CN106834340A - A kind of method for obtaining the rape material for being adapted to high-density planting and mechanized harvest - Google Patents
A kind of method for obtaining the rape material for being adapted to high-density planting and mechanized harvest Download PDFInfo
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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Abstract
The present invention relates to a kind of method of the rape material for obtaining high-density planting and mechanized harvest, belong to plant genetic engineering and biological technical field；The present invention is transformed into rape using Agrobacterium flower-dipping method by label carrier pCB260 is activated first；Transformed plant seed is screened using green fluorescent protein GFP；And using careless fourth phosphine herbicide sieve agent screening rape transformant；Transformed plant is finally identified using PCR method；Phenotypic analysis discovery to transformed plant, finally gives few branch Rape Mutant, is designated asSfz,The plant meets mendel's law in F2 generation growths, and Seedling Stage blade surface is shinny, and maturity period branch is less, and effective branch highly increases, and silique concentrates on plant top；The present invention is studied for mechanization large-scale production and provides new material, with important application prospect using Rape Mutant is obtained after activating label carrier pCB260 conversion rapes.
Description
Technical field
The present invention relates to a kind of method of the rape material for obtaining high-density planting and mechanized harvest, belong to plant gene
Engineering and biological technical field.
Background technology
Rape (Brassica napus) belong to Cruciferae, be the fifth-largest crops of China be only second to paddy rice, wheat,
Corn and soybean.China is vegetable oil consumption big country, and domestic vegetable seed oil yield is only capable of meeting the 2/3 of people's consumption demand, largely
Fuel consumption dependence on import.Other rape is paid attention to as biodiesel raw material crop by increasing country, is expected to turn into
The important substitute of fossil energy crisis is solved, therefore the yield of raising rape has important strategic meaning to China.
Chinese society is quickly grown in recent years, and rural labour is reduced, and the price of labour power goes up, and the cost for planting rape is carried
Height, cultivated area is increasingly reduced.On the other hand, China's Rape-seed production mode tradition, Mechanization Level can not show a candle to wheat, paddy rice and
Seeding corn and other crops are, it is necessary to a large amount of labours, so as to cause China's Rape-seed production efficiency low, international competitiveness is weaker.Therefore, one is entered
The Mechanization Level of step enhancing Rape-seed production, reduces production cost, improves peasant planting enthusiasm, is the head that China needs to solve
Want problem.From breeding angle, the rape variety of the excellent suitable highly dense plantation of cultivation and mechanized harvest is basic to solve oil
Dish harvesting mechanization problem, as one of breeding man primary goal.
Rape branch plays an important role in not only being formed in plant above ground form during plant strain growth, and can lead to
Cross influence individual plant hip number and further have influence on rape total output, therefore there is very important status in Rape-seed production.I
State's rape variety principal character has that branch is more, cane is sturdy, plant type is larger, silique dispersion and easily split, and causes mechanical harvesting tired
Difficult big efficiency is low, and individual plant silique mature period is different in addition, and the maturity period differs greatly between each position silique, and prematurity silique takes off
Grain is difficult and cause rape harvest loss late to be up to 10% ~ 15%, improve rape harvest in the urgent need to the excellent rape variety of seed selection
Obtain Mechanization Level.
It was found that and cultivate that plant type is compact, branch highly increases, the florescence concentrates, the medium new oil vegetable kind of the layer collection that bear pods is with suitable
Answer the development trend that dense planting and mechanized harvest are domestic and international rape variety improvement.
Fu Tingdong etc.（Application number CN200810048092.5）Disclosure of the invention is a kind of to adapt to high-density planting and mechanization
The seed selection of new compact rape of short stem and authentication method are harvested to obtain, the method is hybridized the F1 generation for obtaining by different rape varieties and planted
Strain, by F1 generation plant bud colchicin doubling chromosome, microspores culture obtains dihaploid segregating population.Then to dividing
The body that peels off carries out the compact rape variety of short stem that many years of field observation, phenotypic screen obtain the suitable mechanized harvest.The party
It is owned by France in field of plant breeding, obtain that the rape variety time is very long, workload is big, phenotypic difference is not obvious.
At present, the present invention obtains the mutant that a kind of primary branch highly increases, branch concentrates on top, the layer that bears pods is concentrated
Plant, fixes tentatively entitledsfz（Few branch）.
The content of the invention
The present invention is to overcome technological deficiency present in prior art, it is therefore intended that provides one kind and is suitable for rape high density
Plantation and the preparation method of mechanized harvest material.The oil for adapting to high-density planting, mechanized harvest obtained using the method
Vegetable kind can effectively solve the problems, such as that the harvesting loss rate faced in current Rape-seed production is high, planting cost is high.
The invention also discloses the activation label carrier PCB260 of CaMV 35S enhancer sequences of being connected containing 4 repetitions
（Guan Yanlong etc., Activation tagging builds Arabidopsis Mutants storehouse and its phenotypic analysis, Yunnan plant research, 2010）Obtained in conversion
Obtain the application in Rape Mutant strain.
A kind of preparation method of rape material for being suitable to high-density planting and mechanized harvest of the present invention, specific behaviour
Make as follows：
（1）It is transformed into label carrier is activated in rape using Agrobacterium flower-dipping method：
Activation label carrier is transformed into Agrobacterium competent cell, the identification successful bacterium of conversion is prepared into bacteria suspension；By mesh
The bud and silique bloomed on mark thing rapeseed plants cut, and then carry out dip-flower process.
Wherein described activation label carrier is pCB260；
Described Agrobacterium competent cell is GV3101；
The dip-flower process is as follows：
（1）First time dip-flower：Open bud is removed, entirely non-open bud is infiltrated on the agriculture containing recombinant plasmid pCB260
5 minutes in bacillus bacterium solution, bacterium solution is gently rocked in dip-flower, beneficial to raising conversion ratio；The bud that infiltration is finished is inserted in sheepskin bag
It is interior.
（2）Second dip-flower：1-2 days after first time dip-flower, repeat step（1）, this step is without the open flower of removal
Flower bud.
（3）Third time dip-flower：1-2 days, repeat step after second dip-flower of distance（2）.
（4）Managed after dip-flower：After dip-flower terminates, plant to be planted pollination is finished, and removes sheepskin paper bag, and not timing is cut and newly born
Side shoot inflorescence and by its top of the inflorescence of dip-flower newly sprout bud, directly with water pour culture plant, until harvest.
Note：The Agrobacterium bacterium solution containing pCB260 plasmids will be again shaken as stated above before each dip-flower, it is ensured that bacterium solution
OD values are 2, and fresh bacterium solution growth vigor is conducive to improving transformation efficiency by force.
（2）Rape transformant is screened using green fluorescent protein GFP：
（3）Using careless fourth phosphine herbicide sieve agent screening rape transformant；
（4）PCR method identifies transformed plant：
The Rape Mutant plant leaf containing activation label carrier pCB260 that obtains as experiment material, CTAB methods will be screened
Genomic DNA, the bar fragments on PCR amplification activation label carriers PCB260 are extracted in a small amount.
PCR primer：
Bar-R：5’- CACCATCGTCAACCACTACATC-3’
Bar-F：5’- ACTTCAGCAGGTGGGTGTAGAG-3’
PCR programs：95 DEG C, 5min, predegeneration；95 DEG C, 1min；56 DEG C, 50s；72 DEG C, 30s；32 circulations；72 DEG C, 5min,
Extend.
（5）The phenotypic analysis of transformed plant：
In a large amount of transformants, it was found that few branch Rape Mutant, it is designated asSfz,And the plant meets Meng in F2 generation growths
Dare law of inheritance, phenotypic analysis is carried out to it, compared with peaceful oily 12 plant of wild type, Seedling Stage mutantsfzBlade surface
It is shinny, maturity period mutantsfzBranch it is less, effective branch highly increases, and silique concentrates on plant top.
United for maturity period few branch Rape Mutant and peaceful 20 plants of plant one-level branch amounts of oily 12 WT lines
Meter, few branch mutant one-level branch amount is slightly less than WT lines, and branch is relatively concentrated and invalid branch is reduced.
For harvest time few branch Rape MutantsfzEffective branch of 20 plants of plant is highly counted, and finds few point
Effective branch height of branch mutant plant height and peaceful oily 12 no significant difference averagely higher than wild type rather 12 about 20cm of oil,
Few branch mutant branch is focused on plant top.
Advantages of the present invention：
1. agriculture bacillus mediated external employed in the present invention dips osmosis conversion rape, speed is fast, low cost, operation
Simply.
2. the activation label carrier pCB260 employed in the present invention contains 4 repetition series connection CaMV 35S enhancer sequences
Row, be inserted into rapeseed gene group, the gene near insertion point can be activated and obtain overexpression, the gene being activated with
T-DNA is closely connected and is easily isolated out the gene.
3. the overexpression plant that application activating label carrier pCB260 is obtained is dominant mutant, and mutation type surface is in T1
Can observe directly, thus greatly compensate for the deficiency of the stealthy mutant of conventional method structure.
4. the present invention utilizes the efficient preliminary screening transformants of basta and green fluorescent protein GFP, further extracts plant
Tender leaf genomic DNA enters performing PCR identification, finally determines transformant, time-saving and efficiency, low cost, easily operation.
5. present invention utilization activates the effective branch obtained after label carrier pCB260 conversion rapes and highly increases, point
Branch concentrates on few branch Rape Mutant at topsfz, for mechanization large-scale production research provides new material, with important
Application prospect.
6. branch Rape Mutant is lackedsfzIt is gain-of-function type mutant, for research plant branching developmental regulation mechanism is carried
Splendid research material is supplied.
Brief description of the drawings
Fig. 1：The activation label carrier PCB260 of CaMV 35S enhancer sequences of being connected containing 4 repetitions.
Fig. 2：Using green fluorescent protein GFP primary dcreening operation Rape Mutant seeds；Wherein, A：Seed green with plumular axis is glimmering
Light screens composite diagram, B：Seed green fluorescence screening figure with plumular axis
Fig. 3：Careless fourth phosphine herbicide sieves agent primary dcreening operation Rape Mutant plant procedure chart；Wherein A：Careless fourth phosphine herbicide sieve agent is not sprayed
Rape Mutant plant, B：The Rape Mutant plant that careless fourth phosphine herbicide sieves agent, C are sprayed for the first time：Spray grass second
Fourth phosphine herbicide sieves the Rape Mutant plant of agent.
Fig. 4：Rape Mutant plant PCR is identified；Wherein, M: DL2000 DNA Marker；1-12:Mutant is planted
Strain；—:Negative control genomic DNA；+ :Positive control.
Fig. 5：Few branch Rape MutantsfzThe shinny phenotype of Seedling Stage blade surface.
Fig. 6：Few branch Rape MutantsfzFlorescence plant and peaceful oily 12 comparison diagram of WT lines.
Fig. 7：Few branch Rape MutantsfzRipe plant one-level branch amount statistical chart.
Fig. 8：Few branch Rape MutantsfzRipe plant and the peaceful oily effective branch height statistical chart of 12 plant of wild type.
Specific embodiment
According to following examples, the present invention can be better understood from, but described embodiment is to preferably explain this
Invention, rather than limitation of the present invention.
Activation label carrier pCB260 involved in the present invention is article（Guan Yanlong etc., Activation tagging builds arabidopsis
Mutant library and its phenotypic analysis, Yunnan plant research, 2010）Open, its building process, identification, property are disclosed, are known
Material, the present invention is used for author presents.
Embodiment one：Activation label carrier pCB260 is transformed into the peaceful oil 12 of wild type rape
1. activation label carrier pCB260 is transformed into Agrobacterium competent cell GV3101（Being purchased from Shanghai and stepping its biotechnology has
Limit company）
（1）The μ L of activation label carrier pCB260 about 5 are taken, 50 μ L Agrobacterium competent cells GV3101 are added to
In bacterium solution, slowly mix.
（2）Ice bath half an hour in ice chest is put in, is quickly removed to be transferred in liquid nitrogen and is freezed 1 minute（Or be positioned over
In -70 DEG C of refrigerators, 3-5 minutes）, rapid 37 DEG C of placement water-bath 5 minutes.
（3）Add 800 μ L LB fluid nutrient mediums（Without antibiotic）, 250 rpm are placed in 28 DEG C of constant temperature and shake
Bed 4-5 hour of culture.3500 rpm, are centrifuged 30 seconds, suction out supernatant liquid and give up.
（4）Add 100 μ L LB fluid nutrient mediums（Without antibiotic）Gently inhale and beat suspended bacteria block, until bacterium block
Disappear, draw about 80 μ L bacterium solutions, kanamycins containing 0.1g/L, 0.1g/L gentamicins and 0.1g/L are applied to spreading rod
Rifampin（It is purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd）LB flat boards on, 28 DEG C of incubated 24-48
Hour.
The pretreatment of experiment material
Before Agrobacterium dip-flower is carried out, should be to the peaceful oil 12 of viewing WT lines in Jiangsu University's rape experimental plot（Ny12, is purchased from
Academy of agricultural sciences of Jiangsu Province）The situation of blooming of rape；The bud bloomed on rapeseed plants and silique are cut, dip-flower is then carried out.
Agrobacterium bacterium solution pretreatment containing PCB260 plasmids
Bacterium solution PCR identifies the bacterial plaque containing PCB260 plasmids, chooses spot, is put in kanamycins containing 0.1g/L, 0.1g/L and celebrates big mould
Cultivated in the test tube of the fluid nutrient medium of element and 0.1g/L rifampin antibiotic, be then inoculated in 250 mL LB by 1% inoculum concentration
In fluid nutrient medium, Agrobacterium bacterium solution OD is determined600About 2.0 when carry out next step experiment.
It is added in 250mL bacterium solutions by following consumption, is formulated as follows：（Silwet, 6-BA 6-benzyl aminopurine, AS acetyl
Syringone is purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd）.
Cabbage type rape dip-flower process
（1）First time dip-flower：Open bud is removed, entirely non-open bud is infiltrated on the agriculture containing recombinant plasmid pCB260
5 minutes in bacillus bacterium solution, bacterium solution is gently rocked in dip-flower, beneficial to raising conversion ratio.The bud that infiltration is finished is inserted in sheepskin bag
It is interior.
（2）Second dip-flower：1-2 days after first time dip-flower, repeat step（1）, this step is without the open flower of removal
Flower bud.
（3）Third time dip-flower：1-2 days, repeat step after second dip-flower of distance（2）.
（4）Managed after dip-flower：After dip-flower terminates, plant to be planted pollination is finished, and removes sheepskin paper bag, and not timing is cut and newly born
Side shoot inflorescence and by its top of the inflorescence of dip-flower newly sprout bud, directly with water pour culture plant, until harvest.
Note：The Agrobacterium bacterium solution containing pCB260 plasmids will be again shaken as stated above before each dip-flower, it is ensured that bacterium solution
OD values are 2, and fresh bacterium solution growth vigor is conducive to improving transformation efficiency by force.
Embodiment two, the screening of rape transformed plant
1. the seed for being harvested using green fluorescent protein GFP screening dip-flowers（Method 1）
（1）Treatment rape seed：About 1000, the seed of the transformed plant that embodiment one is obtained is chosen, is placed in and is completed the big of filter paper
In number culture dish, seed is uniformly arranged, and adds a small amount of water until seed is infiltrated, dark treatment 2-3 days.
（2）Observation fluorescence：After seed sends tender shoots, you can be placed in light image analysis system（Being purchased from Shanghai day energy science and technology has
Limit company）Observation, adjusts the time for exposure for 50s during observation.As shown in Fig. 2 compared with the peaceful oil 12 of wild type, carrier containing pCB260
Mutant plants substantially send white point-like in green fluorescence i.e. figure.
（3）Transplant：The seed of fluorescence will be sent, is gently picked out, be placed in small side's basin and cultivate, after growing 3-4 piece true leaves,
It is transferred in experimental plot, attention action is treated without proper respect prevents blade broken.
2. careless fourth phosphine herbicide sieves agent mutant plants（Method 2）
Prepare careless fourth phosphine herbicide mother liquor A（As solvent）1000 mL, it is as follows：（Tween-20, Basta are purchased from Beijing Baeyer
Enlightening Bioisystech Co., Ltd）
Vortex concussion is mixed, instant to match somebody with somebody.
Prepare mother liquor B（As solute）150 mL, it is as follows：
Fully mix, it is instant to match somebody with somebody.
Nursery to rape grows 3-4 piece true leaves first, is carrying out Basta screenings.Detected through fully experiment, Basta work
The concentration of liquid is adapted to the most 0.01%.Use to take after 180mL mother liquor A and 20mL mother liquors B is fully mixed every time and use.Every time
Blade is sprayed to moisten completely, every sprinkling in three days once, about 5 times altogether（Specific number of times observation growth of rape situation）, until only
There is small part rape to survive.
The above method 1 and 2 can be applied individually to any screening the plant containing pCB260 carriers, also can will both connected applications,
To improve accuracy rate.
Identification Brassica napus Mutant Cr plant
The Rape Mutant plant leaf containing activation label carrier pCB260 for obtaining is screened with two methods of the above as reality
Material is tested, CTAB methods extract genomic DNA, the bar genetic fragments on PCR amplification activation label carriers pCB260 in a small amount.
PCR primer：
Bar-R：5’- CACCATCGTCAACCACTACATC-3’
Bar-F：5’- ACTTCAGCAGGTGGGTGTAGAG-3’
PCR programs：95 DEG C, 5min, predegeneration；95 DEG C, 1min；56 DEG C, 50s；72 DEG C, 30s；32 circulations；72 DEG C, 5min,
Extend.
Testing result shows（Fig. 4）, positive control（PCB260 plasmids）And #2, #5, #7, #9, #11, #12, this 6 conversions
Plant can amplify electrophoretic band（252bp）, after sequencing with ncbi database in bar alignments be correct sequence
Row, and negative control（The peaceful oil 12 of wild type, is purchased from academy of agricultural sciences of Jiangsu Province）Then without electrophoretic band, show transgene rape gene
Activation label carrier pCB260 sequences are contained in group.
Embodiment three, few branch Rape MutantsfzPhenotypic analysis
By the above method, in a large amount of transformants, it was found that few branch Rape MutantSfz,And the plant grows in F2 generations
In meet mendel's law, phenotypic analysis is carried out to it, compared with peaceful oily 12 plant of wild type, Seedling Stage mutantsfz
Blade surface is shinny（Fig. 5）, maturity period mutantsfzBranch it is less, effective branch highly increases, and silique concentrates on plant
Strain top.
Fig. 6 is few branch Rape MutantsfzFlorescence plant and WT lines comparison diagram；Few branch Rape Mutant
Inflorescence and silique concentrate on plant top, effective branch height apparently higher than peaceful oily 12 plant of wild type, be very suitable for dense planting and
Mechanized harvest.
Few branch rapesfzBranch amount is counted
Counted for maturity period few branch Rape Mutant and peaceful 20 plants of plant one-level branch amounts of oily 12 WT lines（Figure
Shown in 7）, lacking branch mutant one-level branch amount and be slightly less than WT lines, branch is relatively concentrated and invalid branch is reduced.Because one
Level branch amount and second branch number are reduced, and can be suitable for high-density planting, so make up because branch less caused by silique number subtract
Few defect, further increases the total yield, simultaneously because high-density planting, the cane of rape is thinner, is suitable for harvester cutter
Cutting.
Fig. 7 is few branch Rape MutantsfzRipe plant one-level branch amount statistical chart；One-level branch amount and second branch
Number is reduced, and can be suitable for high-density planting, so make up because branch less caused by the defect that reduces of silique number, further improve
Total output, simultaneously because high-density planting, the cane of rape is thinner, is suitable for the cutting of harvester cutter.
Few branch rapesfzEffective branch is highly counted
For harvest time few branch Rape MutantsfzEffective branch of 20 plants of plant is highly counted（Fig. 8）, find few point
Effective branch height of branch mutant plant height and peaceful oily 12 no significant difference averagely higher than wild type rather 12 about 20cm of oil,
Few branch mutant branch is focused on plant top.Research shows that effective branch height is to yield of rape under high-density planting
Influence be only second to knot mil(unit of angular measure) be second largest material impact proterties.Few branchsfzEffective branch highly increase, in Branching Sets
In plant top, the feature that florescence more concentrates is the optimal Cultivars of mechanization high-density planting rape.
Fig. 8 is few branch Rape MutantsfzRipe plant and the peaceful oily effective branch height statistical chart of 12 plant of wild type.
Few branchsfzEffective branch highly increase, branch concentrates on plant top, and the feature that florescence more concentrates is that mechanization is high
Density plants the optimal Cultivars of rape.
SEQUENCE LISTING
<110>Jiangsu University
<120>A kind of method for obtaining the rape material for being adapted to high-density planting and mechanized harvest
<130>A kind of method for obtaining the rape material for being adapted to high-density planting and mechanized harvest
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence
<400> 1
caccatcgtc aaccactaca tc 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<400> 2
acttcagcag gtgggtgtag ag 22
Claims (6)
1. a kind of preparation method of the rape material for being suitable to high-density planting and mechanized harvest, it is characterised in that concrete operations
It is as follows：
（1）It is transformed into label carrier is activated in rape using Agrobacterium flower-dipping method：
Activation label carrier is transformed into Agrobacterium competent cell, the identification successful bacterium of conversion is prepared into bacteria suspension；By mesh
The bud and silique bloomed on mark thing rapeseed plants cut, and then carry out dip-flower process；
（2）Rape transformant is screened using green fluorescent protein GFP：
（3）Using careless fourth phosphine herbicide sieve agent screening rape transformant；
（4）PCR method identifies mutant plants：
The Rape Mutant plant leaf containing activation label carrier pCB260 that obtains as experiment material, CTAB methods will be screened
Genomic DNA, the bar fragments on PCR amplification activation label carriers pCB260 are extracted in a small amount；
（5）The phenotypic analysis of transformed plant, it is determined that the transformed plant for obtaining is the oil for being suitable to high-density planting and mechanized harvest
Dish material.
2. method according to claim 1, it is characterised in that step（1）Described in activation label carrier be pCB260；Institute
The Agrobacterium competent cell stated is GV3101.
3. method according to claim 1, it is characterised in that step（1）Described in dip-flower specific operation process it is as follows：
（1）First time dip-flower：Open bud is removed, entirely non-open bud is infiltrated on the agriculture containing recombinant plasmid pCB260
In bacillus bacterium solution, bacterium solution is gently rocked；The bud that infiltration is finished is inserted in sheepskin bag；
（2）Second dip-flower：1-2 days after first time dip-flower, repeat step（1）, this step is without the open bud of removal；
（3）Third time dip-flower：1-2 days, repeat step after second dip-flower of distance（2）；
（4）Managed after dip-flower：After dip-flower terminates, plant to be planted pollination is finished, and removes sheepskin paper bag, and the new side born is cut in not timing
Branch inflorescence and the bud newly sprouted by its top of the inflorescence of dip-flower, directly pour culture plant with water, until harvesting.
4. method according to claim 1, it is characterised in that step（3）Described in PCR amplification activation label carrier
The primer sequence of the bar fragments on pCB260 is：
Bar-R：5’- CACCATCGTCAACCACTACATC-3’；
Bar-F：5’- ACTTCAGCAGGTGGGTGTAGAG-3’.
5. method according to claim 1, it is characterised in that step（5）Described in mutant plants contain pCB260 load
Body.
6. method according to claim 1, it is characterised in that step（5）Described in obtain be suitable to high-density planting and
The transformed plant phenotype of mechanized harvest is that Seedling Stage blade surface is shinny, and maturity period branch is less, and effective branch highly increases,
And silique concentrates on plant top.
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| CN201710025164.3A CN106834340A (en) | 2017-01-13 | 2017-01-13 | A kind of method for obtaining the rape material for being adapted to high-density planting and mechanized harvest |
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| CN115232833A (en) * | 2022-09-01 | 2022-10-25 | 深圳大学 | Agrobacterium-mediated efficient genetic transformation method for brassica crops |
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| CN109554371A (en) * | 2018-11-07 | 2019-04-02 | 江苏大学 | BnGRF7a gene and application thereof |
| CN115232833A (en) * | 2022-09-01 | 2022-10-25 | 深圳大学 | Agrobacterium-mediated efficient genetic transformation method for brassica crops |
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Application publication date: 20170613 |