EP0585275A1 - Ameliorations relatives a des composes organiques - Google Patents

Ameliorations relatives a des composes organiques

Info

Publication number
EP0585275A1
EP0585275A1 EP92909693A EP92909693A EP0585275A1 EP 0585275 A1 EP0585275 A1 EP 0585275A1 EP 92909693 A EP92909693 A EP 92909693A EP 92909693 A EP92909693 A EP 92909693A EP 0585275 A1 EP0585275 A1 EP 0585275A1
Authority
EP
European Patent Office
Prior art keywords
protoplast
donor
diploid
fertile
eggplant
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP92909693A
Other languages
German (de)
English (en)
Inventor
Arnoldus Johannes Kool
Elizabeth Mariette Van-Vuure-Rietveld
Carin Ingegärd JARL-SUNESSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sandoz AG
Original Assignee
Sandoz AG
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.)
Filing date
Publication date
Application filed by Sandoz AG filed Critical Sandoz AG
Publication of EP0585275A1 publication Critical patent/EP0585275A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • A01H6/826—Solanum melongena [eggplant]

Definitions

  • the present invention relates to diploid eggplants (S ⁇ lanum melongena) having traits from other Solanaceae and a method of producing such plants.
  • Eggplants do not have natural resistance against certain fungal diseases. They are very susceptible to fungal diseases such as Verticillium wilt and Fusarium wilt. Sources of natural resistance against such diseases are available in other Solanaceae such as Solanum torvum which shows natural resistance against Verticillium and Fusarium. Interspecific sexual crossings between eggplant and other Solanaceae give sterile progeny. This is i.a. known for sexual crossings between eggplant and S. torvum. Furthermore, such crossings are inefficient and laborious, even when embryo culture is used (K.R. McCammon et al in Hort Science 18(6) 894-895 (1983). Also, this cross can only be made when S. torvum is used as pollinator.
  • Protoplast fusion between eggplant and S. torvum has for various reasons not given the desired result.
  • the protoplast fusion is inefficient, resistant plants whenever obtained are aneuploid, i.e. not diploid, thus the progeny shows a fertility ranging from low fertility to sterility.
  • the fusions between eggplant and S. torvum protoplasts reported in literature (e.g. A. Guri et al in Theor. Appl. Genet 76 (1988) 490-496) have been effected with plant material of the same kind (mesophyll x mesophyll) and without irradiation of the donor.
  • a method of preparing fertile, diploid eggplants including a trait found naturally in species of the family Solanaceae, said trait not being found naturally in eggplant which comprises:
  • Fragmentation of the chromosomes of the Solanaceae protoplast donors showing resistance against fungal diseases can be obtained in a manner known per se e.g. by irradiation.
  • the irradiation can be effected with the aid of gamma, UV or X-rays.
  • chromosome fragmentation will in general be obtained by applying a dose of from 20 to 100 krad.
  • a lethal dose is used, in which case at least 50 krad must be applied.
  • the protoplasts are conveniently in suspension in an appropriate protoplast suspension medium, such as a protoplast washing medium.
  • the donor and acceptor protoplasts employed herein as starting material may be obtained in a manner known per se from appropriate plant cells. It is thereby essential, that the plant materials employed for the preparation of the protoplasts of the donor and the acceptor are of different origin or type.
  • plant material types suitable for use as starting material for the preparation of protoplasts are leaf material, stem material, root material, cotyledon and the like.
  • acceptor protoplasts are derived from cotyledons.
  • Donor protoplasts are preferably derived from stem material.
  • the plant material is conveniently obtained by germination of sterilised seeds in an appropriate hormone-free basal nutrient medium, e.g. an hormone-free Murashige and Skoog (MS) basal medium containing salts, vitamins supplemented with a carbohydrate such as sucrose under seed germination conditions. Such conditions are known per se.
  • the germination temperature lies conveniently in the range of from 22° C to 28°C.
  • cotyledons of ca. 9 to 17 days are suitable for use as protoplast starting material.
  • Protoplasts from cotyledons are for example obtained by treating finely diced cotyledons with an enzyme solution comprising appropriate enzymes for destruction of the cell walls such as hemicellulase, cellulose and/or pectinase in a manner known per se.
  • Cell suspension from stem, leaf or root material are likewise obtained in a manner known per se, e.g.
  • the parental protoplasts are for example stained with fluorescent dyes, e.g. fluorescein isothiocyanate (FITC) fluorescein diacetate (FDA), or for one of the fusion partners auto-fluorescence of the chlorophyll may be used.
  • fluorescent dyes e.g. fluorescein isothiocyanate (FITC) fluorescein diacetate (FDA)
  • FITC fluorescein isothiocyanate
  • FDA fluorescein diacetate
  • Such staining may be carried out in a manner known per se.
  • the protoplast fusion in the method of the invention can be carried out in a manner known per se, e.g. by so-called chemical fusion or by electrofusion under conditions known in the art.
  • the protoplasts of different origin are mixed and preferably concentrated to a final concentration of from 10 5 to 10 6 protoplasts per ml.
  • the fusion partners are preferably mixed in a 1:1 ratio.
  • the chemical protoplast fusion is conveniently effected by agglutination of the protoplasts in an appropriate protoplast agglutination solution followed by membrane fusion in a high pH solution.
  • the protoplast agglutination solution comprises essentially polyethylene glycol (PEG) causing agglutination, an osmoticum, e.g. a carbohydrate such as mannitol or glucose to avoid bursting of the cells, and calcium salts.
  • PEG has preferably a molecular weight of from 1500 to 6000.
  • agglutination is obtained in a solution having a final concentration of from 10 to 16 % by weight of PEG.
  • the thus obtained suspension of agglutinated protoplast cells is slowly diluted with CaCl 2 -high pH solution to let the membranes fuse.
  • Electrofusion is conveniently effected in the presence of an osmoticum, preferably a carbohydrate such as mannitol, and calcium salts.
  • an osmoticum preferably a carbohydrate such as mannitol, and calcium salts.
  • the pH is preferably in the range of from 7.2 + 0.1.
  • chains of protoplasts are formed and such chains are subjected to a direct current (DC).
  • DC direct current
  • Protoplast chain formation can for example be induced by subjecting the protoplasts to an alternating current (AC) electric field having a frequency of around 1 MHz and an electric fieldstrength in the range of from 50 to 150 V/cm, more preferably of from 50 to 100 V/cm.
  • AC alternating current
  • protoplast chains of ca. 5 to 8 protoplasts are obtained within a few minutes.
  • the protoplast chains are subjected to a DC-pulse ranging from e.g. 0.4 to 1.7, more preferably 1.3 to 1.7 kV/cm with a pulse duration of e.g. 10 to 50 us.
  • the fusion products may be regenerated in the presence of non-fused parental protoplasts or after optical selection from the culture.
  • the optical selection may be performed by micro-manipulation of the cells, e.g. according to the procedure disclosed by Patnaik et al, Plant Science Letters 24 (1982) 105, for the manual isolation and identification of plant heterokaryons, or by using a cell sorter, e.g. according to the procedure disclosed by Glimelius et al, Plant Science 45 (1986) 133, for the selection and enrichment of plant protoplast heterokaryons by cell sorting.
  • Regeneration of the fusion products to mature plants can be carried out in conventional manner, e.g. as follows:
  • the fusion products are cultivated in an appropriate culture medium comprising a well-balanced nutrient supply for protoplast growth, containing micro- and macro-elements, vitamins, amino acids and carbohydrates, e.g. sugars including glucose and mannitol.
  • the sugars serve as a carbon source as well as an osmoticum.
  • Appropriate culture media are known in the art, e.g. B5 medium and KM8p medium.
  • Such culture media comprise plant hormones (auxins and cytokinins) which are able to regulate cell division and shoot generation.
  • auxins are naphthyl acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA).
  • cytokinins examples include benzyl aminopurine (BAP) and zeatin (Z).
  • BAP benzyl aminopurine
  • Z zeatin
  • NAA and 2,4-D are used in combination with BAP to initiate cell division.
  • the ratio auxin/cytokinin must then be high, e.g. greater than 1.
  • the microcalli When icrocalli are formed, in general after 2 to 4 weeks, the mannitol concentration is reduced further. About 4 to 6 weeks after the protoplast fusion, resp. ca. 1 to 2 weeks after microcalli formation, the microcalli are transferred to solid medium lacking or substantially devoid of auxins to allow callus growth. Where auxin is employed it is preferably NAA.
  • the medium comprises preferably BAP as a cytokinin.
  • the weight ratio of auxin: cytokinin present in the callus regeneration medium is below 1, more preferably below 1:5.
  • the calli are conveniently grown under low light intensity , e.g. 3 W ⁇ r 2 . When the calli have attained the desired size, ca.
  • the calli are transferred to a shoot inducing medium. While the calli are in the shoot inducing medium, the light intensity is increased stepwise for example by ca. 3 W ⁇ r 2 every 2 weeks up to a maximum of 18 W ⁇ r 2 .
  • the calli are transferred regularly, e.g. every 2 to 3 weeks to fresh shoot inducing medium.
  • Plant development is obtained in a shoot regeneration medium comprising auxins and cytokinins in a weight ratio auxin: cytokinin which is below 1, preferably below 1:5.
  • auxins and cytokinins in a weight ratio auxin: cytokinin which is below 1, preferably below 1:5.
  • auxin IAA in combination with the cytokinins Z and/or BAP for shoot regeneration.
  • the shoots and shoot primordia are dissected out and then rooted on a basic rooting medium such as MS, devoid of cytokinins and lacking or substantially devoid auxins.
  • a basic rooting medium such as MS, devoid of cytokinins and lacking or substantially devoid auxins.
  • glass jars to decrease vitrification.
  • the plantlets obtained according to the method of the invention can be regenerated in a manner known per se to mature fertile diploid eggplants having in their genome genes from other Solanaceae.
  • Such mature plants can be used in a breeding program employing breeding techniques known per se, including in vitro and/or crossing techniques using eggplants having desired traits.
  • breeding techniques known per se including in vitro and/or crossing techniques using eggplants having desired traits.
  • Preferred fertile diploid eggplants of the invention have reduced susceptibility to Verticillium wilt and/or Fusarium wilt.
  • B5 refers to basic medium disclosed by Gamborg, O.L., Miller, R.A. and Ojima, K. ; Exp. Cell. Res. 50:151-158 (1968)
  • CPW refers to basic medium disclosed by Banks N.S. and Evans P.K.; Plant Science Letter No. 70: 409-416 (1976)
  • KM8p refers to basic medium disclosed by Kao, K.N. and Michayluk, M.R. ; Planta 126:105-110 (1975)
  • Morel refers to basic medium disclosed by Morel, G. and Wetmore, R.M.; Am. J. Bot. 38: 141-143 (1951)
  • MS refers to basic medium disclosed by Murashige, T. and Skoog, F.; Physiol. Plant. 15: 473-497 (1962)
  • W5 refers to basic medium disclosed by Negrutiu I. et al; Plant Md. Biol. No. 8: 363-373 (1987) Example 1. Selection of fusion parents
  • Seeds of Solanum torvum from an accession, tested in vivo for resistance against Verticillium dahliae and Fusarium oxysporum, are used. Sterilized seeds are germinated on a MS medium [culture medium (17)] with 1 X sucrose. Calli are induced from stems of 3-months-old plants. The stems are cut into 1 cm pieces and put on culture medium (1) [Table 1]. After 2 months, 2 g of calli are used for 10 ml of culture medium (2) [Table 1] to induce a cell suspension. The cell suspensions are grown in darkness at 28°C on a rotary shaker (100 rpm). Once a week, 10 ml of packed cells are transferred to 75 ml new media. Another 30 ml new media are added after 3 days. For protoplast isolation, 4-5-days-old cell suspensions are used.
  • the cotyledons of 2-weeks-plants are used for the isolation of protoplasts. About one g cotyledons are used per 10 ml enzyme solution [culture medium (3), Table 1]. The cotyledons are diced finely in the enzyme solution, and incubated 16 h in the dark at 24°C. If needed, the solution is then incubated for 30 min on a rotary shaker (30 rpm). The protoplasts suspension is then filtered through a 70 ⁇ m filter and washed with culture medium (4) .[Table 3] , using 5 ml per 10 ml enzyme solution. A fraction of pure protoplasts can be collected by flotation after centrifugation at 600 rpm for 15 min. This fraction is then washed in W5 medium, after which the density of protoplasts is adjusted with W5.
  • a cell suspension at the begin of the exponential growth phase is used. This phase occurs at 4-5 d after transfer.
  • 4 ml of packed cells are used per 10 ml enzyme solution [culture medium (5), Table 1] .
  • the suspension is filtered through 3 subsequent sieves (200 ym, 100 ⁇ m, 70 ⁇ m).
  • the protoplast suspension is washed with an equal volume of washing solution [culture medium (6), Table 1].
  • the pellet is then resuspended in culture medium (7) [Table lj .
  • a fraction of pure protoplasts can be collected by flotation after centrifugation at 600 rpm for 15 min. The following steps are basically identical to those described for the acceptor protoplasts.
  • FITC Fluorescein isothiocyanate
  • Fluorescein diacetate (FDA) 50 ⁇ l stock [1 mg/ml acetone] in 5 ml of 0.5 M mannitol solution
  • FDA Fluorescein diacetate
  • 0.3 ml FDA-solution is added per 2 ml protoplast-suspension and incubated for 1 h. Washing is done with W5 medium.
  • X-ray irradiation is used for the fragmentation of the donor chromosomes prior to fusion.
  • Two ml of protoplasts (1 x 10 6 protoplast/ml) in W5 medium are irradiated with 50 to 100 krad.
  • Protoplasts of the two fusion partners are suspended in W5 medium at a density of 7 x 10 5 protoplasts/ml, and mixed in equal volumes.
  • the protoplast suspension is distributed as 4 x 150 ⁇ l droplets per petridish, and left to settle for 5 min.
  • 50-100 ⁇ l PEG solution medium (8) [Table 3] are added to give a final concentration of 10 - 16 % PEG.
  • the PEG solution is slowly diluted during 20 min with 3-4 ml CaCl 2 -high pH-solution medium (9) [Table 3].
  • the fused protoplasts are washed once with W5.
  • 2 ml culture medium (11) [Table 2] is added to give a final concentration of 1.5 x 10 5 protoplasts/ml.
  • a square-wave pulse generator (C0MPTEX R ) is used together with a stainless steel fusion-chamber.
  • the protoplasts Prior to the fusion, the protoplasts are washed three times with a washing solution [culture medium (10), Table 3], containing mannitol and calcium. Finally, the protoplasts are diluted with this medium to a concentration of 5 x 10 5 proto ⁇ plasts/ml. Protoplasts of the two fusion partners are mixed 1:1 and kept on ice. For each fusion, 0.8 ml protoplasts are used.
  • the protoplasts are cultured at 1.5 x 10 5 protoplasts/ml culture medium (11) [Table 2] in 5 cm TC-petri dishes. One ml new medium is added after 3 days. After another 6 days, each culture is divided into two dishes and diluted with 1 ml culture medium (11). Henceforth, 1 ml of culture medium (12) having a lower mannitol concentration and without 2,4-D is added twice a week or less, depending on the growth. The protoplasts are distributed in new dishes as needed. Microcalli can be observed after 3 weeks. At this point, the mannitol concentration is lowered even further by addition of culture medium (13).
  • the microcalli are transferred, together with some liquid culture medium to solid culture medium (14) and grown under a low light intensity (3 W ⁇ r 2 ). After 6 weeks, the calli will be 3-5 mm. They are then transferred to a shoot inducing culture medium (15). The light intensity is now increased stepwise: 2 weeks at 3 W ⁇ r 2 , 2 weeks at 9 W ⁇ r 2 , and finally at 18 W ⁇ r 2 . Every two to three weeks, the calli are transferred to new medium. Shoots and shoot primordia are dissected out and put on a shoot developing culture medium (16), followed by culture medium (17) without hormones to induce root formation. To decrease vitrification, glass jars are used during the regeneration process.
  • Regenerated plants are tested for resistance by inoculation with the pathogen. The tests are carried out ca. two weeks after transfer of the plants to soil. Reduced susceptibility of the diploid eggplants to fungal diseases of Verticillium dahliae and Fusarium oxysporum is observed. The plants are fertile and diploid.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Physiology (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)

Abstract

Procédé de préparation d'aubergines diploïdes fertiles caractérisées par une particularité rencontrée naturellement dans des espèces de la famille des Solanaceae, telle qu'une faible susceptibilité à contracter des maladies fongiques provoquées par Verticillium et/ou Fusarium, ledit ou lesdites particularités n'étant pas rencontrées naturellement dans des aubergines, ledit procédé consiste à: i) à irradier un protoplaste donneur de Solanaceae ayant une particularité voulue avec une dose d'irradiation suffisante pour provoquer la fragmentation des chromosomes du protoplaste donneur ii) à fusionner ledit protoplaste donneur irradié avec un protoplaste d'aubergine servant d'accepteur, ledit protoplaste donneur et ledit protoplaste accepteur étant dérivés de différents types d'explants, et iii) à régénérer les protoplastes fusionnés obtenus afin d'amener des plantes à maturité. L'invention concerne également des aubergines diploïdes fertiles caractérisées par des particularités désirables rencontrées naturellement dans des espèces de la famille des Solanaceae autres que des aubergines.
EP92909693A 1991-05-20 1992-05-11 Ameliorations relatives a des composes organiques Withdrawn EP0585275A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB919110855A GB9110855D0 (en) 1991-05-20 1991-05-20 Improvements in or relating to organic compounds
GB9110855 1991-05-20

Publications (1)

Publication Number Publication Date
EP0585275A1 true EP0585275A1 (fr) 1994-03-09

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EP92909693A Withdrawn EP0585275A1 (fr) 1991-05-20 1992-05-11 Ameliorations relatives a des composes organiques

Country Status (5)

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EP (1) EP0585275A1 (fr)
JP (1) JPH06508510A (fr)
AU (1) AU1697892A (fr)
GB (1) GB9110855D0 (fr)
WO (1) WO1992020215A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CO6210117A1 (es) * 2009-04-21 2010-10-20 Garces Lucia Atehortua Metodo para multiplicar tejido celular de jatropha curcas

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9220215A1 *

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Publication number Publication date
JPH06508510A (ja) 1994-09-29
WO1992020215A1 (fr) 1992-11-26
AU1697892A (en) 1992-12-30
GB9110855D0 (en) 1991-07-10

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