CN1213404A - Genetic transformation of trees - Google Patents
Genetic transformation of trees Download PDFInfo
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- CN1213404A CN1213404A CN97192880A CN97192880A CN1213404A CN 1213404 A CN1213404 A CN 1213404A CN 97192880 A CN97192880 A CN 97192880A CN 97192880 A CN97192880 A CN 97192880A CN 1213404 A CN1213404 A CN 1213404A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- 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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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
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Abstract
The invention is an improvement in the transformation of trees and provides a method which is less stressful to the tree being transformed. Notoriously difficult trees such as Eucalyptus globulus can be successfully transformed. An incision is made into a site capable of shoot initiation, disarmed Agrobacterium cells carrying one or more exogenous DNA sequences are introduced into the incision and the site is subjected to a selection step for a time period sufficient to produce transformed shoots.
Description
The present invention relates to by importing the conversion of the seeds that the heterologous gene gene of certain tree other approach in addition itself (promptly from) carries out.
With the existing many treatises of new gene transfered plant species, become standard practice for the many annual and living throughout the year crop that comprises tomato, potato, rape and paddy rice.The gene that imports has been used to modify multiple specific and general proterties, comprises, for example, and the quality of protein and carbohydrate, post-harvest physiology, photosynthetic capacity and pest-resistant characteristic all can be expressed whole strain plant and plant privileged site.According to the natural characteristic of plant species, developed the method for the new gene of various importings, comprise that dna direct shifts (microinjection, biological emission (biolistics), primitive fiber method, chemistry perforation, electric shock) and carry out the class natural infection with microorganism agrobacterium tumefaciens and Agrobacterium rhizogenes.
The use that the gene transformation of forest species up to now is labeled gene is greatly limiting, and comprises and influences insect-resistant, herbicide tolerant, the more business-like correlation properties of response to traume and lignified gene though imported in some instances.
The gene transformation of seeds exists specific question (Jouanin etc., 1993), and one of them is its natural stubborn susceptibility that has limited transforming of organizing self.Many trees are not good in growth in vitro, comprise the low breeding potential of external seedling system, and the low regeneration power of bud and root.In addition, the living then field crops of cultivating than process usually according to the idiotype tree demonstrates the external behavior of wider scope.Also have, exist different between very big kind and in planting for the susceptibility of gene transformation, some are set the increase susceptibility that also demonstrates along with physiological age and obviously reduce.Can obtain the moment importing of new DNA to vegetable cell, but DNA can not go into Plant Genome by stable integration, the expression of quiding gene is of short duration.Further problem is before conversion, is difficult to keep when cultivating and breed tissue during the transformant screening and during the whole strain plant regeneration that transforms.The health of tissue and survival are also to transforming and described above being integrated with concerns and relevant with the object of the invention.
The method that obtains the most common utilization of stable conversion is the Agrobacterium by modifying.Many seeds are responsive but only obtain in the minority kind with the stable conversion of other gene to the infection of wild-type agrobacterium tumefaciens, as Brackpool etc. 1990; DeBlock, 1990 and McGranahan etc., 1990.Success only is limited to the specific gene type of these species, mainly is that those are easy to the genotype that transforms and breed.
The method of using needs series of steps usually, for example:
ⅰ) under plant tissue cutting-out target, plant body, for example, the leaf dish, the root piece, the stem section, plumular axis, cotyledon, embryo or cell suspension thing,
ⅱ) explant pre-cultivation a few hours or time a couple of days on the substratum that is fit to,
ⅲ) explant is cultivated altogether with the vegetable cell by cut wound with the bacterial cell that contains dna vector and is infected,
ⅳ) go to the selection substratum with a) growth of the residual bacterial cell of prevention, and b) screen by the plant transformed cell, and
ⅴ) the one or more substratum that go to make its callus from the explant source begin to be regenerated as complete plant.
Basic skills comprise make organize explant be subjected to the cutting cause the physical property wound, with bacterium cultivate, place chemical selective agent altogether; each step all applies serious pressure in transformant and tissue, can the bacterium regenerative power that tissue is had be had a negative impact usually.Use and health if get rid of, the isolating necessity of organizing explant that viability and regenerative power are relevant, the influence of these treatment steps will reduce.
In the tree crop, the sexual or vegetative propagation of following improvement germplasm is a mature operation especially, and this fecundity is held as a key property for the purpose of cloning afforestation (forestry) in the improvement material.Produce the process that transforms plant and should not get rid of the propagation method of the conversion species that can normally breed originally by following any generation.
The method for transformation of purpose of the present invention for providing one plant tissue and transformant only to be caused the tree of slight pressure.
The present invention also wishes to obtain the method for transformation of a tree, this method of utilization in plant tissue, their performance with regenerative power that higher survival becomes reconciled help to guarantee through transform or improvement after the fecundity of germplasm still keep.
Further target be decide target in the layer of predetermined seedling energy for growth to improve the seedling regenerated possibility that contains transformant to greatest extent.
The invention provides a kind of method that any DNA sequence is imported tree.More specifically, this method can use those can influence phenotypic characteristic, for example, and silviculture characteristic, timber, the mass property of the fruit or the leaf course of processing, or the gene or the dna sequence dna of the phenology of ripe trees regeneration product.So the present invention will make by clonal propagation improvement tree and produce and the syngenesis result in seed garden controls better and becomes possibility.The offspring of containing the conversion tree of new gene also can further transform, if need the improvement that can improve gradually.
Ellis etc., 1992 have described a kind of acerose method for transformation, and it comprises the otch that the wild-type Agrobacterium rhizogenes is imported European larch seedling cotyledon joint.But this method has only produced the tumour group of bud original hase, does not obtain the complete transformed plant of regenerated.
The present invention has overcome the problem of tumor growth, and provides suitable conversion plant to transform difficulty up to now and still do not have other seeds regenerated method of openly reporting its complete transformed plant from least a.
The invention provides the method for transformation that transforms root class or other class tree, this method comprises following step: introduce otch in the site that bud can be sprouted, incision import be loaded with one or more exogenous DNA arrays unload first (disarmed) agrobatcerium cell, and screening step, wherein make this site and contain nutritive substance, the substratum of growth regulatory factor and selective agent (transformant has resistance to this) contacts for some time to be enough to produce the conversion seedling that contains exogenous DNA array.
Be preferably placed at the sprout otch in site of bud and form a crack.According to the size of handled tree, otch can be about 1mm or darker.Term used herein " tree " comprises seedling, seedling (obtaining from beta pruning or alternate manner) and beta pruning, or bigger plant.
The bud site of sprouting can suitably be cotyledonary node, axillary meristem or an apical meristem.
When being a seedling by the tree that transformed, the preferred bud site of sprouting is a cotyledonary node.The preferred method that transforms sapling further is included in and imports before the agrobatcerium cell, among or remove all already present these steps of bud of sprouting afterwards.This importing can be known as inoculation step.
When being one section sapling by the tree that transformed, the preferred bud site of sprouting is an axillary meristem or apical meristem.In this special methods need not before the inoculation step, among or remove already present merismatic cell afterwards.
Preferably make before the step bud site of sprouting contact for some time with the substratum that contains nutrition and growth regulator in screening.This step is called as common culturing step.When finding that in a seedling bud is sprouted, preferably culturing step carries out with vertical seedling altogether.Being fit to altogether, culturing step carries out in test tube.
The substratum of preferred screening step also contains microbiotic to kill agrobacterium strains.
Preferred tree preferably shows as living plant germination on the ground.More preferably this method is applied to as eucalyptus genus, Populus, Malus and Prunus, and the seeds of any infection sensitivity to agrobacterium tumefaciens or Agrobacterium rhizogenes.This method is especially useful to some kind that transforms eucalyptus, blue gum for example, and it is famous to be difficult to successfully transform and produce transformed plant up to now.
Preferably when infected and/or when cultivating altogether, tree has complete root system.
Preferred tree is blue gum or alpine ash.
The present invention further provides the tree that transforms according to aforesaid method.
The present invention further provides a kind of cell that transforms the back carrying genes by above method.The propagulum that the present invention also provides method in view of the above to transform the back tree, the seed of the seedling of the tree after transforming according to this method, the tree after transforming according to this method, and the germplasm of the tree after transforming according to this method.
For making easy to understand of the present invention and convenient enforcement now by following examples reference is provided.
The conversion of blue gum seedling is carried out according to following program: the preparation that embodiment 1 blue gum seedling transforms
With the household bleach solution that contains clorox and stain remover the blue gum seed is carried out surface sterilization.Used concentration and disinfecting time length are unimportant, usually seed are hatched in the aseptic reverse osmosis water of 10% (v/v) household bleach dilution, constantly stir on the orbital oscillation device.Upgrade sterile liquid after 30 minutes and carry out hatching 60 minutes second time.Between the incubation period first time, sodium hypochlorite solution often becomes very black color.At last, jolting seed in clean sterilized water, repeated washing is repeatedly with the smell of removal chlorine and the foam of residual stain remover on demand.
Seed after the sterilization is spread and places the Magenta culture dish to contain 0.4% sterilized water nutrient agar surface of 5g/L sucrose.Leave suitable space between the seed that media surface is broadcasted sowing, density is 25 seeds of every ware.Place a growth case to carry out long low light photograph of 16 hours days in 20 ℃ in culture dish, make seed germination and growth 10 days.Most during this period seedling should reach cotyledon and fully stretch the stage of just having exposed with plumule.
Seedling is in such seedling age makes them provide a healthy and strong environment for the survival and the propagation of transformant.Seedling also is enough big or small so that operation.Embodiment 2 is used for the preparation of the agrobacterium tumefaciens cell of common cultivation
Cultivate altogether is to be loaded with coding beta-glucuronidase (GUS) gene and the plasmid p35S-GUSint that kantlex forms the NPT II gene of resistance is carried out by agrobacterium tumefaciens bacterial strain EHA105.Can produce this two kinds of marker gene expression in vegetable cell with this plasmid vector to the conversion of blue gum tissue, therefore, make that the healthy tissue that contains independent or most of transformant of growth performance obtains screening when containing kantlex, upward carry out the evaluation of these specific transformants by the catalytic reaction of β-Pu Taotanggansuanmei by being added with color emissivity substrate X-gluc (5-bromo-4-chloro-3-indyl-β-D-glucuronide).The L-broth culture that contains the 50mg/L kantlex with some bacterial cultures inoculation 50ml prepares cell.Centrifugal collecting cell also is resuspended in 10ml 1% glucose before use after 28 ℃ of overnight growth.
L-meat soup 10g/L bacterium Tryptones (bactotryptone)
The 5g/L yeast extract
The common cultivation of 5g/L sodium oxide embodiment 3 blue gums and agrobacterium tumefaciens
Under aseptic condition by sampling in the sprouting seedling of the method for embodiment 1 preparation with contain the bacterium that transforms carrying agent and cultivate altogether.Be the suitableeest co-culture method that the decision blue gum transforms, to three kinds of different methods of the agrobacterium tumefaciens infection of the preparation of the method among the embodiment 2 as evaluation.Bottom and root are cut and discarded to method 1. along plumular axis from nearly middle part with seedling.Cut plumule with the dissection point of a knife, remove petiole.The plumular axis that downcuts is placed horizontally on the solid medium 1 of culture dish.By making Agrobacterium inoculation tissue on the plumular axis that in the disposable dropper bacterial suspension is dripped to level.Method 2. along plumular axis near middle part cutting seedling and discard the bottom and root.Plumule removes with the dissection point of a knife.With crack that 1mm is dark of the scalper cutting of the top from cotyledonary node to each plumular axis vertically.Before each cutting, at once scalpel blade is immersed in the bacterial suspension to finish the inoculation of agrobatcerium cell simultaneously.Then the bottom of postvaccinal each plumular axis is erect in the solid medium 1 that inserts in the culture dish.Every ware is placed 20 plumular axis.Method 3. usefulness are dissected point of a knife and are downcut plumule from seedling, but keep the complete seedling of root system.With crack that 1mm is dark of the scalper cutting of the top from cotyledonary node to each plumular axis vertically.Before each cutting, scalpel blade is immersed in the bacterial suspension at once to finish the inoculation of agrobatcerium cell simultaneously.Postvaccinal degerming seedling is gone in the other test tube of diameter 25mm and with its root system and is vertically inserted in the 20ml substratum 2.
Prepared 100 seedling with method 1 and 3, and prepared 200 seedling with method 2.
24 ℃ of days long 16 hours low light according under continue to cultivate altogether 10 days.Afterwards, will from separately culture dish or test tube, take out and accept further operation by the explant of method 2 and method 3 preparations.
Downcut the cotyledon blade for preparing with method 2, cotyledon blade, root and the plumular axis lower part of from the seedling of method 3 preparations, taking out from plumular axis.
The armpit of any explant after handling in three kinds of modes is sprouted and is also all removed.The plumular axis that cuts in three kinds of methods is placed horizontally at contains microbiotic Cefotaxime (cefotaxime) and express with screening on the selection substratum 1 of transformed plant cells of NPT II gene to remove residual agrobatcerium cell and to contain kantlex.Every plate is placed the plumular axis after 20 cuttings.After 10 days they are gone to selection substratum 2, after 4 weeks, go to again in the fresh ware that contains same medium.24 ℃ are carried out common cultivation under day, long 16 hours low light shone.
After four weeks, those outer plant materialss that turn green (natural colored) are gone in the new ware that contains same medium.Organize further to forward on the fresh dish and kept what screen by certain interval.
The result
| Handle | ????1 | ????2 | ????3 |
| The regenerate that plumular axis 8 week survival 15 week survival 22 week survival is set up | ????100 ????0 ????0 ????0 ????0 | ????200 ????9 ????4 ????3 ????1 | ????100 ????7 ????4 ????1 ????1 |
From the conversion of blue gum, only obtained the regenerate of small number, do not obtained regenerate (method 1) from contact the cutting plumular axis that carries out common cultivation with the substratum level, and the very fast decline of healthy state of tissue.All obtained regenerate from method 2 and 3.The survival number reduces the regeneration site that may show that some are formed by non-transformed cell (being unable to undergo the screening of kantlex) along with the prolongation of time.With the regeneration rate of method 2 preparation and the tissue cultivated altogether a shade below regeneration rate with the tissue of method 3 preparations.
The characteristics of method 3 are that tissue wounds is small but accurate, and the vascular system between root and plumular axis keeps, and plumular axis slightly directly contacts with the substratum that contains the external source growth substance, and the bacterial cell inoculation accurately, the less excess growth of bacterium.Method 3 is selected as the method that transforms the blue gum tissue.Embodiment 4. method for transformation are revised and transgene expression
Press embodiment 1 and 2 preparation blue gum seedling and agrobacterium suspensions.3 kinds of modification assessments of best inoculation method to embodiment 3 are as follows:
1. decaptitate and crack (promptly removing whole cotyledonary nodes and cotyledon)
2. degerming and crack (as embodiment 3, method 3)
Degerming and indentation (with scalpel blade along plumular axis at its length part surface indentation).
In addition, also three kinds of modifications are investigated:
4. degerming and stab with hypodermic needle
5. degerming, indentation and with the tweezers top of crushing
6. degerming and crushing
After the inoculation, all seedling are all changeed plant in the independent test tube that contains substratum 3, and place on the dark frame of growth room and carry out common cultivation.
After three weeks, the seedling sampling of handling through three kinds of main modes detects by the following β-Pu Taotanggansuanmei activity (GUS+) of doing with x-gluc.The 3mg x-gluc that is dissolved in the 100ml dimethyl formamide is complemented into 10ml with the 50mM sodium phosphate buffer (pH7.0) that contains 0.5%Triton x 100 and 1mM EDTA.Groups of samples is woven in 37 ℃ of cultivations reach 16 hours in the dark in this reagent of small volume.Cultivate in Virahol in room temperature afterwards and decolour 4 hours to a couple of days.The β-Pu Taotanggansuanmei activity blue region occurs and detected obtaining by tissue.
| Handle | ????1 | ????2 | ????3 | ????4 | ????5 | ????6 |
| Numbering 3 week survival GUS+ (3 week) | ????120 ????68 ????(57％) ????8/12 ????(67％) | ????200 ????123 ????(62％) ????10/11 ????(91％) | ????200 ????88 ????(44％) ????21/31 ????(68％) | ????40 ????10 ????(25％) | ????40 ????0 ????(0％) | ????40 ????2 ????(5％) |
Only on the sampling seedling, also carried out the x-gluc detection after the week.Most of sample of selecting from six kinds of methods has shown the GUS activity.
The method of selecting in embodiment 3 is to handle 2 in this example, has high viability and maximum conversion or most sustained reaction.This is that foreign gene is stable, but not the performance of transient expression.
The main blue part of dying that the chances are in all are handled distinguishes out transformant is distributed in around the crack.Handling in 1, is the discrete point that forms independent cell, and in that to handle the darker blue look zone of dyeing bigger in 2 more remarkable, is showing from these regional regenerated seedlings may have grown into transformed plant.Through the conversion of the plumular axis of indentation generally than those cut open on the top joint-cutting for poor.
For doing further comparison, the method for transformation (embodiment 3, method 1) of cutting plumular axis some careful improvement have also been done.Improvement comprises with scalper indentation plumular axis to produce the wound of a plurality of different lengthss, also with the shallow brush of sterilization at plumular axis tissue acanthopore.All these handle the regeneration that does not all produce successful transformant.The regeneration of embodiment 5. express transgenic seedlings
By the method preparation of describing among the embodiment 3 with cultivated 120 seedling altogether.After being total to cultivation stage, having selected 16 plumular axis to carry out x-gluc at random and detected.All the other plumular axis stay does screening and regeneration.Remove lower part and root and cotyledon blade and all cotyledon armpits of having sprouted of plumular axis.The remainder of plumular axis is placed ware with the level of density of 10 in every ware.Seal ware and place on the dark frame of growth room with sealing film.All around plumular axis is gone in the new ware that contains same medium, and check green callus Growth.Then all green callus are gone to and select substratum 3, and those are seemed to select substratum 4 near going to of bud differentiation.
Carry out in the plumular axis sample that x-gluc detects at 16, remove an exception and shown that all indigo plant dyes the zone.
After one week, all plumular axis explants all seem to bleach fully and do not observe a sub-axil bud.The back has 8 and shows very bottle-green callus and healthy appearance on the selection substratum in 100 plumular axis all around, and other has 10 Yu's injured tissues with medium green.13 callus are gone to selection substratum 3 and go to selection substratum 4 with 5.
Go to select substratum 4 after, 4 callus regenerations sprout, and spend for six weeks again after x-gluc detects them is GUS+.
The present invention limits used dna sequence dna or binding sequence, so can be used to import the foregoing characteristic of any range.The present invention also can be used for generation or the previous material that had been transformed.
To another method of substitution of above seedling method is the bud the sapling of vitro culture can be sprouted the site, as axillary meristem or apical meristem, cuts the processing in meristematic tissue zone and uses the agrobacterium strains through genetic modification to infect with same way as.Transformant can separatedly produce plants transformed as explant and cultivation after for some time.
Reference Brackpool A.L., Ward M.R.﹠amp; Weir A.F. (1990) " is belonged to the regeneration of seedling of acquisition of plumular axis seedling and the top condition of conversion " by eucalyptus
7thInternational Congress of Plant Tissue and Cell Culture, Amsterdam, 24-26 June, 1990.Poster A1-23DeBlock, M. (1990) " influence the factor that tissue culture and Agrobacterium tumefaciens mediated hybridization aspen and poplar clones transform " Ellis D.D., Diner A.M.﹠amp; Huang Y. (1992) " importance of engineered acerose regeneration-biosystem "
Southern Regional Information ExchangeGroup Biennial Symposium, Huntsville, AL, 8-10 July, 1992Jouanin L., Brasileiro A.C.M., Leple J.C., Pilate G.﹠amp; CornuD. (1993) " gene transformation: method, application, result and and to the summary of the prospect of forest-tree "
Ann.Sci.For.50 325-336.McGranahan G.H., Leslie C.A., Uratsu S.L.﹠amp; Dandekar A.M. (1990) " the English walnut body blastema of improvement is because of the effectiveness of transfer system "
Plant Cell.Rep.8 512-516.Linsmaier E.M.﹠amp; Skoog F. (1965) " the organic growth factor that the tobacco tissue culture is required " Physiol Plant.18100-127
Appendix
Used substratum-all is based on the substratum of full strength Linsmaier and Skoog (1965), uses half intensity substratum 2 and uses except the substratum 3 of 1/4th intensity.Substratum contains following additive: substratum 1 15g/L glucose
10 μ M zeatin
5 μ M indyl-3-acetate
0.01％????pluronic?F-68
4g/L nutrient agar 2 10g/L sucrose
4g/L nutrient agar 3 10g/L sucrose
4g/L agar
The 1g/L gac is selected substratum 1 15g/L glucose
10 μ M zeatin
5 μ M indyl-3-acetate
The 250mg/L Cefotaxime
The 30mg/L kantlex
7g/L agar is selected substratum 2 15g/L glucose
4 μ M zeatin
1 μ M indyl-3-acetate
The 250mg/L Cefotaxime
The 35mg/L kantlex
7g/L agar is selected substratum 34 μ M zeatin
2 μ M indyl-3-acetate
The 250mg/L Cefotaxime
The 30mg/L kantlex
7g/L agar is selected substratum 42 μ M 6-benzylaminopurines
1 μ M indyl-3-acetate
The 250mg/L Cefotaxime
The 30mg/L kantlex
7g/L agar
Claims (19)
1. one kind transforms tool root tree or other method, the step that described method comprises has: introduce otch at the position of can bud sprouting, Yu Jia (disarmed) agrobatcerium cell that is loaded with one or more exogenous DNA arrays is imported this otch, screen step, in this step described site is contacted with the substratum that contains nutrition, growth regulator and selective agent, cell is a resistance to this selective agent, and for some time is enough to produce the conversion seedling that contains exogenous DNA array.
2. by the process of claim 1 wherein that described otch forms a joint-cutting in bud sprouting site.
3. by claim 1 or 2 method, the about 1mm of wherein said otch deeply or darker.
4. by the method for claim 1,2 or 3, described site of can bud sprouting is cotyledonary node, axillary meristem or an apical meristem.
5. according to the method for claim 4, when the tree that is transformed was a seedling, the bud site of sprouting was a cotyledonary node.
6. according to the method for claim 5, before importing agrobatcerium cell, among or remove all existing (perexisting) bud of sprouting afterwards.
7. according to the method for claim 4, when the tree that is transformed is one section sapling, the bud site of sprouting is axillary meristem or apical meristem.
8. according to the method for claim 5, culturing step adopts vertical seedling altogether.
9. according to any one method of front claim, tree has complete root system as infected (injected) and/or when cultivating.
10. according to any one method of front claim, the substratum of screening step also comprises microbiotic to kill agrobacterium strains.
11., can be applicable to the tree of the seeds of the tree of any or multiple eucalyptus genus, Populus, Malus and Prunus and any infection sensitivity to agrobacterium tumefaciens or Agrobacterium rhizogenes according to any one method of front claim.
12. according to any one method of front claim, wherein said tree is blue gum or alpine ash.
13. the tree that transforms according to any one the described method in the claim 1 to 12.
14. the cell of the carrying genes that transforms according to any one the described method in the claim 1 to 12.
15. the propagulum of the tree that transforms according to any one the described method in the claim 1 to 12.
16. the seedling of the tree that transforms according to any one the described method in the claim 1 to 12.
17. the seed of the tree that transforms according to any one the described method in the claim 1 to 12.
18. the germplasm of the tree that transforms according to any one the described method in the claim 1 to 12.
19. serve as with reference to setting the method for conversion mainly with embodiment described above.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9600698.6 | 1996-01-13 | ||
| GBGB9600698.6A GB9600698D0 (en) | 1996-01-13 | 1996-01-13 | Genetic transformation of trees |
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| Publication Number | Publication Date |
|---|---|
| CN1213404A true CN1213404A (en) | 1999-04-07 |
Family
ID=10786998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97192880A Pending CN1213404A (en) | 1996-01-13 | 1997-01-09 | Genetic transformation of trees |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0880592A2 (en) |
| JP (1) | JP2000517162A (en) |
| CN (1) | CN1213404A (en) |
| AP (1) | AP9801292A0 (en) |
| AR (1) | AR005443A1 (en) |
| AU (1) | AU720610B2 (en) |
| BR (1) | BR9706942A (en) |
| GB (1) | GB9600698D0 (en) |
| NZ (1) | NZ325384A (en) |
| OA (1) | OA10709A (en) |
| UY (1) | UY24435A1 (en) |
| WO (1) | WO1997025434A1 (en) |
| ZA (1) | ZA97192B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1094037C (en) * | 1999-11-23 | 2002-11-13 | 雷州市科技服务中心 | Ultra-low temperature breeding method |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9818808D0 (en) * | 1998-08-29 | 1998-10-21 | Cambridge Advanced Tech | Modification of plant fibres |
| JP4099898B2 (en) | 1999-05-07 | 2008-06-11 | 王子製紙株式会社 | Methods for transforming adult Eucalyptus plants |
| IT1317038B1 (en) * | 2000-06-05 | 2003-05-26 | Vitroplant Vivai Di Zuccherell | METHOD FOR THE REGENERATION OF PLANTS AND ITS USES FOR THE MULTIPLICATION AND / OR TRANSFORMATION OF PLANTS. |
| JP3985474B2 (en) | 2001-07-24 | 2007-10-03 | 日本製紙株式会社 | Gene transfer method to Eucalyptus plants with improved gene transfer efficiency |
| JP4170078B2 (en) * | 2002-11-25 | 2008-10-22 | クミアイ化学工業株式会社 | Implant plant transformation method of kenaf plant by Agrobacterium tumefaciens |
| EP3265569B1 (en) | 2015-03-02 | 2021-07-28 | Stora Enso Oyj | Generation of biomass |
| CN112626105B (en) * | 2020-12-13 | 2022-07-26 | 山东农业大学 | Method and device for obtaining transgenic apple tissue culture seedlings by apple leaf infection |
| CN115896160B (en) * | 2022-10-28 | 2023-07-04 | 西北农林科技大学 | Method for efficiently and rapidly obtaining apple stable transgenic plants by using agrobacterium rhizogenes |
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|---|---|---|---|---|
| JPH0611209B2 (en) * | 1985-09-04 | 1994-02-16 | 王子製紙株式会社 | Mass growth method for woody plants |
| US5177010A (en) * | 1986-06-30 | 1993-01-05 | University Of Toledo | Process for transforming corn and the products thereof |
| GB2211204B (en) * | 1987-10-20 | 1992-05-20 | Oji Paper Co | Process for production of plant transformant |
| EP0652965A1 (en) * | 1992-07-27 | 1995-05-17 | Pioneer Hi-Bred International, Inc. | An improved method of agrobacterium-mediated transformation of cultured soybean cells |
| FR2709496B1 (en) * | 1993-08-30 | 1995-11-03 | Limagrain Holding Groupe | Process for the production of transgenic plants, entirely transformed into generation T0, from meristems. |
| JP4075081B2 (en) * | 1994-01-25 | 2008-04-16 | 王子製紙株式会社 | Method for producing transformed Eucalyptus plants |
| JP3565284B2 (en) * | 1994-09-22 | 2004-09-15 | 王子製紙株式会社 | Method for producing transformed Eucalyptus plant |
| GB2298205A (en) * | 1995-02-17 | 1996-08-28 | Shell Int Research | Genetic transformation of eucalyptus |
-
1996
- 1996-01-13 GB GBGB9600698.6A patent/GB9600698D0/en active Pending
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1997
- 1997-01-09 AP APAP/P/1998/001292A patent/AP9801292A0/en unknown
- 1997-01-09 AU AU13883/97A patent/AU720610B2/en not_active Ceased
- 1997-01-09 CN CN97192880A patent/CN1213404A/en active Pending
- 1997-01-09 ZA ZA97192A patent/ZA97192B/en unknown
- 1997-01-09 EP EP97900294A patent/EP0880592A2/en not_active Withdrawn
- 1997-01-09 BR BR9706942A patent/BR9706942A/en unknown
- 1997-01-09 WO PCT/GB1997/000042 patent/WO1997025434A1/en not_active Application Discontinuation
- 1997-01-09 JP JP09524975A patent/JP2000517162A/en active Pending
- 1997-01-09 NZ NZ325384A patent/NZ325384A/en unknown
- 1997-01-10 UY UY24435A patent/UY24435A1/en not_active Application Discontinuation
- 1997-01-10 AR ARP970100116A patent/AR005443A1/en not_active Application Discontinuation
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1998
- 1998-07-03 OA OA9800107A patent/OA10709A/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1094037C (en) * | 1999-11-23 | 2002-11-13 | 雷州市科技服务中心 | Ultra-low temperature breeding method |
Also Published As
| Publication number | Publication date |
|---|---|
| OA10709A (en) | 2001-05-09 |
| UY24435A1 (en) | 1997-07-01 |
| AU1388397A (en) | 1997-08-01 |
| AU720610B2 (en) | 2000-06-08 |
| JP2000517162A (en) | 2000-12-26 |
| GB9600698D0 (en) | 1996-03-13 |
| NZ325384A (en) | 2000-01-28 |
| ZA97192B (en) | 1997-07-23 |
| BR9706942A (en) | 1999-04-06 |
| EP0880592A2 (en) | 1998-12-02 |
| WO1997025434A1 (en) | 1997-07-17 |
| AR005443A1 (en) | 1999-06-23 |
| AP9801292A0 (en) | 1998-09-30 |
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