CN106676131A - Method for generating alfalfa genome edited homozygous plant - Google Patents

Abstract

The invention discloses a method for generating an alfalfa genome edited homozygous plant, and belongs to the technical field of plant genetic engineering. The method mainly comprises: establishing an alfalfa hairy root system; converting alfalfa on a CRISPR/Cas9 over-expression vector, and obtaining resistant hairy roots; quickly filtering a homozygous hairy root system having an alfalfa genome edited by a fixed point; establishing a hairy root regeneration system, and obtaining a genome edited homozygous alfalfa plant. The method is mainly used for solving the problems of large workload and long period for obtaining a CRISPR/Cas9 genome edited homozygous plant. By means of the method of the invention, an alfalfa genome edited homozygous hairy root system can be obtained within 1-2 months; all alfalfa plants obtained by regenerating the homozygous hairy root system are homozygous edited plants.

Description

A kind of method for producing Genomic DNA editor's homozygous plants

Technical field

The invention belongs to field of plant genetic, clover CRISPR/Cas9 genes are produced more particularly to one kind The method of group editor's homozygous plants.

Background technology

Clover plays an important role as a kind of high-quality legume forage in Production of Livestock and Poultry.Its strong adaptability, cultivation are gone through History is long, have a very wide distribution, with productivity stabilization, utilization periods are long, be of high nutritive value, good palatability, Effect of feeding livestock and poultry are good, Conserve water and soil, check winds and fix drifting sand, infertile field of improving the soil, it is rear make to increase production and recover vegetation, the application value such as protecting ecology, be described as " herbage King ", irreplaceable effect is played in agriculture-stock production.

Bioactive ingredients in clover, such as Alfalfa Polysaccharide, alfalfa saponin and clover flavones are improving the healthy shape of animal Condition, raising breeding performonce fo animals and immunologic function aspect have preferable effect.Additionally, leaf protein, natural pigment in clover and The beneficiating ingredients such as chlorogenic acid equally have effect to the health status and production performance of livestock and poultry, it is believed that with the development of science and technology and Clover research is deepened continuously, its bioactive ingredients will hold out broad prospects as feed addictive, clover will be herding Bigger contribution is made in the development of industry and feed industry.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is into Cluster, regular interval, short palindrome repetitive sequence.The discovery of CRISPR/Cas systems is derived to bacterial defenses systematic research.Make It is an emerging technique for gene engineering, CRISPR/Cas systems can be carried out by transgenic technology means to crop gene group Precisely editor, obtains the germplasm new resources similar with natural mutation, so as to effectively evade market and the environment of transgenic product Risk.Meanwhile, CRISPR/Cas9 technologies are simple because of design, easy to operate, and a few years has fallen over each other the heat of research as researcher Point.

At present, CRISPR/Cas9 technologies are obtained in the genome editing process such as arabidopsis, paddy rice, willow, soybean The mutant plant of stabilization, and in the blade or protoplast of the various plants such as tobacco, soybean, sweet orange, wheat, sorghum, corn Deng genome editor has been carried out in transient expression system, demonstrate CRISPR/Cas9 systems carries out genome editor in plant Feasibility.At present, first case genome editor mushroom has come out, and it need not pass through the supervisory process of Code of Federal Regulations mechanism, It is used directly for planting and sells.However, the material produced in CRISPR/Cas9 genome editing process is generally heterozygosis compiling Volume, how homozygosis editor is obtained with a set of succinct quickly method, extend experiment week because growth cycle is long for solving plant The problem for needing Large-scale Screening to identify after phase, genome editor, asks as very important in genome editor's research work Topic.

The content of the invention

It is an object of the invention to provide a kind of method for producing clover CRISPR/Cas9 genome editor's homozygous plants, carry For a kind of system of rapidly and efficiently generation clover CRISPR/Cas9 genome editor's homozygous plants, for clover Important Economic The genetic improvement and MOLECULE DESIGN of shape.

The present invention is achieved by the following technical solution：

A kind of method for producing clover CRISPR/Cas9 genome editor's homozygous plants, its content includes：

The foundation of clover hairy root genetic conversion system；

The Screening and Identification of Genomic DNA editor's homozygosis hairy root root system；

The acquisition of Genomic DNA editor's homozygous plants.

Further, the foundation of described clover hairy root genetic conversion system：GRNA is designed for genes of interest, and is built Into CRISPR/Cas9 genome editor's expression vectors, be then transferred to agrobacterium rhizogenes is used to infect as engineering strain Clover explant, and clover hairy root is produced in induction screening and culturing medium (SHS) culture 10-14d inductions, incubator temperature is 25 ± 2 DEG C, light culture.

Further, the Screening and Identification of described Genomic DNA editor's homozygosis hairy root root system：The hairy for producing will be induced Root carries out random number, and S1 and S2 generations then select 5-10 single hairy root and carry out continuous squamous subculture, incubator temperature at random It is 25 ± 2 DEG C, light culture；Remaining root segment carries out DNA extractions, PCR mesh of the amplification comprising gRNA after collecting subculture simultaneously Gene, and carry out single endonuclease digestion identification, the PCR primer of the hairy root that selection has been edited carries out sequencing analysis, quick screening Obtain homozygosis root system.

Further, the acquisition of described Genomic DNA editor's homozygous plants：Genome editor's homozygosis of Screening and Identification Root system subculture was produced on calli induction media (SHC) through 3-5 weeks callus induction cell, and then subculture is to differential medium (MSR) on, regeneration bud was obtained through 3-5 weeks, then subculture obtained regrowth, incubator to root media (MS0) through 3-4 weeks Temperature is 25 ± 2 DEG C, and daily 16h light/8h is dark for light application time.

Further, described agrobacterium strains type is agrobacterium rhizogenes；Wherein in addition to carrying Ri plasmids, also carry outer Source CRISPR/Cas9 genome editor's expression vector plasmids；Described CRISPR/Cas9 carriers are pYLCRISPR/Cas9 series Carrier.

Further, resistance screening reagent is phosphine oxamate (PPT), hygromycin (Hyg) or kanamycins (Kan).

Further, the described clover explant for hairy root induction is clover aseptic seedlings complete stool or vegetative growth phase Outdoor plant leaf.

Further, described clover aseptic seedlings are seed seedling or the numerous seedling of aseptic expansion.

Further, the preparation method of the seed seedling is：Mature seed carries out disinfection using the following method：70% alcohol does not surpass 1min is crossed, the seed after sterilization is inoculated in 1/2MS culture mediums by 0.1% mercuric chloride 5-10min, aseptic washing 5 times, and light culture is urged Bud, then moves to photoperiod 16h light/8h and secretly continues culture, is taken at any time after 10d；

Further, the preparation method of the aseptic numerous seedling of expansion is：Aseptic seedling is cut under apical growing point at number 2-3 sections, It is inoculated in MS0 medium cultures to take root, continues to cultivate to normal seedling is grown, cuts stem and be outer including the blade including petiole Implant is standby.

Further, the sterilization method of the outdoor plant leaf is：Washing blade 2min containing 0.1% tween, 30% NaClO 5-10min, it is aseptic be washed to it is tasteless.

Heretofore described SHS is that SH basal mediums add 400mg/L Ticarcillin/Clavulanate Acids, and 15g/L sucrose, 4g/L plants coagulate Glue；According further to according further to add PPT 1.5mg/L or Hyg respectively using the class of antibiotic of plant expression vector 15mg/L or Kan50mg/L is used as screening reagent, pH5.8,121 DEG C of autoclaving 15min；

Heretofore described SHC be SH basal mediums add 2-4mg/L 2,4- dichlorphenoxyacetic acids (2,4-D), 0.5mg/L 6- benzyls aminoadenine (6-BA), 400mg/L Ticarcillin/Clavulanate Acids, 30g/L sucrose, 4g/L plant gels；According further to institute PPT 1.0mg/L or Hyg 10mg/L or Kan 50mg/L are added respectively as sieve using the class of antibiotic of plant expression vector Reagent, pH5.8,121 DEG C of autoclaving 15min, each components and concentration of the above is selected to be each composition final concentration in the medium；

Heretofore described MSR is that MS basal mediums add 0.5-1.0mg/L 6-BA, 1.0mg/L kinetins (KT), 400mg/L Ticarcillin/Clavulanate Acids, 30g/L sucrose, 7.5g/L agar, pH5.8,121 DEG C of autoclaving 15min, each composition of the above is dense Degree is each composition final concentration in the medium；

Heretofore described MS0 be 1/2MS culture mediums add 1.0mg/L indolebutyric acids (IBA), 400mg/L Ticarcillin/Clavulanate Acids, 10g/L sucrose, 7.5g/L agar, pH5.8,121 DEG C of autoclaving 15min, each components and concentration of the above is each composition in culture Final concentration in base.

The concentration of the above each composition is the final concentration in culture medium.

Described CRISPR/Cas9 carriers can connect multiple gRNA target sequences, and state-of-the art can connect at least 8 Individual gRNA, can significantly improve the efficiency of genome editor.

Heretofore described growth of hair root is rapid, can significantly shorten the screening cycle of genome editor's homozygous line.

Heretofore described hairy root induction, screening and expand in breeding culture medium without hormone addition.

Contain restriction endonuclease sites near the PAM areas of heretofore described gRNA.

The heretofore described explant abundance for hairy root induction, including the root of clover aseptic seedlings, stem, The positions such as leaf, and vegetative growth phase outdoor plant leaf.

Heretofore described gene cloning, gRNA designs, vector construction and Agrobacterium are infected and genetic engineering can be used The standard method of technology and CRISPR/Cas9 designs is realized.

The present invention can be used to successfully carry out CRISPR/Cas9 genome editors in clover.

Central characteristics of the invention and inventive concept include：

If 1) crop gene group editor can be in the strain of present age acquisition homozygosis, then will greatly reduce screening of future generation The cycle of identification and workload, but as other crops, the whole cycle of the classical leaf disc method for transformation of clover needs 6-8 month, the strain of genome editor's homozygosis once genome editing sites fail or can not obtain will certainly increase genome Edit the workload of clover and extend its cycle for producing, it is therefore desirable to develop fast and reliable Genomic DNA editor's system, Editorial efficiency gRNA high is filtered out rapidly, and produces the plant of genome editor's homozygosis.Based on foregoing invention theory, the present invention Central characteristics be：The current hairy root culture systems set up have that induced efficiency is high, growth is rapid and every root system is most Advantage from individual cells, can be used in clover CRISPR/Cas9 genome editor's gRNA editorial efficiencies Rapid identification and The stabilization generation of gene pure system.

2) which kind of edit mode no matter genome produce after being edited by fixed point, and gRNA sequences can change, therefore base If because of a group neighbouring restriction enzyme site of the PAM areas containing restriction enzyme for editor's target site, then the hairy root of homozygosis editor DNA would not be cut by restriction enzyme, and the hairy root without editor or heterozygosis editor can then be cut open wholly or in part, from And quickly Screening and Identification can go out the homozygosis root system of genome editor.Based on foregoing invention theory, central characteristics of the invention exist In：In the gRNA sequences Designs of genome editor prioritizing selection adjacent to the restrictive restriction enzyme site in PAM areas sequence, pole The big workload for simplifying later stage screening-gene group editor homozygosis hairy root root system.

Present invention beneficial effect compared with prior art：

1) Screening and Identification cycle time：Heretofore described agrobacterium rhizogenes starts to produce for 10-14 days after infecting explant Setation shape root.Growth of hair root 10-14d can a breeding generation, at most the needs time of 2 months as S0 generations, later 10-14d S2 generations can be bred.Homozygosis hairy root root system is obtained so being identified by 1-2 month after infecting explant through agrobacterium rhizogenes. Significantly shorten the cycle for obtaining genome editor's homozygous line.

2) homozygosis editor root system obtains efficiency high：The present invention is obtained after the digestion identification by S0, S1 and S2 for hairy root The probability for obtaining homozygosis editor's hairy root root system is up to more than 80%.

3) the acquisition amount of homozygosis editor plant is big：By homozygosis editor's hairy root root system callus induction, regenerated, obtained Regeneration plant almost 100% be all homozygosis editor, compared to a large amount of screening converting materials for, the workload of follow-up screening subtracts Few, hairy root regeneration obtains the increasing number of homozygosis editor plant.

Brief description of the drawings

Clover pYLCRISPR/Cas9P35S-B-tRNA merges the over-express vector sketch of fragment in Fig. 1 embodiments 1.

M. truncatula R108 hairies root morphology figure in Fig. 2 embodiments 1.

Homozygosis hairy root root system rapid screening method sketch in Fig. 3 embodiments 2.

Digestion identification electrophoretogram in Fig. 4 embodiments 2.WT is wild type, and 1 is small part editor, and 2 edit for most of, and 3 are Homozygosis editor.

Sequencing result analysis chart in Fig. 5 embodiments 2.Left figure is the homozygosis editor of various edit modes, and right figure is single volume The homozygosis editor of the mode of collecting.

The regeneration sketch of clover hairy root in Fig. 6 embodiments 3.

The hy brid albino of homozygosis hairy root root system induction in Fig. 7 embodiments 3.

Specific embodiment

It is below embodiment pair in the method for producing M. truncatula (R108) CRISPR/Cas9 genome editor's homozygous plants Principle of the invention and feature are described, and the example is served only for explaining the present invention, is not intended to limit the scope of invention.Under Material used in embodiment, reagent, agrobacterium rhizogenes and carrier etc. are stated, unless otherwise specified, can be from company by business Approach is bought, and the CRISPR/Cas9 expression vectors described in the present embodiment are presented by Agricultural University Of South China teacher Liu Yaoguang and carried Structural reform is made.

Embodiment 1：CRISPR/Cas9-PDS expression vector establishments and conversion obtain M. truncatula (R108) hairy root root System, it is comprised the following steps：

1) clone of M. truncatula (R108) PDS genes designs with gRNA：

The clone of PDS genes：Searched for by NCBI and Phytozome, only one of which is complete in M. truncatula genome PDS gene orders (gene number is Medtr3g084830).Primer is designed as standard with the sequence of this gene, with M. truncatula (R108) DNA enters performing PCR and expands for template, sequencing.

GRNA is designed：Using CRICPR-P websites, with Medicago truncatula (Mt4.0v2) as Target Genome, is Locus Tag, Submit with Medtr3g084830 genes.Attentional selection has suitable digestion in neighbouring PAM areas The target site that the gRNA in site is edited as fixed point.

2) structure of genome editor expression vector pYLCRISPR/Cas9P35S-B-tRNA-PDS：

PUC57 vector plasmids are extracted：The gRNA+tRNA sequences (strain name is Top10) that the carrier contains synthesis, upgrading Grain, it is standby.

The acquisition of purpose fragment (tRNA-PDS-gRNA fragments)：Respectively with F1 and R1, F2 and R2 as primer, with pUC57 matter Grain is template, and the KOD high-fidelity enzymes from flat end enter performing PCR amplification, reclaim purpose fragment, standby.Contain on wherein R1 and F2 There is the sequence of gRNA；The purpose fragment that F1 and R1 is expanded is 117bp for 111bp's, F2 and R2, and fragment is too small, during gel extraction It is careful to need.

PYLCRISPR/Cas9P35S-B vector plasmids are extracted：Resistance of the carrier in Agrobacterium is Kan, is in plant PPT。

Purpose fragment is connected with expression vector：Reclaim fragment to mix with pYLCRISPR/Cas9P35S-B carriers, build pYLCRISPR/Cas9P35S-B-tRNA-PDS.PYLCRISPR/Cas9P35S-B carrier digestions use BsaI restriction endonucleases, The two connection uses the T4DNA ligases, two-step reaction to be carried out in same reaction system.

Connection product converts Escherichia coli, identifies positive colony, sequencing.

Recombinant plasmid pYLCRISPR/Cas9P35S-B-tRNA-PDS transforming agrobacterium rhizogenes, identification is positive, deposits -80 DEG C, It is standby.

3) prepared by engineering strain：The agrobacterium rhizogenes streak inoculation preserved at -80 DEG C for obtaining in step (2) in On the solid medium of YEP culture mediums addition 50mg/L rifampins (Rif), 28 DEG C of incubated overnights.Picking single bacterium colony is inoculated in YEP Add Rif fluid nutrient medium incubated overnight, next day with 1% inoculum concentration Amplification Culture, to OD₆₀₀18 DEG C during for 0.6-0.8 3500rpm is centrifuged 15min, abandons supernatant, and OD is diluted to conversion fluid₆₀₀For 0.2-0.4 is standby.

4) preparation of explant：The whole plant of M. truncatula (R108) aseptic seedlings can be used as explant, its root system Position is used directly for infecting conversion, and stem or petiole are cut into the segment of 1.5cm or so, and then slightly trimming edge is allowed to produce wound blade Mouthful.

5) agrobacterium rhizogenes is infected：The explant obtained in step (4) is put into the bacterium solution for filling step (3) preparation In, in jog 20min on decolorization swinging table.

6) co-culture：In exhaustion step (5) infect liquid after, explant is placed on aseptic filter paper, in ultra-clean work Dried in platform, be then placed on co-cultivation culture medium, 25 DEG C of dark culturing 24-48h.

7) induction of hairy root：Explant after being co-cultured in step (6) is transferred to hairy root induction screening and culturing medium (SHS) on, dark culturing, about 10-14d can grow hairy root.Incubator temperature is 25 ± 2 DEG C, light culture.

Described clover PDS mrna lengths are 1347bp, and gene order, primer sequence and target site sequence are shown in SEQ NO.1-3。

The gRNA target site sequences of described M. truncatula (R108) PDS genes contain the identification of MwoI restriction enzymes Site is SEQ NO.4,5.

Described gRNA, tRNA and F1, R1, F2, R2 primer sequence are shown in SEQNO.6-11.

The sketch of described Genomic DNA editor's carrier pYLCRISPR/Cas9P35S-B-tRNA fusion fragments is shown in accompanying drawing 1。

Described agrobacterium rhizogene strain is LBA9402.

Described M. truncatula explant can come from the outdoor plant leaf of seed seedling vegetative growth phase, it is also possible to come from The whole plant of aseptic seedlings, including root, stem and different tissues organ etc. blade.

It is that SH minimal mediums add acetosyringone (As) 100mg/L that described clover co-cultures culture medium.SHS is SH Minimal medium adds 400mg/L Ticarcillin/Clavulanate Acids, 15g/L sucrose, 4g/L plant gels.Another addition PPT 1.5mg/L.PH5.8, 121 DEG C of autoclaving 15min, each components and concentration of the above is each composition final concentration in the medium.

The described M. truncatula R108 hairy root morphologies for inducing are shown in accompanying drawing 2.

Embodiment 2：The quick screening of the homozygosis hairy root root system that M. truncatula (R108) PDS genes are edited by fixed point, its Main operational steps are as follows：

1) expansion of hairy root is numerous：The resistance hairy root random number that will be induced in embodiment 1, and in sequence by root system Random number 1S0,2S0,3S0 ..., are easy to follow the trail of and purify.Resistance root growth is rapid, and 10-14d subcultures are once.S1 and S2 In generation, then selects 5-10 single hairy root and carries out continuous squamous subculture at random, while remaining root segment carries out DNA after collecting subculture Extract, stay and do identification use.Incubator temperature is 25 ± 2 DEG C, light culture.

2) the digestion identification of hairy root：Resistance hairy root sample extraction DNA, the PCR amplification that will be obtained in step (1) is included GRNA carries out single endonuclease digestion, electrophoretic analysis in interior genes of interest with the MwoI enzymes chosen.

3) sequencing and analysis of homozygosis hairy root root system：Step (2) is entered through the PCR primer that PCR/RE methods are accredited as the positive Row sequencing analysis, quick screening obtains homozygosis root system.This PCR primer is connected into carrier T simultaneously, picking single bacterium colony PCR is accredited as sun Property, each root system at least 10 monoclonals of sample presentation are sequenced, and determine editorial efficiency and edit mode.

It is identical with hairy root induction screening and culturing medium (SHS) that the hairy root expands breeding culture medium.

After described PDS genetic fragments are by MwoI digestions, wild type is edited, and should occur two after digestion in theory Band, respectively 978bp and 369bp.

Described homozygosis hairy root system rapid screening method sketch is shown in accompanying drawing 3.

, there are wild type, three kinds of possible versions of heterozygosis and homozygosis in editor's situation of described PDS genetic fragments, if The fragment is not edited successfully, then can be cut by MwoI restriction enzymes as the wild type version for compareing, and is edited Version, whether heterozygosis or purified fragments, the recognition site of MwoI restriction enzymes have partly or entirely occurred prominent Becoming, therefore be compiled into the fragment of work(will not be cut by MwoI restriction enzymes, i.e., can still retain single of 1347bp Section (see Fig. 4).

Described sequencing analysis, are further smart to the identification successful fragment of genome editor on the basis of digestion identification Really analyze editorial efficiency and edit mode (see Fig. 5).

Embodiment 3：Hairy root regeneration Establishing and PDS gene pure editor M. truncatula (R108) Albino Seedling plant Obtain, its main feature includes following operating procedure：

1) hairy root induction callus：Genome editor's homozygosis root system of Screening and Identification is transferred to calli induction media (SHC) on, induce within 3-5 weeks, the callus of generation can carry out the differentiation and regeneration of next step.Incubator temperature is 25 ± 2 DEG C, Light culture.

2) hairy Furcation defects regeneration：The callus obtained in step (1) is transferred on differential medium (MSR), one Can break up within week and sprout a little, regeneration bud is obtained through differentiation in 3-5 weeks.Incubator temperature is 25 ± 2 DEG C, and light application time is daily 16h light/8h is dark (see Fig. 6).

3) hairy root regeneration bud is taken root：The regeneration bud subculture that will be obtained in step (2) to root media (MS0), through 3-4 Week obtains M. truncatula (R108) regrowth of albefaction.Incubator temperature is 25 ± 2 DEG C, and daily 16h light/8h is dark for light application time.

Described SHC is that SH basal mediums add 2-4mg/L 2,4-D, 0.5mg/L6-BA, 400mg/L Ticarcillin/Clavulanate Acid 30g/L sucrose, 4g/L plant gels；Another addition 1.0mg/L, pH5.8,121 DEG C of autoclaving 15min of PPT, the above.PH5.8, 121 DEG C of each components and concentrations of autoclaving 15min are each composition final concentration in the medium..

Described MSR is that MS basal mediums add 0.5-1.0mg/L 6-BA, 1.0mg/LKT, 400mg/L Ticarcillin/Clavulanate Acids, 30g/L sucrose, 7.5g/L agar, pH5.8,121 DEG C of autoclaving 15min, each components and concentration of the above is each composition in culture Final concentration in base.

Described MS0 is that 1/2MS culture mediums add 1.0mg/L IBA, 400mg/L Ticarcillin/Clavulanate Acids, 10g/L sucrose, 7.5g/L Agar, pH5.8,121 DEG C of autoclaving 15min, each components and concentration of the above is each composition final concentration in the medium.

The regrowth that described homozygosis editor's hairy root is produced is all Albino Seedling (see Fig. 7).

It is more than 30% using M. truncatula (R108) hairy root induction frequency in embodiment 1.

The cycle time of homozygosis editor's root system is obtained using M. truncatula (R108) in embodiment 2, digestion was identified to S2 generations, The pick-up rate of homozygosis hairy root root system is higher than 80%.

The use of the regeneration frequency of M. truncatula (R108) hairy root in embodiment 3 is higher than 50%, Albino Seedling pick-up rate is almost It is 100%.

Technical solution of the present invention is applicable not only to annual self-compatible M. truncatula, applies also for alfalfa etc. many Year is conigenous the acquisition of the genome editor's homozygous plants for handing over not affine leguminous forage.

Finally it should be noted that above example is merely to illustrate technical scheme and unrestricted, above-mentioned implementation Example has been described in detail to the present invention, therefore any modification or equivalent to the present invention program, all without departing from the present invention The spirit and scope of technical scheme.

SEQUENCE LISTING

<110>Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences

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Claims (10)

1. it is a kind of produce clover CRISPR/Cas9 genome editor's homozygous plants method, it is characterised in that its content includes：

The foundation of clover hairy root genetic conversion system；

The Screening and Identification of Genomic DNA editor's homozygosis hairy root root system；

The acquisition of Genomic DNA editor's homozygous plants.

2. method according to claim 1, it is characterised in that the foundation of described clover hairy root genetic conversion system：Pin GRNA is designed genes of interest, and is built into CRISPR/Cas9 genome editor's expression vectors, be then transferred to agrobacterium rhizogenes It is used to infect clover explant as engineering strain, and clover hair is produced in induction screening and culturing medium culture 10-14d inductions Shape root, incubator temperature is 25 ± 2 DEG C, light culture.

3. method according to claim 1, it is characterised in that described Genomic DNA editor's homozygosis hairy root root system Screening and Identification：The hairy root for producing will be induced carries out random number, and S1 and S2 generations then select 5-10 single hairy root and enter at random The continuous squamous subculture of row, incubator temperature is 25 ± 2 DEG C, light culture；Carried while collecting remaining root segment after subculture and carrying out DNA Take, PCR genes of interest of the amplification comprising gRNA, and carry out single endonuclease digestion identification, the PCR of the hairy root that selection has been edited Product carries out sequencing analysis, and quick screening obtains homozygosis root system.

4. method according to claim 1, it is characterised in that the acquisition of described Genomic DNA editor's homozygous plants： The genome editor homozygosis root system subculture of Screening and Identification was produced, so on calli induction media through 3-5 weeks callus induction cell In follow-up generation, on differential medium, regeneration bud was obtained through 3-5 weeks, and then subculture was regenerated to root media through 3-4 weeks Seedling, incubator temperature is 25 ± 2 DEG C, and daily 16h light/8h is dark for light application time.

5. method according to claim 1, it is characterised in that resistance screening reagent is phosphine oxamate, hygromycin or to block that mould Element；The described clover explant for hairy root induction is clover aseptic seedlings complete stool or the outdoor Plant Leaf of vegetative growth phase Piece.

6. method according to claim 2, it is characterised in that described agrobacterium strains type is agrobacterium rhizogenes；Wherein In addition to carrying Ri plasmids, external source CRISPR/Cas9 genome editor's expression vector plasmids are also carried；Described CRISPR/ Cas9 carriers are pYLCRISPR/Cas9 serial carriers.

7. method according to claim 2, it is characterised in that described clover hairy root induction screening and culturing medium is SH bases Basal culture medium adds 400mg/L Ticarcillin/Clavulanate Acids, 15g/L sucrose, 4g/L plant gels；According further to used plant expression vector Class of antibiotic adds phosphine oxamate 1.5mg/L or hygromycin 15mg/L or kanamycins 50mg/L as screening reagent respectively, PH5.8,121 DEG C of autoclaving 15min, each components and concentration of the above is each composition final concentration in the medium.

8. method according to claim 4, it is characterised in that described calli induction media adds for SH basal mediums Plus 2-4mg/L 2,4- dichlorphenoxyacetic acid and 0.5mg/L 6- benzyl aminoadenines, 400mg/L Ticarcillin/Clavulanate Acids, 30g/L sucrose, 4g/L plant gels；According further to add phosphine oxamate 1.0mg/L or tide respectively using the class of antibiotic of plant expression vector Mycin 10mg/L or kanamycins 50mg/L is used as screening reagent, and pH5.8,121 DEG C of autoclaving 15min, each composition of the above is dense Degree is each composition final concentration in the medium.

9. method according to claim 4, it is characterised in that described differential medium is the addition of MS basal mediums 0.5-1.0mg/L 6- benzyl aminoadenines, 1.0mg/L kinetins, 400mg/L Ticarcillin/Clavulanate Acids, 30g/L sucrose, 7.5g/L agar, PH5.8,121 DEG C of autoclaving 15min；Each components and concentration is each composition final concentration in the medium above.

10. method according to claim 4, it is characterised in that described root media is the addition of 1/2MS culture mediums 1.0mg/L indolebutyric acids, 400mg/L Ticarcillin/Clavulanate Acids, 10g/L sucrose, 7.5g/L agar, pH5.8,121 DEG C of autoclaving 15min； Each components and concentration is each composition final concentration in the medium above.