JPH06508510A - Improvements related to organic compounds - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Improvements related to organic compounds The present invention is directed to a diploid eggplant (Solanum melongii) that has characteristics of other Solanaceae plants. f) and a method for producing such a plant.

background Eggplants have no natural resistance to certain fungal diseases. These are Very susceptible to fungal diseases such as Ertinrium wilt and Fusarium wilt . Sources of natural resistance to such diseases are Vuelticillium and Other Solanaceae such as Solanum torvum that exhibit natural resistance to Fusarium Available in plants. Interspecific dioecious hybrids between eggplants and other Solanaceae plants result in sterility. Give sexual offspring. This applies especially to female-male hybrids between eggplants and Niss tolzim. It is known as Moreover, such crosses are ineffective even when using embryo culture. The rate is poor and difficult [K, R, Manrikimon et al., Holt Science, 18(6) Vol. 894-895 (1983)]. In addition, this cross was produced by Nis torvum. This can only be done when used as a flour-feeding plant.

Protoplast fusion between Solanum and Nis torvum may not be the desired result for various reasons. brought no fruit. This protoplast fusion is inefficient and the resulting resistance Plants are always aneuploid, i.e. not diploid, and their offspring are therefore low or sterile. Shows a range of fertility. Solanum and Nis torvumprot reported in the literature Fusion between blasts [e.g. A, Cali, etc., Theoretical and Applied ・Noe Futenochus Vol. 76 (1988) pp. 490-496] is the same type of plant material. (mesophyll x mesophyll) and without donor irradiation.

detailed description According to the present invention, it is found naturally in species of the Solanaceae family and is naturally found in eggplants. 1. A method for producing fertile diploid eggplant containing characteristics that A sample with the desired characteristics at a dose sufficient to cause fragmentation of rotoplast chromosomes. ii) irradiating a protoplast donor of a plant belonging to the family Succedeaceae; By fusing plasts with eggplant protoplasts, which have the role of acceptors ( wherein the donor protoplast and acceptor protoplast are different 1ii) the resulting fused protoplasts A method is provided comprising: regenerating into a mature plant.

Chromosomes from a Solanaceae protoplast donor showing resistance to fungal diseases The cleavage can be achieved in a manner known per se, for example by irradiation. This light The radiation can be carried out using gamma, UV or X-rays. Where irradiation is carried out by an X-ray source chromosomal fractionation, generally by applying a dose of 20 to 100 krad. You can get a clear answer. Preferably a lethal dose is used, in which case at least 50 krad must be applied. For irradiation purposes, protoplasts are conveniently Suspend the protoplasts in a suitable protoplast suspension medium, such as a plast washing medium.

Donor and acceptor protoplasts used herein as starting materials can be obtained by methods known per se from suitable plant cells. This results in The plant materials used for the preparation of donor and acceptor protoplasts are It is essential that it be of the origin or type that it became.

Examples of types of plant material suitable for use as starting material for the preparation of protoplasts are stem material, stem material, board material, cotyledon, etc. Preferably, the acceptor proto Plasts are derived from cotyledons. Donor protoplasts are preferably from stem material be guided. The plant material is grown in a suitable hormone-free basic nutrient medium, e.g. Hormone-free milk containing salts and vitamins with added carbohydrates such as Sterilized under seed germination conditions in Lasige and Skoog (MS) basal medium. It can be easily obtained by germinating seeds. Such conditions are known per se. germination The temperature conveniently ranges from 228C to 28C. Allow seedlings to develop properly Therefore, sufficient light must be provided, equivalent to daylight on an average summer day ( Approximately 16 hours) is appropriate. Generally, cotyledons that are about 9 to 17 days old are protoplasts. Suitable for use as starting material. Cotyledon-derived protoplasts are e.g. Diced cotyledons are treated with hemicellulase, cellulose and/or pectin. The enzyme solution containing appropriate enzymes to destroy the cell wall, such as tinase, can be used to obtained by processing the body in a known manner. For example, 2 to 5 month old plants The material is placed in callus induction medium and the resulting callus material is suspended in a suitable cell suspension medium. Cell suspensions derived from stem, leaf or plate materials can be made automatically by preparing cell suspensions from can be similarly obtained by known methods. Then the cell suspension at the beginning of the exponential growth phase Protoplasts can be obtained by treating with an appropriate enzyme solution.

Enzymes suitable for the preparation of callus induction media, cell suspension media and protoplast suspensions Solutions are known per se.

For selection purposes, parent protoplasts may be dyed with fluorescent dyes, e.g. Cein isothionate (FTTC) Fluorescein diacetate (FD A) or use chlorophyll autofluorescence for one of the fusion partners can do. Such staining can be carried out by methods known per se.

Protoplast fusion in the method of the present invention can be carried out using methods known per se, such as so-called fusion. carried out by chemical fusion or electrofusion under conditions known in the art. Can be done.

Mix protoplasts of different origins, preferably 105 to 10'/ml Concentrate to a final concentration of protoplasts. The fusion partners are preferably in a ratio of 1.1 Mix.

Chemical protoplast fusion involves merging protoplasts in a suitable protoplast aggregation solution. Conveniently carried out by aggregating the membranes and then membrane fusion in a high pH solution. can. The protoplast flocculation solution essentially consists of polyethylene that induces flocculation. glycol (PEG), osmoticum, e.g. consisting of carbohydrates such as nitol or glucose, as well as calcium salts . PEG preferably develops a molecular weight of 1500 to 6000. In general, the desired aggregation was obtained in solutions with a final concentration of PEG of 10 to 16% (by weight). It will be done. The suspension of aggregated protoplast cells thus obtained was mixed with CaCl2-high p Fuse the membrane by gradual dilution with H solution.

Electrofusion is carried out with osmoticum, preferably carbohydrates such as mannitol, and It is conveniently carried out in the presence of calcium salts. pH is preferably 7.2±0°1 is within the range of

For the purpose of electrofusion, chains of protoplasts are formed and the chains are exposed to direct current (DC ). Formation of protoplast chains can be achieved by, for example, converting the protoplasts into IMH frequency around z and 50 to 150 V/am, more preferably 50 to 1 induced by exposure to an alternating current (AC) electric field with an electric field strength in the range of 00 V/cm. can be guided. have about 5 to 8 protoplasts under such conditions. Protoplast chains are obtained within a few minutes. This protoplast chain is pulsed More preferably, the duration is e.g. 10 to 50 μs, e.g. 04 to 1,7, or to a DC pulse in the range of 1.3 to 1.7 kV/cm. In general, 2 Six pulses result in a good frequency of protoplast fusion. Do it like this The fused protoplasts are held in an electric field for some time, e.g. 1 to 2 seconds. But after this, when the electric field is stopped, the protoplasts eventually regain their spherical shape. .

Fusion products are determined by visual selection in the presence of unfused parental protoplasts or from culture. It can be played after. Visual selection can be done by plant support, e.g. Patnaik. According to the method disclosed in Jens Letters, Vol. 24 (1982), p. 105, By micromanipulating cells or by Based on the cell sorting method disclosed in Science Vol. 45 (1986) p. 133, According to the method for selection and enrichment of plant protoplast heterozygotes, This can be done by using a Rousoter.

Regeneration of the fusion product into a mature plant is carried out by conventional methods, for example as follows. be able to.

The fusion product is combined with trace (micro) and macroscopic (macro) elements, vitamins, and amino acids. containing amino acids and carbohydrates, such as sugars, including glucose and mannitol. culture in a suitable culture medium containing balanced nutrients for protoplast growth. nourish Sugars have a role as a carbon source and osmoticum. suitable Culture media are known in the art, such as B5 medium and KM8p medium. be. Such culture media can regulate cell division and shoot development. Contains plant hormones (auxin and cytokinin). suitable oak Examples of nons include naphthylacetic acid (NAA), 2,4-dichlorophenoxyacetic acid ( 2,4-D) and indoleacetic acid-(IAA). Preferred cytokinins Examples include benzylaminopurine (BAP) and zeatin (Z). one Generally, NAA and 2.4-D are combined with BAP to initiate cell division. used for. In this case, the oquinone/cytokinin ratio is high, e.g. It shouldn't be big.

For culture and regeneration of protoplasts, add fresh culture medium regularly to Add every 4 days.

Once the first cell division has occurred (i.e. this is generally about 5 minutes after protoplast fusion) (occurs after 10 days), does not contain 2.4-D to induce callus formation, and Additional culture medium with a lower concentration of mannitol (to reduce the osmolarity of the culture medium) Add. Such callus induction medium is added regularly, for example every 2 to 4 days. In addition, callus is formed.

Generally after 2 to 4 weeks, once a minute callus has formed, concentrate the mannitol concentration. further reduce the degree of Approximately 1 to 2 weeks after the formation of micro callus , approximately 4 to 6 weeks after protoplast fusion, this microcallus was cultured in the absence of auxin. The callus is then transferred to a solid medium substantially free of auxin or auxin to grow callus. Oh If a quinone is used, this is preferably NAA. This medium is preferably Contains BAP as a cytokinin. Ouchinone present in callus regeneration medium /cytokinin weight ratio is less than 1, more preferably less than 1:5. callus It is convenient to grow under low light intensity, e.g. 3 Wm. When the desired size is reached, approximately 3-5 mm (this is generally not 6 mm in the callus growth medium) After 8 weeks of growth), the callus is transferred to shoot induction medium. callus is seedling While in the row induction medium, the light intensity may be increased stepwise, e.g. by approximately 3 Wm-" every two weeks. Increase up to 18 Wm-2. Calluses are removed periodically, e.g. every 2 to 3 weeks. Transfer to fresh shoot induction medium.

Shoot development is achieved when the auxin:cytokinin weight ratio is less than 1, preferably 1.5. This is accomplished in a shoot regeneration medium containing less than 10% auxin and cytokinin.

Generally, for shoot regeneration, auxin IAA is combined with cytokinin Z and/or or in combination with BAP.

Shoots and primordial shoots are excised and then depleted of cytokinin and auxin. or root on a basic rooting medium such as substantially fire<MS.

During the regeneration process, it is preferable to use glass bottles to reduce vitrification. .

Seedlings obtained according to the method of the present invention can be prepared by a method known per se. can be regenerated into mature, fertile diploid eggplants with genes derived from other Solanaceae plants. I can do it. Such mature plants can be grown in vitro and/or with the desired characteristics. Breeding methods using known breeding techniques, including cross-pollinating techniques that use It can be used for drawings. The product obtained in this way does not exist naturally in eggplant. However, other Solanaceae plants have naturally occurring characteristics such as low resistance to fungal diseases. The diseased eggplant is new and forms part of the present invention.

Preferred fertile diploid eggplants according to the present invention have Vueltinorium wilt and/or has low susceptibility to Fusarium wilt.

The following examples illustrate the invention: The following abbreviations are used in the examples and tables: use.

B5: ○, L, Gamborg, R, A Mirror and Ni, Onma, Expedition Disclosed by Remental Cell Research, Vol. 50, pp. 151-158 (1968) refers to the basic medium used in

6-BA: 6-benzylamitubulin.

CPW: N.S. Banks and P.K. Evans, Plant Scien. Basal medium disclosed in S. Letter, No. 70, pp. 409-416 (1976). means.

2, 4-D.(2,4-nochlorophenoxy)acetic acid.

IAA: Indole-3-acetic acid.

KM8p: K, N, Kao and M, R, Michailuk, blank, 126 vol. 105-110 (1975).

MES: 2-(N-morpholino)ethanesulfonic acid.

Morel, G. Morel and R.M. Wetmore, American Journala The basic culture disclosed in Book of Botany, Vol. 38, pp. 141-143 (1951). means earth.

MS, T, Murashige and F, Skoog, Physiologia Plantarum. 15, pp. 473-497 (1962).

NAA: 1-naphthaleneacetic acid.

ST: J, E, Shepherd and R, E, Traten, Plant Physiolo. No. 60, pp. 313-316 (1977).

W5　■　Nongluteyou etc., Plant Molecular Biology, No. 8 ．． 363-373 (1987).

Example 1 Selection of fusion parent 1.1 Acceptor The following commonly available genotypes/variants were selected from Solanum melongina eggplant. I used Ponica (Slice and Groot) and Regal (North Slack). King). Sterilized seeds were placed in a solid MS medium (culture medium) containing 1% sucrose. (17). See Table 2] at 25°C with a light intensity of 4 Wm-2 and a photoperiod. Germinate at 16/8 hours. The cotyledons are ready for use after about two weeks.

12 Donor Vuelticillium dahliae and Fusarium oxysporum in vivo. Use Solanum torvum seeds from our collection that have been tested for resistance to . Sterilized seeds were grown on MS medium [culture medium (17)] supplemented with 1% sucrose. Germinate with. Callus is induced from the stems of 3 month old plants. Cut this stem into 1cm pieces. Cut into pieces and place on culture medium (1) [Table 1] After 62 months on the device, 2g of callus was placed in 10m1 Culture medium (2) [Table 1] is used to induce a cell suspension. This cell suspension , grown in the dark at 28°C on a rotary shaker (100 rpm). Once a week, 1 Transfer 0 ml of compacted cells to 75 ml of fresh medium. Furthermore, a new 30m1 Add medium after 3 days. For isolation of protoplasts, 4-5 day old cell suspensions should be used. Use a suspension.

Example 2 Isolation of protoplasts 2.1 Acceptor protoplast 2. Cotyledons of appropriate-age plants are used for protoplast isolation. per 10ml of enzyme solution Approximately 1 g of cotyledons are used [culture medium (3). Table 1. ]. The cotyledons were placed in an enzyme solution. Cut into cubes and incubate for 16 hours in the dark at 24°C. If necessary , then incubate this solution for 30 minutes on a rotary shaker (30 rpm), Ru. Next, this protoplast suspension was filtered through a 70 μm filter, and the enzyme solution was Wash with culture medium (4) [Table 3] using 5 ml per 10 ml. pure pu The rotoplast fraction is collected by flotation after centrifugation at 600 rpm for 15 minutes. be able to. Both parts were then washed in W5 medium, and then the protoplasts were Adjust the density with W5.

2.2 Donor protoplast Use cell suspensions at the beginning of the exponential growth phase. This phase lasts 4-5 days of migration. will appear later. For isolation, add 4 ml of compacted cells per 10 ml of enzyme solution. Use [Culture medium (5). Table 1. ]. While shaking slowly (40 rpm) Protoplasts are collected after 16 hours of incubation in the dark at 25°C. suspension The liquid is filtered three times in succession (200 μm, 100 μm, 170 μm). protopra Wash the stock suspension with the same volume of washing solution (culture medium (6), Table 1). Re-suspend the pellet in culture medium (7) [Table 1]. Pure protoplast fraction can be collected by flotation after centrifugation at 600 rpm for 15 minutes.

The following process is essentially the same as that described for acceptor protoplasts. are the same.

Example 3 Donor fusion pretreatment fluorescent staining Fluorescein isothionoanate (FITC) solution 8 μm (5 mg FITC /ml acetone) is added to 10 ml of enzyme solution before incubation.

Fluorescein diaceter) (FDA) 1ml (0.5M mannitol solution A stock solution of 50 μm in 5 ml [1 mg/ml acetone] was added to the incubator. Add to 10 ml of cell suspension before incubation. For staining after protoplast isolation, Add 13 ml of FDA solution per 2 ml of toplast suspension and incubate for 1 hour. start. Wash with W5 medium.

Example 4 Nuclear division Prior to fusion, X-ray radiation is used to disrupt the donor chromosomes. in W5 medium 2ml of protoplasts (1xlO' protoplasts/ml) 50 to 10 Irradiate at 0 krad.

Example 5 Protoplast fusion 51 Chemical fusion The protoplasts of the two fusion partners were brought to a density of 7xlO5 protoplasts/ml. 5 medium and mix the same volume. Add this protoplast suspension to 4x150μ Dispense as a droplet in a Petri dish and leave for 5 minutes to settle. In each droplet, P 50-100 μm PEG solution such that the final concentration of EG is 10-16%. Add medium (8) [Table 3] After incubation for 5-10 minutes, PE G solution was slowly added to 3-4 ml of CaC]□-high pH solution medium for 20 minutes. After that, add 2 ml of culture medium (11) [Table 2] and add 1.5xlO5 protoplasts. /ml final concentration.

5.2 Electric fusion Stainless steel fusion chamber with square wave pulse generator (Combutex(TM)) Use with. Protoplasts were treated with mannitol and calcium before fusion. A washing solution containing [culture medium (10)]. Table 3. ] Wash 3 times. Finally a pro Toplasts are diluted with this medium to a concentration of 5xlO5 protoplasts/ml.

The two fusion partner protoplasts are mixed 1:1 and kept on ice. each melt Use 0.8 ml of protoplasts per batch. Dielectrophoresis of protoplasts or IMHz, 8O-130V/cm AC electric field to achieve chain formation. Apply 4-6 pulses (10-50 μsec x 1.3-1. 7kV/cm). After fusion, 1.6 ml of culture medium (11) [Table 2] was added to the fused protoplasts for a final concentration of 1.6 x 105 protoplasts/ml. Get the concentration.

Example 6 Protoplast culture and regeneration 1.5x in 5 cm TC-Petri dishes 105 protoplasts/ml culture medium (11) [Culture the protoplasts in Table 2 do. Add 1 ml of fresh medium after 3 days. After another 6 days, divide each culture into two dishes. Separate and dilute with 1 ml of culture medium (11).

From now on, 1 ml of culture medium (12) containing low concentration of mannitol and not containing 2.4-D Add twice a week or less frequently depending on growth. protoplus Divide the ingredients into new plates if necessary. Minute callus can be observed after 3 minutes. child At the time point, the mannitol concentration was further reduced by the addition of culture medium (13). let After another week, micro calli were grown in solid culture medium (1 4) and grow under low light intensity (3Wm). After 6 weeks, the callus will grow at 3Wm. It will be 5mm. These are then transferred to the shoot induction culture medium (15). light here The intensity was increased to 3 Wm-2 for 2 weeks, 9 Wm-2 for 2 weeks, and finally 18 Wm''. In this way, it will be strengthened in stages. Transfer callus to new medium every 2 to 3 weeks . Shoots and primordial shoots are cut out and placed on a shoot growth medium (16), followed by hoisting. Induce root formation by placing on culture medium (17) lacking rumons. Reduce vitrification. To prevent this, glass bottles are used during the recycling process.

Test the regenerated plants for resistance by inoculating them with the pathogen. This test , about two weeks after transferring the plants to soil. Fungus Vulticillium da This diploid eggplant against diseases caused by Fusarium oxysporum and Fusarium oxysporum A decrease in susceptibility is observed. This plant is fertile and diploid.

Table 1 1 Macro MS MS Its CPW CPf CPW-Micro MS MS 115M5 CPW CPW CPWN^^ (mg/l) 1.0 - ■^^ (mg/1) 6-BA (mg/l) 0.1 0.1 0.2 - Agar Merck 0.8% - 7 Cero No. Im (R100 Nos' Power) -〇　8%　-Cerulino (H Rubio Chem) -〇1% -Macerase (Calbiochem) -0,025% pH5 , 75, 75, 55, 55, 85, 8 Table 2 One Maca KM8p KM8p KM8p 85 B5 113 MS Manito (g/l) 70 60 10 36.4 - -Nucrose (g/l) 10 10 10 5 20 10 10 Osmolarity 550 450 200 500 15ONAA (mg/l) LO1,01,00,05-- IAA (II1g/l) -0,20,1-6-BA (a+g/l) 0.5 0.5 0.5 0.5 - -ZEA (mg/l) -2,01,0-a Gallose --0.7% -- nubrak Table 3 PEG1500 40% Grinone 5QmM - Glucose 0.3M O, 21M -CaC1□ (mM) 15 50 50 1pH5,810,5- Table 4 Basic medium・ Salt (mg/ml)! IIs B5 K1118pCPW W5KNO3190 0,02500,01900,0101,0NH4NO*1650.0 Kl12P0. 170.0 170.0 27.2CaC122Hzo 44 0.0 150.0 600.0 1480.0 18.300Mg5047H 20370,0250,0300,0246,(]-KCI　300.0　3 70.0 NaH2PO42H20150,0 (NH4)2SO4134,0 NaC19,000,0 micro CoC1□6B20 0.025 0.025 CUSO45 [1200,025 0,0250,025-1(,Bo, 6.2 3.0 KI　O,830,750,16- MnS0.4H2022,310,O Na2MoOJ(200,250,25246,0ZnSO47H208,62 ,0 FeS (L7H2027,827,8 Na2EDTA 37.3 37.3 Glucose 9000 p8 5.8 Table 5 Basic medium: Organic matter addition MS ST KM8p Morel ascorbic acid 20 Biotin 001 Zein hydrolyzate 1000.0 250.0 Choline ct 1,0 Citric acid 40.0 Coconut water ml/1 20.0 Nostain 0.2 Calcium d-hentothenate 10 1.0 Folic acid 0.5 0.4 Fumaric acid 40.0 Grinnon 2.0 2.0 Inositol 100.00 100.0 100°0 100. OL-Gluta Min 5/6 Malic acid 40.0 Sodium pyruvate 20.0 Nicotinamide 5.0 5.0 1.0 1. Op-aminobenzoic acid 0.0 2 Pyridoquinone hydrochloride 5.0 0.5 1.0 1.0 Ribo 7 Uto 7 0.2 Thiamine hydrochloride 1.0 0.5 1.0 1.0 Vitamin 812 0.02 Cellobiose 250.0 Fructose 250.0 Manonose 2500 Ramnose 2500 Ribose 2500 1 lupitor Quinolose -2500 1--N, PCT/εP 92no01035 Front page continuation (51) Int, C1,5 identification code Office mT1 number //Cl2N 5/14 (72) Inventor: Jarrus Souneson, Karlin Ingeierd Sweden, Nis-2225 Furund, Jurivwegen 22 I

Claims (13)

[Claims] 1. Characteristics naturally found in species of the Solanaceae family that are not naturally found in eggplants. 1. A method for producing fertile diploid eggplant comprising: i) donor protoplasts; generation of solanaceous plants with the desired characteristics at a dose sufficient to cause chromosome fragmentation. ii) irradiating such irradiated donor protoplasts; fused with eggplant protoplasts that have a role as receptors (here, the donor - Are protoplasts and acceptor protoplasts different types of explants? and iii) regenerate and mature the resulting fused protoplasts. A method consisting of making a plant. 2. Donor protoplasts are derived from Solanum torvum explant material. The method according to claim 1. 3. Claim 1 or 2, wherein the donor protoplast is derived from stem explant material. The method described in. 4. Claim 1, wherein the acceptor protoplasts are derived from cotyledon explant material. The method according to any one of item 3. 5. Claim 1, wherein the desired characteristic is reduced susceptibility to fungal diseases. The method according to any one of item 4. 6. The desired characteristics are induced by Verticillium and/or Fusarium. 6. The method of claim 5, wherein the method has a reduced susceptibility to fungal diseases. 7. The method according to claims 1 to 6, wherein the irradiation dose is an X-ray dose of 20 to 100 krad. The method described in any one of the above. 8. Characteristics naturally found in species of the Solanaceae family that are not naturally found in eggplants. fertile diploid eggplant, including; 9. 9. The method of claim 8, wherein the characteristics include reduced susceptibility to fungal diseases. Fertile diploid eggplant. 10. This feature is effective against fungal diseases caused by Verticillium. A fertile diploid eggplant according to claim 9, comprising reduced susceptibility. 11. A fungal disease caused by Verticillium dahliae, The fertile diploid eggplant according to claim 10. 12. Claims 8 to 11, wherein the fungal disease is caused by Fusarium. The fertile diploid eggplant described. 13. Fungal disease caused by Fusarium oxysporum, claim The fertile diploid eggplant according to item 12.