WO1985003085A1 - Procede de production de cultures de tissus mixotrophiques et de plantes resistant aux herbicides - Google Patents

Procede de production de cultures de tissus mixotrophiques et de plantes resistant aux herbicides Download PDF

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Publication number
WO1985003085A1
WO1985003085A1 PCT/HU1985/000003 HU8500003W WO8503085A1 WO 1985003085 A1 WO1985003085 A1 WO 1985003085A1 HU 8500003 W HU8500003 W HU 8500003W WO 8503085 A1 WO8503085 A1 WO 8503085A1
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WO
WIPO (PCT)
Prior art keywords
photosynthesis
culture medium
sugar
cultivation
plants
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Application number
PCT/HU1985/000003
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English (en)
Inventor
Ágnes CSÉPLO^"
Pál MALIGA
Péter MEDGYESY
László MENCZEL
László MÁRTON
Original Assignee
Mta Szegedi Biológiai Központja
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Mta Szegedi Biológiai Központja filed Critical Mta Szegedi Biológiai Központja
Publication of WO1985003085A1 publication Critical patent/WO1985003085A1/fr
Priority to DK418185A priority Critical patent/DK418185D0/da

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues

Definitions

  • Tissue cultures are sterile, homogeneous, can be maintained both in a stationary and exceptionally growing phase, and are suitable for micro-testing. The problems of penetration and transport as associated with the whole plant can be avoided with the use of cell cultures.
  • tissue culture techniques The main problem with tissue culture techniques is that the tissue cultures of plants are heterotrophic; plant cells cannot grow in tissue cultures without sugar. Even the green tissue cultures are not capable of supporting their growth from photosynthesis (their photosynthetic activity is very low). Photosynthesis-inhibiting herbicides are therefore in-effective in tissue-cultures under standard laboratory conditions (a culture medium containing 3 % sugar and a light intensity of. 1000 to 2000 Ix). More particularly, in the effective concentrations the herbicides also inhibit the growth of cultures grown in the dark; the effect observed is not the primary photosynthesis inhibiting activity.
  • the invention relates to a process for the preparation of raixotrophic tissue cultures induced from seeds, plant parts, protoplasts or single cells on a known culture medium containing less than 1 % sugar which is capable of the needed amount of photosynthesis for selection.
  • raixotrophic tissue culture is used to refer to cultures in which the growth can substantially be attributed to both sugar content and light conditions of the culture medium.
  • seed When preparing the cultures, one can start from any plant material conventionally used for this purpose. It should be noted that the term "seed" referred to as a possible starting material includes the embryo as well.
  • a plant part for example, leaves, stems, roots, blooms, etc. or part thereof can be used.
  • Protoplasts are plant cells without a cell wall which are referred to from herein as to include the protoplasts modified by fusion or other cell genetic methods.
  • the single cells are plant cells having a cell wall, including pollen.
  • sugars accessible to the plants e.g., saccharose and glucose (Plant Sci. Letters, 21, 209-214 (1981) 7.) .
  • the raixotrophic tissue cultures are preferably prepared by starting from tissue cultures prepared from sterile leaf or stem parts, on a known culture medium having a normal sugar concentration. More specifically, on a culture medium containing approximately 1 % sugar, callus formation is induced under a light intensity of approximately 1000 Ix at a temperature of approximately 25 After several weeks of cultivation inoculae selected from the calli are placed on a culture medium having a low (less than 1 %) sugar concentration and cultivation is continued under light intensity of about
  • Cultivation is performed on a known culture medium containing 0.2 to 0.8 % , more preferably 0.2 to 0.5 %, most preferably 0.2 to 0.3 % of sugar.
  • a known culture medium containing 0.2 to 0.8 % , more preferably 0.2 to 0.5 %, most preferably 0.2 to 0.3 % of sugar.
  • the process can be carried out under conventional, sterile laboratory conditions, i.e. under approximately 1000 Ix of continuous light at about 25 oC.
  • the process is equally suitable for the cultivation of tissues capable or uncapable of autotrophic growth.
  • the raixotrophic tissue cultures obtained proved suitable for testing the primary effects of photosynthesis-inhibiting herbicides including fading due to photodestruction.
  • tissue cultures can be used to distinguish the effect of photosynthesis-inhibiting herbicides from the effect of other types of herbicides. Thus, it can be determined with a very simple method if a given herbicide exerts its activity via inhibition of photosynthesis and in an affirmative case if it has a secondary effect at higher concentrations.
  • the mixotrophic tissue cultures can be used for the production of herbicide-resistant plants which is the most important achievement of the invention.
  • tissue cultures prepared by the process, according to the invention are suitable for testing the effect of a large variety of herbicides such as urea triazine, uracyl, diphenyl ether, pyridazine, hydroxybenzonitrile, nitrophenol, tridiazine, bispyridine, amide, anilide, triazole, thiocarbamate, glycine and chlorosulfurone and for the production of plants which are resistant to photosynthesis inhibiting herbicides.
  • herbicides such as urea triazine, uracyl, diphenyl ether, pyridazine, hydroxybenzonitrile, nitrophenol, tridiazine, bispyridine, amide, anilide, triazole, thiocarbamate, glycine and chlorosulfurone
  • the calli cultivated under a defined light intensity, on a culture medium having a low sugar concentration fade under the effect of photosynthesis inhibiting herbicides; except for herbicide-resistant calli which can easily be selected on the basis of this property.
  • the selection method according to the invention is equally suitable for the production of mono- and dicotyledonous herbicide-resistant plants.
  • plants illustrated by the Examples include practically all cultivated plants such as cereals (corns), maize, tomato, potato, sugar beet, sun-flower, soya, cotton, grapes, etc. Most of our tests have, however, been performed on tobacco plants since tobacco plant is the most widely used model plant for such kind of experiments.
  • resistancy can be transplanted from resistant plant species into sensitive plants., preferably by protoplast fusion.
  • Herbicide-resistance is preferably tested by protoplast technique since this method is suitable for testing the most cells in a time unit. In a Petri dish only about 9 calli can be tested, whereas by the protoplast method about 1000 colonies can be examined simultaneously.
  • a further advantage of the colonies which originate from protoplasts is that they are derived from a single cell therefore, they are much more homogeneous than the calli of multiple cell origin which are mixed genetically. Accordingly, the most advantageous way to produce resistant plants is the cultivation in cultures of protoplast origin.
  • Example 1 Example 1
  • saccharose 3 % (w/v) agar 0.8 % (w/v) 1 1 of culture medium is prepared by admixing 250 ml of the stock-solution RM I, 5 ml of the stock-solution RM II and 10 ml of the stock-solution RM III.
  • RM-salts Components RM I, RM II and RM III together are called RM-salts (Murashige et al. : Physiol. Plant 15, 473-497 (1982). Plants were cultivated under a light intensity of 1000 1x and a relative humidity of 70 % at 25 °C.
  • RM-salts (RM I + RM II + RM III) in the above proportions, i.e. 250 ml of RM I + 5.0 ml of RM II + 10 ml of RM III for one 1 of culture medium; inosite 100 mg/1 thiamine 1 mg/1 naphthylacetic acid 0.1 mg/1 benzyladenine 1.0 mg/1 saccharose 1 % (w/v)
  • the controls are cultures grown under the same conditions but without light.
  • the growth rate is satisfactory for experimental purposes, for example on a culture medium containing 0.3 % of saccharose about half of the weight measured after growth on a culture medium containing 2.0 % of saccharose is obtained.
  • the experimental data clearly show that on culture media having a low saccharose concentration the calli, although require an external saccharose supply for their growth, show a substantial photosynthesis-dependant growth, i.e. are mixotrophic.
  • Example 5 Regeneration of plants resistant to photosynthesis-inhibiting herbicides.
  • 1 g of sterile Nicotiana plumbaginifolia leaves was cut to about 1 mm pieces and were sterilized in 10 ml of a filter sterile enzyme solution in dark at a temperature of 25 oC without shaking. The incubation time was 18 hours.
  • KCL 400 pH 5.6 The suspension obtained was centrifuged again (50 g, 2 minutes).
  • the protoplasts collected from the bottom of the centrifuge tube were cultivated in a K3 nutrient solution (1 x 10 5 protoplasts/ml) under illumination ( 100 1x, 25 oC).
  • the cultures were diluted with K3 nutrient solutions having gradually decreasing glucose concentrations (0.4, 0.3, 0.2, 0.1 M) every 7 to 10th days until a 30-40 times final dilution.
  • Example 1 About 6 weeks after starting the cultivation the cell colonies were added to a culture medium containing 0.3 % of saccharose and 0.4 M of terbutryne, and otherwise having the same composition as given in Example 1 ( 10-fold dilution which in the end corresponded to a colony density of about 1000 colonies/Petri dish having a diameter of 10 cm).
  • the colonies were cultured at 28 oC in a light intensity of 1500 1x. After 1 to 2 months the colonies capable of greening were isolated and grown to calli on the same medium. From the calli obtained plants were regenerated on a culture medium containing 2 % of saccharose, no terbutryne and otherwise having the same composition as the culture medium used for the selection. After a one-month growth, sprouts were appearing which were rooted and cultivated on a culture medium described in Example 1. Cultivation was continued until 10 cm plants were obtained. Small pieces of the leaves of 10 cm plants were placed on a culture medium containing 0 3 % of saccharose and 0.4 M of terbutyne but otherwise having the same composition as given in Example l.
  • the test was repeated using methobrorauron as a herbicide in place of terbutryne. There could be isolated resistant cell lines also in this case and the resistance was expressed in the regenerated plants as well. The frequency of the appearance of resistant cell lines was about 1/10 5 colonies.
  • the herbicide sensitivities of TBR plants and wild-type N. plumbaginifolia were compared in isolated chloroplasts by measuring the inhibition of the photosynthetic electron transport from water to ferricyanide (Hill-reaction) in the presence of various herbicides.
  • the half inhibitory concentrations (I 50 ) and the relative resistances (R/S) are shown in Table 6.
  • RF 1 resistance in the TBR plants was inherited maternally.
  • Chloroplasts were isolated as described by Reeves and
  • Example 6 Transmission of herbicide-resistance between plant species by protoplast fusion
  • atrazine-resistant chloro plasts were transplanted from a mutant weed (Solanum nigrum) into tobacco (Nicotiana plumbaginifolia) and potato, respectively.
  • the transmission was carried out by the process described in Example 5 for the cultivation of protoplasts by a chloroplast transfer method based on protoplast fusion (Menczel et al. : Genetics, 100, 487-95 (1982); Medgyesy et al. : Molec. Gen. Genet. 179, 693-698 (1980)).
  • This procedure we made use of the possibility of the selection of cell lines containing resistant chloroplasts on low sugar concentrations.
  • the appearance of the resistance in the cultivated plants was controlled as disclosed in Example 5.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
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  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Developmental Biology & Embryology (AREA)
  • Plant Pathology (AREA)
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  • Biophysics (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Abstract

Procédé de préparation de cultures de tissus mixotrophiques en les cultivant dans une culture de tissus capable de photosynthèse ou contenant des parties capables de photosynthèse, préparée à partir de semences, d'une partie quelconque de la plante, de protoplastes ou d'une cellule unique sur un bouillon de culture connu contenant du sucre et exposé à un éclairage. Le procédé se caractérise par le fait que la culture est exécutée sur un bouillon de culture contenant moins de 1% de sucre, par exemple du saccharose ou du glucose. Est également décrit un nouveau procédé de sélection et de reproduction de plantes résistant aux herbicides inhibant la photosynthèse. Pour la sélection de plantes résistant aux herbicides ou utilise le bouillon de culture susmentionné.
PCT/HU1985/000003 1984-01-16 1985-01-16 Procede de production de cultures de tissus mixotrophiques et de plantes resistant aux herbicides WO1985003085A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DK418185A DK418185D0 (da) 1984-01-16 1985-09-13 Fremgangsmaade til fremstilling af mixotrofiske vaevskulturer og frembringelse af herbicidresistente planter

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
HU152/84 1984-01-16
HU84152A HU190751B (en) 1984-01-16 1984-01-16 Process for producing mixotroph tissue-cultures and herbicide-resisting plants

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WO1985003085A1 true WO1985003085A1 (fr) 1985-07-18

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PCT/HU1985/000003 WO1985003085A1 (fr) 1984-01-16 1985-01-16 Procede de production de cultures de tissus mixotrophiques et de plantes resistant aux herbicides

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EP (1) EP0169230A1 (fr)
JP (1) JPS61501187A (fr)
DK (1) DK418185D0 (fr)
ES (1) ES539629A0 (fr)
HU (1) HU190751B (fr)
IT (1) IT1183269B (fr)
WO (1) WO1985003085A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0364580A1 (fr) * 1987-06-01 1990-04-25 Hokko Chemical Industry Co. Ltd. Procede de production de riz resistant aux herbicides, plante de riz et graine de cette plante
GB2203022B (en) * 1987-03-23 1991-11-20 Imp Tobacco Co Ltd Smoking material and process for making the same
GB2234663B (en) * 1987-03-23 1991-12-04 Imp Tobacco Co Ltd "process for producing tobacco cells"
WO1993008683A1 (fr) * 1991-10-31 1993-05-13 Zeneca Limited Plantes resistantes aux herbicides
WO1993023529A2 (fr) * 1992-05-12 1993-11-25 Champagne Moët & Chandon Culture stabilisee d'agregats cellulaires et procede de developpement des embryons a partir d'une souche proembryogene destine a la regeneration de la vigne

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0009319A1 (fr) * 1978-08-16 1980-04-02 The British Petroleum Company p.l.c. Culture de cellules des végétaux supérieurs
DE3126001A1 (de) * 1980-07-01 1982-03-11 Nippon Paint Co., Ltd., Osaka Verfahren zur selektiven zuechtung bzw. veredelung von pflanzenzellenkulturen
EP0050562A1 (fr) * 1980-10-22 1982-04-28 Synthelabo Procédé de production ou de biotransformation de métabolites par des cellules végétales in vitro
EP0052001A1 (fr) * 1980-11-06 1982-05-19 Albright & Wilson Limited Culture de callus dans un courant nutritif
EP0071999A2 (fr) * 1981-08-11 1983-02-16 Mitsui Petrochemical Industries, Ltd. Procédé de production de métabolites secondaires de plantes

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0009319A1 (fr) * 1978-08-16 1980-04-02 The British Petroleum Company p.l.c. Culture de cellules des végétaux supérieurs
DE3126001A1 (de) * 1980-07-01 1982-03-11 Nippon Paint Co., Ltd., Osaka Verfahren zur selektiven zuechtung bzw. veredelung von pflanzenzellenkulturen
EP0050562A1 (fr) * 1980-10-22 1982-04-28 Synthelabo Procédé de production ou de biotransformation de métabolites par des cellules végétales in vitro
EP0052001A1 (fr) * 1980-11-06 1982-05-19 Albright & Wilson Limited Culture de callus dans un courant nutritif
EP0071999A2 (fr) * 1981-08-11 1983-02-16 Mitsui Petrochemical Industries, Ltd. Procédé de production de métabolites secondaires de plantes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PLANTA, volume 144, 1978/79 (Springer-Verlag, Berlin-Heidelberg-New York), K. KOTOH et al. "Photoautotrophic Growth of Marchantia polymorpha L. Cells in Suspension Culture", see pages 509-510, whole article. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2203022B (en) * 1987-03-23 1991-11-20 Imp Tobacco Co Ltd Smoking material and process for making the same
GB2234663B (en) * 1987-03-23 1991-12-04 Imp Tobacco Co Ltd "process for producing tobacco cells"
EP0364580A1 (fr) * 1987-06-01 1990-04-25 Hokko Chemical Industry Co. Ltd. Procede de production de riz resistant aux herbicides, plante de riz et graine de cette plante
EP0364580A4 (fr) * 1987-06-01 1990-06-26 Hokko Chem Ind Co Procede de production de riz resistant aux herbicides, plante de riz et graine de cette plante.
WO1993008683A1 (fr) * 1991-10-31 1993-05-13 Zeneca Limited Plantes resistantes aux herbicides
WO1993023529A2 (fr) * 1992-05-12 1993-11-25 Champagne Moët & Chandon Culture stabilisee d'agregats cellulaires et procede de developpement des embryons a partir d'une souche proembryogene destine a la regeneration de la vigne
WO1993023529A3 (fr) * 1992-05-12 1994-01-20 Moet & Chandon Culture stabilisee d'agregats cellulaires et procede de developpement des embryons a partir d'une souche proembryogene destine a la regeneration de la vigne
US5968827A (en) * 1992-05-12 1999-10-19 Champagne Moet & Chandon Cellular aggregate stabilized culture and process for the development of embryos from a proembryogenic strain for use in vine regeneration techniques

Also Published As

Publication number Publication date
DK418185A (da) 1985-09-13
ES8601646A1 (es) 1985-11-16
HUT37648A (en) 1986-01-23
IT1183269B (it) 1987-10-22
IT8519118A0 (it) 1985-01-16
ES539629A0 (es) 1985-11-16
JPS61501187A (ja) 1986-06-19
EP0169230A1 (fr) 1986-01-29
DK418185D0 (da) 1985-09-13
IT8519118A1 (it) 1986-07-16
HU190751B (en) 1986-11-28

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