CN103993038B - Method for guiding exogenous gene into cleistogamous indica rice by using PMI (phosphomannose isomerase) selection marker - Google Patents
Method for guiding exogenous gene into cleistogamous indica rice by using PMI (phosphomannose isomerase) selection marker Download PDFInfo
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Abstract
The invention provides a method for guiding an exogenous gene into cleistogamous indica rice by using a PMI (phosphomannose isomerase) selection marker. Specifically, the exogenous gene is guided into a cleistogamous indica rice variety 9m90 by using mannose as a selecting agent through mediation of a pCAMBIA1381-PMI carrier-containing agrobacterium tumefaciens strain EHA105. After guiding is ended, PCR (polymerase chain reaction) detection and identification prove that the exogenous gene is guided into the cleistogamous indica rice variety 9m90. The method successfully realizes the genetic transformation of the cleistogamous indica rice variety and does not need to use antibiotics or weed killers for screening; the gene drift caused by external spreading of pollen is avoided while the character improvement is accelerated; and the environmental protection degree is increased.
Description
Technical field
The present invention relates to biotechnology and field of plant genetic.Specifically, the present invention relates to a kind of utilize
Pmi selection markers, foreign gene are imported to the genetic transforming method closing grain husk pollination rice variety 9m90.
Background technology
Paddy rice (oryza sativa l.) is China or even most important cereal crops in the world, in the world more than 60%
Population with rice as staple food.In recent years in the face of the impact of the increasingly frequently abiotic stress such as arid, extreme temperature, population increasing
The long pressure with ecological environment, modern biotechnology means, particularly transgenic technology are important in terms of crop genetic improvement
Property day by day highlights.Transgenic technology is that yield lifting, quality-improving and the resistance enhancing of crops provide new route, open up
New space.
Planting range with genetically modified crops expands rapidly, the security of genetically modified crops and products thereof, causes wide
General concern.For example transformed variety pollen unofficial biography can bring genetic drift, it is possible to create " superweed " equivalent risk, but at present by
Overcome in the technological means lacking practicability and effectiveness, such as physical isolation needs certain space to stop pollen transmission (paddy rice
Need to be in more than 100m), it is adapted only to small area experiment, be unpractical in large area produces.And close clever pollinated variety whole
In individual pollinating process, grain husk flower does not open, and flower pesticide does not expose, and pollen will not outwards be propagated, and can effectively suppress pollen unofficial biography, it is to avoid
Gene contamination.Solve the problems, such as gene contamination by closing clever acceptor, be to be presently considered to most viable technological means, but give birth at present
Produce and also lack practical and close clever rice varieties.9m90 of this laboratory initiative etc. closes grain husk pollination rice variety, not only closes completely
Grain husk pollination, and the Main Agronomic Characters such as setting percentage, plant height are preferably, can be applied to kind as preferable transgene receptor quick
Genetic improvement, reduces ecological risk.
Existing method for transformation, is mainly used antibiotics resistance gene as selection markers, this genoid may enter with food
Enter enteron aisle, there is the potential risk producing antibody-resistant bacterium with enteric microorganism gene swapping, so that the medical effect of impact antibiotic
Really.For substitute antibiotics resistant gene, other types riddled basins are researched and developed successively, such as Herbicid resistant base
Cause, amino acid metabolism screening-gene, visable indicia gene, or but there are Similar Problems in these riddled basins, or sieve
Efficiency and cost is selected to be unsuitable for scale application.
Therefore, still there is no a kind of selection markers base of the genetic transforming method being suitable for closing grain husk pollination rice variety at present
Cause and corresponding method for transformation.
Content of the invention
Pmi is a kind of glucose metabolism genes, is widely present in algae and some legumes.The higher plant cells such as paddy rice
Though mannose can be converted into 6- phosphomannose, because cell itself lacks pmi it is impossible to further by 6- phosphomannose
It is converted into fructose-1, 6-diphosphate, is utilized hence into glycolytic pathway, therefore pmi can be used as riddled basins.With anti-
The negative selections such as raw element, herbicide are different, and mannose is by positive selection, the cell expression pmi gene of conversion, it is possible to use sweet dew
Sugar normal growth for carbon source, screening efficiency is high.Meanwhile, mannose is the hexose being widely present in the plants such as marine alga, fern,
And the catalysate of pmi is fructose-1, 6-diphosphate, is the Main Ingredients and Appearance of honey and pulp, the two is all eco-friendly natural goods
Matter.
For making full use of, there is the unique genetic resources closing grain husk pollination proterties, reduce simultaneously and resisted using antibiotic or herbicide
Property gene as selection markers potential risk, it is contemplated that set up a kind of pmi as selection markers it is adaptable to close grain husk pollination
The genetic transforming method of rice variety.
Specifically, it is an object of the invention to provide one kind is by agriculture bacillus mediated, with pmi (phosphomannose
Isomerase, phosphomannose isomerase) as selection markers, foreign gene is imported to and closes grain husk pollination rice variety 9m90
Genetic transforming method, and using the method prepare transgenosis rice plant method.
The invention provides following technical proposal is realizing this purpose.
In one aspect, the present invention provides a kind of importing foreign gene using pmi selection markers to close grain husk pollination long-grained nonglutinous rice
Method is it is characterised in that methods described comprises the steps:
Step 1: isolate the embryo of rice paddy seed, be placed on callus inducing medium to produce secondary callus；
Described secondary callus is transferred to new inducing culture and carries out preculture, to obtain callus by step 2；
Step 3 by step 2 obtain described callus with carry phosphomannose isomerase pmi
The Agrobacterium of (phosphomannose isomerase) riddled basins contacts the first predetermined amount of time；
Described callus after step 3 is processed by step 4 transfers to Agrobacterium suspension medium, cultivates the second pre- timing
Between section；
Described callus after step 4 is processed by step 5 is placed in culture the 3rd predetermined amount of time on front screening and culturing medium；
Described callus after step 5 is processed by step 6 is transferred on screening and culturing medium, to obtain kanamycin-resistant callus tissue group
Knit；
Described resistant calli is transferred to seedling differentiation in differentiation and regeneration culture medium by step 7；With
Step 8 is transferred to described seedling in root media and is taken root,
Wherein, [no in described callus inducing medium3 -]/[nh4 +]=20mm/20mm.
Preferably, the nucleotides sequence of described pmi marker gene is classified as: the nucleotide sequence shown in seq id no:1.
Preferably, described rice varieties are 9m90.
Preferably, in described step 4, described callus is transferred to Agrobacterium suspension medium to include described callus
Tissue is transferred in the culture dish being lined with one or more aseptic filter paper thereon 21-23 DEG C and is cultivated 48 hours, described aseptic filter paper
Middle addition 2.5-3.5ml Agrobacterium suspension medium, [no in this Agrobacterium suspension medium3 -]/[nh4 +]=20mm/20mm；
And/or
[no in described front screening and culturing medium3 -]/[nh4 +]=20mm/20mm, and contain 30 μm of cobalt chlorides；And/or
[no in described screening and culturing medium3 -]/[nh4 +]=20mm/20mm, and contain 7.5g/l maltose, 12.5g/l
Mannose, 30 μm of cobalt chlorides；And/or
[no in described differentiation and regeneration culture medium3 -]/[nh4 +]=20mm/20mm, and contain 30 μm of cobalt chlorides, 1mg/l
Kt and 1mg/l naa；And/or
Naa containing 0.4mg/l in described root media.
Preferably, described inducing culture includes: the n6 a great number of elements of optimization, b5 trace element, ms molysite, b5 dimension life
Element, 500mg/l proline, 500mg/l glutamine, 300mg/l casein enzyme hydrolysate, 30g/l maltose, 2mg/l 2,4-
D, 30 μm of cobalt chlorides, 8g/l agar；And/or
Described step Agrobacterium suspension medium includes: the n6 a great number of elements of optimization, b5 trace element, ms molysite, b5 dimension
Raw element, 500mg/l proline, 500mg/l casein enzyme hydrolysate, 2mg/l2,4-d, 20g/l maltose, 10g/l glucose,
100 μm of ol/l acetosyringones；And/or
Described front screening and culturing medium includes: the n6 a great number of elements of optimization, b5 trace element, ms (murashige ＆
Skoog) molysite, b5 vitamin, 500mg/l proline, 500mg/l glutamine, 300mg/l casein enzyme hydrolysate, 30g/l
Maltose, 2mg/l 2,4-d, 30 μm of cobalt chlorides, 8g/l agar, 250mg/l carbenicillins；And/or
Described screening and culturing medium includes: the n6 a great number of elements of optimization, b5 trace element, ms molysite, b5 vitamin, 500mg/
L proline, 500mg/l glutamine, 300mg/l casein enzyme hydrolysate, 7.5g/l maltose, 12.5g/l mannose, 30 μm
Cobalt chloride, 250mg/l carbenicillin, 8g/l agar；And/or
Described differentiation and regeneration culture medium includes: the n6 a great number of elements of optimization, b5 trace element, ms molysite, b5 vitamin,
1g/l ch, 30g/l sucrose, 30g/l sorbitol, 500mg/l mes, 2.5mg/l cuso4,1mg/l kt, 1mg/l
Naa, aa amino acid, 125mg/l carbenicillin, 30 μm of cobalt chlorides, 8g/l agar；And/or
Described root media includes: 1/2ms basis salt (2.l65g/l), ms vitamin, 20g/l sucrose, 0.4mg/l
Naa, 125mg/l carbenicillin, 3.5g/l plant gel.
Preferably, described first predetermined amount of time is 15 minutes；Described second predetermined amount of time is 48 hours；Described 3rd
Predetermined amount of time is 5-7 days.
On the other hand, the present invention provides a kind of method producing transgenic paddy rice, comprising:
1) by above-mentioned method by foreign gene Introduced into Rice cell；With
2) cultivate rice plant using the rice cell being obtained.
Wherein, the embryo isolating rice paddy seed refers to rice paddy seed shells, sterilize after embryo is separated.Preferably
Ground, described step 4 specifically includes to transfer to the callus of step 3 and is lined with three aseptic filter papers (addition 2.5-3.5ml thereon
Agrobacterium suspension medium) culture dish in, 21-23 DEG C cultivate 48 hours.
In preferred embodiments, described paddy rice is long-grained nonglutinous rice, it is highly preferred that described paddy rice is to close grain husk pollination rice variety
9m90.
In a kind of preferred implementation, culture medium adopts the culture medium in following table.
The exemplary formulations of table 1 culture medium
The n6 a great number of elements of the optimization being previously mentioned in literary composition refers to [no in this n6 a great number of elements3 -]/[nh4 +]=20mm/
20mm.
In one implementation, the nucleotides sequence of the pmi marker gene being adopted is classified as:
atgcaaaaactcattaactcagtgcaaaactatgcctggggcagcaaaacggcgttgactgaactttat
ggtatggaaaatccgtccagccagccgatggccgagctgtggatgggcgcacatccgaaaagcagttcacgagtgca
gaatgccgccggagatatcgtttcactgcgtgatgtgattgagagtgataaatcgactctgctcggagaggccgttg
ccaaacgctttggcgaactgcctttcctgttcaaagtattatgcgcagcacagccactctccattcaggttcatcca
aacaaacacaattctgaaatcggttttgccaaagaaaatgccgcaggtatcccgatggatgccgccgagcgtaacta
taaagatcctaaccacaagccggagctggtttttgcgctgacgcctttccttgcgatgaacgcgtttcgtgaatttt
ccgagattgtctccctactccagccggtcgcaggtgcacatccggcgattgctcactttttacaacagcctgatgcc
gaacgtttaagcgaactgttcgccagcctgttgaatatgcagggtgaagaaaaatcccgcgcgctggcgattttaaa
atcggccctcgatagccagcagggtgaaccgtggcaaacgattcgtttaatttctgaattttacccggaagacagcg
gtctgttctccccgctattgctgaatgtggtgaaattgaaccctggcgaagcgatgttcctgttcgctgaaacaccg
cacgcttacctgcaaggcgtggcgctggaagtgatggcaaactccgataacgtgctgcgtgcgggtctgacgcctaa
atacattgatattccggaactggttgccaatgtgaaattcgaagccaaaccggctaaccagttgttgacccagccgg
tgaaacaaggtgcagaactggacttcccgattccagtggatgattttgccttctcgctgcatgaccttagtgataaa
gaaaccaccattagccagcagagtgccgccattttgttctgcgtcgaaggcgatgcaacgttgtggaaaggttctca
gcagttacagcttaaaccgggtgaatcagcgtttattgccgccaacgaatcaccggtgactgtcaaaggccacggcc
gtttagcgcgtgtttacaacaagctgtaa
Technique effect
The method of the present invention is successfully realized the genetic transformation closing grain husk pollination rice variety, and without using antibiotic or removes
Careless agent screening, is accelerating character improvement process simultaneously, it is to avoid the genetic drift that pollen unofficial biography lead to, is improving environmental friendliness degree.
The rice transformation method based on pmi marker gene that the present invention sets up, not only transformation efficiency is no less than antibiosis
Plain resistant gene, and selective agent mannose and pmi catalysate fructose-1, 6-diphosphate are eco-friendly natural materials, keep away
Exempt from the potential risk that may bring using antibiotics resistance gene etc.；Mannose screening simultaneously is to carry out key player on a team to positive cell
Select, screen cycle is short, more cost-effective than using other selective agents.At present although existing pmi is applied to as selection markers
The report of crop genetic conversion, but operating process loaded down with trivial details (generally needing more than 90 days), transformation efficiency relatively low (6% about), no
It is suitable to large-scale production.The genetic transforming method that the present invention sets up, by adjusting training method, changes medium component, makes step
Suddenly simpler, significantly improve transformation efficiency, scale genetic improvement needs can be met completely.Thus what this project was set up
Genetic transforming method achieves the unification of security, high efficiency and economies.
By the invention it is possible to realize paddy rice is especially closed high transformation frequency, the agriculture of high duplication of grain husk pollination long-grained nonglutinous rice
Bacillus mediated transformation；On the other hand, step of the present invention simple (being such as not required to wash bacterium and pre- differentiation, only need a wheel screening), shortening week
Phase；The present invention selects without using antibiotic or herbicide, cost-effective, suitable high flux operation, is easy to scale application.From
And be to develop plant-scale, eco-friendly, no genetic drift transgenic paddy rice preparation technology to provide possibility.
Brief description
Fig. 1 is pcambia1381-pmi vector plasmid schematic diagram.
Fig. 2 is the callus of cleistogamous rice kind 9m90, after agrobacterium mediation converted, in mannose screening
Pressure, kanamycin-resistant callus tissue growing state.
Fig. 3 is the callus of cleistogamous rice kind 9m90, after agrobacterium mediation converted, in differentiation and regeneration training
In foster base, regenerate seedling.
Fig. 4 is that the pcr of transfer-gen plant detects electrophoretogram.Amplified fragments are pmi gene, and clip size is 490bp.M is
dl 2kb marker；Pc is positive control；Nc is negative control；1-13 is the transfer-gen plant selected at random.
Specific embodiment
In the case of not having other to illustrate, the operation in following specific embodiments is all using generally in the art
Routine operation is carrying out.Those skilled in the art can easily obtain with regard to such routine operation from the prior art
Teaching, for example, be referred to textbook sambrook and david russell, molecular cloning:a
laboratory manual,3rd ed.,vols 1,2；charles neal stewart,alisher touraev,
Vitaly citovsky and tzvi tzfira, plant transformation technologies etc..Following enforcements
In example, medicinal raw material used, reagent material etc., if no special instructions, are commercially available purchase product.
Embodiment with mannose as selective agent close grain husk pollination long-grained nonglutinous rice 9m90 genetic transforming method
1st, the induction of mature embryo callus and preculture
Grain husk pollination long-grained nonglutinous rice 9m90 will be closed, and (Paddy Rice Inst., Anhui Agriculture Science Academy preserves, and genetic background is rice variety
Early Xian 788) mature seed shell, choose that outward appearance is normal, the seed of clean no mildew, use 70% alcohol, rock 90sec,
Fall alcohol；With 50% sodium hypochlorite containing tween20, (stoste effective chlorine density is more than 4%, and every 100 milliliters add 1 again
Tween20) solution cleaning seed, rocks 45min (180r/min) on shaking table.Outwell sodium hypochlorite, aseptic washing 5-10 time
To no sodium hypochlorite smell, it is eventually adding sterilized water, 30 DEG C of soaked overnight.With knife blade along aleurone separation embryo, scultellum court
On be placed on inducing culture (composition is shown in Table 1), 12/ware, 30 DEG C of light culture are with evoked callus.
Spherical, coarse, lurid secondary callus occurred after two weeks, preculture operation can be carried out, will be secondary
Callus goes on new inducing culture, 30 DEG C of light culture precultures 5 days.After preculture terminates, will be vigorous in good condition, division
Little particle spoon collect to the sterile centrifugation tube of 50ml, infect for Agrobacterium.
2nd, the culture of agrobacterium strains and suspension prepare
By agrobacterium strains eha105 (the Academy of Agri-Science and Technology Anhui Province rice research containing pcambia1381-pmi carrier
Preserved) in the flat lining out of the lb containing 50mg/l kanamycins (composition is shown in Table 1), 28 DEG C of dark culturing, with aseptic after 24h
The Agrobacterium inoculation that oese will activate, to the lb flat board of fresh 50mg/l kanamycins, carries out second activation, 28 DEG C
Dark culturing is overnight.Add 20-30ml Agrobacterium suspension medium (composition is shown in Table 1) in the sterile centrifugation tube of 50ml, with connecing
Plant ring to scrape the Agrobacterium activating 2 times, adjustment od660 (optical density 660nm, 660nm light absorption value) is to about
0.10-0.25, room temperature stands more than 30min.
3rd, infect and co-culture
To in ready callus (see step 1), plus agrobacterium suspension, soak 15min, frequently gently shake therebetween
Dynamic.Liquid (as far as possible dripping liquid only) is outwelled in immersion after terminating, suck the unnecessary agriculture bar on callus surface with aseptic filter paper
Bacterium bacterium solution, and dried up with sterile wind in super-clean bench.Three aseptic filters on the disposable sterilized culture dish pad of 100 × 25mm
Paper, adds 2.5ml Agrobacterium suspension medium, the callus after blotting is dispersed on filter paper, 23 DEG C of dark culturing
48h.
4th, front screening and screening and culturing
After co-cultivation terminates, the callus through co-culturing is dispersed evenly in front screening and culturing medium (composition is shown in Table 1),
30 DEG C of dark culturing 5 days.After front screening and culturing terminates, callus is gone to (composition is shown in Table 1) on screening and culturing medium, each training
Foster ware connects 25 callus, 30 DEG C of dark culturing, and after 2-3 week, resistant calli growth substantially, can carry out differentiation and regeneration behaviour
Make.
5th, differentiation and regeneration
After screening and culturing, each independent transformants selects 3 good, fresh little particles of growth conditions, goes to point
Change on regeneration culture medium (composition is shown in Table 1).Every culture dish connects 5 independent transformants.28 DEG C of illumination cultivation, periodicity of illumination is 16h
Illumination 8h is dark, and luminous intensity is 3000-6000lx.
6th, take root and transplant
When the bud length of resistant calli differentiation is to about 2cm, each independent transformants only takes one plant of well-grown seedling,
Move to (composition is shown in Table 1) on root media, 28 DEG C of illumination cultivation, periodicity of illumination is that 16h illumination 8h is dark, and luminous intensity is
3000-6000lx.After two weeks, select the seedling of well developed root system, wash culture medium with water, transplanting is buried.
7th, Molecular Identification
Before transplanting, take rice leaf sample, carry out with ctab method that dna is little to be carried.By obtained genome dna sample
Product are used for pcr and analyze.For expand pmi pcr primer be 5 '-ccgccggagatatcgtttcactg-3 ' and 5 '-
Cacggttcaccctgctggctatc-3 ', produces the fragment that length is 490bp.Pcr component is kept 5 points at 95 DEG C first
Clock, then carry out 32 circulation: 94 DEG C 45 seconds, 56 DEG C 45 seconds, 72 DEG C 45 seconds, finally 72 DEG C extend 10 minutes.Select at random
13 transfer-gen plants, identified, wherein 12 plants is the positive, and positive rate reaches 92.3%.
Implementation result and transformation efficiency
The present invention passes through to separate embryonal induction callus, compares with complete Seed inducement callus, not only callus growth
Very fast, state preferably, and can effectively reduce bacterium, fungal contamination, reduce and waste, cost-effective.In addition after wash seeds,
With 30 DEG C of soaked overnight of sterilized water, the abundant imbibition of seed can be made, promote the metabolism of embryo, improve callus induction rate,
Improve callus status.
The present invention passes through lb solid plate activation culture twice, then prepares suspension, rather than chooses single bacterium colony and use liquid again
Culture and Amplification Culture, can simplify operation, be easy to batch application, reduce the possibility of pollution simultaneously, cost-effective.Agrobacterium bacterium
Liquid concentration affects larger on transformation efficiency.Too high Agrobacterium bacterial concentration, not only unnecessarily, and can make Agrobacterium excessively numerous
Grow, increase the injury to callus for the Agrobacterium, reduce transformation efficiency.
The present invention selects 50ml centrifuge tube, rather than culture dish is infected, and both facilitated operation, and was conducive to callus group simultaneously
Knit and being fully contacted of Agrobacterium, thus improving transformation efficiency.Select the side of suspension medium is added on three aseptic filter papers
Formula, rather than solid medium mode co-cultures；Select 23 DEG C of dark culturing, rather than such as 28 DEG C of higher temperature；Can keep away
Exempt from Agrobacteriuna overgrowth hence it is evident that improving callus status, thus significantly improving transformation efficiency.
Because when infecting, bacterial concentration is relatively low, co-cultivation temperature is relatively low, and filter paper co-cultures mode, co-cultures after terminating almost
There is no the situation of Agrobacteriuna overgrowth, because without callus is carried out wash with bacterium operation.Not only save cost, reduced
Pollution may improve transformation efficiency it is thus also avoided that wash the injury to callus for the bacterium process, also allows for scale operation.
Take a wheel screening, rather than two-wheeled screening, experimental period can be shortened, cost-effective.
Using pmi as riddled basins, when positive transformed cells are screened by mannose, the selection that properly screening is pressed
Transformation efficiency is affected larger.If mannose concentration is too high, both increased cost, and also made cell growth slow, significantly affect
Transformation efficiency；If mannose concentration is relatively low, false-positive possibility can be increased.The present invention is directed to 9m90 kind, systematic comparison
The impact of the proportioning of different maltose-mannoses, the final proportioning determining 7.5g/l maltose and 12.5g/l mannose,
It is suitable for carrying out Effective selection to 9m90 positive transformed cells.Wherein 7.5g/l maltose can guarantee that the growth speed of positive transformed cells
Rate, and positive transformed cells can effectively be distinguished by 12.5g/l mannose, significantly reduce cost again.
Take direct differentiation, rather than first pre- differentiation, workload can be mitigated, simultaneously effectively save cost.Select culture
Ware is as the group culture container of differentiation and regeneration, rather than triangular flask, is easy to operate, at utmost utilizes the sky of illumination cultivation frame simultaneously
Between, it is easy to scale application.
The present invention adds the agar of 8g/l as coagulator in induction, front screening, screening and differentiation and regeneration culture medium, and
It is not the plant gel being usually used, has not only saved cost, what is more important callus can preserve relatively dry, thus
It is more conducive to keep the kilter of callus, reduce browning necrosis.Train in induction, front screening, screening and differentiation and regeneration simultaneously
Add 30 μm of cobalt chlorides in foster base, can effective ethene suppressing generation, the proliferative induction of promotion callus or differentiation.
Add appropriate naa (0.4mg/l) in root media, root growth can be promoted, thus improve transplanting into
Motility rate.
By foregoing invention, in conjunction with inventor to induction, Agrobacterium suspension, front screening, screening and differentiation and regeneration culture medium
In nitrogen source optimization ([no3 -]/[nh4 +]=20mm/20mm), maltose and mannose proportioning (7.5g/l wheat in screening and culturing medium
Bud sugar and 12.5g/l mannose), the hormone combination (1mg/l kt, 1mg/l naa) in differentiation and regeneration culture medium, the present invention is real
Show to the High-efficient Genetic Transformation closing grain husk pollination rice variety 9m90, concrete outcome is shown in Table 2.
The transformation efficiency closing grain husk pollination rice variety 9m90 with mannose as selective agent for the table 2
It should be understood that specific embodiment described herein is intended only as example to help those skilled in the art preferably
Understand the present invention, and any restriction is not constituted to the scope of the present invention.Those skilled in the art can make respectively according to the present invention
Plant and change or deform, without departing from the spirit of the present invention, scope of the following claims of the present invention all should be belonged to.
Claims (4)
1. a kind of utilizationpmiForeign gene is imported the method closing grain husk pollination long-grained nonglutinous rice it is characterised in that methods described by selection markers
Comprise the steps:
Step 1: isolate the embryo of rice paddy seed, be placed on callus inducing medium to produce secondary callus；
Described secondary callus is transferred to new inducing culture and carries out preculture, to obtain callus by step 2；
Step 3 by step 2 obtain described callus and the agriculture carrying phosphomannose isomerase pmi riddled basins
Bacillus contacts the first predetermined amount of time；
Described callus after step 3 is processed by step 4 transfers to Agrobacterium suspension medium, cultivates for second scheduled time
Section；
Described callus after step 4 is processed by step 5 is placed in culture the 3rd predetermined amount of time on front screening and culturing medium；
Described callus after step 5 is processed by step 6 is transferred on screening and culturing medium, to obtain resistant calli；
Described resistant calli is transferred to seedling differentiation in differentiation and regeneration culture medium by step 7；With
Step 8 is transferred to described seedling in root media and is taken root,
Wherein, [no in described callus inducing medium3 -]/[nh4 +]=20mm/20mm,
In described step 4, described callus is transferred to Agrobacterium suspension medium to include for described callus transferring to it
On be lined with the culture dish of one or more aseptic filter paper 21-23 DEG C and cultivate 48 hours, add 2.5- in described aseptic filter paper
3.5ml Agrobacterium suspension medium；
Described inducing culture includes: the n6 a great number of elements [no of optimization3 -]/[nh4 +]=20mm/20mm, b5 trace element, ms iron
Salt, b5 vitamin, 500mg/l proline, 500mg/l glutamine, 300mg/l casein enzyme hydrolysate, 30g/l maltose,
2mg/l 2,4-d, 30 μm of cobalt chlorides, 8g/l agar；
Described Agrobacterium suspension medium includes: the n6 a great number of elements [no of optimization3 -]/[nh4 +The micro unit of]=20mm/20mm, b5
Element, ms molysite, b5 vitamin, 500mg/l proline, 500mg/l casein enzyme hydrolysate, 2mg/l 2,4-d, 20g/l malt
Sugar, 10g/l glucose, 100 μm of ol/l acetosyringones；
Described front screening and culturing medium includes: the n6 a great number of elements [no of optimization3 -]/[nh4 +]=20mm/20mm, b5 trace element, ms
(murashige＆skoog) molysite, b5 vitamin, 500mg/l proline, 500mg/l glutamine, 300mg/l casease
Hydrolysate, 30g/l maltose, 2mg/l 2,4-d, 30 μm of cobalt chlorides, 8g/l agar, 250mg/l carbenicillins；
Described screening and culturing medium includes: the n6 a great number of elements [no of optimization3 -]/[nh4 +]=20mm/20mm, b5 trace element, ms iron
Salt, b5 vitamin, 500mg/l proline, 500mg/l glutamine, 300mg/l casein enzyme hydrolysate, 7.5g/l maltose,
12.5g/l mannose, 30 μm of cobalt chlorides, 250mg/l carbenicillin, 8g/l agar；
Described differentiation and regeneration culture medium includes: the n6 a great number of elements [no of optimization3 -]/[nh4 +]=20mm/20mm, b5 trace element,
Ms molysite, b5 vitamin, 1g/l ch, 30g/l sucrose, 30g/l sorbierite, 500mg/l 2- (n- morpholine) ethyl sulfonic acid (mes),
2.5mg/l cuso4, 1mg/l kt, 1mg/l methyl α-naphthyl acetate, aa amino acid, 125mg/l carbenicillin, 30 μm of cobalt chlorides, 8g/l
Agar；
Described root media includes: 1/2ms basis salt 2.165g/l, ms vitamin, 20g/l sucrose, 0.4mg/l methyl α-naphthyl acetate,
125mg/l carbenicillin, 3.5g/l plant gel.
2. method according to claim 1 is it is characterised in that describedpmiThe nucleotides sequence of marker gene is classified as: seq id
Nucleotide sequence shown in no:1.
3. method according to claim 1 is it is characterised in that described first predetermined amount of time is 15 minutes；Described second
Predetermined amount of time is 48 hours；Described 3rd predetermined amount of time is 5-7 days.
4. a kind of method producing transgenic paddy rice, comprising:
1) pass through in claim 1-3 method described in any one by foreign gene Introduced into Rice cell；With
2) cultivate rice plant using the rice cell being obtained.
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| CN104928252A (en) * | 2015-04-21 | 2015-09-23 | 安徽省农业科学院水稻研究所 | Application of arabidopsis thaliana phosphomannose isomerase gene to plant genetic transformation |
| CN105580736B (en) * | 2016-02-29 | 2017-07-28 | 海南波莲水稻基因科技有限公司 | A kind of Rice Callus differential medium and its preparation method and application |
| CN106234228A (en) * | 2016-09-09 | 2016-12-21 | 江西农业大学 | A kind of method cultivating Indica Rice Callus |
| CN109042336A (en) * | 2018-09-30 | 2018-12-21 | 天津大学 | With the method for the sterile callus of rice paddy seed culture rice containing endogenous bacterium |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1711831A (en) * | 2004-06-24 | 2005-12-28 | 华中农业大学 | Cut culture of indica rice |
| WO2007134234A2 (en) * | 2006-05-12 | 2007-11-22 | Monsanto Technology Llc | Methods and compositions for obtaining marker-free transgenic plants |
| CN102337293A (en) * | 2011-10-09 | 2012-02-01 | 安徽省农业科学院水稻研究所 | Transformation method of rice cells |
| CN102337294A (en) * | 2011-10-09 | 2012-02-01 | 先正达生物科技（中国）有限公司 | Transformation method of rice cells |
| CN102787133A (en) * | 2011-05-19 | 2012-11-21 | 华中农业大学 | Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast |
| CN103710381A (en) * | 2013-12-23 | 2014-04-09 | 中国农业大学 | Method of obtaining transgenic indica type rice by utilizing agrobacterium and special-purpose culture medium thereof |
-
2014
- 2014-06-04 CN CN201410245313.3A patent/CN103993038B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1711831A (en) * | 2004-06-24 | 2005-12-28 | 华中农业大学 | Cut culture of indica rice |
| WO2007134234A2 (en) * | 2006-05-12 | 2007-11-22 | Monsanto Technology Llc | Methods and compositions for obtaining marker-free transgenic plants |
| CN102787133A (en) * | 2011-05-19 | 2012-11-21 | 华中农业大学 | Method for screening transgenic paddy plant by phosphomannose-isomerase (PMI) gene of yeast |
| CN102337293A (en) * | 2011-10-09 | 2012-02-01 | 安徽省农业科学院水稻研究所 | Transformation method of rice cells |
| CN102337294A (en) * | 2011-10-09 | 2012-02-01 | 先正达生物科技（中国）有限公司 | Transformation method of rice cells |
| CN103710381A (en) * | 2013-12-23 | 2014-04-09 | 中国农业大学 | Method of obtaining transgenic indica type rice by utilizing agrobacterium and special-purpose culture medium thereof |
Non-Patent Citations (5)
| Title |
|---|
| NH4NO3及碳源对红掌愈伤组织诱导影响的研究;刘炤 等;《北方园艺》;20111231;第152-154页 * |
| 以pmi为筛选标记的水稻转基因研究;梁永亚 等;《安徽农业科学》;20111231;第39卷(第17期);第10143-10146页 * |
| 新型筛选标记磷酸甘露糖异构酶基因在转基因植物中的应用;郭倩倩 等;《中国农业科技导报》;20111231;第13卷(第6期);第12-19页 * |
| 氮源和碳源对冬凌草愈伤组织生长及迷迭香酸的积累的影响;李汉伟 等;《时珍国医国药》;20101231;第21卷(第6期);第1348-1350页，尤其是摘要 * |
| 通过氮源比较试验建立一种较好的水稻花药培养基;朱至清 等;《中国科学》;19750930;第484-490页 * |
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