WO1992020215A1

WO1992020215A1 - Improvements in or relating to organic compounds

Info

Publication number: WO1992020215A1
Application number: PCT/EP1992/001035
Authority: WO
Inventors: Arnoldus Johannes Kool; Elizabeth Mariette VAN VUURE-RIETVELD; Carin Ingegärd JARL-SUNESSON
Original assignee: Sandoz Ltd.; Sandoz-Patent-Gmbh
Priority date: 1991-05-20
Filing date: 1992-05-11
Publication date: 1992-11-26
Also published as: EP0585275A1; GB9110855D0; JPH06508510A; AU1697892A

Abstract

A method of preparing fertile, diploid eggplants including a trait found naturally in species of the family Solanaceae, such as reduced susceptibility to the fungal disease caused by Verticillium and/or Fusarium, said trait(s) not being found naturally in eggplants which comprises: i) irradiating a Solanaceae protoplast donor having a desired trait with an irradiating dose sufficient to cause fragmentation of the chromosomes of the donor protoplast, ii) fusing said irradiated donor protoplast with an eggplant protoplast serving as an acceptor wherein the donor protoplast and the acceptor protoplast are derived from different explant types and, iii) regenerating the fused protoplasts obtained to mature plants. Also fertile, diploid eggplants comprising desirable traits found naturally in species of the family Solanaceae other than egg plants.

Description

IMPROVEMENTS IN OR RELATING TO ORGANIC COMPOUNDS

The present invention relates to diploid eggplants (Sόlanum melongena) having traits from other Solanaceae and a method of producing such plants.

Background

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.

Detailed Description

According to the present invention there is provided 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:

i) irradiating a Solanaceae protoplast donor having- a desired trait with an irradiating dose sufficient to cause fragmentation of the chromosomes of the donor protoplast

ii) fusing said irradiated donor protoplast with an eggplant protoplast serving as an acceptor wherein the donor protoplast and the acceptor protoplast are derived from different explant types and

iii) regenerating the fused protoplasts obtained to mature plants.

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. Where irradiation is effected with an X-ray source, chromosome fragmentation will in general be obtained by applying a dose of from 20 to 100 krad. Preferably a lethal dose is used, in which case at least 50 krad must be applied. For the purpose of irradiation 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.

Examples of 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. Preferably, 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. Sufficient light must be supplied to allow normal development of plantlets, suitably the equivalent of daylight for a period of an average summer day (ca. 16 hours). In general, 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. by placing 2 to 5 months old plant material in a callus induction medium, preparing a cell suspension from the obtained callus material in an appropriate cell suspension medium. Protoplasts can then be obtained by treating cell suspensions at the beginning of the exponential growth phase with a suitable enzyme solution. Callus induction media, cell suspension media and enzyme solution suitable for the preparation of protoplast suspensions are known in the art.

For selection purposes 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. 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 10⁵ to 10⁶ 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. The PEG has preferably a molecular weight of from 1500 to 6000. In general desired 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 CaCl₂-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. The pH is preferably in the range of from 7.2 + 0.1.

For the purpose of electrofusion, chains of protoplasts are formed and such chains are subjected to a direct current (DC). 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. Under such conditions 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. In general 2 to 6 pulses will result in a satisfactory protoplast fusion frequency. The thus fused protoplasts are conveniently retained for some time in the electric field, e.g. 1 to 2 seconds, before the electric field is turned off to give the protoplasts some time to regain their spherical shape.

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. Examples of suitable auxins are naphthyl acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). Examples of suitable cytokinins include benzyl aminopurine (BAP) and zeatin (Z). In general 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.

For protoplast culture and regeneration fresh culture medium is added regularly, every 2 to 4 days. When the first cell divisions have taken place i.e. which in general occurs about 5 to 10 days after the protoplast fusion, further culture medium is added without 2,4-D, to induce callus formation and with reduced mannitol concentration (to reduce the osmotic pressure of the culture medium). Such callus inducing medium is added regularly, e.g. every 2 to 4 days, to allow the formation of calli.

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 Wπr². When the calli have attained the desired size, ca. 3-5 mm, which is in general after 6 to 8 weeks growth in the callus growth medium, 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 Wπr² every 2 weeks up to a maximum of 18 Wπr². The calli are transferred regularly, e.g. every 2 to 3 weeks to fresh shoot inducing medium.

Shoot 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. In general it will be preferred to employ the 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. During the regeneration process it is preferred to use 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. Thus obtained eggplants having traits not naturally occurring in eggplants but naturally present in other Solanaceae, such as reduced susceptibility to fungal diseases are novel and are part of the invention.

Preferred fertile diploid eggplants of the invention have reduced susceptibility to Verticillium wilt and/or Fusarium wilt.

The following examples illustrate the invention:

The following abbreviations are used in the Examples and Tables:

B5: refers to basic medium disclosed by Gamborg, O.L., Miller, R.A. and Ojima, K. ; Exp. Cell. Res. 50:151-158 (1968)

6-BA: 6-benzylaminopurine

CPW: refers to basic medium disclosed by Banks N.S. and Evans P.K.; Plant Science Letter No. 70: 409-416 (1976)

2,4-D: (2,4-dichlorophenoxy) acetic acid

TAA indole-3-acetic acid

KM8p: refers to basic medium disclosed by Kao, K.N. and Michayluk, M.R. ; Planta 126:105-110 (1975)

MES: 2-(N-morpholino) ethane sulphonic acid

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)

NAA: 1-Naphthaleneacetic acid

ST: refers to basic medium disclosed by Shepard, J.E. and Totten, R.E.; Plant Physiol. 60: 313-316 (1977)

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

1.1 The acceptor

From eggplant, Solanum melongena, the following publicly available genotypes / varieties have been used: Bonica (Sluis & Groot) and Regal (Northrup King). Sterilized seeds are germinated on a solid MS medium [culture medium (17), see Table 2] with 1 X sucrose at 25°C with a light intensity of 4 Wrn^-2 and a photoperiod of 16/8 h. The cotyledons can be used after ca 2 weeks.

1.2 The donor

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.

Example 2: Protoplast isolation

2.1 Acceptor protoplasts

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 μm 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.

2.2 Donor protoplasts

A cell suspension at the begin of the exponential growth phase is used. This phase occurs at 4-5 d after transfer. For the isolation, 4 ml of packed cells are used per 10 ml enzyme solution [culture medium (5), Table 1] . After incubation 16 h in the dark at 25°C with slow shaking (40 rpm), the protoplasts are collected. The suspension is filtered through 3 subsequent sieves (200 ym, 100 μm, 70 μm). 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.

W^! Example 3. Pre-fusion treatment of donor

Fluorescent staining

8 μl of Fluorescein isothiocyanate (FITC) solution (5 mg FITC/ml acetone) is added to 10 ml enzyme-solution before incubation.

1 ml Fluorescein diacetate (FDA) (50 μl stock [1 mg/ml acetone] in 5 ml of 0.5 M mannitol solution) is added to 10 ml cell suspension before incubation. For staining after protoplast isolation, 0.3 ml FDA-solution is added per 2 ml protoplast-suspension and incubated for 1 h. Washing is done with W5 medium.

Example 4: Nuclear fragmentation

X-ray irradiation is used for the fragmentation of the donor chromosomes prior to fusion. Two ml of protoplasts (1 x 10⁶ protoplast/ml) in W5 medium are irradiated with 50 to 100 krad.

Example 5: Protoplast fusion

5.1 Chemical fusion

Protoplasts of the two fusion partners are suspended in W5 medium at a density of 7 x 10⁵ protoplasts/ml, and mixed in equal volumes. The protoplast suspension is distributed as 4 x 150 μl droplets per petridish, and left to settle for 5 min. To each of the droplets, 50-100 μl PEG solution medium (8) [Table 3] are added to give a final concentration of 10 - 16 % PEG. After 5-10 min incubation, the PEG solution is slowly diluted during 20 min with 3-4 ml CaCl₂-high pH-solution medium (9) [Table 3]. The fused protoplasts are washed once with W5. Finally, 2 ml culture medium (11) [Table 2] is added to give a final concentration of 1.5 x 10⁵ protoplasts/ml. 5.2 Electrofusion

A square-wave pulse generator (C0MPTEX^R) is used together with a stainless steel fusion-chamber. 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 10⁵ 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. To obtain dielectrophoresis, or chain-formation, of the protoplasts, a 1 MHz, 80-130 V/cm AC field is applied, for the fusion, 4-6 pulses (10-50 μsec x 1.3-1.7 kV/c ) are used. After the fusion, 1.6 ml culture medium (11) [Table 2] is added to the fused protoplasts, giving a final concentration of 1.6 x 10⁵ protoplasts/ml.

Example 6: Protoplasts culture and regeneration

The protoplasts are cultured at 1.5 x 10⁵ 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). After another week, the microcalli are transferred, together with some liquid culture medium to solid culture medium (14) and grown under a low light intensity (3 Wπr²). 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 Wπr², 2 weeks at 9 Wπr², and finally at 18 Wπr². 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.

TABLE 1

Medium

Basic medium (see hereinaf er) :

- macro MS MS

- micro MS MS

- Org. add. MS MS

mannitol (g/1) 90 90 sucrose (g/1) 30 30 136 150

2,4-D (mg/1) 0.5 0.5 0.1 NAA (mg/1) 1.0 IAA (mg/1) 6-BA (mg/1) 0.1 0.1 0.2 agar merck 0.8 % MES (mM) 3.0 3.0

Cellulase (R50nozuka)

Macerozyme (RlOOnozuka)

Cellulysine (Calbiochem)

Macerase

(Calbiochem)

pH 5.7 5.7 5.5 5.5 5.8 5.8 TABLE 2

Medium 11 12 13 14 15 16 17

Basic medium (see hereinafter):

- macro KM8p KM8p KM8p

- micro B5 B5 B5

- Org. add. KM8P KM8p KM8p

pH 5.6 5.6 5.6 5.7 5.7 5.7 5.7 TABLE 3

Medium 8 10

pH 5.8 10.5

TABLE 4

BASIC MEDIA:

Salts MS B5 KM8p CPW W5 (mg/1) (mod)

Macro

18.300

370.0

9.000.0

Micro

900.0

5 ,8 TABLE 5

Morel

1.0

100.0

1.0

Claims

CLAIMS: -

1. 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 eggplants which comprises:

i) irradiating a Solanacea protoplast donor having a desired trait with an irradiating dose sufficient to cause fragmentation of the chromosomes of the donor protoplast.

iii) regenerating the fused protoplasts obtained to mature plants.

2. A method according to Claim 1, wherein the donor protoplast is derived from Solanum torvum explant material.

3. A method according to Claim 1 or Claim 2, wherein the donor protoplast is derived from stem explant material.

4. A method according to any one of Claims 1 to 3, wherein the acceptor protoplast is derived from cotyledon explant material.

5. A method according to any one of Claims 1 to 4 wherein the desired trait is reduced susceptibility to fungal disease.

6. A method according to Claim 5, wherein the desired trait is reduced susceptibility to fungal diseases caused by Verticillium and/or Fusarium.

7. A method according to any one of Claims 1 to 6, wherein the irradiating dose is an X-ray dose of from 20 to 100 krad.

8. A fertile, diploid eggplant comprising a trait or traits found naturally in species of the family Solanaceae, said trait or traits not being found naturally in eggplants.

9. A fertile, diploid eggplant according to Claim 8, wherein the said trait or traits comprise reduced susceptibility to fungal disease.

10. A fertile, diploid eggplant according to Claim 9, wherein the said trait or traits comprise reduced susceptibility to fungal diseases caused by Verticillium.

11. A fertile, diploid eggplant according to Claim 10, wherein the fungal disease is caused by Verticillium dahliae.

12. A fertile, diploid eggplant according to Claims 8 to 11, wherein the fungal disease is caused by Fusarium.

13. A fertile, diploid eggplant according to Claim 12, wherein the fungal disease is caused by Fusarium oxysporum.