CN107034222A - A kind of method for improving strawberry drought-resistant ability - Google Patents
A kind of method for improving strawberry drought-resistant ability Download PDFInfo
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Abstract
The present invention provides a kind of method for improving strawberry drought-resistant ability, pass through genetic engineering means, the rty genes for regulating and controlling root system development are successfully imported in strawberry, the Transgenic Strawberry plant that drought-resistant ability is improved is obtained, it is to improve the drought-resistant ability of strawberry by strengthening the rugosity and quantity of strawberry root system.This method has that the cycle is short, positive rate is high, it is simple to operate the characteristics of, new germ plasm resource is provided for strawberry genetic breeding.
Description
Technical field
The present invention relates to technical field of plant transgene and field of crop genetic breeding, specifically, it is related to a kind of raising
The method of strawberry drought-resistant ability.
Background technology
Strawberry (Fragaria ananassa Duch) is rose family Fragaria herbaceos perennial, is that a kind of world is each
The fruit that state cultivates extensively, have the title of fruit queen.Due to its high heterozygosity and polyploidy so that the crossbreeding work period
Long, workload is big.Breeding effect can be greatly improved with the existing kind of orderly improvement by cultivating strawberry cultivars using gene regulation technology
A kind of rate, the important means that will be improved as strawberry cultivars is mainly Agrobacterium applied to the electroinjection on strawberry at present
The leaf disk method of mediation.Due to the scarcity of water resource, improve the drought resistance of plant turns into the common recognition of water-deficient area people.
The content of the invention
It is an object of the invention to provide a kind of method for improving strawberry drought-resistant ability.
In order to realize the object of the invention, present invention firstly provides rty genes improve plant drought ability in application, from
The rty genes that acquisition is cloned in wildtype Arabidopsis thaliana blade are：i)SEQ ID NO:Nucleotide sequence shown in 1；Or ii) SEQ
ID NO:Nucleotide sequence shown in 1 is substituted, lacks and/or increased one or more nucleotides and expression identical function albumen
The nucleotide sequence of matter；Or iii) under strict conditions with SEQ ID NO:Sequence hybridization shown in 1 and expression identical function albumen
The nucleotide sequence of matter, the stringent condition is molten in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC containing 0.1%SDS
In liquid, hybridize at 65 DEG C, and film is washed with the solution；Or iv) and i), ii) or nucleotide sequence iii) have more than 90%
Homology and the nucleotide sequence for expressing identical function protein.
Plant of the present invention includes but is not limited to strawberry.
The present invention also provides a kind of method for improving strawberry drought-resistant ability, comprises the following steps：
(1) the tissue-cultured seedling immersion of strawberry is carried into rty genes and selectable marker gene (such as kalamycin resistance gene
NptII infected in Agrobacterium bacterium solution) 3-6 minutes (preferably 5 minutes)；
(2) tissue-cultured seedling is moved on regeneration culture medium and cultivated；
(3) tissue-cultured seedling is moved on differential medium and cultivated, obtain resistant budses；
(4) hardening, transplanting, obtain Transgenic Strawberry plant after resistant budses are taken root on root media；
(5) the positive transgenic plant containing target gene rty is detected using PCR methods.
The preparation method of Plantlets of Strawberry is in foregoing method, step (1)：The bud on strawberry stolon is adopted, is rushed with water
Wash after (rinse 3 hours), be put into 4-6 minutes (preferably 5 minutes) of processing in 0.1% mercuric chloride, then with aseptic water washing 3-6 times
(preferably 6 times), blot after the residual moisture on bud, bud are inoculated into Initial culture base, and after cultivating 10-12 days, bud starts to sprout
Tissue-cultured seedling is moved to culture growth 30-35 days on subculture medium by hair, the big tissue-cultured seedling of 2-4cm (preferably 3cm or so) to be grown up to
(preferably 35 days), take stretching, extension blade to be cut into leaf dish, prepare to infect.
The preparation method for carrying the Agrobacterium bacterium solution of rty genes and selectable marker gene is：
1) plant expression vector pBI121-rty is built：By in rty gene clonings to T-easy carriers, carrier construction T-
Rty is small by what is reclaimed after T-rty digestions then with restriction endonuclease Xba I and Sac I difference double digestion carrier T-rty and pBI121
Fragment and the large fragment reclaimed after pBI121 digestions, build in the presence of ligase and obtain plant expression vector pBI121-rty
(SEQ ID NO:2)；
2) Agrobacterium-mediated Transformation：With pBI121-rty convert agrobacterium tumefaciens lba4404, under 28 ± 2 DEG C (preferably 28 DEG C)
Containing 100mgL-1Cultivated in the LB fluid nutrient mediums of kanamycins after 15-16 hours (preferably 16 hours), use MS fluid nutrient mediums
Continue to cultivate 2-4 hours (preferably 2 hours), treat bacterium solution OD600For 0.3-0.5 (preferably OD600For 0.4) when be used for infect.
Tissue-cultured seedling after being infected in foregoing method, step (2) removes unnecessary bacterium solution, is seeded on regeneration culture medium,
28 ± 2 DEG C of light cultures 2-3 days (preferably 28 DEG C light culture 2 days).
The tissue-cultured seedling after regeneration culture is transferred on differential medium in foregoing method, step (3), 28 ± 2
DEG C light culture 10-12 days (preferably 28 DEG C light culture 12 days), then 25 ± 2 DEG C, 16 hours photoperiods illumination/8 hour it is dark,
25-35 μm of olm of intensity of illumination-2·s-1(preferably 30 μm olm-2·s-1) under cultivate 30-40 days, acquisition resistant budses.
Foregoing method, step cuts resistant budses in (4), is seeded on root media, in 25 ± 2 DEG C, photoperiod
16 hours illumination/8 hour are dark, 25-35 μm of olm of intensity of illumination-2·s-1(preferably 30 μm olm-2·s-1) under cultivate 28-
(preferably 30 days) take root within 35 days, obtain the resistance strawberry taken root.
Using-the GATGAGCGAAGAACAACCACAC-3 ' of rty upstream region of gene primer 5 ' and anti-sense primer 5 '-
TTCAAACCCAGAGCATCCCCTG-3 ', the target gene rty of the resistance strawberry using PCR method to taking root carries out sun
Property identification, obtain the strawberry that is handled by gene means, compared with the control, adventitious root is sturdy and radical is more.
30 plants of the Strawberry Seedlings obtained by said gene engineering means are with compareing 30 plants of seedling, while being transplanted to containing Nutrition Soil
(turf, vermiculite, rural area soil mass ratio are 1：1：1) in alms bowl, a water is poured weekly, and water 300mL is resisted for 90 days to be grown
Drought experiment.After the water for pouring 300mL, do not rewater, continuous observation, at the 10th day, Transgenic Strawberry plant is not wilted, and is compareed
(non-transgenic strawberry) 100% wilts, serious control seedling dead.Further identification finds to pass through present invention side
The strawberry drought-resistant ability that method is obtained is greatly improved.
The strawberry cultivars being related in the present invention include strawberry ' Hani '.
The culture medium being related in the present invention is as follows：
Initial culture base is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 0.5-
1.0mg·L-16-benzyladenine and 0.1-0.4mgL-1Indolebutyric acid, pH5.6.Preferably, Initial culture base is：MS bases
30gL is added in basal culture medium-1Sucrose, 6.5gL-1Agar powder, 1.0mgL-16-benzyladenine and 0.2mgL-1Yin
Diindyl butyric acid, pH5.6.
Subculture medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 0.1-
0.5mg·L-16-benzyladenine and 0.1-0.4mgL-1Indolebutyric acid, pH5.6.Preferably, subculture medium is：MS bases
30gL is added in basal culture medium-1Sucrose, 6.5gL-1Agar powder, 0.2mgL-16-benzyladenine and 0.1mgL-1Yin
Diindyl butyric acid, pH5.6.
Regeneration culture medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 2.0-
4.0mg·L-16-benzyladenine and 0.05-0.2mgL-12,4- dichlorphenoxyacetic acids, pH5.6.Preferably, regeneration culture
Base is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 3.0mgL-16-benzyladenine and
0.1mg·L-12,4- dichlorphenoxyacetic acids, pH5.6.
Differential medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 300-
500mg·L-1Cephalosporin, 5-12mgL-1Kanamycins, 2.0-4.0mgL-16-benzyladenine and 0.05-0.2mg
L-12,4- dichlorphenoxyacetic acids, pH5.6.Preferably, differential medium is：30gL is added in MS minimal mediums-1Sucrose,
6.5g·L-1Agar powder, 400mgL-1Cephalosporin, 7.5mgL-1Kanamycins, 3.0mgL-16-benzyladenine and
0.1mg·L-12,4- dichlorphenoxyacetic acids, pH5.6.
Root media is：Added in 1/2MS minimal mediums 20-35gL-1 sucrose, 5-7gL-1 agar powders and
0.1-0.4mgL-1 indolebutyric acids, pH5.6.Preferably, root media is：30gL is added in 1/2MS minimal mediums-1
Sucrose, 6.5gL-1Agar powder and 0.2mgL-1Indolebutyric acid, pH5.6.
LB solid mediums are：10g·L-1Peptone, 5gL-1Yeast extract, 10gL-1NaCl、6.5g·L-1Fine jade
Cosmetics, pH7.0.
LB fluid nutrient mediums are：10g·L-1Peptone, 5gL-1Yeast extract, 10gL-1NaCl, pH7.0.
The formula of the MS minimal mediums is as follows, and wherein each component unit is mgL-1：
The present invention is successfully imported the rty genes for regulating and controlling root system development in strawberry by genetic engineering means, obtains drought resisting
The Transgenic Strawberry plant that ability is improved, it is to improve the drought resisting energy of strawberry by strengthening the rugosity and quantity of strawberry root system
Power.This method has that the cycle is short, positive rate is high, it is simple to operate the characteristics of, new germ plasm resource is provided for strawberry genetic breeding.
Brief description of the drawings
The structure flow chart that Fig. 1 is plant expression vector pBI121-rty in the embodiment of the present invention.
Fig. 2 is strawberry ' Hani ' leaf dish for preparing in the embodiment of the present invention.
The resistant budses that Fig. 3 is obtained for 40 days after being handled for Agrobacterium tumefaciems in the embodiment of the present invention.
Fig. 4 is the positive agarose gel electrophoresis result for turning rty gene strawberries of PCR identifications in the embodiment of the present invention.
Wherein, M is DNA molecular amount standard, and 1-9 is respectively that the different positives turns rty gene strawberries, and 10 be negative control, and 11 are
Positive control.
Fig. 5 be the embodiment of the present invention on root media cultivate 30 days after Transgenic Strawberry plant (on) and control it is non-
Transgenic Strawberry plant (under) take root and compare.
Fig. 6 is that the increased Transgenic Strawberry plant (left side) of the sturdy radical of root system obtained in the embodiment of the present invention and control are non-
The root system of Transgenic Strawberry plant (right side) compares.
Fig. 7 is the Transgenic Strawberry plant (left side) that the drought-resistant ability that obtains is improved in the embodiment of the present invention and control is non-turns base
Because of the comparison on strawberry (right side).
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular cloning:A laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.
The culture medium being related in following examples is as follows：
Initial culture base is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 1.0mgL- 16-benzyladenine and 0.2mgL-1Indolebutyric acid, pH5.6.
Subculture medium is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 0.2mgL- 16-benzyladenine and 0.1mgL-1Indolebutyric acid, pH5.6.
Regeneration culture medium is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 3.0mgL- 16-benzyladenine and 0.1mgL-12,4- dichlorphenoxyacetic acids, pH5.6.
Differential medium is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 400mgL-1
Cephalosporin, 7.5mgL-1Kanamycins, 3.0mgL-16-benzyladenine and 0.1mgL-12,4- dichlorphenoxyacetic acids,
pH5.6。
Root media is：30gL is added in 1/2MS minimal mediums-1Sucrose, 6.5gL-1Agar powder and
0.2mg·L-1Indolebutyric acid, pH5.6.
LB solid mediums are：10g·L-1Peptone, 5gL-1Yeast extract, 10gL-1NaCl、6.5g·L-1Fine jade
Cosmetics, pH7.0.
LB fluid nutrient mediums are：10g·L-1Peptone, 5gL-1Yeast extract, 10gL-1NaCl, pH7.0.
The method that embodiment improves strawberry drought-resistant ability
1st, the preparation of the tissue-cultured seedling of strawberry
Operation obtains tissue culturing seedling's (tissue-cultured seedling) of strawberry ' Hani ' according to the following steps：Autumn adopts satisfies on strawberry stolon
Full bud, flowing water is rinsed 3 hours, with 0.1% mercuric chloride (HgCl2) handle 6 minutes, aseptic water washing 6 times uses sterile blotting paper
Residual moisture is blotted, bud is inoculated into Initial culture base.The 2nd day after inoculation begins with portion of material and starts browning, one
Denier finds the material for having browning, and new Initial culture base is replaced by once, until browning is eliminated；When being inoculated with 10 days, full bud
All start to sprout, about 3cm or so tissue-cultured seedling is grown up at 30 days.Monthly subculture is once on subculture medium later.' it will breathe out
Buddhist nun ' tissue-cultured seedling cultivated on subculture medium growth 35 days, you can cut leaf dish prepare infect.
2nd, plant expression vector pBI121-rty is built
Rty genes (the SEQ ID NO obtained will be cloned from wildtype Arabidopsis thaliana blade:1) T-easy carriers are linked to
On, carrier construction T-rty, then with restriction endonuclease Xba I and Sac I difference double digestion carrier T-rty and pBI121, by T-rty
The small fragment reclaimed after digestion and the large fragment reclaimed after pBI121 digestions, build in the presence of ligase and obtain plant expression
Carrier pBI121-rty.Carrier pBI121-rty structure flow chart is shown in Fig. 1, sequence such as SEQ ID NO:Shown in 2,
3rd, Agrobacterium-mediated Transformation
10uL carrier pBI121-rty and 100uL Agrobacterium competence LBA4404 is taken to be added in 1.5uL centrifuge tube respectively
In, 30 minutes on ice after mixing；It is quick-frozen 2-3 minutes in liquid nitrogen, 37 DEG C of water-baths 3 minutes；Add 800uL LB fluid nutrient mediums
(being free of antibiotic), under the conditions of 28 DEG C, 180 turns are cultivated 2-4 hours；Then 5000 the heart is left 5 minutes, centrifugation bottom of the tube is stayed
100uL, mixing coated plate, (LB solid mediums contain 100mgL in culture dish-1Kanamycins)；Incubated overnight, chooses single bacterium colony,
The agrobacterium tumefaciens lba4404 for being accredited as the positive is the Agrobacterium for carrying target gene rty.
Carry external source target gene rty and selectable marker gene nptII agrobacterium tumefaciens lba4404, at 28 DEG C
Containing 100mgL-1After cultivating 16 hours in the LB fluid nutrient mediums of kanamycins, continued to cultivate crown gall agriculture with MS fluid nutrient mediums
Bacillus 2 hours, bacterial concentration reaches OD600For 0.4, bacterium solution is standby.The squamous subculture tissue-cultured seedling of 35 days is taken on superclean bench
Blade is stretched, about 1-3mm is cut into scissors2Leaf dish, be put into 5 minutes (Fig. 2) in standby bacterium solution.Unnecessary bacterium solution is removed, is handled
Leaf dish afterwards is seeded on regeneration culture medium 28 DEG C of light cultures 2 days.
4th, differentiation, culture of rootage and the identification of positive transgenic strawberry
By leaf dish switching light culture 12 on 400mg/L containing cephalosporin, kanamycins 7.5mg/L differential medium
My god；Then in (25 ± 2) DEG C, 16 hours photoperiods dark, 30 μm of olm of intensity of illumination in illumination/8 hour-2·s-1Lower culture
It is differentiated to form within 30-40 days resistant budses (Fig. 3).The resistance adventitious bud of acquisition is chosen, be seeded in the 400mg/L containing cephalosporin after
Grow up to plantlet for culture medium, then plantlet is seeded on root media, 25 ± 2 DEG C, 16 hours photoperiods illumination/8
Hour dark, 30 μm of olm of intensity of illumination-2·s-1Lower culture completes to take root for 30 days.The now root of partial transgenic strawberry
Compared with compareing (non-transgenic strawberry), adventitious root is thick (Fig. 4).
Using-the GATGAGCGAAGAACAACCACAC-3 ' of rty upstream region of gene primer 5 ' and anti-sense primer 5 '-
TTCAAACCCAGAGCATCCCCTG-3 ', positive identification (Fig. 5) is carried out using PCR method to target gene rty.At 90 days,
The strawberry handled by gene means is obtained, compared with the control, adventitious root is sturdy and radical is more (Fig. 6).
Further by the strain of the seedling of Transgenic Strawberry plant 30 with compareing 30 plants of seedling, while being transplanted to containing Nutrition Soil (turf, leech
Stone, rural area soil volume ratio are 1：1：1) in alms bowl, a water is poured weekly, and irrigation amount 300mL carries out drought resisting in 90 days to be grown real
Test.After the water for pouring 300mL, do not rewater, continuous observation, at the 10th day, Transgenic Strawberry plant is not wilted, and compares (non-
Transgenic Strawberry plant) 100% wilt, serious control seedling dead.Further identification finds to pass through the inventive method
The strawberry drought-resistant ability of acquisition is greatly improved (Fig. 7).
Same batch experiment is using 5 culture dishes in the present embodiment, and each ware places 30 or so leaf dishes, and experiment repeats 6
Secondary, leaf dish total amount is 916 altogether, obtains 32 parts of the plant (table 1) that drought-resistant ability is improved.
The experimental result of the strawberry cultivars of table 1 ' Hani '
Note：PCR positive plants described in table 1 are identical with the increased plant numbering of drought resistance.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
- Application of the 1.rty genes in plant drought ability is improved, it is characterised in that the rty genes are：i)SEQ ID NO:Nucleotide sequence shown in 1；Orii)SEQ ID NO:Nucleotide sequence shown in 1 is substituted, lacks and/or increased one or more nucleotides and expression The nucleotide sequence of identical function protein；OrIii) under strict conditions with SEQ ID NO:Sequence hybridization shown in 1 and the nucleotides sequence for expressing identical function protein Row, the stringent condition is in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, at 65 DEG C Hybridization, and wash film with the solution；OrIv) and i), ii) or nucleotide sequence iii) there is more than 90% homology and the nucleosides of identical function protein is expressed Acid sequence.
- 2. application according to claim 1, it is characterised in that the plant includes strawberry.
- 3. a kind of method for improving strawberry drought-resistant ability, it is characterised in that comprise the following steps：(1) the tissue-cultured seedling immersion of strawberry is carried and infected 3-6 minutes in the Agrobacterium bacterium solution of rty genes and selectable marker gene；(2) tissue-cultured seedling is moved on regeneration culture medium and cultivated；(3) tissue-cultured seedling is moved on differential medium and cultivated, obtain resistant budses；(4) hardening, transplanting, obtain Transgenic Strawberry plant after resistant budses are taken root on root media；(5) the positive transgenic plant containing target gene rty is detected using PCR methods；Wherein, the definition of rty genes is with the description in claim 1.
- 4. method according to claim 3, it is characterised in that the preparation method of Plantlets of Strawberry is in step (1)：Adopt grass Bud on certain kind of berries stolon, after being rinsed with water, is put into 0.1% mercuric chloride and handles 4-6 minutes, then with aseptic water washing 3-6 times, Blot after the residual moisture on bud, bud is inoculated into Initial culture base, after cultivating 10-12 days, bud starts to sprout, 2- to be grown up to Tissue-cultured seedling big 4cm, moves to culture growth 30-35 days on subculture medium by tissue-cultured seedling, takes stretching, extension blade to be cut into leaf dish, prepare Infect.
- 5. method according to claim 3, it is characterised in that carry rty genes and selectable marker gene in step (1) The preparation method of Agrobacterium bacterium solution is：1) plant expression vector pBI121-rty is built：By in rty gene clonings to T-easy carriers, carrier construction T-rty, so Afterwards with restriction endonuclease Xba I and Sac I difference double digestion carrier T-rty and pBI121, by the small fragment reclaimed after T-rty digestions with The large fragment reclaimed after pBI121 digestions, builds in the presence of ligase and obtains plant expression vector pBI121-rty；2) Agrobacterium-mediated Transformation：Converted with pBI121-rty at agrobacterium tumefaciens lba4404,28 ± 2 DEG C containing 100mgL-1Block that After being cultivated 15-16 hours in the LB fluid nutrient mediums of mycin, continued to cultivate 2-4 hours with MS fluid nutrient mediums, treat bacterium solution OD600 It is used to infect during for 0.3-0.5；Wherein, the Initial culture base is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 0.5-1.0mg·L-16-benzyladenine and 0.1-0.4mgL-1Indolebutyric acid, pH5.6；Preferably, Initial culture base is： 30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 1.0mgL-16-benzyladenine and 0.2mg L-1Indolebutyric acid, pH5.6；The subculture medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 0.1- 0.5mg·L-16-benzyladenine and 0.1-0.4mgL-1Indolebutyric acid, pH5.6；Preferably, subculture medium is：MS bases 30gL is added in basal culture medium-1Sucrose, 6.5gL-1Agar powder, 0.2mgL-16-benzyladenine and 0.1mgL-1Yin Diindyl butyric acid, pH5.6.
- 6. method according to claim 3, it is characterised in that the tissue-cultured seedling after being infected in step (2) removes unnecessary bacterium Liquid, is seeded on regeneration culture medium, 28 ± 2 DEG C of light cultures 2-3 days；The regeneration culture medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 2.0- 4.0mg·L-16-benzyladenine and 0.05-0.2mgL-12,4- dichlorphenoxyacetic acids, pH5.6；Preferably, regeneration culture Base is：30gL is added in MS minimal mediums-1Sucrose, 6.5gL-1Agar powder, 3.0mgL-16-benzyladenine and 0.1mg·L-12,4- dichlorphenoxyacetic acids, pH5.6.
- 7. method according to claim 3, it is characterised in that turn the tissue-cultured seedling after regeneration culture in step (3) Be connected on differential medium, 28 ± 2 DEG C of light cultures 10-12 days, then 25 ± 2 DEG C, 16 hours photoperiods illumination/8 hour it is black Secretly, 25-35 μm of olm of intensity of illumination-2·s-1Lower culture 30-40 days, obtains resistant budses；The differential medium is：20-35gL is added in MS minimal mediums-1Sucrose, 5-7gL-1Agar powder, 300- 500mg·L-1Cephalosporin, 5-12mgL-1Kanamycins, 2.0-4.0mgL-16-benzyladenine and 0.05-0.2mg L-12,4- dichlorphenoxyacetic acids, pH5.6；Preferably, differential medium is：30gL is added in MS minimal mediums-1Sucrose, 6.5g·L-1Agar powder, 400mgL-1Cephalosporin, 7.5mgL-1Kanamycins, 3.0mgL-16-benzyladenine and 0.1mg·L-12,4- dichlorphenoxyacetic acids, pH5.6.
- 8. method according to claim 3, it is characterised in that step cuts resistant budses in (4), is seeded in culture of rootage On base, in 25 ± 2 DEG C, 16 hours photoperiods dark, 25-35 μm of olm of intensity of illumination in illumination/8 hour-2·s-1Lower culture 28- Take root within 35 days；The root media is：20-35gL is added in 1/2MS minimal mediums-1Sucrose, 5-7gL-1Agar powder and 0.1- 0.4mg·L-1Indolebutyric acid, pH5.6；Preferably, root media is：30gL is added in 1/2MS minimal mediums-1Sugarcane Sugar, 6.5gL-1Agar powder and 0.2mgL-1Indolebutyric acid, pH5.6.
- 9. method according to claim 3, it is characterised in that enter performing PCR detection the primer in step (5) as follows：Sense primer：5’-GATGAGCGAAGAACAACCACAC-3’Anti-sense primer：5’-TTCAAACCCAGAGCATCCCCTG-3’.
- 10. the method according to claim any one of 3-9, it is characterised in that the strawberry cultivars include strawberry ' Hani '.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107663524A (en) * | 2017-09-20 | 2018-02-06 | 沈阳农业大学 | The FvGAIP genes and its application that a kind of regulation and control strawberry stolon occurs |
| CN116686708A (en) * | 2023-06-13 | 2023-09-05 | 武汉生物工程学院 | Method for open tissue culture of strawberry virus-free seedlings |
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| US8039690B2 (en) * | 2000-08-24 | 2011-10-18 | The Scripps Research Institute | Stress-regulated genes of plants, transgenic plants containing same, and methods of use |
| CN103509822A (en) * | 2013-10-25 | 2014-01-15 | 北京市农林科学院 | Method for obtaining strawberry plant having purple leaf back |
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| US8039690B2 (en) * | 2000-08-24 | 2011-10-18 | The Scripps Research Institute | Stress-regulated genes of plants, transgenic plants containing same, and methods of use |
| CN103509822A (en) * | 2013-10-25 | 2014-01-15 | 北京市农林科学院 | Method for obtaining strawberry plant having purple leaf back |
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| HARPER等: ""Sequence 1984 from patent US 8039690",GenBank:GY500055.1", 《NCBI BLAST》 * |
| 无: ""Arabidopsis thaliana SUR1 (SUPERROOT 1); S-alkylthiohydroximate lyase/ carbon-sulfur lyase/ transaminase (SUR1) mRNA, complete cds"，NCBI Reference Sequence：NM_127622.3", 《NCBI BLAST》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107663524A (en) * | 2017-09-20 | 2018-02-06 | 沈阳农业大学 | The FvGAIP genes and its application that a kind of regulation and control strawberry stolon occurs |
| CN116686708A (en) * | 2023-06-13 | 2023-09-05 | 武汉生物工程学院 | Method for open tissue culture of strawberry virus-free seedlings |
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