DD274234A5 - Method for gene transfer into plants - Google Patents

Abstract

The invention relates to a method for gene transfer in plants, in which mana) removes immature pollen grains from nutrients in a nutrient solution and removes the surrounding tissue. B) cultivating the isolated immature pollen grains in a nutrient solution. C) into the pollen cores during the in vitro cultivation and maturation of foreign genetic material transferring) the transformed pollen grains in vitro to complete maturation) and pollinating recipient plants with the transformed pollen grains and obtaining from these seeds transgenic seeds and propagation products thereof.

Description

Field of application of the invention

The invention relates to a method for gene transfer into plants by means of isolated, immature and in vitro cultured pollen plants and seed and propagation products thereof produced therefrom. Field of application of the invention is therefore the agricultural industry.

Characteristic of the known technical solutions

There are already several techniques of gene transfer in plants. Each has its advantages and nac '. parts (Goodman et al, Science 236: 48-53, 1987). Agrobacterium tumefaciens is currently the most common vector. However, it is limited to a specific host range. Still, the use of A. tumefaciens as a vector depends on the regeneration of in vitro cultured somatic cells to produce transgenic plants capable of forming transgenic progeny. Direct gene transfer by electroporation, liposomes, microinjection and other physico-chemical methods is largely dependent on the use of protoplasts as target cells. However, regeneration from protoplasts is difficult and even impossible in many species.

As an alternative to these gene transfer methods, another target cell, the pollen grain, has been proposed. According to Hess D., Plenum Press, New York, 519-537 (1957), it is possible for mature pollen to take up foreign DNA and to introduce this foreign DNA into the egg cell as a 'supervector' by means of natural pollination and fertilization. Similarly, X-irradiated pollen should be capable of transporting fragments of the irradiated genome into the oocyte (Pandey, Nature 256: 310-313, 1975). DeWet (WO 85/01856) reports that maize pollen treated with exogenous DNA picks up this DNA upon germination and transports it into the egg after pollination.

Despite the potential great importance of gene transfer into and through pollen, it has never been possible to reproduce the results of Hess, P indey and DeWet in other laboratories. For example, Engvild (Theor Appl Genet 69: 457-461, 1985) could not reproduce Pandey's experiments. In the experiments of Hess (1975) and DeWet, the inclusion of exogenous DNA in the pollen grain and the genetic and molecular detection of the transferred DNA is the critical point of evidence. Phenotypic and physiological evidence is insufficient (see: Genetic manipulation in plant breeding, de Gruyter, Berlin, New York 803-811, 1986). The experiments of Sanford et al (Diotechnology and ecology of pollen (Mulcahy D, ed.), Springer, Heidelberg, New York, 71-75, 1968) showed that cocultivation of mature Nicotiana langsdorfii pollen with Agrobacteria to no gene transfer in Pollen led. In extensive research by Negrutiu, Keberle-Bors and Potrykus (65-70, 1986), the neomycin phosphotransferase gene conferring resistance to kanamycin has not been successfully isolated (Shillito et al, Biotechnology 3: 1099-1103, 1985) to carry mature pollen. So, using state-of-the-art methods, it was not possible to confirm the claims of Hess and DeWet. The fact that the results of Hess and DeWet are incomprehensible can be explained by the fact that mature pollen grains immediately begin to form a pollen tube as soon as they enter an aqueous medium necessary for gene transfer. Apparently they are then no longer fertile, or it is the time that is available for gene transfer, too short.

Pareddy et al., Planta 170: 141-143 (1987), describe the cultivation and ripening of immature maize banners ("tassel", inflorescences) in vitro, dusting with the isolated mature pollen, and collecting seeds from the pollinated plants. A proof of successful fertilization could not be provided.

Object of the invention

The aim of the invention is to provide a simple and economical method for introducing genes into germ cells of plants and thus enable the production of transgenic plants.

Explanation of the essence of the invention

The object of the present invention is to make it possible, in a simple manner by introducing foreign genes into pollen, to use a system which can be used in a manner suitable for the production of transgenic plants. This is done by KuIiI ^! by introducing foreign genes into these isolated, immature pollen grains during certain stages of ripening, by dusting plants with the mature pollen grains, by fertilization, normal seed formation, germination and proliferation.

The present invention accordingly provides a method for gene transfer into plants, which is characterized in that

a) removing immature pollen grains in nutrient solution from stamens and removing the surrounding tissue,

b) cultivating the isolated immature pollen grains in a nutrient solution

c) transfers into the pollen grains during the in vitro cultivation and maturation of foreign genetic material

d) DIFC * ^r ansformierten Pollenkörnbr in vitro for complete maturation brings

e) pollinated with the transformed pollen grains recipient plants and from these seeds wins.

Gene transfer into isolated, immature pollen grains, in vitro maturation and the use of pollen as a universal supervector, opens up a completely new strategy of gene transfer into plants. Compared to other methods, it is considerably simplified, as the cell culture phase is greatly shortened and the regeneration with the unpleasant slippery appearance of the somaclonal variation is eliminated. With this new method plants can be transformed, which were previously inaccessible to successful gene transfer: So z. For example, many types of cereals, legumes and trees can not be regenerated from tuberous cells or protoplasts.

The potential benefits of gene transfer technology are of great economic, environmental and social value. It is desired to genetically modify plants so as to increase the yield, to make the plants resistant to diseases and pests, to tolerate cold, heat, drought, salinisation, nutrient deficiency, to obtain a greater nutritional quality produce new indutrial raw materials, or that they fix their own nitrogen or otherwise become independent of fertilizers.

It is essential to the invention that pollen bodies are cultivated without the tissue surrounding them, ie in isolation, so that the gene material to be transferred has direct contact with the pollen grain coupled to a vector or in naked form. Namely, if the complete male inflorescence is cultured, it is not possible because of the obstruction by the extensive enveloping tissue, using common transfer methods (A. tumefaciens, electroporation, microinjection and the like) genes to transfer into the pollen body.

A much more important feature of the invention is the use of immature pollen bodies. As a result, the entire time of in vitro maturation for gene delivery is available. The transmission into the immature pollen grain can take place in different stages of maturity, depending on the plant species. In particular, in the case of gene transfer with vectors, it is important that the genetic material must have as few cell walls as possible in order to enter the genome of the sperm nuclei and become part of the zygote genome when fertilized. The transmission is preferably carried out in mononuclear microspores, but may also during the first PollenmitOi'e, in the early binuclear stage, as long as the generative cell still adheres to the pollen wall, or in addition in those plants that have trinuclear pollen in the stage of maturity (cereals), in the stage just before or during the second pollen mite. respectively.

More specifically, the process is carried out following vvio, using tobacco (Nicotiana tabacum) as the model system.

The system is applicable to all pollination-propagating crops, in particular to monocotyledonous and dicotyledonous crops such as wheat, corn, rice, legumes, oilseeds, vegetables, fruit and forest plants.

In one example, the developmental stage of the pollen of the desired plant is usually determined by isolation of an anther, preparation of a crush, and observation in the microscope after addition of e.g. Carminetic acid determined.

Once the pollen has reached the desired stage, the boll grains are isolated under aseptic conditions by squeezing them out of the anthers in a nutrient medium, passing through a sieve, washing, centrifuging and resuspending.

The cell density should be higher for pollen bodies in the younger stage of development than for pollen grains in the older stage of development. It is about 10 ⁵ / ml in the tobacco of 2kernigen pollen grains, for mononuclear microspores it is about

twice as high. '

The suture medium used contains all the nutrients and essential nutrients essential for the cultivation and maintenance of the maturity of the pollen bodies. Depending on the plant, different compositions may result, so that it fulfills the function of the Nänrgewebes (Tapetum). As main components, the nutrient medium contains sugar, nutrients, mineral salts and Vitarr ine, wherein the pH is about 6.5 to 7.5.

In the case of mononuclear microspores, cultivation takes place up to the binuclear stage in one of sugars. As sucrose and other nutrients significantly enriched medium. For example, the additional nutrients in the form of coconut water zugefi. be heard. Once the pollen grains reach the binuclear stage, they can be transferred to a less nutritious medium.

After completion of the in vitro sterilization, the pollen grains were obtained for pollination. For this they are centrifuged off, washed and applied for pollination in an aqueous medium or dried on flowers from which the anthers were previously removed. From the pollinated with the in vitro ripened pollen grains plants seeds can be obtained in a common way, which are germinable.

For gene transfer during in vitro cultivation, the foreign gene material to be transferred can be used in conjunction with e'.iem usual vector, such. B. A. tumefaciens or as naked DNA by direct transfer by electroporation, microinjection or other physico-chemical methods are introduced into the pollen grains. The term "foreign gene material" means any genetic material produced outside the pollen grain to be transformed, and after maturation, the pollen grains are obtained for pollination in the manner described above.

As proof of the successful in vitro maturation, pollen grains of a tobacco plant with 2 marker genes (KAN ⁿ , NOS) were ripened in vitro and pollinated with these pollen grains normal wild-type plants. The recovered seeds were sterilized and germinated in a kanamycin-containing germination medium. The Mendelian segregation of the marker genes was detected by counting the seedlings in the selective medium. Seedlings germinated on kanamycin-free medium also showed Mendelian segregation of the NOS gene after nopaline detection in high-tension paper electrophoresis. This is proof that it was the in vitro-ripened pollen grains that carried out the fertilization and not any pollen decays from other plants.

As evidence of the expression of the foreign gene introduced by transformation during in vitro culture, chloramphenicol acetyltransferase (CAT) activity was detected in an enzyme test in a homogenate from the transformed pollen grains.

In addition to the CAT gene, a cytochemically detectable gene, the beta-glururonidase (GUS) gene, was also used. The agrobacteria were removed after cocultivation with the pollen by daily washing with the antibiotic claforan or killed. In the cytochemical test (blue colouration of the pollen) over 50% of blue, in vitro mature pollen could be detected. Uninfected pollen, or pollen infected with agrobacteria without or with non-functional GUS gene, did not show any blue color after substrate addition. A fluorimetric assay showed strong GUS activity in GUS-transformed pollen as well as in pollen of GUS-transformed plants which served as a positive control. Pollen that was not infected or infected with GUS gene-free agrobacteria did not show any GUS activity. Also infection with Agrobacteria without infection genes, but with complete GUS gene in the T-DNA led to no GUS activity in the pollen (cytochemically as fluorimetrisch).

It can be concluded that a ° < gene transfer into the pollen by Agrobaktorium tumefaciens. Another indication of successful gene transfer into the rollers is the recognizable attachment of Agrobacteria to the pollen surface with formation of cellulose fibrils (electron micrographs).

Example 1: Determination of Pollen Development Stage

Tobacco flowers were harvested in various lengths, the anthers were aseptically isolated, a five anthers were placed on a microscope slide together with a drop of carmine acetic acid (4% carmine in 45% acetic acid) and a squeeze prepared. The developmental stage of the pollen grains was determined after half an hour under the microscope.

Example 2: Isolation of pollen grains

The tobacco pollen grains were isolated by gently squeezing anthers under aseptic conditions in AMGLU medium (1) with glass rod and microscopy cup, and passing the resulting pollen suspension through a 75 μm sieve and washing twice in AMGLU medium. Finally, the pollen suspension was removed by centrifugation at 6500 rpm in an Eppendorf centrifuge.

Example 3: Pollen culture for maturation of early dinuclear pollen grains

Early dicotyledonous tobacco pollen grains were isolated and the cell density in AMGLU medium was adjusted to 10 ⁵ / ml. One ml of the pollen suspension was cultured in 35 mm Petri dishes at 25 ^° C. in the dark. After 2 to 5 days, depending on the exact stage of development of the pollen grains, the pollen grains were ripe and could be harvested.

Example: pollen culture for the maturation of mononuclear microspores

Mononuclear tobacco microspores were cultured in MR24 medium (2) at a cell density of 2.10 ⁵ / ml. Once the pollen grains reached the binuclear stage (after 2 to 5 days, depending on the exact age), they were spun down and further cultured in M1S medium (3) until maturation was complete.

Very good results were also obtained when culturing in MR26 medium (2 '). As soon as the pollen grains reached the binuclear stage (after 3 days), the same volume of M2S-Medi'jm was added. After another day, the pollen grains were spun down and cultivated in M2S medium (3 ') at a cell density of 10 ml until maturation was complete (one day).

Example 5: Pollination and Proof of Fertilization

The tobacco pollen bodies were centrifuged off, washed in BK medium (Brewbaker and Kwack 1963) and the cell density was adjusted to 1.25.10 ^e / ml. The still closed anthers were removed from flowers that were just opening (red flower tip). Once the flower had opened, a 4-ml drop of the pollen suspensions was applied to the scar of the flower using a 20 μΙ-ΗρβΗβ so that the scar was completely covered with pollen spike. These operations were performed under conditions of no air movement and of other plants of the same species. Once the drop had dried on the scar, the scar and stylus were covered with a 4cm straw to prevent cross-fertilization. The plants were then returned to the glasshouse.

Example 6: Seed harvest, seed germination and genetic test

As evidence that the in vitro mature pollen grains had pollinated and pollinated, pollen grains of a transgenic plant were matured in vitro and pollinated with these pollen grains normal wild-type plants. As a marker gene, the transgenic plant contained the neomycin phospho-transferase gene (resistance to kanamycin) and the nopaline synthase gene. Self-fertilization and reciprocal crossing with in vitro matured wild-type pollen were also performed.

The mature seed capsules (brown and dry) obtained in Example 5 were harvested and the seeds were isolated by cutting the tip of the capsule and placing the seeds directly in an Eppendorf cap. Seeds were surface sterilized in NaOCl solution (3% free chlorine) for 5 minutes, washed twice with sterile water, and placed on a seed germination medium with kanamycin (4). 1: After four weeks, the number of channels is "and Kan ^s seedlings counted In the above crossing experiment, the two reciprocal crosses showed a Segration Kan." Kan ^s = 1. Seedlings that grew up without kanamycin showed a segregation of Nos *: Nos ~ = 1: 1 after a nopaline test by high voltage paper electrophoresis. Self-fertilization with in vitro matured wild types yielded, as expected, 0: 1 segregation for both marker genes. Self-pollination with in vitro matured pollen of the transgenic plant gave a 3: 1 segregation.

Example 7 Transient Expression of the CAT Gene

Agrobacteria (non-tumorigenic A. tumefaciens, with CAT gene linked to 35S promoter) were preincubated in Luria broth one day. Pollen grains in the early binuclear stage were isolated and cultured in AMGLU medium. The bacterial suspension was adjusted to an OD ₆₈₀ of 0.2 with AMGLU medium. After further dilution with AMGLU medium 1: 10, 20μl of the bacterial suspension was added to 1 ml of the pollen suspension. After 24 hours of coking, 1 μΙΟΙβίοΓθη (1 g / 2 ml) per ml was added to kill the agrobacteria. After two more days, the pollen grains were harvested and an extract made. For this purpose, 1.5 ml of calcium washing solution (5), pH 5.6 were added per ml of pollen suspension, centrifuged at 4000 rpm for 5 minutes and washed with a Tris buffer (0.25M, pH 7.8). After centrifugation, the pollen pellet was spiked with 200μΙ Tris buffer and homogenized by sonication (3x15sec) on ice. After 10 min on ice was centrifuged off and the supernatant stored at -2O ⁰ C.

The CAT assay was performed as described by Sleigh (Anal. Biochem., 56: 251-256, 1986). 30μΙ of the extract was mixed with 20μΙ chloramphenicol (8mM), 30μΙ Tris buffer and 20μΙ ^M C-labeled acetyl-CoA (5μCi / ml in 0.5mM cold acetyl-CoA).

After incubation at 37 ° C. for 1 hour, the acetylated chloramphenicol was shaken out with ethyl acetate (2 × 100 μl) and the radioactivity was measured in the scintillation counter.

Radioactivity (cpm) in the CAT test of tobacco pollen extracts after co-cultivation with A. tumefaciens

cpm

Pollen in AMGLU medium 400

Pollen with Agrobacteria in AMGLU-Medium 6000

Polymers with acetosyringone-activated agrobacteria in AMGLU medium 6000

only agrobacteria in AMGLU medium 500

only acetosyringone-activated agrobacteria in AMGLU medium 500

The strong radioactivity signal indicates that the agrobacteria have successfully infected the pollen grains and that the T-DNA must have entered the cell nucleus of the growing cell for expression (transcription).

As a further control to show that the agrobacteria itself did not have CAT activity,. For example, an agrobacterial culture in Luria-broht shows no CAT activity over a complete growth cycle.

Example 8: Expression of the GUS gene in tobacco pollen

Agrobacteria of strain LBA 4404 (A tumefaciens, disarmed, with beta-glucuronidase (GUS) gene coupled to 35S promoter and terminator, (Matzke and Matzkein Plant Molecular Biology 7: 357-365 [1986]) were grown in Luria. Pollen bodies in the late mononuclear stage were isolated and adjusted to an OD ₅₈ O of 0.2 in MR26 medium After further dilution with MR26 medium of 1:10, 20 μl of the bacterial suspension were added to 1 ml of the pollen suspension. After 14-20 hours coculture was centrifuged off and the pollen was washed three times with claforan containing (1 g / 2 ml) MR26 medium and further cultivated until the pollen maturation was changed to the claforan containing medium 40μΙ of final medium (M2S with claforan). were added to a Luria broth and no bacterial growth developed.

The mature pollen was spun down and a portion was taken up in MR26 medium. After addition of X-Glu (5-bromo-4-chloro-3-indolyl-glucuronide) to a final concentration of 1 mM and incubation at 37 ° C for 4-12 hours ( Jefferson in Plant Molecular Biology Reporter, Vol. 5, No.4,387-405, [1987]), the formation of indigo (product of the beta-glucuronidase from X-Glu) could be detected by the blue staining of the pollen under light microscopy. More than 50% of the live, mature pollen showed blue coloration.

A second portion of the cocultivated and in vitro ripened pollen was germinated in GK medium (like BK medium but double the concentration of boric acid). GUS activity could also be detected in the pollen tubes of germinated pollen.

In a control experiment, pollen was infected with an Agrobacterium strain of LBA 4404 containing in its T-DNA the GUS gene without promoter (provided by Drs. Matzke, Salzburg). These pollen showed no blue coloration after in vitro maturation and X-Glu addition. Agrobacteria which did not contain a GUS gene but also contained the KAN _35S gene showed no blue color after cocultivation with pollen and X-Glu addition. Even pollen that was not infected with Agrobacterium did not show any blue coloration after X-Glu addition.

Pollen from a CIS ₃ 5 _S -transformed plant (by leaf-disk), which served as a positive control showed blue staining. Another strain of Agrobacter which, although a functional GUS-ss-Ger. contained in the T-DNA, but was disturbed in its virulence function (binary vector without Ti plasmid), showed no GUS activity in the pollen.

One third of the pollen produced an extract. For this purpose, 4.1 × ⁵ pollen were centrifuged off and taken up in extraction buffer (6). The pollen was broken up with glass beads and simultaneous ultrasound treatment. The fluorometric GUS test was as described by Jefferson. 50μΙ extract was made up with extraction buffer a'jf 1 ml and mixed with MUG (4-methyl-umbelliferyl-glucuronide) to a final concentration of 1mM. Every 10 or 30 seconds 20ϋμΙ aliquots were taken and the enzyme reaction was stopped with 800 μΙ Na ₂ CO ₃ (0.2M). The extinction was measured in a fluorometer at 455 nm after excitation of 365 nm and converted to MUG in concentrations in μΜ / ml using standard solutions.

10min 20 min 30 min 1.0 1.0 1.0 0.1 0.1 0.1 0.016 0,020 0.019 0.057 0.078 0.109 0.023 0.019 0,021 0,022 0.023 0,018 0,051 0.085 0.121

Fluorimetrically measured enzyme activity in μΜ 'ml after 10.20 and 30 minutes of beta-glucuronidaso from tobacco pollen extracts after cocultivation with A. tumefaciens:

Standard 1

Standard 2

Polion without Agrobacteria Pollen co-cultivated with GUSaBsAgrobacteria Pollen with KAN ₃₅₅ Agrobacteria cultured Pollen with GUSj ₅ S with "binary vector" without Ti plasmid

Pollen from trangenic CIS ⁺ plant

Culture Media:

(1) AMGLU Medium Miller Macrosalts MS Microsalze

Glutamine (440mg / l)

pH 7 (2) MR24 medium

MS Macrosalts MS Microsalze Sucrose (0.5M) Glutamine (440mg / l) Coconut Water (2Vol .-%) Lactalbumir. Hydrolysate (200mg / L) Inositol (100mg / L)

pH 7 (2 ') MR26 medium

like MR 24 medium, but lactalbumin hydrolyzate (1 g / L) (3) M1S medium

Miller Macrosalts MS Micro Salts FeEDTA (IO- ⁴ M) sucrose (0.25M)

pH 7 (3 ') M2S medium

Kyo and Harada salts (in Planta 186: 427-432 11986)) sucrose (0.25M) pH 7

(4) Seed germination medium

MS Macrosalts MS Micro Salts FeEDTA (IO- ⁴ M) sucrose (1 wt.%) Agar (0.8 wt.%) Kanamycin .SO ₄ (50 mg / l) pH 5.5

(5) calcium washing solution

CaCl ₂ -2H ₂ O (0.16M) MES buffer (0.5Gcw%) pH 5.6

(6) extraction buffer

NaPO ₄ (50 mM, pH 7) 2-mercaptoethanol (10 mM) Na ₂ EDTAdOmM) Sodium lauryl sarcosine (0.1%) Titron X-100 (0.1%)

Claims (8)

1. A method for gene transfer into plants, characterized in that a) extracts from stamens immature pollen grains in Mährlösung and surrounding tissue entfe ^r nt b) cultivating the isolated immature pollen grains in a nutrient solution c) transfers into the pollen grains during the in vitro cultivation and maturation of foreign gene material d) brings the transformed pollen grains in vitro to complete maturation e) pollinated with the transformed pollen grains recipient plants and from these seeds wins. 2. The method according to claim 1, characterized in that the transfer of foreign genetic material takes place in the stage of mononuclear microspores. 3. The method according to claim 1, characterized in that the transfer of foreign genetic material takes place during the first pollen mitosis. 4. The method according to claim 1, characterized in that the transfer in the early binuclear stage, as long as the generative cell still adheres to the pollen wall occurs. 5. The method according to claim 1, characterized in that the transmission of foreign Genmateru-is done shortly before or during the second Pollenmitose. 6. The method according to claim 1, characterized in that the nutrient solution contains all essentiol'en nutrients and growth substances for the cultivation and maintenance of the maturity of the pollen grains. 7. The method according to claim 2, characterized in that the gene transfer takes place in the stage of mononuclear microscopes in an enriched in sugar and other nutrients medium. 8. The method according to claim 1, characterized gakennzeichnet that the transfer of foreign gene material is done by cocultivation with Agrobacterium tumefaciens.