CN86107575A - Be used to improve modification method and the substratum that somatic embryo takes place - Google Patents
Be used to improve modification method and the substratum that somatic embryo takes place Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Abstract
Provide and improved embryo's quantity and method for quality and the substratum that obtains by inducing plant somatic tissue.Be added with a certain amount of amino acid through selecting in the substratum, its quantity is enough to improve the number of the somatic embryo that is produced.The present invention also provides the substratum that comprises ammonium ion source and the method for culturing plants somatic tissue.
Description
The present invention is the further part of submit May 19 nineteen eighty-three No. 496186 applications.
In general, the present invention relates to the cultivation of embryoid cell and tissue, more particularly, the present invention relates to be particularly suitable for to keep by the embryo's of in-vitro inducing somatic tissue generation improved culture medium and the method for using this substratum.
People know that the latest developments of genetic engineering make the plant breeder can avoid the consuming time oversize problem in the inherent crop improvement in the traditional breeding technology.Yet, because unicellular and multicellular organism need go to change whole genetic information with diverse ways, so, use these technology and still have any problem.For microorganism, a kind of trial is exactly to change in the cell levels influence of getting on, and surely makes it amplification by the successive passage.
For multicellular organism, as plant, it is comparatively favourable carrying out genetic manipulation on cell levels, makes it then to regenerate and cultivate into the maturation plant of expression new features.Can insert so that special change to be provided by plasmid, or merge to carry out large quantities of genetic manipulations, foreign heredity substance is mixed in the host cell by protoplastis.Use traditional plant breeding technology, make further genetic manipulation with maturation plant and can mix new characteristic to kind with agriculture meaning.〔Allard，R.W.，Principles of Plant Breeding，John Wiley and Sons，New York，1960;Simmons，N.W.，Principles of Crop Lmprovement，Langman Group.Ltd.London，1972〕。
Isolated culture vegetable cell and tissue must remain on culture in the substratum, nutrition is provided and is kept viability by substratum.The example of tissue culture medium (TCM) commonly used had literature survey and (saw Huang, L. and T.Murashige, " Plant Tissue Culture Media; Major Constituents, their Preparation, and Some applications "; Tissue Culture Association Manual 3: 539～548; Tissue Culture Association; Rockville; M.D., 1977 and de Fossard, R.A.; Tissue Culture for Plant Propagators, University of New England Printery, Armidale, N.S.W., Australia, 1976).This culture is kept performance and can be obtained promoting in plant organ, tissue or cell culture.
In somatic embryo is cultivated, generally be that the inducing plant somatocyte produces cell fission repeatedly on nutritional medium, produce the amorphous cell agglomerate, promptly so-called callus.This callus can be through subculture to reach a large amount of propagation.But also evoked callus differentiation is to produce the differentiated tissues and the organ of maturation plant.Also can be in culture by other existing embryo's organizer cell stage.Parental generation embryo comprises that the immature spherical stage (sees Lupotto to sophisticated, germination embryo, E, " Propagation of an embryonic Culture of Medicago Sativa L. ", Zeit, Pflanzenphysiol.111:95-104,1983).Therefore, somatic embryo can be produced by existing embryo in undifferentiated callus or the plant tissue cultures.
In this way, can on cell or embryo's level, influence hereditary coup, then by continue after growth keep it, have the whole crop of same hereditary feature with generation.This just makes the plant breeder can walk around the normal hereditary barrier in the plant propagation, and obtains more homogeneous and favourable field-crop.
Use current technology, the method that might utilize somatic embryo to take place produces thousands of strain plants by a gram cell.〔Evans，D.A.，W.R.Sharp，and C.E.Flick，“Growth and behavior of cell Cultures：embryo genesis and Organogenesis”，in Plant Tissue Culture-1981，T.A.Thorpe ed.，Academic Press，45-113，1981〕。These embryos can germinate and transfer in greenhouse or the farmland, and develop into maturation plant.The plant breeder can use these plants to reclaim useful heritable variation, or breeds kind for the plant breeding program by cloning.
Some mensuration partly cultivates the quality of gained embryonic tissue by plant soma and the technology of quantity is known.Can be by the productive rate of mensuration with embryo's relevant structure of etap, come the quantity of detection bodies cell stage (to see Fujimura, T., and A.Komamine, " Synchronization of Somatic embryogenesis in a Carrot Cell Suspension Culture ", Plant Physiology, 64:162-164,1979; Verma, D.C. and D.K.Dougall, " Influence of Carbohydrates on quantitative aspects of Growth and embryo Formation in Wild Carrot Suspension Cultures ", Plant Physiology, 59:81-85,1977).This mensuration generally is to use dissecting microscope that structure is counted and finishes.
Available several different methods is come the quality of estimated body cell stage.Be typically with visual observations globular stage, torpedo stage and plantlet phase and determine fetal development.〔Ammirato，P.V.“The effects of abscisic acid on the development of Somatic embryos from Cells ofCaraway（Carum Carri L.）”，Botanical Gazette，135：328-337，1974〕。Also can determine fetal development or quality according to the productive rate of the plantlet that derives from single somatic embryo.〔Drew，R.L.W.，“The development of Carrot（Daucus Carota L.）embryoids（derived from Cell Suspension Culture）into Plantlets on a Sugar-free basal medium”，Horticultural Research 19：79，1979〕。Although very for important, people are difficult to detect the formation of plantlet to the mensuration that plantlet forms for the embryo's who determines to be suitable for to use in the farmland productive rate.
The technology of improving embryo's quantity or productive rate had description.For example, the formation of known somatic embryo need have a kind of ammonium source in substratum, so just can have the embryo to take place.(Halperin, W. and D.F.Wetherell, " Ammonium requirement for embryogenesis in vitro ", Nature 205: 519-520,1965; Walker, K.A., S.J.Sato, " Morphogenesis in Callus tissue of Medicago Sativa:the role of ammonium ion in Somatic embryogenesis ", Plant Cell Tissue Organ Culture 1:109-121,1981).Because most of plant cell culture mediums all contain a certain amount of ammonium, so think and ammonium concentration is adjusted to one the proper level of high embryo's productive rate and quality is arranged is very important.(see the above-mentioned document of Walker and Sato, and Wetherell, D.F. and D.K.Dougall, " Sources of nitrogen Supporting growth and embryogenesis inCultured Wild Carrot tissue ", Physiologia Plantarum 37:97-103,1976).
In plant cell cultures and sophisticated plant, ammonium and glutamine or L-glutamic acid can promptly be transformed mutually by cell.(see Ojima, K and K.Ohira, " Nutritional requirements of callus and Cell Suspension Cultures ", from Frontiers of Plant Tissue Culture-1978, T.A.Thorpe ed., University of Calgary Offset Printing Services, 265-275,1978; Miflin, B.J. and P.L.Lea, " Ammonium assimilation ", from The Biochemistry of Plants, P.Stumpf and E.Conned., Academic Press, Vol.6,169-201,1980, Dougall, D.K., " Current Problems in the regulation of nitrogen metabolism in Plant Cell Cultures " is from Plant Tissue Culture and its Biotechnological Application, W.Barz, E.Reinhard and M.H.Zenk eds., Springer-Verlag, Berlin, 76-81,1977).
Contemplated ammonium approaching with glutamine and L-glutamic acid in plant metabolism is to reflect from the systematic study scheme of amino acid to the influence of somatic embryo generation.Research is to use the carrot cell of having removed ammonium from plant cell culture medium to carry out, and the embryogenetic influence of test sheet monoamino-acid pair cell.(see the aforementioned article of Wetherell and Dougall; Kamada, H. and H.Harada, " Studies on the Organogenesis in Carrot tissue Cultures II .Effects of amino acids and inorganic nitrogenous Compounds on Somatic embryogenesis ", Zeit.Pflanzenphysiologie 91:453-463,1979).Wetherell and Dougall find, and adds ammonium ion relatively, adds amino acid and can not improve the embryo and take place.On the other hand, Kamada and Harada then find, do not have ammonium or do not have ammonium and the situation of nitrate under, L-Ala and glutamine play the effect that the reduction nitrogenous source is provided for the embryo well.A report thinks, the amino acid concentration of adding is 20mM or is disadvantageous when higher.(see Reinert, J. and M.Tazawa, " Wirkung von Stickstoffverbindungen und von Auxin auf die Embryogenese in Gewebekulturen ", Planta, 87:239,1969) (original text is a German).
Above-mentioned studies show that is at the reduction nitrogenous source, as existing a kind of relation of equivalence between ammonium and the amino acid.(Wetherell and Dougall, above-mentioned article; Kamara and Harada, above-mentioned article).TaZaWa, M, and Reinert, J. research has further shown the metabolism relation of equivalence (Tazawa between ammonium and amino acid, M. and Reinert, J., " Extracellular and intracellular Chemical environments in relation to embryogenesis in vitro ", Protoplasma 69:157-173,1969).In a research of carrying out intrinsic ammonium level in the Radix Dauci Sativae culture that somatic embryo takes place, find no matter whether add ammonium, amino acid or nitrate in the culture, always the level of ammonium is mutually related with embryogenetic amount in the culture.Conclusion is NH
+ 4The key factor of intrinsic level for stimulating somatic embryo to take place.NH
+ 4Inherent level derive from the NH that the external world provides
+ 4Or amino acid, or the NH that obtains through biology reduction nitrate
+ 4Intrinsic NH
+ 4Can be converted into organic nitrogen compound, so that the needed amino acid of normal cell (seeing the above-mentioned document of Tazawa and Reinert) to be provided.Therefore be sure of that amino acid works by discharging ammonium, is used to stimulate the embryo to take place.
The factor of the improved fetal development of knowing has dormin, zeatin, Plant hormones regulators,gibberellins, high concentration sucrose and light.(see Ammirato, P.V. and F.C.Steward, " Some effects of environment on the development of embryos from Cultured free Cells ", Botanical Gazette, 132:149-158,1971; Ammirato, P.V., " Hormonal Control of Somatic embryo development from Cultured Cells of Caraway; Interactions of abscisicacid; Zeatin and gibberellic acid ", Plant Physiology 59:579-586,1977).Ammonium and amino acid it be unclear that the influence of embryo quality.〔Ammirato，P.V.，“The regulation of Somatic embryo development in Plant Cell Cultures：Suspension Culture techniques and honmone requirements”，Bio/Technology 1：68-74，1983〕。
In addition, transformation frequency-, be not used for systematically improving fetal development as the Shang Buming Liao of a kind of calibrating of embryo quality-up to now.By observing the mature condition that embryo morphology is examined and determine the embryo, but this method can not detect the frequency that single embryo forms plant.
For this reason, an object of the present invention is to provide method and the material of increase by the quality and quantity of the somatic embryo of plant tissue generation.
Another object of the present invention is to provide optimum reduction nitrogenous source for somatic embryo.
Another object of the present invention provides method and the material that a large amount of propagation various plants take place by somatic embryo.
But a further object of the present invention provides method and the material that produces a large amount of around body cell stages with same heredity and phenotypic characteristic.
Other purpose of the present invention, advantage and feature will be found out from following description and corresponding embodiment.
The invention provides amino acid and reduction nitrogenous source, produce new and the method and the material improvement of a large amount of high-quality somatocyte embryo tires by plant tissue by adding optimal dose.One aspect of the present invention provides a kind of plant cell culture medium, it comprises a kind of matrix with ammonium ion source, also be added with at least a amino acid that is selected from proline(Pro), L-Ala, arginine, glutamine, aspartic acid, Serine, ornithine, L-glutamic acid and acid amides thereof, alkyl ester and dipeptidase derivant thereof, add the embryo who produces when not adding above-mentioned material in quantity and the substratum and compare, enough increase the number and the quality of the somatic embryo that is produced.
Another aspect of the present invention provides a kind of plant cell culture medium that does not have ammonium ion basically, but comprising at least a amino acid that is selected from proline(Pro), arginine, aspartic acid, ornithine, Methionin and acid amides thereof, alkyl ester and dipeptidase derivant thereof that adds with sufficient amount, compare with the situation that does not add above-mentioned material in the substratum, can significantly improve the quality and quantity of the somatic embryo that produces.
In addition, the present invention also provides the method for using this substratum.
Individual accompanying drawing be graphic representation as the function that in substratum, adds amino acid concentration, the increase of the somatic embryo number that is produced.
The invention provides the method for the quality and quantity that increases the embryo who is produced by the plant tissue, these methods provide a kind of culture medium of cultivating described cell and tissue, and it contains the amino acid through selecting of sufficient amount, take place to stimulate somatic embryo.
The present invention also by provide a kind of contain sufficient amount through selecting amino acid and be added with this class cell of cultivation of ammonium ion source and the culture medium of tissue, with the raising of the quality and quantity that stimulates somatic embryo. A kind of method of using this plant tissue culture media is provided simultaneously.
Although the front has been be sure of amino acid and can be used as the simple equivalent of needed ammonium medium component, when making us finding uncannily that amino acid uses with the same concentrations of ammonium, can be used as the substitute of the ammonium ion that improves the somatic embryo generation. Find uncannily that also the amino acid through selecting can provide the benefit of remarkable increase together with the ammonium ion source that adds, and this point can not be simply from the interpolation effect forecast that increases ammonium concentration to.
Found a kind of culture medium that is selected from proline, arginine, lysine, aspartic acid, ornithine and these amino acid whose acid amides, Arrcostab and these amino acid whose dipeptidase derivants that contains, this culture medium does not have ammonium ion basically, can be used for improving the quality and quantity of the somatic embryo that obtains from somatic tissue's culture.
Also find a kind of culture medium that contains ammonium ion and be selected from proline, alanine, arginine, glutamine, lysine, aspartic acid, serine, ornithine, glutamic acid and these amino acid whose acid amides, Arrcostab and these amino acid whose dipeptidase derivants, add material quantity be enough to stimulate the embryo to be taken place or cell transformation, this culture medium can produce similar embryo's enhancement effect.
Made us finding uncannily that culture medium of the present invention can increase the productive rate of the somatic embryo that obtains from callus, be better than being used for before this inducing, regenerate and keep the typical culture of embryonic tissue. In each stage of these embryo's generating processes, use culture medium of the present invention to obtain much bigger embryonic tissue productive rate than the culture medium that provides before using. In addition, in various useful plant kinds-comprise in the application practice of clover, celery, cotton, corn and paddy rice, prove that culture medium of the present invention has many-sided advantage.
The plant cell culture medium that can be improved by practice of the present invention comprises such as Huang the previously known plant tissue that L. and T.Murashige commented Culture medium (aforementioned documents and Cloning agriculfural Plants Viain Vitro techniques, B.V.Conger ed., CRC Press, 172, its disclosed content and prescription are all classified this paper list of references as). In general, plant culture provides plant nutrient, the energy (such as sugar), plant hormone and is used for the buffering salt of control solution ph and osmotic equilibrium.
Representational in this class plant cell culture medium is so-called Schenk and Hildebrandt(SH) culture medium (sees Schenk, R.U. and A.C.Hildebrandt, " Medium and techniques for induction and growth of monocotyledonous and dicotyledonous Plant Cell Cultures ", Can.J.Bot.50: 199,1972; Its disclosed content and the equal Lie Wenbenwen list of references of prescription). To be that this article is disclosed comprise main salt, vitamin and sucrose to no hormone SH culture medium hereinafter described, but do not have 2,4-D, the culture medium of pCPA and kinetin.
Other has so-called Murashige and Skoog(MS) substratum can (see Murashige in order to replace the SH substratum, T. and F.Skoog, " Arevised medium for rapid growth and bioassays With Tobacco tissue Culture ", Physiologia Planta, 15:473-497,1962; Disclosed content and prescription are all classified this paper reference as in the literary composition).To be that this article is disclosed comprise main salt, VITAMIN and sucrose to no hormone MS substratum hereinafter described, but do not contain the substratum of indole-3-acetic acid and kinetin.
In practice of the present invention, selection to used basic plant cell culture medium, part is to determine De ， And to think that this is that an experienced those of ordinary skill is known in the tissue culture of vegetable cell and body embryo put into practice by the kind of selected plant soma tissue.
A large amount of important crops and garden-variety all show and can make it propagation by tissue culture and somatic embryo.These kinds include but are not limited to following:
Table 1
Vegetable crop fruit and nutwood
The clover Prunus amygdalus
The asparagus apple
The beet banana
Soup dish coffee
The Radix Dauci Sativae nipa palm
The Cauliflower grapefruit
The eggplant lemon
The onion olive
The spinach orange
The sweet potato peach
The tomato bulb
Fruit and berry lily
The blackberry, blueberry Radix hemerocalis plicatae
The grape easter lily
The pineapple jacinthe
Strawberry
The trophophyll African violet
Silver Vase Anthurium
The Flower of Evans Begonia Chrysanthemum
The Crytanthus African daisy belongs to
The Dieffenbachia gloxinia belongs to
The dracaena Petunia
The eiddleleaf rose
The Pointsettia orchid
Weeping fig medicinal plant
The insane Solanum of rubber producting plant
The pteridophyte genseng
Australia's tree-fern Pyrethrum
Boston fern afforestation plant (forestry)
The maidenhair Pseudotsuga
Rabbitsfoot fern pine tree
The lycopod quaking aspen
Knife-edge fern Chinese larch
The cereal rubber tree
Barley
Corn
Millet
Pennisetum
Wheat
The more detailed plant variety that can carry out the somatic embryo generation can be referring to Evans, D.A. wait the people: " Growth and Behavior of Cell Cultures:Embryogenesis and Organogenesis ", from Plant Tissue Culture:Methods and
Applications in Agriculture, Thorpe compiles, and Academic Press publishes, 45 pages (1981); Its relevant portion is classified this paper reference as.
Many amino acid are exactly known in the prior art, all have a common feature in them except indivedual, a free carboxy is promptly arranged and a unsubstituted amino is arranged on alpha-carbon atom.Proline(Pro) is a very significant exception, because the alpha-amino group of proline(Pro) is substituted, so it is actually alpha-imino acid.
Amino acid generally can be divided into protein and nonprotein amino acid, and wherein gal4 amino acid comprises 20 kinds of modal amino acid.These amino acid comprise four subgroups again: have nonpolarly or hydrophobic substituent, comprise L-Ala, leucine, Isoleucine, Xie Ansuan, proline(Pro), phenylalanine, tryptophane and methionine(Met); Amino acid with uncharged polar R gene comprises Serine, Threonine, tyrosine, phenylalanine, glutamine, halfcystine and may also have glycine; Amino acid with electronegative R gene comprises aspartic acid and L-glutamic acid; With and the amino acid of positively charged R gene is arranged, comprise arginine, Methionin and may also have Histidine.
Except 20 kinds of common amino acids, also have many amino acid that other seldom occurs or do not have fully in protein.The rarely found ， And of hydroxylysine and oxyproline exists only in the scleroproein.In addition, known have other amino acid more than 150 kinds to be present in different cells and the tissue with free or bonded form, comprising modal citrulline and ornithine, and the common amino acid of many β, γ and △ form.
Except primary amino acid structure discussed above, can also multiple mode modified amino acid and do not change the function that it is had in the present invention.These modifying method comprise and add amino and carboxyl respectively, form amino acid amide and aminoacid alkyl ester.In addition, can connect two amino acid by α-carboxyl and alpha-amino group and form amino acid whose dipeptidase derivant.Be readily appreciated that each all has two kinds of possible dipeptidase derivants to amino acid.
Also it is important for the present invention, be containing of the present invention of amino acid whose culture medium supplemented ammonium ion (NH
+ 4) source.Ammonium ion source also is known in the plant tissue culture field.Such ammonium ion normally provides by the non-toxicity ammonium salt that adds some amount in substratum, it with the negatively charged ion formation of balance ammonium ion electric charge as ammonium chloride, ammonium phosphate or ammonium sulfate etc.Walker, K.A. and S.J.Sato have introduced some other ammonium ion source in " Plant Cell Tissue Organ Culture " (1:109-121,1981) literary composition (this article relative section is listed in this paper reference).
Provide following enforcement to be intended to further illustrate all respects of the present invention.The a little Shi Shi of Zhe Li And is not the claim that limiting the scope of the invention ， Ji And does not limit the application's issued for approval.
In general, the method that takes place with vegetable cell and tissue culture deposits yields somatic embryo is known, need only change a little promptly applicable to selected plant variety.(for example see Plant Tissue Culture:Methods and Applications in Agriculture, Thorpe compiles, (1981); This article relevant portion is classified this paper reference as).
Clover for example, in the Regen of Saunclers and Bingham S strain, can take place (to see " Production of Altalfa Plants from Callus Tissue " by the ordinary method inducing embryo, Crop Sci., 12:804-808,1972).
Utilize the Cultivar Regens of alfalfa (Medicago Sativa), it is to obtain second cycle from Vernal and Saranac mixing breed repeating to select.The generation of callus is carried out as follows; Use 50%Clorox
RTo petiole surface sterilization 5 minutes, Yong Shui Xi Di And coated on the SH culture plate of no hormone, and this substratum contains Schenk-Hildebrandt substratum (Schenk, R.U. and A.C.Hildebrandt, document is the same, (1972)) contained salt, VITAMIN and sucrose.This substratum contains 25 μ M α-naphthylidene acetate, 10 μ M kinetin and 0.8%(W/V) agar (be called and keep substratum).Isolate the callus ， And that forms in the above cultivates repeatedly keeping on the substratum from the plant in vitro tissue of callusization not yet.Callus is cultivated ， And with 3 weekly intervals and is made it in 27 ℃ of growths under indirect light shines.
In cultivating the back again 17-24 days, collect 3-9 gram callus ， And from the flat board of keeping substratum and be transferred to and contain 50 μ M2, the A-dichlorophenoxyacetic acid (2,4-D) and in the 100ml SH solution of 5 μ M kinetins (B) induce.(seeing Walker, K.A.M.L.wendeln, and E.G.Jaworski, plant sci Lett.16:23-30(1979).Place 500ml to shake in the bottle callus and cultivated 3 days down in 27 ℃, cultivation is under indirect light shines, and carries out on the track electromagnetic shaker of 100K.P.M..
Under slight vacuum, the derivative cell of screening under series of columns sieve (Fisher Scientific company) and aseptic condition.Cell lump sinks by stainless steel sift or (480 μ m) And are collected on 60 orders (the 230 μ m) screen cloth by being pushed through 35 orders.Do not have the hormone nutrient solution with SH and wash the cell of staying on 60 eye mesh screens, every 100ml inducing culture volume washs with 500ml.Vacuum is removed the washing nutrient solution.The Shi Chong And of weighing cell lump with cell suspension in the SH of no hormone substratum, the cell of the every milliliter of 150 milligrams of weight in wet bases that suspend.With 75mg(0.5ml) suspension cell be drawn on about 10ml agar solid medium in 60mm * 15mm culture dish.
In addition, as with 300mg(2ml) the resuspending cell moves into the 8ml that is contained in the 50ml Erlenmeyer flask does not have in the SH liquid nutrient medium of hormone, and somatic embryo in suspension culture, will occur and take place.Embryo's generation substratum contains SH substratum (NH
+ 4Equal 2.6mM), also contain 3%(W/V) sucrose, there is not hormone.No ammonium ion substratum is the NaH with equivalent
2PO
4Replace the NH among the SH
4H
2PO
+ 4Make.25mM NH
+ 4Control medium is by the additional 12.5mM(NH of no ammonium substratum
4)
2SO
4Form.By the organic and inorganic reduction nitrogenous source degerming of 0.2 μ m filter membrane, add to then in the substratum that autoclaving is crossed recently all.
Handle generally for every kind and cultivate with 10 repetition flat boards.The garden dish is incubated 21 days with protective membrane Bao Zhuan And.The suspension bottle clogs and uses Saran Wrap with plastic foam
Seal, insulation is 14 days on the track electromagnetic shaker of 100rpm.Be incubated under 27 ℃ of illumination in 12 hours and carry out, light source is cold white fluorescent tube bulb, and solid culture and light source distance are 28Cm, and suspension culture object distance light source is 200Cm.
After the insulation, the green center with on the stereoscopic microscope counting callus of 10 times of amplifications takes place to detect the embryo.Measure embryo's size with the demarcation range estimation chi that amplifies 10 times.With visual observations to determine embryony.At the 21st day of initial incubation the embryo is handled substratum by amino acid and under aseptic condition, moves into and be added with 25 μ M gibberic acid and 0.25 μ m α-naphthylidene acetate does not also have in the SH substratum of hormone with 0.8% agar solidified half intensity, so that the embryo is converted into the complete plant of root and rudiment axle (the first elementary leaf).
1. the generation of the somatic embryo in containing the ammonium substratum
A. fabaceous somatic cell culture.
Cultivate the representative plant-clover tissue of pulse family by the method for summarizing above, (alfalfa, RegenS kind) ， And is check somatocyte generation in the substratum that contains the 2.6mM ammonium according to the present invention.The amino acid whose experimental concentration of all proteins is 1-100mM.From then on two kinds of reaction types occur in the initial screening process,, further test with the cell of screening based on these results.Table 2 is found these amino acid and SH-control medium (2.6mMNH for not comprising the amino acid of first reaction type
+ 4) relatively take place growing toxic and suppressing the embryo.These amino comprise sulphur and aromatic nucleus De ， And and are branched chain family mostly.These amino acid and SH control medium relatively all do not stimulate the embryo that ， And takes place and all are deleterious, and their concentration is 1 or all suppresses during 10mM to grow or cause brownization of callus.
Compare with SH, second response type of initial screening can stimulate the embryo to take place or cause the embryo to increase (referring to table 2).These amino acid have been done detailed concentration dependent research, the results are shown among Fig. 1.Stimulate in the amino acid that somatic embryo forms proline(Pro) the most effectively, compare 2.6mMNH
+ 4Contrast produces many 3 times embryo approximately, is 25mMNH and render a service
+ 4The twice of suitable ammonium concentration in the-clover (D).(seeing people's such as Walker above-mentioned document).The effectiveness of L-Ala, arginine, glutamine and Methionin is less, but they stimulate the embryo to form near 25mMNH
+ 4Level.Serine and l-asparagine and SH are compared, and it stimulates embryogenetic effect relatively poor, but can increase embryo's volume.
Table 2 has been summarized amino acid and other nitrogenous source of finding the somatic embryo of clover fetal hair is had hormesis.Ester and acid amides that it should be noted that proline(Pro) form, and as dipeptides prolyl L-Ala, have very high activity at stimulation embryo's number with aspect improving the quality.What is interesting is that discovery non-protein amino acid-ornithine has activity.
Table 2
The influence that the reduction nitrogenous source is given birth to the somatic embryo of clover fetal hair
Stimulus
Ammonium
Proline(Pro)
L-Ala
Glutamine
Arginine
L-asparagine
Ornithine
Serine
Methionin
The L-prolineamide
L-prolyl-L-L-Ala
L-proline(Pro) methyl ester
Use above-mentioned technology, use visual observations embryo size to detect embryo quality.Data are listed in the table 3.
Table 3 reduction nitrogen is handled the influence to the somatic embryo size
The treated length width
Contrast (25mMNH
+ 4) 0.805 ± 0.084 0.383 ± 0.019
100mML-proline(Pro) 1.143 ± 0.081 0.867 ± 0.046
30mML-L-Ala 1.163 ± 0.090 0.744 ± 0.037
100mML-L-Ala 1.192 ± 0.102 0.833 ± 0.049
30mML-arginine 1.521 ± 0.142 0.663 ± 0.048
30mML-glutamine 1.342 ± 0.122 0.773 ± 0.051
3mML-Methionin 1.163 ± 0.096 0.652 ± 0.039
3mML-l-asparagine 0.699 ± 0.062 0.446 ± 0.027
10mML-l-asparagine 1.239 ± 0.102 0.610 ± 0.034
According to the data that provide in table 2 and the table 3, the effectiveness of aminoacid addition thing in increasing embryo's volume can be arranged in the following order:
Arginine 〉=glutamine＞L-Ala＞proline(Pro)＞NH
+ 4
Use above-mentioned technology, observed the conversion of embryo to the whole plant that has root and rudiment axle (the first elementary leaf), and the results list is as follows:
Table 4 somatic embryo is to the conversion of clover plantlet
Initial treatment has the plant percentage ratio of the first elementary leaf
25mMNH
+ 433.3%+4.2
100mML-proline(Pro) 54.0%+6.4
50mML-L-Ala 63.5%+4.4
30mML-arginine 59.0%+6.2
30mML-glutamine 67.0%+3.4
According to the data of table 4, the effectiveness that influences the additive that the embryo transforms to plantlet is in proper order:
Glutamine＞L-Ala 〉=arginine＞proline(Pro)＞NH
+ 4
Mutual relationship between these data shows that embryo's volume is the good sign that the embryo transforms to plantlet, therefore also is a good sign of the embryo quality of above-mentioned technology generation.
Having measured stimulates the suitableeest amino acid whose add-on that somatic embryo takes place in agar solid culture and the fluid suspension culture thing.These data under tabulate 5 and table 6 in provide:
Table 5 is to having 2.6mMNH
+ 4The agar solid culture add the hormesis that amino acid is given birth to the somatic embryo of clover fetal hair
Source % maximal stimulation concentration range optimal concentration
（mM） （mM）
Contrast (2.6mMNH
+ 4) 100--
L-proline 3 30 10-300 (100)
L-L-Ala 314 20-150 (75-100)
L-glutaminate 175 20-50 (30-40)
L-arginase 12 44 5-50 (30-40)
Altheine 155 0.5-3 (1)
L-ornithine 156 1-3 (1-3)
L-Serine 160 0.5-2 (1)
L-Methionin 233 1-10 (3)
L-prolineamide 240 30-200 (50-100)
L-proline(Pro) methyl ester 241 5-25 (10)
L-prolyl-L-L-Ala 210 30-200 (50-100)
Table 6 is to having 2.6mMNH
+ 4Add the hormesis that amino acid takes place somatic embryo in the fluid suspension culture thing
Source % maximal stimulation concentration range (mM) optimal concentration (mM)
Contrast (2.6mMNH
+ 4) 100
L-proline(Pro) 528 100-300 (100)
L-L-Ala 240 25-200 (50)
L-glutaminate 243 5-75 (50)
L-arginine 168 5-75 (50)
L-Methionin 180 1-10 (3)
B. the somatic cell culture of samphire
Make the seed germination 1-2 week of umbelliferous representative plant-celery (Apium graveolens, Calmario kind).Use 10%Clorox
Solution with gained seedling sterilization 20 minutes.Remove cotyledon or plumular axis, be placed on and contain 25 μ M2,0.8% agar solidified of 4-D and 5 μ M benzyladenines does not have on the hormone SH substratum.Behind the appearance callus (3-4 week), the callus immigration is contained 2.5 μ M2, in the SH substratum of 4-D and 0.5 μ M kinetin.With filter with the degerming of thermolability additive and add in the hot substratum.During as needs, the drawing together of available improvement smeared device and obtained the tissue of the specified quantitative that is used to inoculate and it is fills up to the homogeneous volume.Cultured calli again on the SH substratum that is added with 1 μ M picloram (Picloram) and 0.5 μ M benzyladenine subsequently.For carrying out somatic embryo production, the 0.8% agar solidified that the 75mg callus cell is moved into the additive that contains the degerming of process filter membrane does not have in the hormone SH substratum, and is cultivating under the somatic similarity condition of clover, and 24 ℃ are incubated 18 to 30 days.
Amino acid-treated embryo and NH such as proline(Pro), L-Ala and glutamine have been compared
+ 4The embryo of control treatment.Handle culture with the 50mM L-Ala and cause higher embryo's occurrence frequency being arranged, and the culture of handling than other method there are better cotyledon, root and elementary leaf development than all other treatment processs.The order of observing formed total embryo number is as follows:
20-100mM L-Ala＞50mM proline(Pro)＞25mM
Glutamine-N＞25mMNH
+ 4
Though stimulate the embryo to take place than better with glutamine with proline(Pro), the latter can cause better seedling sample embryo's growth.The culture that ammonium is handled than all other processing grow lessly and also embryo number less.When L-glutamic acid adds in the celery regeneration culture medium separately with the amount of 30mM, with 25mMNH
+ 4The material compared of handling, it can stimulate celery embryo number purpose to increase.With NH
+ 4The embryo who handles compares, and the L-Ala of above-mentioned concentration, proline(Pro), glutamine and L-glutamic acid can promote the conversion of embryo to plantlet.
C. the somatic cell culture of grass
Use substratum of the present invention, the somatic embryo that carries out representative plant-corn gramineous (Zeamays) takes place.
Be fertilized harvesting corn after ten days fringe and under aseptic condition, therefrom separate and cut immature embryo.The embryo placed be added with 3% sucrose and 5 μ M2, the N-6 mineral salt culture medium (Chu of 4-D, C.C., Wang, C.C., Sun, C.S., Hsu, C., Yin, K.C., Chu.C.Y., 1975.Establishment of an efficient medium for anther Culture of rice through Comparative experiments on the nitrogen Sources Sci.Sin.16:659-688) go up insulation 21 days.The number of the embryo group that forms on each callus when the calibrating of insulation back is with or without the L-proline(Pro).The result provides in table 7, and wherein the percentage reaction is a 287-1165 embryoplastic average frequency that repeats embryo's separate piece.
The influence that table 7 L-proline(Pro) forms corn embryonic callus
Concentration of proline (mM) % embryonic callus forms
0 15.8
6 20.6
12 20.8
24 20.2
As relating to the further example that the grass somatic embryo takes place, be according to practice propagation rice varieties dryland rice (Oryza Sativa) of the present invention.
Dryland rice (Oryza Sativa) seed is shelled, carry out surface sterilization and place being added with 4% sucrose, 0.26mM tryptophane, 5 μ M2,4-D, 1 μ M kinetin, pH6.2, and be added with Murashige and the Skoog(MS of 2.5 grams per liter Gelrite as gel former) (Murashige on the salt substratum, T. and F.Skoog, 1962, on seeing).The embryo who detects in the scutellum district of single seed with dissecting microscope after 15-21 days forms.The result that table 8 has provided usefulness and handled without the L-proline(Pro).
Table 8 L-proline(Pro) is to embryoplastic influence in the rice callus tissue culture
Concentration of proline (mM) % embryo forms
0 57.8
3 59.0
10 77.8
30 64.9
50 70.1
100 68.4
D. the somatic cell culture of Malvaceae plant
As the example of the present invention, in the substratum of foundation the method disclosed in the present preparation, bred the culture of two cotton varieties to the cultivation practice of Malvaceae plant soma tissue.
Upland cotton (Gossypium hirsutum).On Murashige that is added with 3% sucrose and 0.5 μ MNAA, 5 μ M2-isopentyl VITAMIN B4 and 0.8% agar and Skoog salt substratum, will cultivate for 4 weeks again through the initial culture of the seed of surface sterilization by upland cotton (Gossypiumhirsutum).Culture is containing 0.5 μ M2, (wherein adds or does not add proline(Pro)) on the fluid suspension culture base of 4-D and 0.2 μ M kinetin or 1 μ MNAA and 0.5 μ M kinetin and induce 10 days to form the embryo.Then cell is moved into and make it regeneration in the no hormone SH substratum that contains 3% sucrose and 10mML-glutamine.The embryo's who has ripe cotyledon formation is detected in the back all around.The result provides in table 9.
Table 9 adds proline(Pro) in suspension culture influence cotton cotyledon (Gossypium hirsutum) embryo's regenerated
Concentration of proline (mM) belt leaf embryo
0 5.5
24 12.5
Gram labor time base cotton (Gossypium Klotzschianum).On Murashige that contains 3% sucrose, 0.5 μ MNAA and 5 μ M2-isopentyl VITAMIN B4 and Skoog salt substratum, cultivate the initial culture of crossing by surface sterilization of seed again.Callus was suspended 10 days in the MS salt substratum that contains 3% sucrose and 0.2 μ M picloram, make it then be added with on the no hormone culture-medium of L-glutaminate regeneration 21 days.The result provides in table 10.
Table 10 L-glutaminate is to cotton (Gossypium Klotzschianum) the embryoplastic influence of gram labor time base
L-glutaminate concentration (mM) cotyledon embryo
5 0
10 2.0
20 3.5
2. the interaction of amino acid and ammonium ion source
By above-mentioned experimental technique inducing cell, sieve and dull and stereotyped the cultivation.Change proline(Pro), arginine and NH
+ 4Concentration, have the optimal concentration when whether influencing any additive of independent adding to determine other additive.
1. proline(Pro): the concentration of proline scope of test is 30mM to 300mM, and this moment, institute added NH
+ 4The quantitative change scope be 0 to 25mM.Test-results provides in table 11.
2. arginine: be similar experiment, wherein remove the NH that adds substratum
+ 4Outside the change in concentration, arginic concentration also changes.The result provides in table 11.
Amino acid and the interactional influence of ammonium ion during table 11 clover embryo is taken place (embryo's that at least seven tests are produced mean number)
Concentration of proline (mM) 30 100 300
NH
+ 4Concentration (mM)
0 326 470 133
1.0 502 747 541
2.6 753 731 825
10.0 887 811 572
25.0 744 1042 844
Arginine concentration (mM) 0 10 30 100
NH
+ 4Concentration (mM)
0 12 147 126 99
1.0 70 252 246 157
2.6 207 298 306 264
10.0 340 408 411 311
25.0 335 297 233 148
Find out from above each experiment, when adding arginine or proline(Pro) be optimal concentration, and NH
+ 4Then produce synergy during also for optimal concentration.
Repeat the part of the example that table 11 describes, tested NH
+ 4With the various different concns of L-proline(Pro) to somatic embryo quantity and the influence that transforms to plantlet.
Table 12 ammonium and proline(Pro) are to the influence of somatic embryo (SE) quality and quantity
Proline(Pro) (mM) NH
+ 4(mM) SE quantity transforms %
0 0 4±1 3±3
0 2.6 36±5 4±3
0 25 60±6 9±5
10 2.6 95±12 25±5
30 2.6 134±17 42±7
100 2.6 175±19 42±4
30 10 187±24 42±6
Find out that therefrom the substratum that is added with proline(Pro) and ammonium can improve embryo's number; Proline(Pro) can improve embryo quality when high or low concentration ammonium ion exists.
3. in the substratum that does not have ammonium ion basically, add amino acid
Induce the clover cell and carry out the flat board cultivation by the method for the foregoing description, different is to remove NH from culture medium prescription
+ 4Test the influence of the amino acid of a series of concentration to the somatic embryo generation, gained is the result be summarized in the table 13.
Table 13 is to there not being NH basically
+ 4The agar solidified culture of substratum in add the hormesis that amino acid is given birth to the somatic embryo of clover fetal hair
Add % maximal stimulation concentration range (mM) optimal concentration (mM)
Contrast (no NH
+ 4) 100--
L-proline(Pro) 525 6-300 (10-30)
L-arginine 1200 1-100 (20-50)
Altheine 750 1-100 (2-10)
L-ornithine 900 0.3-3 (1)
L-Methionin 500 1-10 (3)
Basically there is not NH
+ 4The time, as unique reduction nitrogenous source, showing has hormesis to somatic embryo with above-mentioned amino acid.In addition, except ornithine,, prove the amino acid that adds above-mentioned various concentration ranges, all can significantly improve the embryo's that produces quality by the mensuration of embryo's size, shape and maturity.
4. unite use amino acid
Following table shows is not having NH
+ 4In time, unites in the clover culture and adds amino acid whose effect.Unite and use amino acid to have synergy improving embryo number.
Table 14 embryo number
Experiment 1.50mML-proline(Pro) 80
30mML-glutamine 50
50mM proline(Pro) and 30mM glutamine 248
Experiment 2.100mML-proline(Pro) 27
100mML-L-Ala 74
100mML-proline(Pro)+50mML-L-Ala 215
30mML-arginine 135
100mML proline(Pro)+30mM arginase 12 15
In the combination treatment of carrying out, maximum and top-quality embryo have been observed with proline(Pro) and other amino acid.
Though for the purpose of clear understanding, by chart and embodiment detailed description has to a certain degree been made in aforementioned invention, in the claim scope of issued for approval, carry out some change and modification, be conspicuous to the one of ordinary skilled in the art.
Claims (31)
1, a kind of plant cell culture medium that is used to induce, regenerate and keeps the plant somatocyte embryo tissue, it is characterized in that it comprises a kind of substratum that contains ammonium ion source, also be added with at least a amino acid that is selected from L-proline(Pro), L-L-Ala, L-arginine, L-glutaminate, altheine, L-Serine, L-ornithine, L-L-glutamic acid and acid amides, alkyl ester and its dipeptidase derivant in the substratum, compare with the embryo who does not add amino acid whose substratum and produced, the amount that is added is enough to increase
The quality and quantity that adds the somatic embryo that produces.
2, according to each substratum in claim 1 or 28, wherein be added with L-proline(Pro), its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-proline(Pro), make its ultimate density in substratum be about 6-300mM.
3, according to each substratum in claim 1 or 28, wherein add L-L-Ala, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-L-Ala, make its ultimate density in substratum be about 10-200mM.
4, according to each substratum in claim 1 or 28, wherein add L-arginine, its acid amides, alkyl ester or contain the arginic dipeptidase derivant of L-with sufficient amount at least, make its ultimate density in substratum be about 3-75mM.
5, according to each substratum in claim 1 or 28, wherein add L-glutaminate, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-glutaminate, make its ultimate density in substratum be about 3-50mM.
6, according to each substratum in claim 1 or 28, wherein add L-Methionin, its acid amides, the alkyl ester of sufficient amount at least or contain the dipeptidase derivant of L-Methionin, make its ultimate density in substratum be about 1-10mM.
7, according to each substratum in claim 1 or 28, wherein add altheine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of altheine, make its ultimate density in substratum be about 0.5-10mM.
8, according to each substratum in claim 1 or 28, wherein add L-Serine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-Serine, make its ultimate density in substratum be about 0.5-2mM.
9, according to each substratum in claim 1 or 28, wherein add L-ornithine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-ornithine, make its ultimate density in substratum be about 1-3mM.
10, according to each substratum in claim 1 or 28, wherein add L-L-glutamic acid, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-L-glutamic acid, make its ultimate density in substratum be about 15-40mM.
11, according to each substratum in claim 1 or 28, wherein add the L-prolineamide with sufficient amount at least or contain the dipeptidase derivant of L-prolineamide, make its ultimate density in substratum be about 1-200mM.
12, according to each substratum in claim 1 or 28, wherein add L-proline(Pro) methyl ester with sufficient amount at least or contain the dipeptidase derivant of L-proline(Pro) methyl ester, make its ultimate density in substratum be about 0.3-40mM.
13, according to each substratum in claim 1 or 28, wherein add L-proline(Pro)-L-L-Ala with sufficient amount at least, make its ultimate density in substratum be about 1-200mM.
14, a kind of plant cell culture medium that is used to induce, regenerate or keeps the plant somatocyte embryo tissue, it is characterized in that this substratum is substantially free of ammonium ion, its improvement comprises the amino acid that adds at least a L-of being selected from proline(Pro), L-arginine, altheine, L-ornithine, L-Methionin and acid amides, alkyl ester and dipeptidase derivant in substratum, do not compare with the embryo who produces in the substratum that has to do so to add, add number or the quality that quantity is enough to improve significantly the somatic embryo that produces.
15, according to the substratum of claim 14, wherein add L-proline(Pro), its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-proline(Pro), make its ultimate density in substratum be about 6-300mM.
16, according to the substratum of claim 14, wherein add L-arginine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-proline(Pro), make its ultimate density in substratum be about 15-100mM.
17, according to the substratum of claim 14, wherein add L-Methionin, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-Methionin, make its ultimate density in substratum be about 1-10mM.
18, according to the substratum of claim 14, wherein add altheine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of altheine, make its ultimate density in substratum be about 2-100mM.
19, according to the substratum of claim 14, wherein add L-ornithine, its acid amides, alkyl ester with sufficient amount at least or contain the dipeptidase derivant of L-ornithine, make its ultimate density in substratum be about 0.3-3mM.
20, according to the substratum of claim 14, wherein add L-proline(Pro) acid amides with sufficient amount at least or contain the dipeptidase derivant of proline(Pro) acid amides, make its ultimate density in substratum be about 1-200mM.
21, according to the substratum of claim 14, wherein add L-proline(Pro) methyl ester with sufficient amount at least or contain the dipeptidase derivant of L-proline(Pro) methyl ester, make its ultimate density in substratum be about 0.3-40mM.
22, according to the substratum of claim 14, wherein add L-prolyl-L-L-Ala at least, the ultimate density that institute's dosage is enough to make it in substratum is about 1-200mM.
23, according to each substratum in the claim 1,14 or 28, wherein plant cell culture medium is to be selected from Schenk-Hildebrandt substratum and Murashige-Skoog substratum.
24, a kind of method that the embryo takes place to produce by somatic embryo from the plant tissue of cultivating, it is characterized in that this method is included in inducing, regenerate or keeping the phase of somatic embryo generation, in the substratum of claim 1, cultivate through inductive plant soma tissue; And recovery body cell stage from said plant culture then.
25, a kind of method that the embryo takes place to produce by somatic embryo from the plant tissue of cultivating, it is characterized in that this method is included in inducing, regenerate or keeping the phase of somatic embryo generation, in the substratum of claim 14, cultivate through inductive plant soma tissue; And recovery body cell stage from said plant culture then.
26, according to the substratum of claim 1, wherein ammonium ion source is to be selected from one group of at least a material that comprises in ammonium chloride, ammonium nitrate, volatile salt, ammonium sulfate, ammonium phosphate and the ammonium citrate.
27, according to the substratum of claim 1, the amount of the ammonium ion source that wherein provides is enough to make it ultimate density in substratum and reaches and be about 0.5-50mM.
28, a kind of plant cell culture medium that is used to induce, regenerate or keeps the plant somatocyte embryo tissue, it is characterized in that this substratum does not have ammonium ion basically, its improvement comprises in substratum and to add L-glutaminate and to be selected from least a amino acid in L-proline(Pro), L-L-Ala, L-arginine, altheine, L-Serine, L-ornithine, L-L-glutamic acid and acid amides, alkyl ester and the dipeptidase derivant, compare with the embryo who produces in the substratum that does not have to do so to add, institute's dosage is enough to improve the number and the quality of the somatic embryo that is produced.
29, according to the substratum of claim 28, wherein the amount of the L-glutaminate that is provided is enough to make it to reach the ultimate density of about 10-50mM in substratum.
30, a kind of method that the embryo takes place to produce from the plant tissue of cultivating by somatic embryo, it is characterized in that this method is included in inducing, regenerate or keeping the phase of somatic embryo generation, in the substratum of claim 28, cultivate through inductive plant soma tissue; And recovery body cell stage from said plant culture then.
31, according to each method in the claim 24,25 or 30, wherein the plant soma tissue is at least a plant that is selected from alfalfa (Medicago Sativa L.), celery (Apium graveolens L.), upland cotton (Gossypium hirsutum), cotton (Gossypium Klotzschianum), the corn (Zea mays L) of gram labor time base and the dryland rice (Oryza sativa).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79025785A | 1985-10-22 | 1985-10-22 | |
| US790.257 | 1985-10-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN86107575A true CN86107575A (en) | 1987-05-27 |
Family
ID=25150117
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN198686107575A Pending CN86107575A (en) | 1985-10-22 | 1986-10-21 | Be used to improve modification method and the substratum that somatic embryo takes place |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0243469A4 (en) |
| JP (1) | JPS63501263A (en) |
| KR (1) | KR900007085B1 (en) |
| CN (1) | CN86107575A (en) |
| AU (1) | AU6521986A (en) |
| WO (1) | WO1987002701A1 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101848925A (en) * | 2007-09-05 | 2010-09-29 | 健泰科生物技术公司 | Biologically active C-terminal arginine-containing peptides |
| CN102892878A (en) * | 2010-04-23 | 2013-01-23 | 生命技术公司 | Cell culture medium comprising small peptides |
| CN108719052A (en) * | 2018-06-11 | 2018-11-02 | 中国农业科学院特产研究所 | The positive and negative first familiar generation rataria rescue culture method of ginseng, American Ginseng |
| CN115885846A (en) * | 2022-08-11 | 2023-04-04 | 南京农业大学 | Rapid tissue culture regeneration system for white carrots |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0740843B2 (en) * | 1987-10-23 | 1995-05-10 | 三井石油化学工業株式会社 | Plant tissue culture method |
| US6753463B1 (en) | 1987-11-18 | 2004-06-22 | Mycogen Corporation | Transformed cotton plants |
| US5244802A (en) | 1987-11-18 | 1993-09-14 | Phytogen | Regeneration of cotton |
| IL88266A (en) * | 1987-11-18 | 1998-03-10 | Phytogen | Method for the regeneration of a cotton plant from somatic cotton cells |
| US5834292A (en) * | 1987-11-18 | 1998-11-10 | J. G. Boswell Company | Method for producing somaclonal variant cotton plants |
| ATE78972T1 (en) * | 1987-11-18 | 1992-08-15 | Ciba Geigy Ag | A POWERFUL WAY TO REGENERATE COTTON FROM CULTURED CELLS. |
| US6340594B1 (en) | 1991-12-19 | 2002-01-22 | Cellfor, Inc. | Production of desiccation-tolerant gymnosperm embryos |
| DE69231890T2 (en) | 1991-12-19 | 2002-03-28 | Univ Saskatchewan Saskatchewan | DESICCATION OF GYMNOSPERM SOMATIC EMBRYONS |
| FR2710233B1 (en) * | 1993-09-22 | 1995-12-15 | Lvmh Rech | Method for promoting secondary somatic embryogenesis and application to the regeneration of plants, in particular of the vine. |
| WO1996037096A1 (en) * | 1995-05-25 | 1996-11-28 | Carter Holt Harvey Limited | Improved embryogenesis process for initiation and muturation |
| US5846797A (en) * | 1995-10-04 | 1998-12-08 | Calgene, Inc. | Cotton transformation |
| US6143563A (en) * | 1997-05-20 | 2000-11-07 | Pioneer Hi-Bred International, Inc. | Cryopreservation of embryogenic callus |
| CN103097516B (en) * | 2010-08-31 | 2015-09-09 | 菲仕兰品牌公司 | Eukaryotic cell substratum |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2559349B1 (en) * | 1984-02-09 | 1986-10-24 | Ecole Nale Ing Travaux Agricol | CRAMBE MARITIME CLONE (CRAMBE MARITIMA L.) AND METHOD FOR ITS VEGETATIVE MULTIPLICATION BY IN VITRO CULTURE |
| US4670391A (en) * | 1984-07-27 | 1987-06-02 | Sungene Technologies Corporation | Sunflower regeneration through embryogenesis and organogenesis |
-
1986
- 1986-10-21 JP JP61505569A patent/JPS63501263A/en active Pending
- 1986-10-21 CN CN198686107575A patent/CN86107575A/en active Pending
- 1986-10-21 KR KR1019870700528A patent/KR900007085B1/en not_active IP Right Cessation
- 1986-10-21 AU AU65219/86A patent/AU6521986A/en not_active Abandoned
- 1986-10-21 WO PCT/US1986/002223 patent/WO1987002701A1/en not_active Application Discontinuation
- 1986-10-21 EP EP19860906652 patent/EP0243469A4/en not_active Ceased
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101848925A (en) * | 2007-09-05 | 2010-09-29 | 健泰科生物技术公司 | Biologically active C-terminal arginine-containing peptides |
| CN104844690A (en) * | 2007-09-05 | 2015-08-19 | 健泰科生物技术公司 | Biologically active C-terminal arginine-containing peptides |
| CN104844690B (en) * | 2007-09-05 | 2019-08-16 | 健泰科生物技术公司 | Peptide that have biological activity, the terminal arginine containing C- |
| CN102892878A (en) * | 2010-04-23 | 2013-01-23 | 生命技术公司 | Cell culture medium comprising small peptides |
| CN108719052A (en) * | 2018-06-11 | 2018-11-02 | 中国农业科学院特产研究所 | The positive and negative first familiar generation rataria rescue culture method of ginseng, American Ginseng |
| CN115885846A (en) * | 2022-08-11 | 2023-04-04 | 南京农业大学 | Rapid tissue culture regeneration system for white carrots |
Also Published As
| Publication number | Publication date |
|---|---|
| KR880700063A (en) | 1988-02-15 |
| EP0243469A4 (en) | 1988-02-16 |
| KR900007085B1 (en) | 1990-09-28 |
| JPS63501263A (en) | 1988-05-19 |
| WO1987002701A1 (en) | 1987-05-07 |
| EP0243469A1 (en) | 1987-11-04 |
| AU6521986A (en) | 1987-05-19 |
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