Physiology and biochemistry
After the weighing of index test demand, freeze in -80 DEG C of refrigerators.Malonaldehyde (MDA) content is measured using thiobarbituricacidα- method；It utilizes
Azanol oxidizing process measures ultra-oxygen anion free radical (O2) generate rate；H is measured using ultraviolet absorption method2O2Content；Using nitrogen
Blue tetrazolium (NBT) method measures superoxide dismutase (SOD) activity；It is living using ultraviolet absorption method measurement catalase (CAT)
Property；Using guaiacol method measurement peroxidase (POD) activity；The survival rate of various dose mutagenic treatment is counted when 40d.
4, Capillary Electrophoresis
Primer Source: since Louisiana iris is by short stem iris (I.Brevicaulis), dark yellow iris
(I.Fulva), hexagonal fruit iris (I.Hexagona), tall and big iris (I.Giganticaerulea) and the gloomy iris of inner ear
(I.Nelsonii) totally 5 wild species hybridization；Therefore, Tang etc. (2009) has been used to pass through short stem iris and dark yellow iris
The SSR marker that the transcript profile data of blade and root system are excavated, 14 pairs of screening is relatively more from 532 pairs of SSR primers of its excavation
The higher SSR primer of state property is used for follow-up test.14 pairs of SSR primers are used for 40 parts of different Louisiana iris materials (no
Same kind, the filial generation of different cultivars, different cultivars mutagenesis system) analysis when, testing result shows wherein 10 pairs of SSR primers
(table 1) specificity is high and polymorphism is preferable, is suitable for subsequent analysis；And there is no polymorphism, item in remaining 4 pairs of primer in the detection
The problems such as band is mixed and disorderly or polymorphism is low, therefore it is not used for the analysis of genetic diversity of test sample.10 pairs of SSR primers filtered out
It is IB127, IB168, IB175, IB179, IB211, IB241, IB314, IB318, IF173, IF194 respectively.
DNA is extracted and detection: extracting leaf DNA using TSINGKE plant DNA extraction kit.Pass through 1.0% agarose
Gel electrophoresis and Nanodrop ND measurement DNA concentration and purity (OD260/OD280), quality meets the DNA of test requirements document, and -20 DEG C
It freezes spare.
SSR-PCR amplification: multiplexed PCR amplification system is 15 μ L, including 7.5 μ L 2*Tsingke Master mix
(green), 1 μ L of DNA (concentration 10ng/ μ L), each 1 μ L of forward and reverse primer (10 μm of ol/L of concentration), ddH2O 4.5μL.PCR is anti-
Answer program are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 61 DEG C~64 DEG C annealing 30s, 72 DEG C of extension 30s, totally 30 recycle；
72 DEG C of extension 5min, last 4 DEG C of preservations amplified production.Product (+5 μ L bromophenol blue of 2 μ L amplified production) after expanding for the first time is logical
1.0% agarose gel electrophoresis (voltage 300V, 12min) detection is crossed, judges whether corresponding primer amplifies purpose according to glue figure
DNA fragmentation；Secondary amplification is then carried out, amplification system and PCR response procedures are same as the first time, and the second wheel annealing temperature is 53 DEG C
~57 DEG C (the specific annealing temperature of different primers is shown in Table 2).
Capillary electrophoresis detection: height deionized formamide is configured to mix with molecular weight internal standard ROX500 according to 130:1
Liquid is closed, then into domestic 96 hole reaction plate, every hole dispenses 10 μ L for packing.0.5 μ L of PCR product is added in every hole, under room temperature
Centrifugation 4000rpm stops.Mixed plate is heated by rich day HB-T2-D metal bath heating instrument, 95 DEG C of initial denaturation 5min, immediately
It takes out and is put into -20 DEG C of rapid coolings, 96 orifice plate, 4 000rpm centrifugation after cooling after thawing and mixing, passes through ABI 3730xl
Genetic analyzer (Applied Biosystems, USA) carries out capillary tube electrophoresis detection.
Data statistics: the initial data of capillary electrophoresis detection uses the (U.S. software Gene mapper V4.1
Applied Biosystems company) carry out data exact site analysis.Detection primer is judged according to peak figure and site information
Whether there is loci polymorphism, chooses specificity height, the good site of polymorphism is as subsequent analysis emphasis.It is soft using PopGen32
Part calculates the site SSR genetic diversity index, and calculates the standard genetic distance between sample；Then, using unweighted mean
It is poly- that method (unweighted pair-group method with arithmetic means, UPGMA) carries out genetic similarity
Alanysis, and draw genetic relationship Cluster tree.
5, physiological and biochemical index and survival rate after mutagenic treatment
After different radioinduction processing, malonaldehyde in blade (MDA) and hydrogen peroxide (H are caused during 0-30d2O2)
Accumulation, superoxide anion (O2) generating rate improves and superoxide dismutase (SOD), catalase (CAT) and peroxide
Compound enzyme (POD) active enhancing；During 30-40d, with new culture medium is forwarded to, the above physiological and biochemical index has
Declined.Identical physiological and biochemical index, its higher malonaldehyde of dose of radiation and content of hydrogen peroxide are higher, and superoxide anion generates
Rate is fast, and activities of antioxidant enzymes (SOD, CAT, POD) significantly increases.2.5, the survival rate of 5,10,20 and 40Gy, 5 dosage
Respectively 21.11%, 13.33%, 12.22%, 3.33% and 1.11%.In conjunction with the testing result of physiological and biochemical index, tentatively
Think that there may be certain correlations between dose of radiation, physiological and biochemical index and survival rate.
6, the correlation analysis of Induced dosage, physiological and biochemical index and survival rate
By 17.0 software of SPSS, to different Induced dosages treated physiological and biochemical index (MDA, H2O2、O2、
SOD, CAT and POD) with survival rate correlation analysis is carried out, correlation analysis result see the table below.Between every physiological and biochemical index,
There are certain correlations, such as MDA content and H2O2Content, O2Generating rate in 0.01 horizontal significant correlation, with SOD,
CAT activity in 0.05 horizontal significant correlation, but with POD non-correlation.It is worth noting that although POD is in variation tendency side
Face is similar to SOD, CAT, but it is to every physiological and biochemical index and survival rate and uncorrelated.
By correlation analysis data as it can be seen that the mutagenesis seedling of various dose, survival rate and MDA content, O2Generating rate exists
0.01 horizontal significant related (negative correlation)；With H2O2Content, SOD to CAT activity are in 0.05 horizontal significant related (negative correlation).
That is MDA content height, O2Generating rate is fast, and SOD and the high mutagenesis shoot survival percent of CAT activity are low.MDA,H2O2、O2, SOD and CAT can
As the index estimated mutagenesis and be tied to form motility rate, for quickly screening suitable dosage.
The every physiological and biochemical index of table 1 and survival rate correlation analysis
7, the mutagenesis system identification based on SSR marker
For ' Louisiana Heather Stream ' iris kind, the lower survival rate of 2.5Gy processing is 21.11%, phase
To higher.10 pairs of SSR markers are based on to the part mutagenesis system of acquisition and carry out analysis of genetic diversity, analysis result is as shown in Figure 7.
Wherein L6 is that ' Heather Stream ', L7-L15 are different mutagenesis systems, and L17 is ♀ ' Heather Stream ' × ♂
The filial generation of ' Pegaletta '.As seen from the figure, L7, L13, L14 and parent's genetic background are more consistent, it is subsequent can be without
Squamous subculture, then there is some difference in genetic background with parent by mutagenesis system L8, L9, L10, L11 and L12, especially L10 and
L12 can emphasis expand numerous and take root, planted after hardening, to observe whether its phenotype has differences with parent, or one could be shown
A little excellent characters.
Belonging to sum up, MDA and H2O2Content, O2It is closely related to generate rate, SOD and CAT activity and mutagenesis shoot survival percent,
Show as that those index values are higher, and survival rate is lower.After radioinduction, certain stress damage is caused to tissue-cultured seedling, is caused above
The value that index measures during 0-30d increases with the extension of time, but in 30d after switching, those refer to during 30-40d
Mark is decreased obviously.Therefore, for various dose60Co- γ processing can pass through the MDA and H after measurement mutagenic treatment when 40d2O2
Content, O2Rate, SOD and CAT activity are generated, index measured value is bigger, and respective material survival rate is lower, and measured value is smaller, at
Motility rate is higher.For Louisiana ' Heather Stream ' kind, when being handled by 2.5Gy, survival rate 21.11%,
When 2.5Gy processing, survival rate is only 13.33%, however when 2.5Gy processing, it is identified through SSR marker, obtains multiple and parent and have
There are the mutant materials of certain hereditary difference.Therefore, it for Louisiana ' Heather Stream ' kind, carries out60At Co- γ
When reason, its MDA, H are measured in 40d2O2、O2, SOD and CAT, when the value that 5 indexs are surveyed is respectively in 2.77-3.24nmol
G-1,0.524-0.564 μm of ol/g, 0.558-0.736nmol/ (g FWmin), 234.44-320.40U/gFW and
When within the scope of 132.33-134.17U/gFW, it is ensured that its certain survival rate (10%-25%) and can get it is more with it is close
This mutant materials that there is some difference.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (5)
1. a kind of iris radioinduction dose screening method, which comprises the following steps:
S1 constructs iris clonal tissue culture seedling；
S2 handles iris clonal tissue culture seedling in S1 with various dose radioinduction, continues to cultivate after radiation treatment；
S3, tissue-cultured seedling when mutagenic treatment 40 days in selecting step 2, measures malonaldehyde (MDA) content, ultra-oxygen anion free radical
(O2) generate rate, H2O2Content, antioxidase (SOD, CAT) activity；
S4, Induced dosage screening, is screened according to the measurement result of the step S3, when by 40 days after measurement mutagenic treatment
MDA and H2O2Content, O2The suitable Induced dosage of rate, SOD and CAT activity judgment is generated, index measured value is bigger, right
Answer material survival rate lower, corresponding radioinduction dosage is more improper, and measured value is smaller, and survival rate is higher, corresponding radiation
Induced dosage is more suitable.
2. the method for one kind according to claim 1, which is characterized in that in step S1, with iris current year new sprouting young shoot
Stem apex be explant, using 0.1%HgCl2 to explant sterilize 7min, Initial culture based formulas be MS+6-BA 1.5mg/L
+ IAA 0.2mg/L, squamous subculture based formulas are MS+6-BA 2.0mg/L+IAA 0.5mg/L.Cultivation temperature is 25 DEG C ± 2 DEG C,
Humidity 60%-70%, intensity of illumination 2000lx, light application time 12h/d.
3. the method for one kind according to claim 1, which is characterized in that in step S2, the mutation source of the radioinduction
For60Co- gamma-rays.
4. the method for one kind according to claim 1, which is characterized in that in step S2, the dosage of the radioinduction is
1-40Gy, dosage rate 1Gymin-1。
5. the method for one kind according to claim 1, which is characterized in that further comprising the steps of
S5, mutant materials of the screening for SSR marker identification: selection MDA, H2O2、O2, 5 index determining values of SOD and CAT
Respectively in 2.77~3.24nmolg-1, 0.524~0.564 μm of ol/g, 0.558~0.736nmol/ (g FWmin),
Mutagenesis seedling within the scope of 234.44~320.40U/gFW and 132.33~134.17U/gFW is used for subsequent identification；
The mutagenesis system identification based on SSR marker: S6 meets the mutagenesis seedling of indication range, by screening and testing in selecting step S5
It demonstrate,proves SSR marker and carries out analysis of genetic diversity, the genetic distance of mutant materials and parent is identified by dendrogram, is therefrom identified
With the consistent material of parent's genetic background, the mutant materials with parent there are hereditary difference are filtered out, the later period is to this some materials
Expand and numerous take root, planted after hardening.
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| 褚润: ""三种湿地植物对UV-B辐射的响应及其生理生化机制"", 《中国博士学位论文全文数据库（电子期刊）基础科学辑》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109819891A (en) * | 2019-02-26 | 2019-05-31 | 黑龙江省科学院自然与生态研究所 | Small stream sweet-smelling grass tissue-cultured seedling60Co- gamma Rays method for mutation breeding |
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