CN105296534B - The method and its application of black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System - Google Patents
The method and its application of black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System Download PDFInfo
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Abstract
The invention discloses the method and its application of black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System.The method of black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System provided by the present invention includes the following steps: that the plasmid containing target DNA is imported Agrobacterium and obtains recombinational agrobacterium by (1)；(2) culture recombinational agrobacterium is used as conversion infected liquid；(3) black fruit fructus lycii explant is impregnated with infected liquid；(4) explant co-culture medium is placed in co-culture；(5) explant after co-cultivation is transferred on callus induction and screening and culturing medium and is cultivated, and remove Agrobacterium；(6) using kanamycins as screening pressure, induction of resistance callus；(7) resistant calli is transferred to differential medium culture, obtains Multiple Buds；(8) to get black fruit fructus lycii resistance regrowth is arrived, resistance Miao Jing PCR is accredited as positive transgenic seedling for root induction.The genetic transforming method of black fruit fructus lycii provided by the present invention, operation is simple, and callus survival rate is high.
Description
Technical field
The present invention relates to the genetic transforming method of biological field, in particular to the method established of black fruit fructus lycii genetic transformation and
It is applied.
Background technique
Black fruit fructus lycii (Lycium ruthenicum Murr.) is a kind of barbed wild bush of Solanaceae Lycium, plant height
About 20~50cm, is distributed mainly on the Desert Areas such as North of Shanxi, Ningxia, Gansu, Qinghai, Xinjiang, Tibet, grows thickly in the form of sheets point
Cloth has very strong adaptability to salinization soil.At present for the research of black fruit fructus lycii be concentrated mainly on fruits nutrition at
Divide, in terms of medicinal ingredient, pigment and polysaccharide extracting process and pharmacology analysis, yet there are no about black fruit fructus lycii genetic conversion system
The report of foundation.
Summary of the invention
The object of the present invention is to provide the methods of black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System.
The method that black fruit fructus lycii genetic transformation provided by the present invention is established, includes the following steps:
(1) plasmid containing target DNA is imported into Agrobacterium and obtains recombinational agrobacterium；The recombinational agrobacterium is being contained
In the LB liquid medium of kanamycins (Kanamycin, Kan) in 28 DEG C shaken cultivation 24-36 hours；
(2) take the bacterium solution of the step (1) in LB liquid medium containing kanamycin, 28 DEG C of shaken cultivations are extremely
OD600Value is 0.5-0.7, and thalline were collected by centrifugation；
(3) thallus that the step (2) is collected, 28 DEG C of continuation shaken cultivations to bacterium solution OD are resuspended with LB liquid medium600
Value is respectively 0.5,0.6 and 0.7, is used as infected liquid；
(4) it is impregnated black fruit fructus lycii explant 10-30 minutes with the infected liquid of the step (3), the black fruit after being infected
Fructus lycii explant；
(5) by the step (4) obtain infect after black fruit fructus lycii explant be placed in co-culture medium in black
It co-cultures 2-4 days in the dark, obtains coculture；
(6) coculture for obtaining the step (5) is used containing 500mg/L cephalosporin (Cefotaxime, Cef)
Callus induction and screening and culturing containing 20mg/L kanamycins and 300mg/L cephalosporin are transferred to after sterile water wash
It is cultivated 10-14 days on base, the callus after obtaining removal Agrobacterium；
(7) callus of the callus after the removal Agrobacterium for obtaining the step (6) in cephalosporin descending concentrations
It is cultivated 2-3 weeks on tissue induction and screening and culturing medium, obtains resistant calli；
(8) resistant calli that the step (7) obtains is transferred on selection and differential medium and is cultivated 6-8 weeks, obtained
To Multiple Buds；
(9) Multiple Buds for obtaining the step (8) on rooting induction culture medium root induction to get arrive black fruit fructus lycii
Resistance regrowth.
In the step (1) in LB liquid medium containing kanamycin each ingredient concentration are as follows: kanamycins 50mg/
L, tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L.
In the step (2) in LB liquid medium containing kanamycin each ingredient concentration are as follows: kanamycins 50mg/
L, tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L.
The OD of bacterium solution described in the step (3)600Value is 0.6.
In the step (4), the black fruit fructus lycii explant is black fruit fructus lycii blade；The soaking time is 20 minutes.
Co-culture medium described in the step (5) are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L agar
Powder and 0.2mg/L 2,4-D.
Callus induction and screening and culturing medium described in the step (6) are as follows: MS minimal medium adds 30g/L sugarcane
Sugar, 6.0g/L agar powder, 0.2mg/L 2,4-D, 20mg/L kanamycins and 300mg/L cephalosporin.
Selection and differential medium described in the step (8) are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L
Agar powder, 2.0mg/L 6-BA, 20mg/L kanamycins and 150mg/L cephalosporin.
Rooting induction culture medium described in the step (9) are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L fine jade
Cosmetics and 0.2mg/L α-naphthylacetic acid.
The method for establishing black fruit fructus lycii genetic conversion system is obtaining the application in black fruit fructus lycii transgenic line
It belongs to the scope of protection of the present invention.
The genetic transforming method of black fruit fructus lycii provided by the present invention, operation is simple, the callus group obtained after conversion
Survival rate height is knitted, material can be provided for the genetic engineering research of black fruit fructus lycii and molecular breeding.
Detailed description of the invention
Fig. 1 is that plasmid extracts testing result；Swimming lane 1-4: plasmid pET21b；Swimming lane M:100bp plus DNA ladder.
Fig. 2 is NahG gene High fidelity PCR result；Swimming lane 1 and 2:NahG band；Swimming lane M:100bp DNA ladder.
Fig. 3 is purpose gene and carrier double digestion；Swimming lane 1:NahG gene double digestion；Swimming lane 2: the bis- enzymes of carrier pBI121
It cuts；Swimming lane M:100bp DNA ladder.
Fig. 4 is the result that Trans1-T1 bacterium solution PCR is carried out using M13F/R primer；Swimming lane 1 and 2:NahG band；Swimming lane
M:DL1000DNA ladder。
Fig. 5 is the GV3101 bacterium solution PCR testing result for carrying NahG；Swimming lane 1 and 2:NahG band；Swimming lane M:100bp
plus DNA ladder。
Fig. 6 is construction of eukaryotic expression vector schematic diagram.
Fig. 7 is black fruit fructus lycii blade Kan resistance test；Wherein, a:A in Fig. 7: control, B:50mg/L Kan, C:100mg/
L Kan, D:200mg/L Kan；B:A:10mg/L Kan in Fig. 7, B:20mg/L Kan, C: control, D:30mg/L Kan, E:
50mg/L Kan。
Fig. 8 is the process that black fruit fructus lycii genetic transformation obtains resistance regrowth；A: the callus group that explant generates after infecting
Knit, B: callus breaks up on screening and culturing medium, C: the resistance Multiple Buds of differentiation, D: the resistance regeneration plant taken root.
Specific embodiment
Embodiment 1 establishes black fruit fructus lycii genetic conversion system
One, the building of carrier for expression of eukaryon
1, the acquisition of target gene NahG
(1) plasmid extracts
E.coli bacterial strain DH5 α containing carrier PET21b-NahG is by Logistics College of Chinese People's Armed Police Force (day
Jinshi City's occupation and environmental hazard Fang Zhi key lab) teacher Zhao Huabing give.NahG gene (AY20891ND09189257-
90561) naphthalene degradation bacteria strains Pseudomonas sp.ND6 (Zhao et al.2005) is come from.DH5 α (pET21b-NahG) is connect
Kind to LB liquid medium, 37 DEG C, stay overnight, and uses the small extraction reagent kit (TIANprep of Tiangeng plasmid by 200rpm/min shaken cultivation
Mini Plasmid Kit) extract plasmid, the method is as follows:
1. the equilibrium liquid BL of 500 μ L, 12,000rpm centrifugations are added in (adsorption column is put into collecting pipe) into adsorption column CP3
1min outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe, equilibrium adsorption column；
2. the bacterium solution for taking 1-5mL to be incubated overnight is added in centrifuge tube, conventional desktop centrifuge, 12,000rpm centrifugations are used
1min, as far as possible absorption supernatant；
3. 250 μ L solution P1 (using preceding addition RNase A), vortex oscillation are added into the centrifuge tube there are bacterial sediment
The thorough suspended bacterial precipitating of device；
4. 250 μ L solution P2 are added into centrifuge tube, leniently spinning upside down 6-8 times cracks thallus sufficiently；
5. 350 μ L solution P3 are added into centrifuge tube, leniently spin upside down 6-8 times, mix well immediately, will go out at this time
Existing white flock precipitate, 12,000rpm centrifugation 10min；
6. the supernatant that previous step is collected is transferred in adsorption column CP3 (adsorption column is put into collecting pipe) with pipettor,
12,000rpm centrifugation 30-60s, outwell the waste liquid in collecting pipe, adsorption column CP3 are put into collecting pipe；
7. 600 μ L rinsing liquid PW (using preceding addition dehydrated alcohol) is added into adsorption column CP3,12,000rpm centrifugation 30-
60s outwells the waste liquid in collecting pipe, and adsorption column CP3 is put into collecting pipe；
8. repetitive operation step 7；
9. adsorption column CP3 is put into collecting pipe, 12,000rpm centrifugation 2min remove rinsing liquid remaining in adsorption column
It removes.Adsorption column CP3 is uncapped, is placed in and is placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
10. adsorption column CP3 is placed in a clean centrifuge tube, 50-100 μ L is added dropwise to the intermediate position of adsorbed film and washes
De- buffer EB, is placed at room temperature for 2min, and plasmid solution is collected into centrifuge tube by 12,000rpm centrifugation 2min.
The plasmid of extraction is subjected to agarose gel electrophoresis testing result such as Fig. 1.
(2) design of primers, synthesis and PCR method amplifying target genes NahG
With Primer Premier5 software Design primers, and add digestion with restriction enzyme corresponding with expression vector
Site, You Shenggong (Sangon Biotec) synthesis, final primer sequence are as follows:
NG-F5’-CGCGGATCCATGAAAAACAATAAACC-3’
NG-R5’-CCAATCTCGAGCCCTTGACGTAGCAC-3’
Wherein underscore part is BamHI restriction enzyme site, and italicized item is XhoI restriction enzyme site.With the plasmid vector of extraction
PET21b is template, carries out PCR with high-fidelity DNA polymerase Pyrobest, obtains target gene NahG, 50 μ L reaction systems are such as
Under:
PCR amplification program:
PCR product agarose gel electrophoresis testing result is shown in Fig. 2.
Target gene fragment is recycled with Transgen DNA plastic recovery kit, the method is as follows:
1. cutting the Ago-Gel containing DNA fragmentation under ultraviolet lamp, it is put into 1.5mL centrifuge tube；
2. the sol solutions GSB of 3 times of gel volumes is added, 55 DEG C of water-baths are until gel dissolves completely；
3. colloidal sol is transferred in DNA adsorption tube centrifugal column after being cooled to room temperature, 1min is stood；
4.12,000rpm, it is centrifuged 30-60s；Abandon efflux；
5. 650 μ L rinsing liquid WB, 12,000rpm, 30-60s of centrifugation, abandoning efflux is added；
6.12,000rpm, sky centrifugation 2min, removal residual WB；
7. uncapping adsorption column in superclean bench 2min, WB is made to volatilize completely；
8. 30-50 μ L eluent TE is added, it is stored at room temperature 1min；Adsorption column is put into new 1.5mL centrifuge tube；
9.12,000rpm, it is centrifuged 2min, -20 DEG C is placed in and saves backup.
(3) PCR product sequencing
3 positive monoclonals are determined, sequence is completely the same, there is the difference of 2 bases with the NahG sequence delivered,
I.e. 43 and 609 (identity=99.58%), it is believed that the gene expanded is NahG.
(4) target gene and expression vector digestion, recycling and connection
PBI121 plasmid and NahG (being connected to pEASY-T3 carrier) are used into restriction enzyme BamH I and XhoI respectively
Double digestion is carried out, 20 μ L digestion systems:
Double digestion result is shown in Fig. 3.
Agarose gel electrophoresis separates and cuts required DNA fragmentation, and with DNA plastic recovery kit recovery product, method is same
On.Segment T will be recycled4DNA ligase connection, 10 μ L linked systems:
3, connection product amplification and sequencing
Connection product is converted to 50 μ L competent escherichia coli cell Trans1-T1 using heat shock method, it is mould that coating blocks that
Plain resistance Selective agar medium plate (LB solid medium+50mg/L Kan).Picking positive single colonie, is seeded to LB Liquid Culture
Base, extracts plasmid and is sequenced by 37 DEG C, 200rpm/min shaken cultivation 14h.
Bacterium solution PCR method identifies positive colony, and 15 μ L systems are as follows:
PCR amplification program:
PCR product is subjected to agarose gel electrophoresis, as a result such as Fig. 4.
4, heat shock method converts Agrobacterium GV3101 competent cell
The connection product of target gene and expression vector is transferred to (purchased from Beijing by Agrobacterium GV3101 bacterial strain using heat shock method
Hua Yue ocean Biotechnology Co., Ltd, catalog number Waryong GT707), the method is as follows:
1) 50 μ L competent cells are added in connection product, soft to mix, ice bath 30min；
2) 500 μ L LB liquid mediums, 28 DEG C of shaken cultivation 4h are added in quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 3min；
3) it takes 500 μ L bacterium solutions to be coated on kanamycin resistance screening culture medium flat plate, dries up, 2-3 is cultivated in 28 DEG C of inversions
It；
4) positive colony is identified using PCR method.
Bacterium solution PCR method is same as above, and agarose gel electrophoresis results are shown in Fig. 5.To positive colony sequencing, column and former sequence are sequenced
Column are consistent.So far, it completes the building of carrier for expression of eukaryon and carries Agrobacterium (engineering bacteria) building of target gene, eukaryotic expression
Carrier schematic diagram is shown in Fig. 6.
Two, the genetic transformation of black fruit fructus lycii
1, resistance test of the black fruit fructus lycii to kanamycins
Black fruit fructus lycii seedling leaves are seeded on the MS culture medium containing various concentration kanamycins, observe blade to card
The tolerance of that mycin.The concentration of kanamycins is respectively set are as follows: 0,50,100,150,200,250 and 300mg/L, as a result: when
When kanamycins concentration is greater than 200mg/L, there is albinism after inoculation 3 days in explant, and almost all albefaction is dead after 5 days
It dies；When kanamycins concentration be 50mg/L when, explant with compare no significant difference；When kanamycins concentration is 100 Hes
When 150mg/L, explant is in inoculation obvious flavescence after two weeks, and the albefaction of half explant is dead after three weeks.As a result see Fig. 7 a.
Later, further implement black fruit fructus lycii explant callus inducing medium (MS+30g/L sucrose+
6.0g/L agar powder+0.2mg/L 2,4-D) on kanamycins resistance test, by explant be seeded in kanamycins concentration according to
On the secondary callus inducing medium for being 0,10,20,30,50mg/L.The result shows that the control group that kanamycins concentration is 0
Explant initially forms callus after being inoculated with 10 days；The explant that kanamycins concentration is 10 and 20mg/L is when being inoculated with 15 days
Start callus occur, and the former callus formation rate is higher than the latter；After kanamycins concentration is higher than 30mg/L, explant
Drying out, callus, which is formed, to be reduced, and it is of poor quality, and the formation time seriously postpones.Therefore this experimental selection 20mg/L kanamycins
As screening pressure, Fig. 7 b is as a result seen.
2, test is infected
It 1) is that the monoclonal of the positive is inoculated in the LB Liquid Culture of the kanamycins containing 50mg/L by PCR detection and sequence verification
Base (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L；PH 7.0) in, 28 DEG C of shaken cultivation 24-36h；
2) take the above-mentioned bacterium solution of 1mL in LB liquid medium (the tryptone 10g/L, ferment of 50mL kanamycins containing 50mg/L
Female extract 5g/L, sodium chloride 10g/L；PH 7.0) in, 28 DEG C of shaken cultivations to OD600Value is 0.5-0.7, then 4,
000rpm, 4 DEG C of centrifugation 10min collect thallus；
3) LB liquid medium (tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L are used；PH 7.0) it is resuspended
Thallus, 28 DEG C of continuation shaken cultivations to OD600Value is respectively 0.5,0.6 and 0.7, is used as infected liquid；
4) the black fruit fructus lycii blade in 20-25 days seedling taking age and stem section impregnate explant with above-mentioned infected liquid as explant
Different time: 10,20 and 30min；It is carried out similarly infecting experiment with the black fruit fructus lycii callus that early period cultivates simultaneously；
5) bacterium solution extra on the blade after infecting is blotted with sterile blotting paper, blade is then placed in co-cultivation training
Support base MS1Appropriate time is co-cultured in dark on (MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L 2,4-D)；It infects
Test is with different explant types, bacterial concentration, time of infection and co-cultures 4 factors progress orthogonal designs of time, to obtain
Most preferably infect scheme (being shown in Table 1).
1. black fruit fructus lycii of table infects test orthogonal design
Note: G0 indicates conversion empty carrier pBI121；GV indicates conversion recombinant vector pBI121-NahG
6) with after the sterile water wash containing 500mg/L cephalosporin after co-culturing, blade is transferred to callus group
Knit induction and screening and culturing medium MS2(MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L 2,4-D+20mg/L kanamycins+
300mg/L cephalosporin), it cultivates 10-14 days, the callus after obtaining removal Agrobacterium；
7) culture medium is replaced after two weeks, cephalosporin concentration in culture medium is down to 200mg/L, later replacement one in every 10 days
Subculture, cephalosporin concentration are successively successively decreased；
8) resistant calli induced is transferred to selection and differential medium MS after one month3(MS+30g/L sucrose+
6.0g/L agar powder+2.0mg/L 6-BA+20mg/L kanamycins+150mg/L cephalosporin) on continue to cultivate, every 20 days are more
Change a subculture；
9) after two months Multiple Buds in rooting induction culture medium MS4(MS+30g/L sucrose+6.0g/L agar powder+0.2mg/L
NAA root induction is on) to get the resistance regrowth for arriving black fruit fructus lycii.
10) resistance seedling PCR is accredited as positive transgenic seedling
Using black fruit fructus lycii resistance regrowth genomic DNA as template, with the Kan resistant gene (npt II) of pBI121 carrier
Primer carries out PCR, to detect whether pBI121 expression vector is integrated into plant genome；Simultaneously with the 35S promoter on carrier
Upstream primer/NahG downstream of gene primer (35SF/NGR) carries out PCR, and whether testing goal gene NahG is integrated into plant gene
Group.
Kan resistant gene (npt II) primer sequence:
Kan-F:5’-TATGACTGGGCACAACAGACAA
Kan-R:5’-AGCGGCGATACCGTAAAGCAC
Target gene detection primer sequence:
35SF:5’-ACCCACAGATGGTTAGAGAGG
NGR:5’-CCAATCTCGAGCCCTTGACGTAGCAC
PCR system and program and connection product above amplification are identical as the system of sequencing and program.
Two above PCR reaction result shows that NahG gene integration enters black fruit fructus lycii genome, obtained resistance regrowth
For transgenic positive seedling.
The process that genetic transformation obtains resistance regrowth is as shown in Figure 8.
3. the determination of black fruit fructus lycii genetic conversion system method
By infecting the result of experiment (table 1) it is found that genetic transformation for black fruit fructus lycii, uses blade as explant progress
Its effect is infected better than stem section, and directly infects that effect is worst with callus, and tracing it to its cause may be callus group after infecting
It is excessive to knit absorption thallus, and is not easy to remove, so that callus is lethal by fouled by microzyme when co-culturing.Bacterial concentration 0.5 infects
Time 30min, the test combinations for co-culturing 4 days and bacterial concentration 0.6, time of infection 20min, the test combinations for co-culturing 2 days
It compares, the two transformation efficiency is not significantly different.Therefore, the operability and time factor of Considering experimental, we establish
The preferred method of black fruit fructus lycii genetic conversion system, it may be assumed that using blade as explant, infect bacterium solution OD600It is 0.6, bacterium solution is impregnated
Time is 20min, and thallus and explant co-cultivation time are 2 days；Each period used medium type and ingredient are respectively as follows:
Co-culture medium MS1: MS minimal medium adds 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L 2,4-
D。
Callus induction and Selective agar medium MS2: MS minimal medium, add 30g/L sucrose, 6.0g/L agar powder,
0.2mg/L 2,4-D, 20mg/L kanamycins and 300mg/L cephalosporin.
Selection and differential medium MS3: MS minimal medium adds 30g/L sucrose, 6.0g/L agar powder, 2.0mg/L
6-BA, 20mg/L kanamycins and 150mg/L cephalosporin.
Rooting induction culture medium MS4: MS minimal medium adds 30g/L sucrose, 6.0g/L agar powder and 0.2mg/L α-
Methyl α-naphthyl acetate.
Claims (2)
1. a kind of method for establishing black fruit fructus lycii genetic conversion system, includes the following steps:
(1) Agrobacterium will be imported containing the plasmid of target gene NahG obtain recombinational agrobacterium；The recombinational agrobacterium is being contained
Have in the LB liquid medium of kanamycins in 28 DEG C shaken cultivation 24-36 hours；The Agrobacterium is Agrobacterium GV3101 bacterium
Strain；The concentration of each ingredient in the LB liquid medium containing kanamycin are as follows: kanamycins 50mg/L, tryptone 10g/
L, yeast extract 5g/L and sodium chloride 10g/L；
(2) take the bacterium solution of the step (1) in LB liquid medium containing kanamycin, 28 DEG C of shaken cultivations to OD600Value
For 0.5-0.7, thalline were collected by centrifugation；The concentration of each ingredient in the LB liquid medium containing kanamycin are as follows: it is mould to block that
Plain 50mg/L, tryptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L；
(3) thallus that the step (2) is collected, 28 DEG C of continuation shaken cultivations to bacterium solution OD are resuspended with LB liquid medium600Value point
Not Wei 0.6, be used as infected liquid；
(4) it is impregnated black fruit fructus lycii explant 20 minutes with the infected liquid of the step (3), the black fruit fructus lycii explant after being infected
Body；The black fruit fructus lycii explant is black fruit fructus lycii blade；
(5) by the step (4) obtain infect after black fruit fructus lycii explant be placed in co-culture medium in dark
It co-cultures 2-4 days, obtains coculture；The co-culture medium are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L
Agar powder and 0.2mg/L 2,4-D；
(6) it will be transferred to and contain after sterile water wash of the coculture containing 500mg/L cephalosporin that the step (5) obtains
Have and cultivated 10-14 days on the callus induction and screening and culturing medium of 20mg/L kanamycins and 300mg/L cephalosporin, is obtained
Callus after removing Agrobacterium；The callus induction and screening and culturing medium are as follows: MS minimal medium adds 30g/L
Sucrose, 6.0g/L agar powder, 0.2mg/L 2,4-D, 20mg/L kanamycins and 300mg/L cephalosporin；
(7) callus of the callus after the removal Agrobacterium for obtaining the step (6) in cephalosporin descending concentrations
It is cultivated 2-3 weeks on induction and screening and culturing medium, obtains resistant calli；
(8) resistant calli that the step (7) obtains is transferred on selection and differential medium and is cultivated 6-8 weeks, obtain clump
It sprouts；The selection and differential medium are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L agar powder, 2.0mg/L
6-BA, 20mg/L kanamycins and 150mg/L cephalosporin；
(9) Multiple Buds for obtaining the step (8) on rooting induction culture medium root induction to get to the anti-of black fruit fructus lycii
Property regrowth；The rooting induction culture medium are as follows: MS minimal medium adds 30g/L sucrose, 6.0g/L agar powder and 0.2mg/
L α-naphthylacetic acid.
2. the method for black fruit fructus lycii Establishment of Agrobacterium-Mediated Transformation System described in claim 1 is in obtaining black fruit fructus lycii transgenic line
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