PROCESS FOR THE PRODUCTION OF MIXOTROPHIC TISSUE CULTURES AND HERBICIDE-RESISTANT PLANTS

Abstract: Process for the preparation of mixotrophic tissue cultures by cultivation in a tissue culture capable of photosynthesis or containing parts capable of photosynthesis, prepared from seeds, any plant part, protoplast or single cell on a sugar containing known culture medium under illumination. The process is characterized in that the cultivation is performed on a culture medium containing less than 1% of sugar, e.g. saccharose or glucose. The invention further relates to a new process for the selection and reproduction of plants resistant to photosynthesis-inhibiting herbicides. For the selection of herbicide-resistant plants the above culture medium is employed.

Description: PROCESS FOR THE PRODUCTION OF MIXOTROPHIC TISSUE CULTURES AND HERBICIDE-RESISTANT PLANTS One of the oat difficult problems of chemical weed control is that the chemicals (her¬ bicides) generally injure not only the undesired weeds but the crop pLants as welL. There have been made many attempts at solving this problem. Selec¬ tivity can or example be improved by appLying the herbicide preemergently (after sowing prior to emergence) or in the case of tall crop plants, by applying the herbicide to the weeds only by guided spraying. The orphoLogical, physiological, biochemical, etc. differences between the useful plants and weeds can also be made use of if unde the given conditions for the given herbicide the cultivated plant is con¬ siderably less sensitive " than the weeds. These methods proved particularly suitable for the treatment of corns (cereals). As a result, the most dangerous weeds (barnyard grass, sorghum, foxtail) belong to the monocotyledons. The effectively controlled weeds are quickly replaced by other weeds which due to their similar properties to the given usefuL plants, escape the injuring effect of herbicidal treatment. The best results have so far been achieved by chemical weed control in maize cultures. Crop rotation, however, is made difficult by the fact that most of the crop plants cannot be sown to the field which had already been subjected to herbicidal treatment as there would be either a total or a substantial reduction in crop yields (Varga, M. JATE, Theoretical bases of chemical weed control, Faculty of Plant Physiology, Szeged, Hungary) . _> To avoid the above mentioned problems, it would be desirable to make the crop plants resistant to herbicides genetically. The most effective was of obtaining plants with new properties is mutant isola- 0 tion in cell culture (Maliga, P., Int. Rev. Cyt. Suppl. 11A, 225-250 (l98θ)). Plant cells are totipotent, i.e. from a single plant cell the whole plant can be regenerated. Therefore, millions of potential plants can be tested by the use of cell cultures in a few 5 petri dishes. This would be especially important in the case of herbicide resistance since the probability of the appearance of one herbicide-res1stant plant is estimated to one to the 10 (Gressel, J. , Outlook on Agriculture £, 283-287 (1978)). 0 There are herbicide-resistant weeds randomly appearing in the fields from which this property could be incorporated into crop plants. This is, however, generally inhibited by the sexual incompatibility of 5 the species. Once the herbicide resistance has been selected in tissue cultures, the property can be transmitted by cell culture, techniques even in the case of non-hydridizable species (Maliga, P. et al. : Cell culture mutants and their uses. In: Plant im¬ provement and somatic cell genetics, Academic Press, New York, 221-237 (1982)). Cell cultures may also have numerous advantages over the whole plants in studying the action mechanism of herbicides. 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 penetra¬ tion and transport as associated with the whole plant can be avoided with the use of cell cultures. It would, therefore, be desirable to use tissue culture techniques to select and regenerate herbicide-resistant plants. Many herbicides exert their activity via the inhibition of photosynthesis (Fedtke, C, Bio- chemistry and physiology of herbicide action, Springer- -Verlag, Berlin-New York (1983)). Plant being auto- trophic, are capable of converting light energy into chemical energy, particularly into high-energy sugar compounds from carbon dioxide and water. The central element of this process called photosynthesis, is an inhibited by photosynthesis-inhibiting herbicides which results in the destruction of plants due to the photodestructive effect of the free radicals formed. As a consequence of the decomposition of chlorophyll molecules, the chlorosis and fading of treated plants are the typical symptoms . The main problem with tissue culture techniques is that the tissue cultures of plants are heterotrophicj plant cells cannot grow in tissue cul- ture-s without sugar. Even the green tissue cultures are not capable of supporting their growth from photo- synthesis (their photosynthetic activity is very low). Photosynthesis-inhibiting herbicides are therefore in- -effective in tissue-cultures under standard labora¬ tory conditions (a culture medium containing 3 sugar and a light intensity of. 1000 to 2000 Ix). More par- ticularly, in the effective concentrations the her¬ bicides also inhibit the growth of cultures grown in the dark; the effect observed is not the primary photosynthesis inhibiting activity. A possibility of solving this problem is the selection of autotrophic plant tissue cultures which are capable of intensive photosynthesis and, therefore, grow in the absence of sugar. Such tissue cultures (Yamada, Y. et al. , Photoautotrophis in green cultured cells. In: Frontiers of plant tissue culture, Univ. of Calgary Press, Calgary, ^53-^62 (1978): Katoh, K. et al. : Planta \____A_, 509-510 (1979) have, however, numerous disadvantages. First highly chlorophyllous cell lines having a high photosynthetic activity have to be produced by a selection process and then grown under extreme conditions (light inten¬ sity: 10 000 Ix, and a C0 concentration of 1 o) to obtain satisfactory growth. The whole procedure is cumbersome, time-consuming, requires many inoculations which impairs the culture properties and therefor in the long run the cultures will not be suitable for the regeneration of pLants. So despite the fact that by selecting the highly chlorophyllous cell lines which show a high photosynthetic activity, the basic proper¬ ties of the plants might also be modified, however, it is not sure that the regenerated plants will carry all the basic properties of the.original plant. We aimed at developing a process by which the selection of plants resistant to photosynthesis- -inhibiting herbicides can be carried out under normal laboratory conditions by tissue culture techniques. We have surprisingly found that by decreas¬ ing the sugar concentration conventionally used in culture media by tissue culture techniques there can be found a low sugar concentration range at which the growth of calli is still satisfactory, and photosynthe¬ sis being essentially responsible for the growth. The obtained tissue cultures which base their metabolism substantially on photosynthesis and only to a small extent on external sugar supply, are called mixotrophxc tissue cultures. This name reflects the intermittant position of these cultures between the autotrophic and heterotrophic systems. Accordingly, 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. In the above de inition and throughout the speci ication the term "raixotrophic tissue cul- ture" 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. 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. As 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. The term "sugar" refers to sugars accessib to the plants, e.g., saccharose and glucose (Plant Sci. Letters, 2_L, 209- 1^ (l98l) 7.). According to the invention, 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 o sugar, callus formation is induced under a light intensity of approx imately 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 cultiva¬ tion is continued under light intensity of about 1000 Ix at a temperature of about 25 °c. Cultivation is performed on a known culture medium containing 0.2 to 0.8 , more preferably 0.2 to 0.5 o, most preferably 0.2 to 0.3 ^ of sugar. For the cultivation no extreme conditions are required. The process can be carried out under conventional, sterile laboratory conditions, i.e. under approximately 1000 Ix of continuous light at about 25 C. The process is equally suitable for the cultivation of tissues capable or uncapable of auto- trophic growth. The raixotrophic tissue cultures obtained proved suitable for testing the primary effects of photosynthesis-inhibiting herbicides including fading due to photodestruction. The tissue cultures can be used to distin— guish the effect of photosynthesis-inhibiting her¬ bicides 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 inhibi¬ tion of photosynthesis and in an affirmative case if it has a secondary effect at higher concentrations. Finally, according to the invention, the mixotrophic tissue cultures can be used for the production of herbicide-resistant plants which is the most important achievement of the invention. More specifically, there is a procedure to obtain plants resistant to photosynthesis-inhibiting herbicides by selection in tissue cultures. The mixotrophic tissue culture prepared in accordance with the invention is then treated with a photosynthes inhibiting herbicide, the parts of the culture which are suitable for greening are isolated and from these plants are regenerated in a defined manner. The tissue cultures prepared by the pro¬ cess, according to the invention, are suitable for testing the effect of a large variety of herbicides such as urea triazine, u acyl, diphenyl ether, pyrid- azine, hydroxybenzonitrile, nitrophenol, tridiazine, bispyridine, amide, anilide, triazole, thiocarbaraate, • glycine and chlorosulf rone and for the production of plants which are resistant to photosynthesis inhibit¬ ing herbicides. 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 inven¬ tion is equally suitable for the production of mono- and dicotyledonous herbicide-resistant plants. In addition to the plants illustrated by the Examples such plants 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. According to the invention using the process, resistancy can be transplanted from resistant plant species into sensitive plants., preferably by proto¬ plast 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 rom . 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. Further details of the invention will now be illustrated by the following non-limiting Examples. Example 1 Preparation of a mixotrophic tissue cul¬ ture of Nicotiana plurabaginifolia: A tissue cuLture was induced from sterile leaf pieces of Nicotiana piumbagini olia on a known cuLture medium. The plants were maintained on the following medium: RM I; MgSO j , (anhydrous) 723 mg/l RM II: FeSO,, x 7H 0 557 mg/lOO ml EDTA ^+5 rag/100 ml RM III: ZnSO^ x H 2 0 86 mg/100 ml Na 2 MoO z+ x 2H 2 0 2.5 mg/100 ml CuSO^ x 5H 2 0 0.25 mg/100 ml C 0 C1 2 x 6H 2 0 0.25 mg/100 ml saccharose 3 * (w/v) agar 0.8 £ (w/v) 1 I of culture medium is prepared by ad¬ mixing 50 ml of the stock-solution RM I, 5 nil of the stock-solution RM II and 10 ml of the stock- -solu ion RM III. O Components RM I, RM II and RM III together are called RM-salts (Murashige et al. : Physio1.Plant 15, ^73-^97 (1982). Plants were cultivated under a light intensity of 1000 Ix and a relative humidity of 0 70 ^ at 25 °C. At the beginning of the experiment callus formation was induced on a culture medium containing 1 'fa of. saccharose, under a light intensity of 1000 Ix, 5 at 25 C. For this purpose the following culture medium was used: RM-salts (RM I + RM II + RM III) in the above proportions, i.e. 250 ml of RM I + 5-0 ml of 0 RM II + 10 ml of RM III for one 1 of culture medium; inosite 100 ' mg/l thiamine 1 mg/l naphthylacetic acid 0.1 mg/l benzyladenine 1.0 mg/l 5 saccharose 1 (w/v) 0.8 (w/v). The controls are cultures grown under the same conditions but without light. After cultivation for four weeks 20 mg inocula were taken from the calli (which had been induced under light and darkness) and were placed onto culture media with various saccharose concent¬ rations and cultivation was continued in light and darkness, respectively. The culture medium had the same composition as described above, except sacc- harose. After four weeks the fresh weight of the calli was measured. The results obtained after growth under light and darkness cultures as a function of saccharose concentration are given in Table 1, where¬ in the mean values of 18 experiments (n=l8) and the standard deviations are indicated. Table I Fresh weight of tobacco calli grown under illumina¬ tion and in dark cultures on culture media with various saccharose concentrations Saccharose Fresh weight of calli (g) concentration in light in dark (1000 Ix) 0.1 0.20 +_ 0.05 0.05 -__ 0.01 0.2 0.18 _+ 0.12 0.11 _+ 0.0^ 0.3 Q . k +_ 0.10 0.L9 + , 0.06 O Λ 0.59 + O.l 0.25 + 0.08 Table 1 ( contd . ) Saccharose Fresh weight of calli (g) concentration in light in dark (1000 Ix) 0.5 0.63 + 0.L6 0.37 + 0.10 0.75 0.76 +_ 0.16 0.53 + , 0.17 1.0 0.82 -__ 0.l6 O.76 Hh 0.21 1.5 0.92 +_ 0.2^ 0.97 i 0.25 2.0 0.86 +■ 0.23 1.01 +_ 0.28 3.0 0.70 _+ 0.16 1.05 0.33 The data in Table 1 clearly show that over a saccharose concentration of 1.0 the growth rate of calli grown under illumination and in dark respecti ly is identical within the limit of error, i.e. the growth is heterotrophic. On media without saccharose practically no growth is observed, indicating that the calli are incapable of autotrophic growth. On media with low saccharose concentration, particular¬ ly between 0.2 and 0.5 ^, the weight of calli grown under illumination is substantially higher than that of the calli obtained in dark cultures. In case of a saccharose concentration of 0.2 d o the difference is almost two-fold, at a saccharose concentration of 0.3 ^ about 2.5-fold, between a saccharose con¬ centration of O. k- fo and 0.5 it is also around 2.0-foLd. At the same time the growth rate is satis¬ factory 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 2 Essentially following the procedure describe in Example I but starting from sterile stem pieces in place of leaves, the following results were obtaxned: (n=!8). Table 2 Fresh weight of tobacco calli grown under illumina- tion and in dark cultures on culture media with variou saccharose concentrations Saccharose Fresh weight of calli (g) concentration in light in dark (#) (1000 lx) o o.o6 +_ o.03 o.o6 +_ 0.03 0.1 0.22 +_ 0.08 0.06 +_ 0.02 0.2 0.^6 + 0.10 0.11 + 0.03 Table 2 (contd. ) Saccharose Fresh weight of calli (g) 5 concentration in light in dark 0.3 0 9 £ 0.17 0.16 + 0.05 0.5 0.69 _*_ 0.27 0.28 +_ 0.11 1.0 0.70 + , 0.35 O.69 £ 0.26 O 2.0 1.13 H- 0.30 1.12 +_ O . kl 3.0 0.93 +.0.33 0.87 _*_ 0 3 From the data set forth in Table 2 the same conclusions can be drawn as from the data of ^-3 Table 1 but here the difference between the weights of the calli grown under illumination and in dark cultures, respectively, are even more expressed (at a saccharose concentration of 0.2 > the difference is more than ^-fold). 0 Repetition of the tests described in Example 1 and 2 on culture media containing glucose in different concentrations gave completely analogous results. 25 Example 3 Screening the primary effect of photosynthe -inhibiting herbicides in raixotrophic tissu cultures . In a Nicotiana plumbaginifolia tissue cul¬ ture prepared according to Example 1 on a culture medium having the same composition as given in Example 1 but containing 0 # 3 f* and 3 $> saccharose, respectively terbutryne, a known photosynthesis- -inhibiting herbicide was tested under light (1500 o Ix, 28 C) and in darkness. The fresh weight of calli was measured as described in Example 1. The herbicide was given to the warm (^5 C) medium from a filter sterile stock solution. The terbutryne concentration -U- -6 was varied between 10 and 10 moles. The results obtained together with the standard errors (n=l8) are given in the following Table 3 - * Table 3 Saccharose concentration: 0.3 Terbutryne cone. (M) Fresh weight of calli (g) under illumination in dark 0 S 0.^8 £ 0.13 0.21 £ 0.06 10~ O . lk £ 0.04 0.18 £ 0.0 lθ "D O.lA £ 0.03 0.27 +_ 0.07 10 0.15 + o.o 0.23 + 0.07 Table 3 (contd. ) Saccharose concentration: 3 Terbutryne cone, (M) Fresh weight of the calli (g) under illumination in dark 0 S 0.89 + 0.25 0.87 + 0.23 -6 x 10 1.06 £ 0.17 0.86 £ 0.26 lO "3 *0.93 + 0.22 1.09 + 0.26 - x 10 0.88 + 0.32 0.82 + 0.15 green callx From the above data it can be seen that on a culture medium containing 3 of saccharose under illumination or in darkness terbutryne was ineffec¬ tive. If, however, the saccharose concentration was decreased to 0.3 ?° } the herbicide inhibited the growth even in a concentration of 10~ M, to the same level as though the calli had been grown in darkness. This effect was not further increased at higher concentra¬ tions. It is to be noted that the calli grown on a culture medium containing 0.3 7° of saccharose were bleached as a result of herbicide treatment which proves that a photosynthetic electron transport phenomena has taken place in the calli, in contrary to the results obtained on media containing 3 7° of saccharose. Example k Essentially following the procedure describe in Example 3 t>ιt carrying out the herbicide treatment with the following herbicides completely analogous results were obtained : atrazine, prometryne, aziprotryne, metribuzin, diuron, chlorobromuron, methabenzthiazuron, Linuron, monolinuron, lenacyl, terbacyl, bromoxynyl. Similar results were obtained if the tests were carried out on Nicotiana tabacum, Solanum nigrum or Solanum tuberosura, instead of Nicotiana plumbagini- folia. Example 5 Regeneration of plants resistant to photo¬ synthesis-inhibiting herbicides. 1 g of sterile Nicotiana plumbaginxfolia leaves was cut to about 1 mm pieces and were sterilize in 10 ml of a filter sterile enzyme solution in dark at a temperature of 25 C without shaking. The incuba¬ tion time was 18 hours. Composition of the enzyme solution: 0.5 of driselase in a K., solution containing 0.4 M of saccharose. Composition of the K~ solution: Substance Concentration (mg/l) KN0, 24-00 CaCl x 2H 2 0 880 H^N0 3 24θ SO^ x H 2 0 10 H 3 B0 3 3 ZnSO^ x 7H 2 0 2.3 a 2 oO^ x 2H 2 0 0.24 CoCl 2 x 6H 2 0 0.0l4 KI 0.75 gSO^ 120 aHgPO^ x H 2 0 156 (NH^SO^ 130 nicotinic acid 1 vitamin Bg 1 vitamin B, 10 Subs tance Concentration 2 , 4-D 0. 1 benzyladenine 0.2 naphthylacetic acid 1 D-xylose 250 inos ite 100 pH 5.8 After incubation the suspension was passed through a nylon filter (63 urn) and was centrifuged (100 g, 3 minutes). Protoplasts floating in the meniscus after centrifugation .were suspended in a W-5 washing solution. Composition of the W-5 washing solution: Substance Concentration (mg/l) NaCL 9000 glucose 1000 CaCl 2 x 2H 2 0 18400 KCL 400 pH 5. 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 (l x 10 protoplasts/ml) under illumination ( 100 Ix, 25 C). 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. 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 C in a light intensity of 1500 Ix. 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 contain¬ ing 2 fa 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 cultivate on a culture medium described in Example 1. Cultivatio 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 d - of saccharose and 0.4 M of terbutyne but otherwise having the same com¬ position as given in Example L. Cultivation was then continued as described in Example 1. After four weeks the resistance of the plants was controlled by visual evaluation. If the plants became resistant both the calli and the leaf-pieces used for cultivation are green, while in the case of non-resistant plants the callus is white and also the leaf piece is bleached. The appearance frequency resistant cell lines was about 1/10" 5 colonies. The test was repeated using methobrorauron as a herbicide in place of terbu±ryne. 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 colonies. xAnalogous results were obtained with Solanum tuberosum as a test plant. Cross-resistance of the terburyne-resistant plants to other photosynthesis inhibitors was also studied. Using the leaf assay, each regenerate ex¬ pressed resistance to the triazine-type atrazine and aziprotryne, and to the uracile-type terbacil and lenacil. No resistance could be observed to the urea- -type diuron, metobromuron and methabenzthiazuron, to the hydroxybenzonitrile-type bromoxynil, or to benta- zone. The herbicide sensitivities of TBR plants and wild-type N„ plumbagini olia 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 (l_ n ) and the relative resistances (R/S) are shown in Table 6. * Crosses (F, ) and reciprocal crosses (RF. ) were made between the TBR regenerates and the wild-type plants. Resistance in the TBR plants was inherited maternally. This genetic behaviour is consistent with the chloroplast location of the terbutryne resistance mutation. Table 6 I_._ values and R/S ratios for various herbicides in DO wild-type (N.p.) and triazine-resistant (TBR) Nicotiana piumbaginifolia Herbicides I concentrations (M) R/S values N.p. TBR -7 -4 Terbutryne 1. x10 ' 1.6x10 910 -7 -4 Atrazine 4.2x10 ' 3.2x10 760 Terbacil 6.6xl0~ 7 2.0xLθ "3 30 Diuron (DCMU) l.OxLO "7 l.8xlO~ 7 1.8 Dinoseb 7 xlθ " 2.5χi0~ 7 0.03 Hill-reaction measurements from water to ferrycianide were made by using a Clark-type oxygen electrode [Droppa et al. Z. Pflanzenphysiol. 105:75-79/ Chloroplasts were isolated as described by Reeves and Hall [Biochim. Biophys. Acta 314:66-78/1973/] from three months old plants grown in the greenhouse. The I_„ value for a herbicide is defined as the concentra- DO tion causing 50 ia inhibition of electron transport, and R/S=I ~_O_ resistant/l_TO- wild-type. Example 6 Transmission of herbicide-resistance between plant species by protoplast fusion In this experiment atrazine-resistant chloro- plasts were transplanted from a mutant weed (Solanum nigrum) into tobacco (Nicotiana pLumbaginx olia) 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)). In 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 resistanc in the cultivated plants was controlled as disclosed in Example 5.

Claims: C laims 1. Process for the preparation of mixo¬ trophxc tissue cultures by cultivation in a tissue culture capable of photosynthesis or containing parts capable of photosynthesis, prepared from seeds, any vegetable part, protoplast or single cell, on a sugar- -containing known culture medium, under illumination, which comprises carrying out the cultivation on a culture medium containing less then 1 ?Q of sugar. 2. A process as claimed in claim i, in which cultivation is carried out on a culture medium contain ing 0.2 to 0.8 of sugar. 3. A process as claimed in claim 1 or claim in which cultivation is carried out on a culture medium containing 0.2 to 0.5 of sugar. 4. A process as claimed in any one of claims 1 to 3, in which cultivation is carried out on a cul¬ ture medium containing 0,2 to 0.3 of sugar. 5. A process as claimed in any one of claims 1 to 4 in which as a sugar saccharose or glucose is employed. 6. Process for the cultivation of plants resistant to photosynthesis-inhibiting herbicides, by selection in tissue cultures, which comprises treating a culture medium prepared according to any one of claims 1 to 5 with a photosynthesis-inhibiting her¬ bicide, isolating the parts capable of greening from the culture, and regenerating plants therefrom in a manner known per se. 7« A process as claimed in claim 6, which comprises carrying out the procedure on a mixσtrophic tissue culture prepared from a starting material modified by cell genetical methods. 8. A process a3 -claimed in claim 6 in which as a photosynthesis-inhibiting herbicide terbutryn is employed. 9- A process as claimed in claim 6 in which as a photosynthesis-inhibiting herbicide methobromuron is employed. 10. A process as claimed in any one of claims 1 to 8, in which as a test plaiit Nicotiana plumbagini- folia or Solanum tuberosum is used.

Inventors: CSEPLOE AGNES, MALIGA PAL, MARTON LASZLO, MEDGYESY PETER, MENCZEL LASZLO, CSEPLOE, AGNES, MALIGA, PAL, MARTON, LASZLO, MEDGYESY, PETER, MENCZEL, LASZLO
Applicants: MTA SZEGEDI BIOLOG KOEZPONTI, MTA SZEGEDI BIOLOGIAI KOEZPONTJA
Application year: 1985
Main classification: C12N5
IPC codes: A01H1, A01H4, A01N25, C12N5, C12N15, C12R1