FR2661421A1 - PROCESS FOR THE TRANSFORMATION OF SOMATIC EMBRYOS OF USEFUL PLANTS. - Google Patents

Abstract

The invention relates to a method for transforming somatic embryos of useful plants. Immature somatic embryos, in a dried state, are incubated with a solution containing DNA or RNA. The phosphinothricin acetyl transferase gene is used, for example, as DNA. The embryos thus transformed can be used for the preparation of artificial seeds.

Description

1 -

  PROCESS FOR THE TRANSFORMATION OF SOMATIC EMBRYOS FROM

USEFUL PLANTS

  Dicotyledonous plants are essentially transformable via Ti plasmid vector systems using Agrobacterium tumefaciens Until now, this pathway has only been adapted to a small number of monocotyledonous plants Direct gene transfer , without bacterial or viral vectors, has heretofore yielded better results with monocotyledonous plants. In particular, the use of protoplasts as objects to be transformed has proved, at least in the case of rice and

  maize, on the cellular level, as a practicable way.

  However, the ability to regenerate protoplasts in

  Fertile plants is problematic.

  In WO 88/09374 there is disclosed a method in which sex embryos of useful plants serve as receptor systems for foreign genetic information. Ripe seed embryos are able, during swelling, to isolated state or mechanically released in the seed, to receive foreign genetic information One to three days after the transformation, it was possible to highlight in the embryos an expression of: transient gene However, it has not managed so far to find transformed cells stably in the plants produced or in their offspring. It has now been found, surprisingly, that immature somatic embryos can be dried and can be rehydrated in a DNA solution,

  foreign genetic information.

  The invention therefore relates to an embryo transformation method which is characterized by incubating immature somatic embryos in the dried state with a solution.

containing DNA or RNA.

  2 Somatic monocotyledonous embryos transformed. 3 Monocotyledonous plants obtainable at

  from transformed somatic embryos.

  4 The use of dried, monocotyledonous, somatic, and possibly transgenic, embryos for the

  preparation of artificial seeds.

  The use of transformed somatic embryos for the regeneration and cultivation of useful plants

fertile transgenic.

  The invention will be explained in detail in the following, in particular, in its modes of

preferred embodiments.

  For carrying out the invention, immature somatic embryos of all plants can be used basically. Useful plant cells, such as grasses such as maize, wheat and barley, woody plants such as lamb, are preferably used. spruce, American larch, or pines, or herbaceous herbaceous weeds such as alfalfa, rapeseed, soybean, cotton, celery, and carrot Corn, canola and alfalfa are preferred. preferably, monocotyledonous embryos are used, grasses, particularly maize, being particularly preferred Immature somatic embryos can be obtained from both embryogenic callus cultures and organ cultures. By "immature embryos" is meant embryos at various stages of development, which differentiate from embryogenic cell associations in plant cell cultures or plant organ cultures and have reached a maximum of 75% of the total population. size of a mature embryo, derived from seed Organ cultures are well-differentiated embryos of zygotic or somatic origin, which form in a suitable medium adventitious embryos "Dried state" means a water content of 20% maximum The water content of the embryos is preferably 5 to 15% starting from a cell in the state

turgid.

  The embryos are subjected to drying in sterile conditions for a period of 10 to 18 days. The cells are preferably previously cultured for at least 7 days, more preferably for about 2 to 8 weeks, in a high-nutrient medium. Sugar or sugar alcohol The concentration of sugar or sugar alcohol should be about 8 to 12% based on the weight of the nutrient medium, preferably 9 to 10%. sucrose, fructose, or sorbitol or mannitol in combination or singly

  preferably uses sucrose.

  Dried embryos, which have been treated as described, may be stored for more than one year at room temperature in the air-dried state. These embryos may also be proposed as certified artificial seeds. then monocotyledonous embryos, for example grasses, in particular maize. For rehydration and for resumption of growth, the dried embryos are deposited on a growth medium with or without agar having a sugar content,

  sucrose in particular, from 2 to 6%.

  For transformation, the dried cells are bathed in an aqueous solution of DNA or RNA, which may contain

  also constituents of the medium and buffer salts.

  Any DNA or RNA can be introduced into the cell. Advantageously, it is possible to use selectable 4-marker genes, such as, for example, the gene for phosphinothricinacetyltransferase, hygromycinephosphotransferase, acetolactatesynthetase,

  5-enolpyruvyl-shikimate-phosphate synthetase (EPSP-

  synthetase), glutaminesynthetase or transaminase It is of course also possible to introduce genes whose expression can not be selected by the growth of the plant. Such genes may, for example, code for Bacillus thuringiensis 5-endotoxin or for resistance. viruses, such as genes for virus coat proteins, particularly cucumber mosaic virus, alfalfa mosaic virus or blackberry mosaic virus. use the chitinase gene (s), which enhance the photosynthetic productivity of the plant, for example the gene for phosphoenolpyruvatecarboxylase or bacterial asparaginesynthetase (Nakamura, MM et al; Nucleic Acids Research 9, 4669 (1981)). with 1 eg phosphinothricinacetyltransferase of Streptomyces viridochromogenes (Wohlleben, W et al; 80, 25-27 (1988)), which can be Suitably modified for expression in plants By incorporation of this gene, maize plants and their offspring become resistant to phosphinothricin (Glufosinate) and Bialaphos. After synthesis or isolation, the gene can be multiplied by conventional methods, for example by transformation of E. coli or by a "polymerase chain reaction". The isolated functional gene can then advantageously be introduced directly or onto any cloning vector, preferably BR 322, p UC 18 or p DH 51 (Pietrzak, M et al. NAR 14, 5857 (1986)) with or without an active promoter in plants, in embryo cells. The DNA promoter can also be used in the form of a complex with histones, as described, for example, in German patent application P 40 05 152 8. somatic embryos to a treatment that causes breaks in DNA strands, for example UV or X-ray irradiation Subsequent selection is not performed, as described in WO 88/09374 in terms of the p whole, but in terms of the unit cell In this measure, we can accumulate and discover even extremely rare transformation phenomena,

for an acceptable cost.

  From transformed somatic embryos, preferably from monocotyledonous embryos, for example from grasses, in particular maize, it is possible to regenerate the

  fertile plants by methods known per se.

  In addition, transformed somatic embryos, in particular monocotyledonous embryos, may

  also be used, as already mentioned above.

  for untransformed embryos, after selection and multiplication which are effected by methods known per se, as artificial seeds.

  Appropriate grass embryos, especially maize.

  The invention further relates to a method for the protection of monocotyledonous plants by selectively destroying weeds with phosphinothricin, by planting on the cultivated surfaces plants which can be obtained by the process according to the invention and equipped with N tty è N ede 1 a

  phosphinothricinacetyltransferase, and their offspring.

  The following examples are given as

  further illustration of the invention.

Examples

  1 Launch of embryogenic corn callus cultures and their preconditioning for the

drying.

  Immature embryos suitable for culturing tissues of tissue culture maize genotypes, for example H 99, B 73, A 188, W 64 A, and their hybrids, are prepared with a size of embryos of 1- 1.5 mm from surface-sterilized caryopses under aseptic conditions and cultured on N 6 medium according to Duncan (Planta 165, 322 (1985)) which contains 9% sucrose and 1 mg / l The embryogenic callus, which forms on the edges of the scutellum, is subcultured on N 6 medium with 1 mg / l of dicamba and 3% of sucrose at an interval of 3 to 4 weeks. embryogenic callus cultures suitable for the transformation process may be subcultured as described herein for one to three years retaining their ability to regenerate. The culture is carried out at 25 ° C, on a light / dark rhythm of 12 hours, the

about 1000-5000 lux.

  Before drying, the calli are cultured for 5 days on a medium N 6 to 9% sucrose. The osmotic force caused by the increased sucrose content increases the survival capacity in the drying process.

ulterior.

  2 Drying embryogenic corn callus The preconditioned callus is transferred to sterile Petri dishes equipped with ventilation cams, without culture medium The Petri dishes are placed in the dark, at room temperature, preferably at 23 c C , 3 C per Petri dish, a minimum of 1 g and a maximum of 2 g of callus tissue are dried. After two weeks, the embryogenic calli have lost more than 80% of their weight.

initial.

  Storage of dried embryogenic calli As soon as the calli still have 15 5% of their starting weight, they can be stored for an unlimited duration at -70 ° C. or -193 ° C. while maintaining their survival capacity. liquid nitrogen can be done directly or through a cooling 7 - progressive Cooling with a drop in temperature

  defined, for example 1c C / minute, is not necessary.

  4.Rehydration of embryos For rehydration and resumption of growth, embryogenic calli are placed on an agar medium (medium N 6 to 3% of sucrose) Rehydration and revitallization are also carried out on other media, by Example on Murashige-Skoog medium (Physiol Plant 15, 473 (1962) and with higher sucrose contents With the exception of autotrophic maize cultures using auxin, a content of 0.5-2 mg / l dichlorophenoxyacetic acid or 3,6-dichloro-anisic acid

  (dicamba) in the middle is advantageous In one half

  hour, the callals take back in the middle the water lost during drying The parts of tissue which consist of somatic embryos at various stages of development are able

to grow again.

  The rehydrated tissues are first cultured for 7 days at 25 ° C in the dark, then under 1000-3000 Lux during a 12-hour illumination period. In the light, the regions of the surviving embryogenic calli green 7-10. days Green tissues or tissues with visible growth are transferred during 2

  at 3 weeks after rewetting on fresh medium.

  5. Transformation of the embryogenic callus On the agar medium, (medium N 6 with 1 mg / l of 2,4-dichlorophenoxyacetic acid and 3% of sucrose) the DNA solution is pipetted in drops of 20-50 g, following the size of the dried callus fragments The transformation solution contains 20 g of plasmid DNA / ml. It is prepared by mixing a fundamental double concentration DNA solution in TE buffer (10 mM Tris / 1 m EDTA). M, pH 7.0) with 1: 1 liquid N 6 medium immediately before use. For transformation, the gene of the

  synthetic phosphinothricin-acetyltransferase indicated below.

  Below under the control of the cauliflower mosaic virus 35 S transcript promoter (see sequence below) or the synthetic gene of European patent application CP O 275 957 This chimeric gene is integrated into the polylinker EcoRI site of p UC 18. In most experiments, an uncut UC 18-35 S acetyltransferase plasmid was used. In the individual experiments, the plasmid was cut with Eco RI and the plasmid mixture puc 18 linearized and insertion segment of the S1 acetyltransferase at 1.1 Kb is used

for the transformation.

  The dried embryogenic calli are placed in the drops containing the DNA on the agar medium The embryogenic callus, which in the dried state is more than 3 mm, is separated through a sieve smaller pieces Small fragments are used without other pretreatment The larger fragments are ground in the dried state either by carefully pounding them in a mortar or pretreating them by immersion in liquid nitrogen. In this way, fissures are formed in the dried embryogenic calli which promote the penetration of the transformation solution into deeper layers of the tissue In -30 minutes, the calli absorb the DNA solution The calli can then be subjected to a subsequent culture as described in Example 4 It s It is found advantageous to irradiate the calluses with a short wavelength UV light 2 to 24 hours after rehydration, for 5 to 20 seconds. s Petri in the open state on a UV light source (254 nm; 8000 1 g W / cm 2), so that the calli are exposed

directly to UV light.

  6 Culture of the DNA-treated tissues The culture is carried out during the first four weeks in a manner identical to that of the non-transformed embryogenic calli (see Example 4). Next, the surviving embryogenic callus fragments are transformed on a modified N 6 medium, which is free of glutamine and

  of casein hydrolyzate, but which contains 20 mg / L of

  phosphinothricin By this amount of phosphinotricin, the untransformed embryogenic tissue is immediately destroyed, but it plays the role of a nourishing culture for cells stably transformed in the tissue assembly In this case, the concentration of phosphinothricin is sufficiently high to allow

  very low growth of untransformed cells.

  Transgenic corn cells, which express the phosphinothricin-acetyltransferase gene, have a selective advantage and can accumulate in the cell population. As the selection for the expression of the marker gene begins no earlier than four weeks after the transformation, a possible transient expression of the transferred DNA only plays a secondary role. After 4 to 6 weeks, the embryogenic calli are transformed on of

  N 6 fresh medium with 20 mg / l of L-phosphinothricine.

  After a culture time of 8 to 10 weeks in total in the presence of 20 mg / l (L-phosphinothricin-NH 4 1 10-4 M), the non-transgenic maize tissue shows no growth on a medium free of amino acids Some records survive this selection These pieces are being transferred to

  fresh N 6 medium with 100 mg / l L-phosphinothricin-

  ammonium Two pieces of callus out of about 10000 show a growth comparable to the control call on a phosphinothricin-free medium These tissues are alone used for the subsequent biochemical characterization and for tests of

regeneration.

  7 Culture of Transgenic Regenerate Plants The transformed embryogenic maize tissue is grown on

  a hormone-free N 6 medium in the presence of L-

  Phosphinothricin 5 10-4 M On this medium, embryos of maize which express in a

  sufficiently marked the phosphinothricin gene

  - acetyltransferase (PAT gene), to give plants Unprocessed embryos or those with very low PAT activity, die As soon as the leaves of plants in vitro have reached a length of 4-6 mm, can be passed through the soil After root washing is washed out, the plants are planted in a mixture of silt, sand, vermiculite, and unitary soil, and are adapted for the first three days. after planting in 90-100% air-moisture culture The crop is grown in an air-conditioned room with a duration of illumination of 14 hours, at about 25000 Lux, at a daytime temperature and at night 23 2/17 10 C Adapted plants are

  grown at 65 5% air humidity.

  8 Demonstration of the expression of the transferred phosphinothricinacetyltransferase gene a) Aspersion of the transgenic plants with phosphinothricin 3 to 4 weeks after transfer to the soil, the transgenic regeneration plants and the control regenerating plants obtained from non-DNA-treated calli are sprayed with phosphinothricin in commercial formulas (Basta R) Phosphinothricin is used which corresponds to treatment with 2.0 kg of active substance / ha The herbicide is applied in a quantity of 50 ml / m 2 (= 500 1 / ha) Plants with no PAT activity are killed by this amount of phosphinothricin in 7-10 days. On the other hand, maize plants with PAT are absolutely undamaged, or, in the case of low PAT activity, have local leaf spots that do not measurably affect this treated plants, according to the degree

expression of the gene.

  b) Each time 100 mg corn and leaf tissue samples were frozen in 1.5 ml reaction vessels in liquid nitrogen and homogenized with a stainless steel micro-pestle. adapted to

  the shape of the container is added 100 μl of Tris buffer

  EDTA (Tris 50 mM / EDTA 2 mM, pH 7.5) The homogenate is centrifuged at 12000 rpm for one minute in an Eppendorf centrifuge. The clear supernatant is pipetted off and the pellet is thrown away. centrifugation The protein content of the crude extract is determined by means of the Bio-Rad protein assay. For the determination of the PAT, aliquots of the crude extracts which contain in each case 20 g of protein are taken. Acetyl-Co-A and 14 C-Lphosphinothricin The final concentration in the incubation mixture is 1 mM for acetyl-Co-A and 0.1 mM for 14 C-Lphosphinothricin On The samples are briefly mixed and incubated for one hour at 370 ° C. The embryo-reaction is then stopped by the addition of a half volume of 12% trichloroacetic acid, mixed thoroughly and placed on ice for 10 minutes. It is then centrifuged at 12000 rpm for 10 minutes and n supernatant liquid is used for thin-layer chromatography On a thin-layer chromatography plate (Cellulose F 254, Merck Company), 6 mixtures are applied each time.

  incubation, and also the reference substances 14 C-

  phosphinothricin and 14 C-acetylphosphinothricin As the eluent for thin layer chromatography,

  either n-propanol: NH 3 at 25% (3: 2), or pyridine: n-

  butanol: glacial acetic acid: water (3: 5: 1: 4).

  The dried thin layer chromatography plate is packed in a plastic sheet and exposed in a X-ray film cassette. On the X-ray film developed, PAT activity is recognizable on the acetylphosphinothricin band (which does not exist transgenic tissue) Phosphinothricin and acetylphosphinothricin are readily separable by thin layer chromatography

  in the eluents indicated above.

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Claims (11)

  A method of transforming embryos, characterized in that immature somatic embryos are incubated in a desiccated state with a solution containing DNA or RNA. Process according to Claim 1, characterized in that somatic embryos of embryogenic callus cultures are used. Process according to claim 1, characterized in that   Somatic embryos of organ cultures are used.   Process according to one or more of claims 1 to   3, characterized in that somatic embryos are used of monocotyledonous plants.   Method according to claim 4, characterized in that   Somatic embryos of grasses are used.   Process according to claim 5, characterized in that   Somatic embryos of maize are used.   Process according to one or more of Claims 1 to 6,   characterized by culturing the somatic embryos prior to incubation with the DNA or RNA solution on a   nutrient medium containing a sugar or sugar alcohol.   Process according to one or more of claims 1 to   7, characterized in that the gene of phosphinothricinacetyltransferase S e of Streptomyces is used as DNA.   viridochromogenes, if appropriate in modified form.   9 Conjugated somatic monocotyledonous embryos.   Somatic embryos of transformed grasses. -   11 Somatic transformed maize embryos.   12 somatic embryos according to one or more of the claims   9-11 containing the phosphinothricinacetyl-   transtérase. 13 Plants that can be obtained from embryos   somatic devices according to claims 9 to 12.   14 Use of transformed embryos that can be   obtained according to one or more of claims 1 to 8,   for the preparation of artificial seeds.   Using dried somatic embryos as artificial seeds.   16 Use of transformed somatic embryos obtainable by one or more of   claims 1 to 8 for regeneration and cultivation of   Useful transgenic plants fertile.   17 A method of protecting monocotyledonous plants by selective destruction of weeds by phosphinothricin on cultivated surfaces, characterized in that the cultivated area is planted with plants obtainable from somatic embryos according to   claim 12 and their offspring.