CN1515671B - Agrobacterium mediated plant nonselective gene conversion method - Google Patents
Agrobacterium mediated plant nonselective gene conversion method Download PDFInfo
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Abstract
The present invention relates to an agrobacterium mediated plant nonselective gene transforming method, including the following steps: constructing gene transforming carrier applicable to agrobacterium mediation, using contracted explant, said explant or infected section of the explant contains 1-20 regenerable cells, pretreating or preculturing explant to reduce the infected cell death due to action of agrobacterium, co-culturing the expant and agrobacterium under the condition of nutritional deficiency, using antibiotic to inhibit the growth of agrobacterium, inducing explant callus in the culture medium containing no selective reagent so as to obtain regenerated plant, utilizing molecular biological or chemical analysis to identify transgenic plant, and the ratio of transgenic plant in all the regenerated plants is above 20%.
Description
Technical field: the present invention does not have the selection gene transformation method for a kind of agriculture bacillus mediated plant, the method for particularly a kind of direct acquisition non selecting sign transgene plant.Relate to genetic engineering technique, special production at the non selecting sign transgene plant.Belong to field of biology.
Background information: transgenic technology had become already and solves multicellular organism, particularly plant important biomolecule knowledge and inscribe strong tool.The task that the traditional genetic method of many utilizations can not be finished then can make it to become a reality by utilizing transgenic technology, as homology or heterologous gene with modification and difference in functionality can make plant obtain the needed proterties of people fast by genetically modified mode introduced plant.
The plant gene transformation technology is developing rapidly closely during the last ten years, the transgenic line that obtains by methods such as Agrobacterium (comprising by suction method in its deutero-), particle gun, protoplastis, acupuncture, ultrasonic wave, pollen tube channels only surpasses 8925 parts so far what the U.S. carried out field experiment, has contained 98 species (www.nbiap.vt.edu/cfdocs/fieldtests1.cfm) such as corn, paddy rice, soybean, rape, potato, tomato.In numerous transgenic technologys, agriculture bacillus mediated gene transformation is the most a kind of gene transformation method of normal use.The successful Application of this technology has benefited from depending in other words the use of selectable marker gene.The use of selectable marker gene makes us can utilize the corresponding selective reagents non-transformed cell that rare transformant and number is huge to make a distinction by suppressing growing of non-transformed cell.Therefore, selectable marker gene is widely used in the plant gene conversion.Utilize selectable marker gene to utilize its expression product to modify exactly, thereby make the transformant that carries this gene produce resistance corresponding selective reagents to corresponding selective reagents.
Coding antibiotics resistance that present widely used selectable marker gene usually is some dominant genes or herbicide resistance gene (Yoder and Goldsbrough, 1994).When carrying out gene transformation, corresponding selective reagents is microbiotic or weedicide.Although these selectable marker genes are very useful concerning the gene transformation process, but also there are some fatal weakness in it: the use of (1) microbiotic or antiweed usually has a negative impact to the differentiation and the regeneration of transformant, it can suppress the differentiation (Ebinuma et al., 1997) of indefinite bud; (2) kind of or anti-selection insensitive for some, these selective reagentss can not be distinguished well and transform and non-transformed cell, thereby can not play its due effect (Ebinuma et al., 1997); (3) these selectable marker genes are usually chain with goal gene, be incorporated in the Plant Genome together, thereby transformed plant being produced a series of bad influences: (a). the constitutive character of these selectable marker genes is expressed, generally can not produce active influence with growth, unnecessarily expend the bioenergy of transfer-gen plant on the contrary the growth of transfer-gen plant; (b). these selectable marker gene expression product existence in food, produced the potential food safety question, make consumers in general produce and guard psychology genetically modified food; (c). these selectable marker genes may cause environment difficult to the appraisal and ecological security problem to the drift of other species, have greatly influenced popularization and the application of genetically modified crops; (d). when same kind is carried out multi-functional modification, may relate to and repeatedly repeat to transform, thereby need a plurality of effective choice marker gene.Therefore, the existence of selectable marker gene in transformant can hinder the stack of a plurality of functional genes on same transformation plant.
For fear of the generation of the problems referred to above, best mode is the transformed plant that replaces containing selectable marker gene with the transformed plant (Marker-free transgenic plants) that does not contain selectable marker gene.In theory, the method for acquisition marker-free gene transformed plant has two kinds: (1) utilizes selectable marker gene and corresponding selective reagents thereof in the gene transformation process; After cell transformed, tissue or plant obtain, excise again or the separation selectable marker gene; (2) by there not being the gene transformation (Nonselective gene transformation) of selection method, promptly when the structure of conversion carrier, just reject selectable marker gene, in the gene transformation process, do not use selective reagents.
1994, Yoder etc. summed up three kinds by utilizing earlier the method for afterwards excising acquisition marker-free gene transfer-gen plant (marker-free transgeni cplants-MFTPs):
(1) cotransformation method: to be the selectable marker gene that will be cascaded traditionally be structured in respectively on different expression vectors or the different T-DNA with goal gene its principle, transforms the common explant simultaneously.By selecting, not only contain selectable marker gene in the hope of obtaining, also contain the transfer-gen plant of goal gene simultaneously; By selecting the syngenesis offspring of this class transfer-gen plant, can obtain not contain the transgenic line that selectable marker gene only contains goal gene.Although this method has obtained MFTPs on crops such as tobacco, rape, efficient is very low, and not seeing has the report that further is applied in the business system.1996, the super binary vector pUC pUC of utilizations such as Komari carried out the research of gene cotransformation to paddy rice, tobacco, has obtained comparatively ideal results, and obtained the patent that the super binary vector of utilization carries out the gene cotransformation in 1998.Its cotransformation rate reaches 50%, separates integration rate and also reaches 50%, be i.e. the separable MFTPs that obtains from the syngenesis offspring of 25% cotransformation plant.
(2) swivel base method: selectable marker gene or goal gene were together changed in the acceptor with first linking to each other of swivel base, utilize swivel base unit to be excited the characteristic (under the inducing of transposase or tissue culture) of swivel base, break the chain state of goal gene and selectable marker gene, thereby can from the filial generation of transfer-gen plant, obtain the transfer-gen plant of marker-free gene.
(3) recombination method: as the Cre/lox system, when the loxp of two 34bp sequence was arranged with identical sequence, the gene of Cha Ruing will be sheared under the effect of Cre enzyme and recombinate therebetween, obtained MFGPs thereby can separate from the sexual hybridization offspring of transfer-gen plant.
1997, Ebinuma etc. utilize prenyltransferase (ipt) gene in the Agrobacterium Ti-plasmids to replace traditional antibiotics resistance gene and anti-herbicide gene as selectable marker gene, and the Ac of itself and corn swivel base unit linked to each other transform, metamorphosis by regeneration bud, i.e. differentiation from the regeneration of the no apical dominance bud of no hormone (phytokinin) substratum to field run plant, judge whether gene has transformed and whether selectable marker gene rejects, thereby set up and from transformant, rejected selectable marker gene, make transformant obtain repeatedly to repeat to transform the system of (multi-auto transformation-MAT), the pick-up rate of MFGPs reaches 5%.The advantage of this system is that the acquisition of MFGPs need not through syngenesis.1999, this research group improved above-mentioned MAT system, replaced Ac swivel base unit with the R/RS recombination system, made the pick-up rate of MFGPs increase substantially.2000, they did further improvement to above-mentioned system again, replaced 35S constitutive character promoters driven R recombinase gene with the sub-GST of a kind of chemical inducible promoter (glutathione-S-transferase), made the pick-up rate of MFGPs reach 14%.With regard to the present circumstances analysis, the GST-MAT system has been represented the highest level of producing MFGPs.
Except that above-mentioned three kinds of methods, also have a kind of method to be exactly gene with a class what is called " harmless " and substitute " being harmful to " gene such as antibiotics resistance as selectable marker gene.As phosphomannose isomerase (phosphomannoseisomerase-PMI) gene.PMI can be converted into fructose with seminose, thereby so available seminose replaces glucose in the general substratum or sucrose to select to have changed over to the cell of PMI gene as carbon source.In sum, for solving the problem of selectable marker gene, above-mentioned thinking is first utilization, and reject the back, as first three methods; Another thinking is to seek the gene of what is called " harmless ".But these two kinds of thinkings are not all fundamentally broken away from the dependency to selectable marker gene.
The inventor thinks that above-mentioned purpose can reach by the mode of not having the selection gene transformation; Promptly when making up the T-DNA of rotaring carrier, excising existing selectable marker gene or looking all genes is goal gene; Can but this have just produced the another one problem: promptly utilizing such carrier to carry out in the conversion process of goal gene, owing to do not have and can obtain to contain the transfer-gen plant of goal gene effectively for the selective reagents that utilizes.Utilizing common agriculture bacillus mediated gene transformation program and method, under the condition that lacks selectable marker gene and corresponding selective reagents, is the transfer-gen plant that can not obtain to contain goal gene effectively.Therefore, must invent new transgenosis program and solve this problem, thereby under nonoptional condition, also can obtain to contain the transfer-gen plant of goal gene effectively.
Common agriculture bacillus mediated gene transformation method comprises following plurality of processes:
(1) contains the cultivation of the Agrobacterium of goal gene and appropriate selection marker gene;
(2) preparation of plant explants;
(3) Agrobacterium is cultivated infecting together of explant;
(4) containing amplification transformant and tissue on the selection substratum of selective reagents;
(5) containing the transformed plant of regenerating on the selection substratum of selective reagents;
Common agriculture bacillus mediated gene transformation method needs selectable marker gene, need to use selective reagents, it is too big that we think to have following three factor: a. to transform explant, can regenerated in an explant transformant shared ratio in can the regenerated non-transformed cell too low, often less than one of percentage and even thousandth, make these transformants as no selective reagents to the growth of non-transformed cell and the inhibition of differentiation, be difficult to or can only become transfer-gen plant with extremely low frequency regeneration.B. most transformant is some cells that fully are exposed to Agrobacterium, and these cell polars are subject to infecting of Agrobacterium and death, and the ratio of transformant and non-transformed cell further descends thereby make.C. Agrobacterium all is to carry out in containing the developing medium of sufficient nutrition with the common cultivation that transforms explant in the common Transformation Program; We think that well-fed like this environment is to be unfavorable for Agrobacterium infecting and the conversion of goal gene vegetable material; We think that Agrobacterium is a power consumption process to infecting of plant, and under the condition that does not jeopardize breeding and existence, Agrobacterium there is no need to carry out the process of this power consumption.
Common agriculture bacillus mediated gene transformation method needs selectable marker gene, and its underlying cause is that transformation frequency is low, and transformant shared ratio in non-transformed cell is too low.If under the condition of no selective reagents, obtain transfer-gen plant, just must improve transformant shared ratio in non-transformed cell, improve the transformation frequency of gene.
Summary of the invention: this patent provides a kind of agriculture bacillus mediated plant not have the selection gene transformation method.Especially, this patented invention provides the method, the inhibition Agrobacterium inductive that prepare the explant that dwindles to be subjected to the method for infected cell death, to be total to cultured method under the nutritional deficiency condition.The invention provides the optimum combination of these methods, and under no selection condition, high frequency ground obtains transfer-gen plant.
The present invention provides a kind of new agriculture bacillus mediated transgenic method for plant genetic engineering.By this method, can directly, high frequency obtain the required transformed plant that contains goal gene; Selectable marker gene is dispensable in this method for transformation, with the corresponding selective reagents of selectable marker gene be unwanted in the method.
The present invention provides the method for a kind of new acquisition non selecting sign transgene plant for plant genetic engineering.By on conversion carrier T-DNA, not adding or excise existing selectable marker gene, utilize Transformation Program of the present invention again, it is the transfer-gen plant that contains goal gene that a certain proportion of plant is promptly arranged in regenerated transgenic plant, and the selection of this class transfer-gen plant can be undertaken by the method for molecular biology or chemistry with evaluation.
The present invention also provides a kind of agriculture bacillus mediated transgenic method that improves transformation frequency for plant genetic engineering, no matter uses or do not use selectable marker gene or selective reagents.
The present invention finishes like this: a kind of agriculture bacillus mediated plant does not have the selection gene transformation method, it is characterized in that said nothing selection is meant that not using selective reagents that the plant transformed cell or tissue is carried out difference selects, and does not then contain selectable marker gene among the T-DNA of Zhuan Huaing in the gene transformation process.Said nothing selects to comprise following content and step: (1) makes up to be applicable to no matter contain or do not contain selectable marker gene by agriculture bacillus mediated gene transformation carrier on T-DNA, and all genes all are considered as goal gene; (2) use the explant that dwindles, usually, 1-20 regenerable cell contained in the cross section of being infected of this class explant or explant; (3) explant reduces the Agrobacterium death that is subjected to infected cell that effect causes by pre-treatment or pre-the cultivation; (4) this class is cultivated under the nutritional deficiency condition of approximate Agrobacterium natural infection altogether by pre-treatment or pre-incubated explant and Agrobacterium; (5) use microbiotic to suppress growth of Agrobacterium; (6) in the substratum that does not contain selective reagents, induce the explant callus; (7) in the division culture medium that does not contain selective reagents, obtain regeneration plant; (8) identify transfer-gen plant by molecular biology or chemical analysis.
High frequency, efficient and can produce transfer-gen plant in large quantities under the condition of do not have selecting, its committed step comprises following four aspects: (1) uses the explant that dwindles, and usually, 1-20 regenerable cell contained in the cross section of being infected of this class explant or explant; (2) explant causes the death that is subjected to infected cell by pre-treatment or pre-the cultivation with the effect that reduces owing to Agrobacterium; (3) this class is cultivated under the nutritional deficiency condition of approximate Agrobacterium natural infection altogether by pre-treatment or pre-incubated explant and Agrobacterium; (4) amplification on the substratum that does not contain selective reagents, differentiation transgenic cell and tissue finally obtain transfer-gen plant.
It is the nucleotide sequence of going up in all senses that a kind of agriculture bacillus mediated plant of the present invention does not have its T-DNA of the gene transformation method of selection, but necessarily comprises border, the T-DNA left and right sides.Obsolete selective reagents, all help pharmaceutical chemicals and the reagent that transformant or tissue growth grow and help transformant or the pharmaceutical chemicals and the reagent of the identification of tissue to comprise microbiotic, weedicide etc.The Agrobacterium of indication is soil Agrobacterium Agrobacteriumtumefaciens; The plant of indication is swede type rape Brassica napus.Under the condition of not using selective reagents, obtain to contain the gene transformation method of goal gene transfer-gen plant effectively; This validity is meant that in the regenerated plant the shared percentage ratio of transfer-gen plant that contains goal gene is 5% to 20%.The method of carrying out gene transformation when on conversion carrier T-DNA, not containing selectable marker gene.
A kind of agriculture bacillus mediated plant of the present invention does not have the transgenosis of selection and marker-free gene method for transformation, and the method that it is characterized in that dwindling explant comprises mechanical or manual cutting, enzymolysis and nutritional deficiency cultivation.Suppress Agrobacterium and infect the vegetable cell that causes, the method for tissue die, comprise the thermal shock processing, interpolation can suppress chemical reagent such as Silver Nitrate, PVP, gac, vitamins C, gsh or the halfcystine of infected cell death.Co-culture method under the nutritional deficiency condition of approximate Agrobacterium natural infection; Comprise the co-culture method that blots substratum or do not contain nutritive substance.
Method for transformation of the present invention is equally applicable to selectable marker gene and has in the transgenosis program of selective reagents, and it helps to improve transformation frequency, and the selection of transfer-gen plant is by molecular biosciences or chemical method with evaluation; Microbiotic be kantlex, Totomycin, gentamicin, bleomycin, Streptomycin sulphate, spectinomycin, paraxin, Xin Meisu etc. all can influence vegetable cell and knit the microbiotic that grows of group; Weedicide be glyphosate, oxalyl phosphine, sulfonylurea etc. all can influence vegetable cell and knit the weedicide that grows of group; Selective reagents comprises that also all can influence vegetable cell and knit the enzyme inhibitors that group is grown as methotrexate sodium etc.; Selective reagents also comprise mannitol etc. all can difference the plant transformed cell with knit pharmaceutical chemicals and the reagent that group is grown; Help the pharmaceutical chemicals of transformant and tissue identification and reagent and be under the effect of certain enzyme, adding lustre to or produce the material of fluorescence.
The agriculture bacillus mediated plant of the present invention does not have the gene transformation method of selection:
1.T-DNA formation
The constructed T-DNA of the structure of T-DNA involved in the present invention and formation and common agriculture bacillus mediated gene transformation does not have special part.But when the method for utilizing this patented invention is carried out the marker-free gene conversion, should on the T-DNA that makes up, not contain or excise existing selectable marker gene.
2. the preparation of the explant that dwindles
The key that transfer-gen plant obtains is that Agrobacterium is to explant somatocyte, effectively infect this rapid amplifying, breeding and reorganizationizations and effective differentiation that is subjected to infected cell to explant regenerable cell (totipotent cell).For achieving the above object, dwindling of explant is an important step.The first, it helps increasing the explant outside surface and is subjected to infected cell to account for the ratio of explant somatocyte sum, helps infecting of Agrobacterium; The second, it helps being subjected to infected cell to be subjected to the influence of infected cell not less and increases more independently, breeds, thereby helps the regeneration of transformant and tissue.The explant that dwindles of this patent invention indication generally refers to the sum of regenerable cell or is infected the explant that the cross section regenerable cell is counted 1-20, and they can obtain by following three kinds of modes:
(1) machinery and manual cut method.Plant plant, organ, the tissue that will contain regenerable cell (totipotent cell) utilize machinery (as homogenizer) or craft to cut at tiny particle under aseptic condition.By the screen cloth of certain pore size, collection is suitable for the particle that Agrobacterium is infected.Usually, these the material diameters size at 35um between the 500um; Better, at 46um between the 230um.
(2) enzymolysis process.Plant plant, organ, the tissue that will contain regenerable cell (totipotent cell) utilize enzyme (normally, be cellulase and polygalacturonase) peel off cell walls and outside organization thereof, form single isolating protoplastis, collect and cultivate such protoplastis, make it to be used for the conversion of Agrobacterium.
(3) nutritional deficiency is cultivated (or hungry cultivation) method.The plant of plant, organ, be organized in the battalion deletion condition under; as at unglazed photograph and do not have when cultivating on the substratum of inorganic salt, no organic component; formed explant incision cell total amount and contrast are (without such processing; under normal matrix and illumination condition, cultivate) compare obvious decline, thus reach the purpose of dwindling explant.
3. suppress the Agrobacterium inductive and be subjected to infected cell death
The death of Agrobacterium inductive infected cell can reduce by the mode that thermal shock is handled plant tissue or explant or suppress.It is that best thermal shock temperature is 30-45 ℃ prior to the infecting of Agrobacterium that this thermal shock is handled, especially, and at 32-37 ℃; Treatment time is generally between 0.1h-24h; Best, between 8h-16h.
The death of Agrobacterium inductive infected cell can be by using chemical inhibitor (as ethylene inhibitor, Silver Nitrate), nonpolar organic matter matter sorbent material (as PVP and gac), active oxygen voltinism material reductive agent (as VC, gsh and halfcystine) to reduce or suppressing.The working concentration of Silver Nitrate is generally at 1-10mg/L; The usage quantity of PVP and gac is at 0.1-0.5g/L; The usage quantity of VC is at 0.5-10g/L; The usage quantity of gsh and halfcystine is generally at 10-100mg/L.The processing of this inhibition is prior to the infecting of Agrobacterium equally, and is extended to the amplification and the breeding stage of common cultivation and transformant; Generally between 16-72h, the aftertreatment time, (and Agrobacterium is cultivated the back altogether) was generally between 7-14 days for pretreatment time (infecting prior to Agrobacterium).
4. cultivate altogether under the nutritional deficiency condition
With preparation and pretreated explant and certain density Agrobacterium be blended in liquid contain the plant cell growth hormone (as 2, substratum 4-D) (as the MS substratum) and lack dip-dye 10-120min in the medium (only containing the medium that hormone, osmotic pressure are regulated composition and above-mentioned Agrobacterium inductive cell death inhibitor composition) of nutrition with normal nutrition; Then, blot liquid medium, under the situation that keeps certain humidity, explant and Agrobacterium were cultivated 2-7 days altogether under the condition of nutritional deficiency.
5. the amplification of transformant and breeding
What the explant after cultivating altogether was transferred to liquid or solid is added with the plant cell growth hormone (as 2,4-D) and on the MS substratum of Agrobacterium growth inhibitor (as Pyocianil) and above-mentioned Agrobacterium inductive cell death inhibitor cultivated 7-20 days, the cell transformed of this moment is amplified tens agglomerates to a hundreds of cell.
6. the regeneration of transformed plant
The explant that has been amplified transformant is transferred on the MS substratum that is added with phytocytomine (as 6-BA, Zt etc.) of liquid or solid to be cultivated 10-40 days.Regeneration techniques is well-known, is unessential for the present invention, and any culture technique that can produce the regeneration plant that can educate can both be used for this specially related nothing and select gene transformation.
7. the Analysis and Identification of transformed plant
By the regeneration plant that aforesaid method produces, owing to do not use any selective reagents in conversion process, some is unconverted plant in the regenerated plant; Have only 5%, best, about 20% regeneration plant is a transfer-gen plant.Utilize molecular biology method (as the dot blot method, the pcr amplification method) and chemical analysis (analyzing genetically modified expression product, meta-bolites etc.) commonly used can distinguish, distinguish these regenerated transgenic plant.
Description of drawings: patent of the present invention will further be illustrated by following example, but it will not produce restriction to the clause in the patent claim.
Fig. 1 adopts the agriculture bacillus mediated plant of the present invention not have and selects the coloration result figure of gene transformation method at the plant leaf material.
The agriculture bacillus mediated plant of Fig. 2 the present invention does not have selects the coloration result figure of gene transformation method at callus stage converting material.
The agriculture bacillus mediated plant of Fig. 3 the present invention does not have the selection gene transformation method, does not have the gene transformation technology of selection route at agriculture bacillus mediated rape.
Embodiment: following example transfer-gen plant can select gene transformation method to obtain by agriculture bacillus mediated nothing.Certainly, this nothing selects gene transformation to need special organization to cultivate and Agrobacterium is infected program.
Example 1: the preparation method 1-cut mechanically method of the explant that dwindles
With the commodity chlorine bleach liquor of mercuric chloride or 20% cabbage type rape variety Wei Sita (the Brassica napuscv Westar) seed of sterilizing, be seeded on the MS substratum and cultivated 7-10 days.The upper half part of seedling 1-2cm band bud point and cotyledon tissue is also collected in cutting, with sterilized stainless steel cell homogenates device in liquid medium with its chopping.Usually, per 200 seedling are used the 30-50ml liquid MS medium.Mixed solution after the homogenate is collected the tissue particles of 46-230um diameter with the strainer filtering in a series of different apertures.With these short grained explants pre-explant that dwindles that Agrobacterium is infected test that can be used for that promptly became suitable size in 1-3 days of cultivating in pre-developing medium (see below and state literal).The result of follow-up transformation experiment shows, explant dwindle obvious improved efficient and the percentage (see Table 1) of transformant in explant that explant is infected.
The size of the explant that table 1 cut mechanically is dwindled is infected the influence of efficient to explant
| The explant granular size | The explant percentage that contains at least one transformant | The percentage that the duty transformant is shared |
| 500-1000um | 90％ | 0.1％ |
| 250-500um | 70％ | 1.0％ |
| 150-250um | 50％ | 5.0％ |
| 100-150um? | 30％? | 20％? |
| 50-100um | 10％ | 40％ |
Above-mentioned experiment be based on that Agrobacterium is infected and cultivate altogether after cultivate a week again, the result who adds up through X-Gluc dyeing back.The shared percentage of transformant is meant the mean value of transformant and non-transformed cell ratio in containing the tissue of transformant.
Example 2: preparation method's 2-enzymolysis process of the explant that dwindles
Cabbage type rape variety Wei Sita is seeded in greenhouse (17-23 ℃).After 20 days, the 3-10 sheet true leaf of the getting plant 5-15min that in the polysorbas20 of 20% commodity chlorine bleach liquor and 0.1%, sterilizes.With the back side of fine sandpaper polishing blade, enzymolysis 8-20h in the enzymolysis solution that contains cellulase 2mg/ml, polygalacturonase 0.25mg/ml and mannitol 100mg/ml.Separate and washing by common protoplastis separable programming, and the protoplastis after will washing was cultivated 3-12 days in the culture dish of 60mm diameter; The protoplastis culture density is 1 * 10
5Individual/ml, substratum is that 1/2MS adds 2,4-D0.5mg/L, NAA0.5mg/L, 6-BA0.5mg/L and mannitol 100g/L.Collect protoplastis and callus thereof after cultivating, the pre-explant that dwindles that Agrobacterium is infected test that can be used for that promptly became suitable size in 1-3 days of cultivating in pre-developing medium (see below and state literal).The result of follow-up transformation experiment shows, efficient that the protoplastis callus explant of different sizes is infected explant and the percentage of transformant in explant have significantly influences (seeing Table 2).
The effectiveness affects that the protoplastis callus explant of the different sizes of table 2 is infected explant
| The explant granular size | The explant percentage that contains at least one transformant | The percentage that transformant is shared |
| 1-2 cell | 20％ | 80％ |
| 4-8 cell | 30％ | 15％ |
| 10-20 cell | 50％ | 6.0％ |
| 20 more than the cell | 70％ | <1.0％ |
Above-mentioned experiment be based on that Agrobacterium is infected and cultivate altogether after cultivate a week again, statistics after X-Gluc dyeing.The shared percentage of transformant is meant the mean value of transformant and non-transformed cell ratio in containing the tissue of transformant.
Example 3: preparation method's 3 one nutritional deficiencies of the explant that dwindles are cultivated (or hungry cultivation) method:
With the commodity chlorine bleach liquor of mercuric chloride or 20% cabbage type rape variety Wei Sita (the Brassica napuscv Westar) seed of sterilizing, be seeded in 1/4MS or do not have dark the cultivation 7-10 days on the substratum of nutritive substance.The hypocotyl of seedling is cut into the fragment of 0.7-1.0cm, the pre-explant that dwindles that Agrobacterium is infected test that can be used for that promptly became suitable size in 1-3 days of cultivating in pre-developing medium (see below and state literal).The result of follow-up transformation experiment shows, adopt or do not adopt nutritional deficiency and cultivate efficient that (CK, with normal MS and contain the substratum of sucrose 10g/L and cultivate having under the condition of periodicity of illumination) infected explant and the percentage of transformant in explant and have significantly and influence (seeing Table 3)
Table 3: nutritional deficiency is cultivated the effectiveness affects that explant is infected
| The explant culture condition | The explant percentage that contains at least one transformant | The percentage that transformant is shared |
| CK | 20％ | 6.0％ |
| 1/4MS? | 90％? | 30％? |
| No nutrition | 70％ | 40％ |
Above-mentioned experiment be based on that Agrobacterium is infected and cultivate altogether after cultivate a week again, the result who adds up through X-Gluc dyeing back.The shared percentage of transformant is meant the mean value of transformant and non-transformed cell ratio in containing the tissue of transformant.
Example 4: the Agrobacterium inductive is subjected to inhibition method 1-thermal shock facture of infected cell death:
That to obtain with aforesaid method or through the pre-incubated explant that dwindles in 30-45 ℃, especially, in 32-37 ℃ incubator, handle 0.1h-24h; Best, 8h-16h.Explant after the processing carries out the amplification (see below and state literal) of infecting, be total to cultivation, transformant of Agrobacterium again, and the result shows the survival rate (seeing Table 4) that thermal shock is handled has increased transformant significantly.
Table 4 thermal shock is handled the effectiveness affects that explant is infected
Above-mentioned experiment be based on that Agrobacterium is infected and cultivate altogether after cultivate a week again, the result who adds up through X-Gluc dyeing back.The shared percentage of transformant is meant the mean value of transformant and non-transformed cell ratio in containing the tissue of transformant.CK represents homologue but handles without thermal shock.
The present invention can suppress the reagent pre-treatment explant that Agrobacterium is induced transformant death by utilizing thermal shock and some, has improved the survival rate of transformant, thereby has improved the ratio of transformant in regenerable cell.
The present invention makes Agrobacterium that the efficient that infects of vegetable cell is improved under the condition of nutritional deficiency by improving the common culture environment and the condition of Agrobacterium and explant.
The present invention has improved the transformation frequency of explant greatly by the change of above-mentioned three conversion conditions, makes it to reach more than 90%.The ratio of transfer-gen plant reaches more than 5% in the regenerated plant under no selection condition, usually, can reach more than 20%.
Especially, of the present inventionly dwindle explant, prevent that Agrobacterium from causing the braking measure that is subjected to infected cell death, in the methods such as common cultivation of nutritional deficiency condition the Plant Transformation beyond the swede type rape all being had useful effect.
The present invention is by reducing outer sum (the advantage ground of growing regenerable cell in the body, between 10-20), as long as the method for promptly dwindling explant makes in case have transformant in explant, its shared ratio can finally become transformed plant to help this transformant greater than 5%.
Claims (7)
1. an agriculture bacillus mediated plant does not have the selection gene transformation method, it is characterized in that said nothing selection is meant that not using selective reagents that the plant transformed cell or tissue is carried out difference selects in the gene transformation process; Do not contain selectable marker gene among the T-DNA that transforms, this method both had been the marker-free gene conversion method; Said nothing selects gene transformation to comprise following content and step:
(1) structure is applicable to agriculture bacillus mediated gene transformation carrier, and all genes all are considered as goal gene;
(2) use the explant that dwindles, 1-20 regenerable cell contained in the cross section of being infected of this class explant or explant;
(3) explant reduces the Agrobacterium death that is subjected to infected cell that effect causes by pre-treatment or pre-the cultivation;
(4) this class is cultivated under the nutritional deficiency condition of Agrobacterium natural infection altogether by pre-treatment or pre-incubated explant and Agrobacterium;
(5) use microbiotic to suppress growth of Agrobacterium;
(6) in the substratum that does not contain selective reagents, induce the explant callus;
(7) in the division culture medium that does not contain selective reagents, obtain regeneration plant;
(8) identify transfer-gen plant by molecular biology or chemical analysis.
2. a kind of agriculture bacillus mediated plant according to claim 1 does not have the selection gene transformation method, it is characterized in that the marker-free gene conversion method is meant when making up T-DNA subsidiary existing selectable marker gene and uses the method that above-mentioned 1-8 step is carried out gene transformation.
3. a kind of agriculture bacillus mediated plant according to claim 1 does not have the selection gene transformation method, it is characterized in that obsolete selective reagents, be meant and do not use all to help pharmaceutical chemicals and the reagent that transformant or tissue growth grow and help transformant or the pharmaceutical chemicals and the reagent of the identification of tissue.
4. do not have the selection gene transformation method according to claim 1 or 3 described a kind of agriculture bacillus mediated plants, it is characterized in that obsolete selective reagents, be meant and do not use microbiotic, weedicide.
5. a kind of agriculture bacillus mediated plant according to claim 1 and 2 does not have the selection gene transformation method, and the Agrobacterium that it is characterized in that indication is soil Agrobacterium (Agrobacterium tumefaciens); The plant of indication is swede type rape (Brassica napus).
6. a kind of agriculture bacillus mediated plant according to claim 1 does not have the selection transgenic method, and the mode that it is characterized in that preparing the described explant that dwindles is that mechanical or manual cutting, enzymolysis and nutritional deficiency are cultivated.
7. a kind of agriculture bacillus mediated plant according to claim 1 does not have the selection genetic method, it is characterized in that the co-culture method under the nutritional deficiency condition of Agrobacterium natural infection, comprises the co-culture method that blots substratum or do not contain nutritive substance.
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| WO1997048814A3 (en) * | 1996-06-21 | 1998-03-19 | Monsanto Co | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
| WO1999010512A1 (en) * | 1997-08-25 | 1999-03-04 | Nunhems Zaden B.V. | Improved agrobacterium-mediated transformation of plants |
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| CN1351668A (en) * | 1999-05-19 | 2002-05-29 | 阿温提斯作物科学公司 | Improved method for agrobacterium mediated transformation of cotton |
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|---|---|---|---|---|
| WO1997048814A3 (en) * | 1996-06-21 | 1998-03-19 | Monsanto Co | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
| WO1999010512A1 (en) * | 1997-08-25 | 1999-03-04 | Nunhems Zaden B.V. | Improved agrobacterium-mediated transformation of plants |
| CN1351668A (en) * | 1999-05-19 | 2002-05-29 | 阿温提斯作物科学公司 | Improved method for agrobacterium mediated transformation of cotton |
| CN1342772A (en) * | 2001-10-24 | 2002-04-03 | 中国科学院成都生物研究所 | Agrobacterium medicated plant transgene techhnique |
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| Moore-Gloria.Transgenic grapefruit plants obtained byAgrobacteriumtumefaciens-mediated transformation..Plant-Cell-Tissue-and-Organ-CultureVol. 57 No. 3.1999,57(3),219-222. |
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| 姚方印等.农杆菌介导将白叶枯病抗性基因Xa21导入水稻获得转基因植株.西北植物学报Vol. 22 No. 5.2002,22(5),1038-1043. * |
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