CN105838734B - A method of wild jujube genetic conversion system is established using blade for receptor - Google Patents
A method of wild jujube genetic conversion system is established using blade for receptor Download PDFInfo
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Abstract
The present invention relates to a kind of methods for establishing wild jujube genetic conversion system using blade for receptor, belong to plant cell engineering and technical field of molecular biology.Wild jujube blade is inoculated in dark culture in WPM adventitious bud Primary culture base and induces 2~4d, obtains explant；Agrobacterium bacterium solution containing plant expression vector is infected into explant, obtains the explant infected；The explant infected is placed on WPM solid medium, 2~4d of dark culture；Blade after dark culture is transferred in WPM solid screening and culturing medium and carries out dark culture, 14~16d of dark culture is moved back to light altogether, the elongation culture of leaf regeneration is carried out in the WPM culture medium of heteroauxin containing 0.1mg/L and 0.5mg/L gibberellin, obtain adventitious bud, adventitious bud is gone in MS culture medium and expand numerous, after obtaining genetic transformation wild jujube plant.Wild jujube genetic conversion system method provided by the invention provides suitable regenerating system for wild jujube, and transformation frequency is high, and repeatability is high, is conducive to the genetic improvement of wild jujube.
Description
Technical field
It is receptor the present invention relates to plant cell engineering and technical field of molecular biology more particularly to a kind of utilization blade
The method for establishing wild jujube genetic conversion system.
Background technique
Wild jujube (Ziziphus jujuba Mill.var.Spinosa) is Rhamnaceae jujube, is the primary of Chinese Jujube
Kind, it is distributed in China very wide.The wild jujube florescence is long, is good nectariferous plant, and kernel can be used as medicine；Jujube fruit nutritive value is high,
Contain the various trace elements such as potassium, sodium, iron, zinc, phosphorus, selenium；More importantly contain a large amount of vitamin C, content in wild jujube
Higher than fruit such as common jujube, citrus, apple, grapes, thus by consumer's pro-gaze, have in the production and selling of fruit tree
Important value.However, the wild jujube maturity period concentrates, storage period is short, is only capable of saving 6-7d under room temperature.If the above difficulty cannot be captured in time
Topic, the then value that can limit wild jujube significantly utilize；In addition, wild jujube tree is small, artificial emasculation is difficult, fruit-setting rate is low and aborted embryo phenomenon
Seriously, make its crossbreeding extremely difficult, cannot achieve the target of directive breeding in a short time.
With the completion of extensive use and jujube tree genome sequencing of the technique for gene engineering on fruit breeding, jujube
It sets breeding research and also enters gene level comprehensively.Although at present transgenic technology achieved in terms of jujube tree breeding work it is some into
Exhibition, but system is still not mature enough, and the genetic transfoumation of same kind and method also have larger difference, poor repeatability, tool
There is significant limitations.
Since jujube tree tissue culture plants regeneration difficulty is larger, lacks suitable regenerating system, turn with other fruit tree genetics
Change compared to making slow progress, there are several correlative study reports so far.Yin Meiqiang has studied isopentenyl transferase genes to pears
The genetic transformation of jujube stem section obtains a small amount of transgenic plant (Yin Meiqiang Agrobacterium tumefaciens mediated prenyltransferase base
Because to Study on Genetic Transformation [D] the Agricultural University Of Shanxi of pear and date-printing blocks, 2002).Meng Hui utilize using Zhanhua winter jujube be material by als with
BetA gene is transferred to winter jujube stem apex, also obtains the (basic research of Meng Hui Zhanhua winter jujube genetic engineering improvement of some transgenic plants
[D] Shandong University, 2005).Hao Zheng (2012) He Huangjian (2006) et al. has carried out pest-resistant and salt-resistance side using Chinese Jujube
The research in face, but conversion ratio is not that very high (jujube tree Study on Genetic Transformation [D] Hebei Agriculture of Hao Zheng mediated by agriculture bacillus is big
Learn, 2012) and (Huang builds research [D] of mediated by agriculture bacillus S6PDH genetic transformation jujube tree (Zyziphus jujuba Mill.)
Xibei Univ. of Agricultural & Forest Science & Technology, 2006).The transgenosis of the Study on Genetic Transformation of other opposite fruit trees, jujube tree works still in exploration
Stage has not been reported the research for being transferred to anti-aging related gene using wild jujube blade for receptor.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for establishing wild jujube genetic conversion system using blade for receptor.This hair
The method transformation frequency of bright offer is high, and repeatability is high, is conducive to the genetic improvement of wild jujube, provides suitable raw body again for wild jujube
System.
The present invention provides a kind of methods for establishing wild jujube genetic conversion system using blade for receptor, including following step
It is rapid:
1) wild jujube blade is inoculated in dark culture in WPM adventitious bud Primary culture base and induces 2~4d, obtain explant, institute
Stating WPM adventitious bud Primary culture base includes 0.8~3.0mg/L Thidiazuron, 0.2~0.5mg/L indolebutyric acid, 28~32g/L sugarcane
Sugar and 5~7g/L agar；
2) the Agrobacterium bacterium solution containing plant expression vector is infected into the explant that the step 1) obtains, obtains being infected
Explant；
3) explant infected for obtaining the step 2) is placed on WPM solid medium, 2~4d of dark culture, institute
Stating WPM solid medium includes 0.5~2.0mg/L Thidiazuron and 0.25~0.4mg/L indolebutyric acid；
4) blade after the step 3) dark culture is transferred to progress 14~16d of dark culture in WPM solid screening and culturing medium,
The WPM solid screening and culturing medium contains 0.8~2.5mg/L Thidiazuron, 0.2~0.4mg/L indolebutyric acid, 180~220mg/L
Cephalosporin and 15~25mg/L kanamycins；
After the dark culture, it is transferred to the elongation culture that leaf regeneration is carried out in WPM elongation medium, it is indefinite to obtain
Bud, the WPM elongation medium contain 0.1mg/L heteroauxin and 0.5mg/L gibberellin；
5) adventitious bud for obtaining the step 4) expands numerous, the wild jujube plant after obtaining genetic transformation.
Preferably, the time infected in the step 2) is 10~20min.
Preferably, during the adventitious bud induction culture of the step 4), the photoperiod of adventitious bud induction culture is 12
~16h/d, the time of the adventitious bud induction culture are 30~40d, and the WPM solid adventitious bud induction culture base includes 0.08
That is mould for~0.12mg/L indolebutyric acid, 0.3~0.6mg/L gibberellin, 180~220mg/L cephalosporin and 15~25mg/L card
Element.
Preferably, the adventitious bud of the step 5) expands numerous specifically:, will not when the stem length of adventitious bud is 15~20mm
Normal bud go in MS culture medium expand it is numerous, the MS culture medium include 0.8~1.2mg/L 6- benzyl aminoadenine, 0.3~
0.6mg/L indolebutyric acid and 18~22mg/L kanamycins.
Preferably, the step 1), 3), 4) He 5) in cultivation temperature be independently selected as 25 ± 2 DEG C.
Preferably, before the step 1) further include: wild jujube blade is carried out pre-treatment；
The process of the pre-treatment specifically: cut wild jujube tissue-cultured seedling morphology the 3rd~5 piece of blade from top to bottom, will cut
Under blade as converting material；
By the converting material along crosscutting 2~4 wounds of blade middle arteries vertical direction, cuts off master pulse but do not cut off blade；
Inoculation in the step 1) is carried out in a manner of paraxial end in contact culture medium.
Preferably, the Agrobacterium bacterium solution preparation method the following steps are included:
The single colonie of agrobacterium strains is inoculated in YEB fluid nutrient medium dark culture to OD600It is 0.5~0.7, it is described
YEB fluid nutrient medium includes 40~55mg/L rifampin, 40~55mg/L cephalosporin and 40~55mg/L kanamycins；
The bacterium solution that the dark culture obtains is inoculated in not antibiotic YEB liquid with 0.02~0.05% inoculum concentration
In culture medium, continue culture to OD600It is 0.4~1.0；
By the OD600It is mixed for 0.4~1.0 bacterium solution with the acetosyringone of 150~300 μm of ol/L, continues to cultivate
0.8~1.5h obtains Agrobacterium bacterium solution.
Preferably, after the step 5) further include: the wild jujube plant after the genetic transformation is carried out Molecular Detection；
The Molecular Detection the following steps are included:
Transgenic plant is detected using PCR method, according to whether there is purpose gene band, determines target gene
The integration of DNA.
Also include after Molecular Detection described in above-mentioned technical proposal: the plant after the detection is subjected to small seedling rooting and shifting
It plants；
The process of the small seedling rooting and transplanting specifically: continue to cultivate by the plant after the detection, to long to plant height
After 1.5~2cm, it is transferred in root media and takes root；After root growth to 2~3cm, continued growth in Nutrition Soil is moved into.
A kind of method for establishing wild jujube genetic conversion system using blade for receptor provided by the invention, blade transformation frequency
Height, repeatability is high, due to avoiding the variation being likely to occur through callus approach by blade Direct Regeneration adventitious bud mode, obtains
To transgenosis wild jujube plant both had the original excellent characteristic of wild jujube, but also with the function of institute's transgenosis, the method for the present invention has
Conducive to the genetic improvement of wild jujube, suitable regenerating system is provided for wild jujube.
Detailed description of the invention
Fig. 1 is sprouting and the growth conditions figure for the adventitious bud that the embodiment of the present invention 1 provides；
Fig. 2 is that the different Agrobacterium bacterial concentrations that the embodiment of the present invention 1 provides influence wild jujube blade genetic transformation rate
Histogram；
Fig. 3 is the map of the expression vector pBI121-SPDS in the agrobacterium strains that the embodiment of the present invention 1 provides；
Fig. 4 is the column that the Agrobacterium time of infection that the embodiment of the present invention 1 provides influences wild jujube blade genetic transformation rate
Figure；
Fig. 5 is the histogram influenced on wild jujube blade genetic transformation rate the co-cultivation time that the embodiment of the present invention 1 provides；
Fig. 6 is that the different Kan concentration that the embodiment of the present invention 1 provides averagely sprout to adventitious bud the influence diagram of number；
Fig. 7 is transformed plant genetic test figure in the embodiment of the present invention 1.
Specific embodiment
The present invention provides a kind of methods for establishing wild jujube genetic conversion system using blade for receptor, including following step
It is rapid:
1) wild jujube blade is inoculated in dark culture in WPM adventitious bud Primary culture base and induces 2~4d, obtain explant, institute
Stating WPM adventitious bud Primary culture base includes 0.8~3.0mg/L Thidiazuron, 0.2~0.5mg/L indolebutyric acid, 28~32g/L sugarcane
Sugar and 5~7g/L agar；
2) the Agrobacterium bacterium solution containing plant expression vector is infected into the explant that the step 1) obtains, obtains being infected
Explant；
3) explant infected for obtaining the step 2) is placed on WPM solid medium, 2~4d of dark culture, institute
Stating WPM solid medium includes 0.5~2.0mg/L Thidiazuron and 0.25~0.4mg/L indolebutyric acid；
4) blade after the step 3) dark culture is transferred to progress 14~16d of dark culture in WPM solid screening and culturing medium,
The WPM solid screening and culturing medium contains 0.8~2.5mg/L Thidiazuron, 0.2~0.4mg/L indolebutyric acid, 180~220mg/L
Cephalosporin and 15~25mg/L kanamycins；
After the dark culture, it is transferred to the elongation culture that leaf regeneration is carried out in WPM elongation medium, it is indefinite to obtain
Bud, the WPM elongation medium contain 0.1mg/L heteroauxin and 0.5mg/L gibberellin；
5) adventitious bud for obtaining the step 4) expands numerous, the wild jujube plant after obtaining genetic transformation.
Wild jujube blade is inoculated in dark culture in WPM adventitious bud Primary culture base and induces 2~4d by the present invention, obtains explant
Body.In the present invention, the time of the dark culture induction is preferably 3d；The temperature of the dark culture induction is preferably 25 ± 2 DEG C,
More preferably 25 DEG C.In the present invention, the inoculation in the step 1) is preferably carried out in a manner of paraxial end in contact culture medium；Each
Culture dish, which is inoculated with, hurts blade at 10 wild jujube quarters.
In the present invention, the WPM minimal medium includes a great number of elements I, microelement I and organic principle I:
Specifically, in the present invention, a great number of elements I preferably comprises the NH of 380~420mg/L4NO3, more preferably
390~410mg/L, most preferably 400mg/L；
The a great number of elements I preferably comprises the Ca (NO of 540~580mg/L3)2·4H2O, more preferably 550~570mg/
L, most preferably 556mg/L；
The a great number of elements I preferably comprises the K of 970~1010mg/L2SO4, more preferably 980~1000mg/L, most preferably
For 990mg/L；
The a great number of elements I preferably comprises the CaCl of 90~100mg/L2·2H2O, more preferably 93~98mg/L, it is optimal
It is selected as 96mg/L；
The a great number of elements I preferably comprises the KH of 150~200mg/L2PO4, more preferably 160~180mg/L, most preferably
For 170mg/L；
The a great number of elements I preferably comprises the MgSO of 350~400mg/L4·7H2O, more preferably 360~380mg/L, most
Preferably 370mg/L；
The microelement I preferably comprises: the Na of 0.2~0.3mg/L2MoO4·2H2O, more preferably 0.22~
0.28mg/L, most preferably 0.25mg/L；
The microelement I preferably comprises the MnSO of 22~24mg/L4·H2O, more preferably 22.2~23.5mg/L, most
Preferably 22.4mg/L；
The microelement I preferably comprises the ZnSO of 8~9mg/L4·7H2O, more preferably 8.2~8.8mg/L, most preferably
For 8.6mg/L；
The microelement I preferably comprises the CuSO of 0.2~0.3mg/L4·5H2O, more preferably 0.22~0.28mg/L,
Most preferably 0.25mg/L；
The microelement I preferably comprises the FeSO of 27~28mg/L4·7H2O, more preferably 27.3~27.9mg/L, most
Preferably 27.8mg/L；
The microelement I preferably comprises the Na of 37~38.5mg/L2EDTA·2H2O, more preferably 37.2~38.3mg/
L, most preferably 37.3mg/L；
The microelement I preferably comprises the H of 6~7mg/L3BO3, more preferably 6.1~6.6mg/L, most preferably
6.2mg/L。
The organic principle I preferably comprises: the inositol of 95~110mg/L, more preferably 98~105mg/L, most preferably
100mg/L；
The organic principle I preferably comprises the vitamin B of 0.8~1.4mg/L1, more preferably 0.9~1.3mg/L is optimal
It is selected as 1.0mg/L；
The organic principle I preferably comprises the niacin of 0.3~0.7mg/L, more preferably 0.35~0.60mg/L, most preferably
For 0.5mg/L；
The organic principle I preferably comprises the vitamin B of 0.35~0.6mg/L6, more preferably 0.4~0.55mg/L, most
Preferably 0.5mg/L；
The organic principle I preferably comprises the glycine of 1.6~2.2mg/L, more preferably 1.8~2.1mg/L, most preferably
For 2.0mg/L.
In the present invention, the WPM adventitious bud Primary culture base is to add other groups on the basis of WPM minimal medium
Point.
In the present invention, the WPM adventitious bud Primary culture base includes 0.8~3.0mg/L Thidiazuron, preferably 1.0mg/
L~2.0mg/L；
The WPM adventitious bud Primary culture base includes 0.2~0.5mg/L indolebutyric acid, preferably 0.3mg/L~0.4mg/
L；
The WPM adventitious bud Primary culture base includes 28~32g/L sucrose, preferably 30g/L；
The WPM adventitious bud Primary culture base includes 5~7g/L agar, preferably 6g/L.
In the present invention, the pH value of the WPM adventitious bud Primary culture base is 5~6.5, more preferably 5.8~6.0.
In the present invention, before the step 1) it is also preferable to include: wild jujube blade is subjected to pre-treatment, is obtained dark for carrying out
Cultivate the material of induction；
The process of the pre-treatment is preferred specifically: wild jujube tissue-cultured seedling morphology the 3rd~5 piece of blade from top to bottom is cut,
Using the blade cut as converting material；
By the converting material along crosscutting 2~4 wounds of blade middle arteries vertical direction, cuts off master pulse but do not cut off blade.
After obtaining explant, the Agrobacterium bacterium solution containing plant expression vector is infected the explant by the present invention.At this
In invention, the plant expression vector is preferably pBI121-SPDS, has nucleotide sequence shown in Pt-SPDS, the expression
Carrier is preferably with spermidine synthase gene SPDS gene as a purpose, and the gene number of logging in is JQ002667.1, with neomycin phosphorus
Sour transferase gene NPT II is used as riddled basins.In the present invention, the time infected is 10~20min, more preferably
For 15min；The temperature infected is preferably 25 ± 2 DEG C, and more preferably 25 DEG C.
In the present invention, the Agrobacterium bacterium solution preparation method the following steps are included:
The single colonie of agrobacterium strains is inoculated in YEB fluid nutrient medium dark culture to OD600It is 0.5~0.7, it is described
YEB fluid nutrient medium includes 40~55mg/L rifampin, 40~55mg/L cephalosporin and 40~55mg/L kanamycins；
The bacterium solution that the dark culture obtains is inoculated in not antibiotic YEB liquid with 0.02~0.05% inoculum concentration
In culture medium, continue culture to OD600It is 0.4~1.0；
By the OD600It is mixed for 0.4~1.0 bacterium solution with the acetosyringone that molar concentration is 150~300 μm of ol/L,
Continue 0.8~1.5h of culture, obtains Agrobacterium bacterium solution.
Agrobacterium strains of the invention are preferably LBA4404 Agrobacterium tumefaciems, purchased from Beijing Hua Yue ocean biology.
The single colonie of agrobacterium strains is inoculated in YEB fluid nutrient medium and carries out dark culture by the present invention, and the dark culture is excellent
It is selected in 28 DEG C of constant temperature and is protected from light speed oscillation culture on shaking table with 180~200rpm, OD is arrived in culture600It is 0.5~0.7.At this
In invention, the YEB fluid nutrient medium preferably includes the rifampin of 40~55mg/L, more preferably 45~50mg/L；
The YEB fluid nutrient medium preferably includes 40~55mg/L cephalosporin, and more preferably 45~50mg/L cephalo is mould
Element；
The YEB fluid nutrient medium preferably includes 40~55mg/L kanamycins, that is mould for more preferably 45~50mg/L card
Element.
In the present invention, the agrobacterium strains in YEB fluid nutrient medium preferred dark culture to OD600For 0.6~
0.65。
After the dark culture, the bacterium solution for the agrobacterium strains that the dark culture obtains is inoculated in without anti-by the present invention
In the YEB fluid nutrient medium of raw element, continue culture to OD600It is 0.4~1.0.In the present invention, the inoculum concentration of the inoculation is excellent
It is selected as 0.02~0.05%, more preferably 0.03%.In the present invention, the not antibiotic YEB fluid nutrient medium with it is upper
It states YEB fluid nutrient medium described in technical solution to compare, other than without containing antibiotic, other components are identical.In the present invention,
The bacterium solution of the agrobacterium strains is preferably cultivated in the not antibiotic YEB fluid nutrient medium to OD600For 0.6~
0.8。
In the present invention, the detection of the OD value is using conventional absorbency detection method, and the present invention is to the absorbance
The equipment of detection does not have special restriction, using spectrophotometer well known to those skilled in the art.
After the completion of culture in the not antibiotic YEB fluid nutrient medium, the present invention by obtained bacterium solution with rub
The acetosyringone mixing that your concentration is 150~300 μm of ol/mL, continues 0.8~1.5h of culture, obtains Agrobacterium bacterium solution.At this
In invention, the molar concentration of the acetosyringone is preferably 180~250 μm of ol/L, more preferably 200 μm of ol/L.In this hair
In bright, the volume ratio of bacterium solution and acetosyringone that the culture in not antibiotic YEB fluid nutrient medium obtains is preferably 400
~600:1, more preferably 500~1, the time for continuing culture is preferably 1~1.2h；The temperature for continuing culture is preferred
It is 27~29 DEG C, more preferably 28 DEG C.
After obtaining the explant infected, the explant infected is inoculated on WPM solid medium by the present invention,
2~4d of dark culture.In the present invention, the WPM solid medium preferably includes the Thidiazuron of 0.5~2.0mg/L, more preferably
1.0~1.5mg/L；
The WPM solid medium preferably includes 0.25~0.4mg/L indolebutyric acid, more preferably 0.3~0.35mg/L.
In the present invention, the temperature for dark culture being carried out on the WPM solid medium is preferably 25 ± 2 DEG C, more preferably
25℃；The time preferred 3d of dark culture is carried out on the WPM solid medium.
It completes after the dark culture on the WPM solid medium, obtained blade is transferred to WPM solid and screened by the present invention
14~16d of dark culture is carried out in culture medium.In the present invention, the WPM solid screening and culturing medium includes 0.8~2.5mg/L's
Thidiazuron, preferably 1.0~2.0mg/L；
The WPM solid screening and culturing medium includes the indolebutyric acid of 0.2~0.4mg/L, preferably 0.3mg/L；
The WPM solid screening and culturing medium includes 180~220mg/L cephalosporin, and preferably 190~210mg/L is more excellent
It is selected as 200mg/L cephalosporin；
The WPM solid screening and culturing medium include 15~25mg/L kanamycins, preferably 18~22mg/L, more preferably
20mg/L。
In the present invention, the temperature for dark culture being carried out in the WPM solid screening and culturing medium is preferably 25 ± 2 DEG C, more excellent
It is selected as 25 DEG C；It is preferably 14~16d that the time of dark culture is carried out in the WPM solid screening and culturing medium, more preferably 15d.
After the dark culture, it is transferred to the elongation culture that leaf regeneration is carried out in WPM elongation medium, it is indefinite to obtain
Bud, the WPM elongation medium contain 0.1mg/L heteroauxin and 0.5mg/L gibberellin；
In the present invention, the photoperiod of the adventitious bud induction culture is preferably 12~16h/d, more preferably 14h/d；Institute
The time for stating adventitious bud induction culture is 30~40d；
In the present invention, the WPM solid adventitious bud induction culture base includes 0.08~0.12mg/L indolebutyric acid, preferably
For 0.09~0.11mg/L, more preferably 0.10mg/L；
The WPM solid adventitious bud induction culture base includes 0.3~0.6mg/L gibberellin, preferably 0.35~0.55mg/
L, more preferably 0.50mg/L；
The WPM solid adventitious bud induction culture base includes 180~220mg/L cephalosporin, preferably 190~210mg/
L, more preferably 200mg/L；
The WPM solid adventitious bud induction culture base include 15~25mg/L kanamycins, preferably 16~22mg/L, more
Preferably 20mg/L.
After obtaining adventitious bud, the adventitious bud is expanded numerous wild jujube plant after obtaining genetic transformation by the present invention.
The present invention by the adventitious bud obtained by adventitious bud induction culture preferably when stem length is 15~20mm, by adventitious bud
Go in MS culture medium expand it is numerous.
In the present invention, the MS minimal medium preferably includes a great number of elements II, microelement II and organic principle II；
Specifically, a great number of elements II preferably comprises: the NH of 1600~1700mg/L4NO3, more preferably 1650mg/L；
The a great number of elements II preferably comprises: the KNO of 1850~1920mg/L3, more preferably 1880~1910mg/L, most
Preferably 1900mg/L；
The a great number of elements II preferably comprises: the CaCl of 420~450mg/L2·2H2O, more preferably 430~445mg/L,
Most preferably 440mg/L；
The a great number of elements II preferably comprises: the MgSO of 350~380mg/L4·7H2O, more preferably 360~375mg/L,
Most preferably 370mg/L；
The a great number of elements II preferably comprises: the KH of 1650~1730mg/L2PO4, more preferably 1680~1720mg/L,
Most preferably 1700mg/L；
The microelement II preferably comprises the KI of 0.75~0.85mg/L, more preferably 0.78~0.84mg/L, optimal
It is selected as 0.83mg/L；
The microelement II preferably comprises the Na of 0.22~0.30mg/L2MoO4·2H2O, more preferably 0.24~
0.28mg/L, most preferably 0.25mg/L；
The microelement II preferably comprises the MnSO of 22.0~23.0mg/L4·4H2O, more preferably 22.1~
22.7mg/L, most preferably 22.3mg/L；
The microelement II preferably comprises the ZnSO of 8.4~8.8mg/L4·7H2O, more preferably 8.5~8.7mg/L,
Most preferably 8.6mg/；
The microelement II preferably comprises the CuSO of 0.02~0.03mg/L4·5H2O, more preferably 0.022~
0.028mg/L, most preferably 0.025mg/L；
The microelement II preferably comprises the CoCl of 0.02~0.03mg/L2·6H2O, more preferably 0.023~
0.029mg/L, most preferably 0.025mg/L；
The microelement II preferably comprises the FeSO of 27~28mg/L4·7H2O, more preferably 2.73~27.9mg/L,
Most preferably 27.8mg/L；
The microelement II preferably comprises the Na of 36.8~38mg/L2EDTA·2H2O, more preferably 37.1~
37.8mg/L, most preferably 37.3mg/L；
The microelement II preferably comprises the H of 6.0~7.2mg/L3BO3, more preferably 6.1~6.9mg/L, most preferably
For 6.2mg/L；
The organic principle II preferably comprises the inositol of 85~110mg/L, more preferably 88~105mg/L, most preferably
100mg/L；
The organic principle II preferably comprises the vitamin B of 0.3~0.6mg/L1, more preferably 0.35~0.55mg/L,
Most preferably 0.5mg/L；
The organic principle II preferably comprises the niacin of 0.3~0.6mg/L, more preferably 0.40~0.55mg/L, optimal
It is selected as 0.5mg/L；
The organic principle II preferably comprises the vitamin B of 0.3~0.6mg/L6, more preferably 0.42~0.58mg/L,
Most preferably 0.5mg/L；
The organic principle II preferably comprises the glycine of 1.8~2.5mg/L, more preferably 1.9~2.4mg/L, optimal
It is selected as 2.0mg/L.
In the present invention, the MS culture medium is to add other components on the basis of MS minimal medium.
In the present invention, the MS culture medium include 0.8~1.2mg/L 6- benzyl aminoadenine, preferably 0.9~
1.1mg/L, more preferably 1.0mg/L；
The MS culture medium include 0.3~0.6mg/L indolebutyric acid, preferably 0.35~0.5mg/L, more preferably
0.4mg/L；
The MS culture medium includes 18~22mg/L kanamycins, preferably 19~21mg/L, more preferably 20mg/L.
In the present invention, the pH value of the MS culture medium is 5~6.5, more preferably 5.8~6.0.
In the present invention, it is described expand it is numerous after it is also preferable to include: by it is described expand it is numerous after wild jujube plant after obtained genetic transformation
Carry out Molecular Detection.
In the present invention, the Molecular Detection preferably includes following steps:
Transgenic plant is detected using PCR method, according to whether there is purpose gene Pt-SPDS (1094bp) item
Band determines the integration of target gene DNA.If there is target gene band, it can tentatively illustrate that SPDS gene has been transferred to wild jujube
Plant.
In the present invention, preferably also include after the Molecular Detection: the transgenic plant obtained after the detection is carried out
Small seedling rooting and transplanting.
In the present invention, the small seedling rooting and the process of transplanting are preferred specifically: are being free of the transgenic plant
Subculture 3 times in the wild jujube subculture medium of antibiotic, after plant height it is long to be transferred in root media after 1.5~2cm carry out it is sterile
Culture of rootage to tissue-cultured seedling root growth stalwartness or is exposed to outside Jiffy breeding block, bottle cap is opened, fits it gradually
External environment condition is answered, adds water or liquid root media in time during which to keep moisture.After hardening 3d, plant is taken out, is moved
Plant to being to be put into growth cabinet culture in the matrix of 1:1 containing vermiculite and soil, condition of culture be 25 DEG C of temperature, humidity 90%,
Intensity of illumination 2000lx, photoperiod 14h light/10h are dark.After 4 weeks, greenhouse continued growth is gone to.
In the present invention, the wild jujube subculture medium is to add other components on the basis of MS minimal medium.
In the present invention, the wild jujube subculture medium includes 0.8~1.2mg/L 6- benzyl aminoadenine, preferably
0.9~1.1mg/L, more preferably 1.0mg/L；
In the present invention, the wild jujube subculture medium include 0.3~0.6mg/L indolebutyric acid, preferably 0.35~
0.5mg/L, more preferably 0.4mg/L；
In the present invention, the wild jujube subculture medium includes the sucrose of 25~35g/L, and preferably 28~32g/L is more excellent
It is selected as 30g/L；
In the present invention, the wild jujube subculture medium includes the agar of 5~7g/L, and preferably 5.2~6.5g/L is more excellent
It is selected as 5.8g/L；
In the present invention, the pH value of the wild jujube subculture medium is 5.0~6.5, more preferably 5.8~6.0.
In the present invention, the root media preferably comprises the 1/2MS liquid training of 0.35~0.5mg/L indolebutyric acid
Base and diameter are supported as the Jiffy nutritive cube of 30mm；The liquid root media preferably comprises 0.35~0.5mg/L indoles
The 1/2MS fluid nutrient medium of butyric acid.
Below with reference to embodiment, to the method provided by the invention for establishing wild jujube genetic conversion system using blade for receptor
It is described in detail, but they cannot be interpreted as to the restriction to the application protection scope.
Embodiment 1
Choose the wild jujube tissue-cultured seedling that grows fine, cut morphology from top to bottom the 3rd~5 piece of blade as converting material,
Along crosscutting 2 wounds of blade middle arteries vertical direction, cuts off master pulse but do not cut off blade, in a manner of paraxial end in contact culture medium (such as
Fig. 1 a) is inoculated in WPM adventitious bud Primary culture base (TDZ1.0mg/L+IBA 0.3mg/L+30g/L sucrose+6g/L agar).?
On WPM adventitious bud Primary culture base after dark culture induction 3d, carries out Agrobacterium and infect.
The pH value of WPM adventitious bud Primary culture base is 5.8.WPM minimal medium formula are as follows:
(1) a great number of elements: NH4NO3(400mg/L)、Ca(NO3)2·4H2O(556mg/L)K2SO4(990mg/L)、
CaCl2·2H2O(96mg/L)、KH2PO4(170mg/L)、MgSO4·7H2O(370mg/L)；
(2) microelement: Na2MoO4·2H2O(0.25mg/L)、MnSO4·H2O(22.4mg/L)、ZnSO4·7H2O
(8.6mg/L)、CuSO4·5H2O(0.25mg/L)、FeSO4·7H2O(27.8mg/L)、Na2EDTA·2H2O(37.3mg/L)、
H3BO3(6.2mg/L)；(3) organic principle: inositol 100mg/L, vitamin B11.0mg/L, niacin 0.5mg/L, vitamin
B60.5mg/L, glycine 2.0mg/L.
The single colonie of agrobacterium strains is inoculated in 20ml rifampin containing 50mg/L, 50mg/L cephalosporin and 50mg/L card
In the YEB fluid nutrient medium of that mycin, 180rpm shaken cultivation is to OD under 28 DEG C of dark600It is 0.6；Take the above-mentioned bacterium solution of 20uL in
In the not antibiotic YEB fluid nutrient medium of 50mL, continue shaken cultivation under similarity condition to OD600Close to 0.4~0.8, no
Result is influenced on wild jujube blade genetic transformation rate with Agrobacterium bacterial concentration as shown in Fig. 2, working as bacterium solution OD600Value is significant when being 0.8
Conversion ratio when lower than 0.4 and 0.6 illustrates that suitable bacterial concentration range is 0.4~0.6.Then to the bacterium in super-clean bench
200 μm of ol/L acetosyringones are added in liquid, continue shaken cultivation 1h.
Agrobacterium strains contain pBI121 carrier, and expression vector map is as shown in Figure 3.Carrier is with spermidine synthase gene
SPDS gene as a purpose, using neomycin phosphotransferase gene NPT II as riddled basins.
Explant infects
1. the collection of explant: the explant in culture dish is added in superclean bench with tweezers the sky of high-temperature sterilization
In triangular flask；
2. explant infects: the Agrobacterium bacterium solution prepared being poured into above-mentioned triangular flask, is gently shaken with hand, institute is made
There is blade to immerse in Agrobacterium bacterium solution, infects 10,15,20min, Agrobacterium time of infection is to wild jujube blade genetic transformation rate shadow
Result is rung as shown in figure 4, the average conversion pole for infecting 20min illustrates that time of infection is significantly lower than 10min and 15min
More transformation seedlings can be obtained when 10min or 15min.
3. the removal of extra bacterium solution: infecting end, explant tweezers are added in the culture dish containing aseptic filter paper；With nothing
Bacterium filter paper blots explant surface residual bacterium solution.
It co-cultures
Metainfective explant is placed on to the WPM culture medium solid medium added with 1.0mg/LTDZ and 0.3mg/LIBA
2d, 3d, 4d respectively are co-cultured under the conditions of upper 25 DEG C, co-culture the time to wild jujube blade genetic transformation rate influence result such as Fig. 5 institute
Show, average conversion highest when co-culturing 3d, that is, the Best Times co-cultured are 3d.
The induction of adventitious bud
1. the induction of leaf regeneration adventitious bud: after co-cultivation, by blade with sterile water wash 3 times, being then transferred to sieve
It selects in culture medium；Screening and culturing medium be WPM solid medium and add 1.0mg/LTDZ, 0.3mg/L IBA, 200mg/LCef and
20mg/L Kan, different Kan concentration averagely sprout to adventitious bud number influence result as shown in fig. 6, when Kan concentration be 20mg/L
When, blade differentiation rate reduces rapidly, and average each blade budding number starts less than 1.And continue growing the dense of kanamycins
It is found when degree is to 25mg/L, blade differentiation rate is extremely low, almost without the adventitious bud to grow fine.Select the kanamycins of 20mg/L
Critical concentration as screening transformed plant.Dark culture under the conditions of 25 DEG C, until the total 21d of blade dark culture.
2. the sprouting and growth (such as Fig. 1, b) of adventitious bud: after dark culture, explant being transferred to adventitious bud induction culture
In base, adventitious bud induction culture base is that WPM solid medium adds 0.1mg/L IBA, 0.5mg/L GA3, 200mg/LCef and
20mg/L Kan；Cultivation temperature is 25 DEG C, photoperiod 14h.
The switching of adventitious bud
Stem length to adventitious bud is that 15~20mm is gone to the benzyl aminoadenine of 6- containing 1.0mg/L and 0.4mg/L indoles
It carries out expanding numerous (such as Fig. 1, c) in the MS culture medium of butyric acid and 20mg/L kanamycins.
MS minimal medium pH value is 5.8, MS minimal medium formula are as follows:
(1) a great number of elements: NH4NO3(1650mg/L)、KNO3(1900mg/L)、CaCl2·2H2O(440mg/L)、
MgSO4·7H2O(370mg/L)、KH2PO4(1700mg/L)；
(2) microelement: KI (0.83mg/L), Na2MoO4·2H2O(0.25mg/L)、MnSO4·4H2O(22.3mg/L)、
ZnSO4·7H2O(8.6mg/L)、CuSO4·5H2O(0.025mg/L)、CoCl2·6H2O(0.025mg/L)、FeSO4·7H2O
(27.8mg/L)、Na2EDTA·2H2O(37.3mg/L)、H3BO3(6.2mg/L)；
(3) organic principle: inositol 100mg/L, vitaminB10 .5mg/L, niacin 0.5mg/L, vitamin B60.5mg/L、
Glycine 2.0mg/L.
Transgenic plant Molecular Detection
Transgenic plant Molecular Detection, which refers to, detects transgenic plant using PCR method, according to whether purposefully
Gene band determines the integration of target gene DNA.
102 kalamycin resistance buds are obtained after screening for the first time in the present invention.Third time has 19 resistant buds after screening
Grow up to intact plant, extract the DNA of these plant, and carry out PCR amplification and electrophoresis detection, as a result as shown in fig. 7, sharing 9 plants
It is positive, accounts for the 47% of resistant plant, positive plant conversion ratio is 8.82%.
Small seedling rooting and transplanting
The plant that detection terminates continues to cultivate, and after length to plant height is 1.5~2cm, is transferred in root media and takes root, root
After growing to 2~3cm, continued growth in Nutrition Soil is moved into.The seedling root media is will be containing the 1/2MS of indolebutyric acid
Fluid nutrient medium pours after Jiffy nutritive cube, is sterilized in high-pressure sterilizing pot, contains in finally obtained culture medium
There is the indolebutyric acid of 0.4mg/L.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of method for establishing wild jujube genetic conversion system using blade for receptor, comprising the following steps:
1) wild jujube blade is inoculated in dark culture in WPM adventitious bud Primary culture base and induces 2~4d, obtain explant, the WPM
Adventitious bud Primary culture base includes 0.8~3.0mg/L Thidiazuron, 0.2~0.5mg/L indolebutyric acid, 28~32g/L sucrose and 5
~7g/L agar；
2) the Agrobacterium bacterium solution containing plant expression vector is infected into the explant that the step 1) obtains, obtains being infected outer
Implant；
3) explant infected for obtaining the step 2) is placed on WPM solid medium, dark culture 2~4d, the WPM
Solid medium includes 0.5~2.0mg/L Thidiazuron and 0.25~0.4mg/L indolebutyric acid；
4) blade after the step 3) dark culture is transferred to progress 14~16d of dark culture in WPM solid screening and culturing medium, it is described
WPM solid screening and culturing medium contains 0.8~2.5mg/L Thidiazuron, 0.2~0.4mg/L indolebutyric acid, 180~220mg/L cephalo
Mycin and 15~25mg/L kanamycins；
After the dark culture, it is transferred to the elongation culture for carrying out leaf regeneration in WPM elongation medium, obtains adventitious bud, institute
It states WPM elongation medium and contains 0.1mg/L heteroauxin and 0.5mg/L gibberellin；
5) adventitious bud for obtaining the step 4) expands numerous, the wild jujube plant after obtaining genetic transformation；
Plant expression vector is pBI121-SPDS in the step 2).
2. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
The time infected in the step 2) is 10~20min.
3. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
During the adventitious bud induction culture of the step 4), the photoperiod of adventitious bud induction culture is 12~16h/d, the adventitious bud
The time of Fiber differentiation is 30~40d, and the WPM solid adventitious bud induction culture base includes 0.08~0.12mg/L indoles fourth
Acid, 0.3~0.6mg/L gibberellin, 180~220mg/L cephalosporin and 15~25mg/L kanamycins.
4. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
The adventitious bud of the step 5) expands numerous specifically: when the stem length of adventitious bud is 15~20mm, adventitious bud is gone to MS culture medium
It is middle expand it is numerous, the MS culture medium include 0.8~1.2mg/L 6- benzyl aminoadenine, 0.3~0.6mg/L indolebutyric acid and 18~
22mg/L kanamycins.
5. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
The step 1), 3), 4) He 5) in cultivation temperature be independently selected as 25 ± 2 DEG C.
6. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
Before the step 1) further include: wild jujube blade is carried out pre-treatment；
The process of the pre-treatment specifically: wild jujube tissue-cultured seedling morphology the 3rd~5 piece of blade from top to bottom is cut, by what is cut
Blade is as converting material；
By the converting material along crosscutting 2~4 wounds of blade middle arteries vertical direction, cuts off master pulse but do not cut off blade；
Inoculation in the step 1) is carried out in a manner of paraxial end in contact culture medium.
7. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
The preparation method of the Agrobacterium bacterium solution is the following steps are included: be inoculated in YEB fluid nutrient medium for the single colonie of agrobacterium strains
Middle dark culture is 0.5~0.7 to OD600, and the YEB fluid nutrient medium includes 40~55mg/L rifampin, 40~55mg/L head
P0-357 and 40~55mg/L kanamycins；
The bacterium solution that the dark culture obtains is inoculated in not antibiotic YEB Liquid Culture with 0.02~0.05% inoculum concentration
In base, continuing to cultivate to OD600 is 0.4~1.0；
By the OD600 be 0.4~1.0 bacterium solution mixed with the acetosyringone of 150~300 μm of ol/L, continue culture 0.8~
1.5h obtains Agrobacterium bacterium solution.
8. the method according to claim 1 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
After the step 5) further include: the wild jujube plant after the genetic transformation is carried out Molecular Detection；
The Molecular Detection the following steps are included:
The wild jujube plant after genetic transformation is detected using PCR method, according to whether there is purpose gene band, determines purpose
The integration of gene DNA.
9. the method according to claim 8 for establishing wild jujube genetic conversion system using blade for receptor, which is characterized in that
Also include after the Molecular Detection: the plant after the detection is subjected to small seedling rooting and transplanting；
The process of the small seedling rooting and transplanting specifically: continue to cultivate by the plant after the detection, be 1.5 to length to plant height
After~2cm, it is transferred in root media and takes root；After root growth to 2~3cm, continued growth in Nutrition Soil is moved into.
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