WO1992014828A1

WO1992014828A1 - Method for genetic transformation of tissue organs from monocotyledonous plants

Info

Publication number: WO1992014828A1
Application number: PCT/SE1991/000102
Authority: WO
Inventors: Anneli Tallberg; Eva THÖRN; Richard Walden
Original assignee: Svalöf Ab
Priority date: 1991-02-14
Filing date: 1991-02-14
Publication date: 1992-09-03

Abstract

A method for genetic transformation of tissues from a monocotyledonous plant is described. Genetic material is inserted into a vector in a bacterium which is then co-cultivated with a microspore culture of the plant to be transformed. Genetically transformed tissues from a monocotyledonous plant, which has been transformed in the manner stated, are also described.

Description

METHOD FOR GENETIC TRANSFORMATION OF TISSUEORGANS FRO MONOCOTYLEDONOUS PLANTS

The present invention relates to a method for genet transformation of tissues from monocotyledonous plants. The invention also comprises tissues from monocotyledons transformed by means of the method according to the inve tion.

Background of the invention Genetic transformation of plant cells, i.e. transfe of genetic material to plants by genetic engineering now is a routine method in plant research. The main part of the gene transfers resulting in regenerated stably trans formed plants have, however, occurred in dicotyledonous species of plants, e.g. Brassica, potatoe and pea. The reason for this is the progress which has been made in t understanding of how the soil bacterium which is charac¬ teristic of dicotyledons, Agrobacterium tumefaciens, transfers and integrates foreign genetic material in a host plant (Zambryski et al 1984). Well-functioning tiss cultivation techniques have also contributed to the fact that up to now, transformation systems have been develop for dicotyledonous, but not for monocotyledonous species of plants. The object of transformation of plant cells in term of genetic engineering is to provide both agronomic end qualitative modifications of species of plants which ere important in agriculture, such as cereals. Modifications of the type here involved are such as are not possible with traditional crossbreeding. Therefore, there is a great need of transformation systems for practical appli¬ cation when improving cereals by breeding.

Since the largest acreage proportion of cereals raised in Sweden consists of barley and, moreover, barley has properties which make it well suited for industrial use, it is of extreme interest to develop transformation strategies for this species.

r>r* Transformed plants are formed by inserting, by means of genetic engineering, genetic material into individual cells (protoplasts) or into an intact tissue. Since the Agrobacterium system has considerable limitations when applied to cereals, transformation systems based on direct gene transfer have been developed for cereals (Paszkowski et al 1986). The drawback of these gene transfer methods, e.g. electroporation and microinjection, is the difficulty of the receptor cells to divide and form intact fertile plants. However, by using standard methods for direct gene transfer to protoplasts from embryogenic suspension cul¬ tures, it has been possible to regenerate fertile trans- genic rice plants (Potrykus 1990).

Progress in the last few years in the field of tissue culture of cereals has substantially increased the possi¬ bilities of obtaining regenerated transformed plants. At present, since embryogenic cultures have a regenerating potential, they represent the best starting material for gene transfer in cereals. Bombarding tissue from embryo- genie cultures with high-speed particles coated with DNA has thus made it possible to regenerate fertile transgenic maize plants (Freiberg 1990). This method suffers from the drawbacks that the inserted foreign DNA can be unstable, which means that either it is rearranged or changed in its structure and, consequently, its message.

The transfer of genetic material by means of Agro¬ bacterium, which is used for dicotyledons, has a great advantage since the inserted DNA does not undergo any rearrangement. It would therefore also be desirable to be able to transform cereals by means of Agrobacterium. The barrier of the monocotyledons against Agrobacterium infection has in fact proved not be absolute. Thus, genes have been transferred to e.g. Asparagus officinalis and Dioscorea bulbifera (Lόrz et al 1988). Brief Description of the Invention

The invention relates to a method for genetic trans¬ formation of tissues from a monocotyledonous plant, in which method genetic material is inserted into a vector i a bacterium, whereupon the bacterium is co-cultivated wit a microspore culture of the plant to be transformed.

The method preferably is applied to cereals, espe¬ cially to barley.

In connection with the present application, by tis- sues are meant seeds, embryos, proembryos, microspores, pollen etc.

The invention also comprises genetically transformed tissues from a monocotyledonous plant which has been transformed by insertion of genetic material into a vecto in a bacterium which is subsequently co-cultivated with a microspore culture of the monocotyledonous plant. Description of the Invention

The inventive method for transforming tissue organs from monocotyledons, especially cereals, is based on tech niques for cultivating isolated microspores. At a specifi stage in the development of pollen, microspores cultivate in vitro have the capability of dividing and differentiat ing to embryos. About 6 days after isolation, the development has reached a stage when the surrounding wall bursts, and a proembryo is released. In transformation experiments in general, the smaller number of cells neede for regeneration, the better is the cultivation system suited for gene transfer. Although a 6-day-old proembryo consists of some hundred cells, it is still very small as compared with a fully developed embryo. A transformation at this stage means good prospects of obtaining embryos which largely consist of transformed tissue.

Thus, the invention is based on co-cultivation of microspores of the plant to be transformed with bacteria cells which contain a vector having the genetic material which is desired to be inserted in the host plant. 4 A bacterium which has appeared to function well in the field of the invention is Agrobacterium, especially Agrobacterium tume aciens.

The transformation process can be divided into four 5 steps, i.e.

1) Insertion of genetic material into a vector in a bac terium

2) Co-cultivation of the bacterium and a microspore cul ture of the plant to be transformed

10 3) Selection and regeneration of transgenic plants

4) Control and analysis of the expression of the geneti material in the transformed plants. For selection of transgenic plants and for control and analysis of the expression of the inserted genetic 15 material in the plants, some form of marker gene is also inserted into the vector in the bacterium.

In the accompanying Figures: Fig. 1 shows the vector pPCV 002, Fig. 2 shows the vector pCVHPT GUS, 20 Fig. 3 shows Southern blot I with the HPT fragment as the probe. Fig. 4 shows Southern blot II 500 bp vector sequence as the probe, Fig. 5 shows Southern blot III with the HPT fragment as 25 the probe, and

Fig. 6 is a photo of a regenerated fertile transgenic bar¬ ley plant. The invention is described in more detail in the Exam ple below which is not limiting. 30 EXAMPLE

Introduction of Genetic Material into an Agrobacterium Vector

Isolated microspores from barley are used as starting material for the experiment, since a well functioning 35 method for cultivating isolated barley microspores and for regenerating plants therefrom is available. As a suitable bacterium for insertion of the genetic material, Agrobac¬ terium tumefaciens was selected.

To begin with, the tolerance of barley microspores for antibiotics was examined, thereby making it possible to select a suitable gene construction for antibiotic resistance to be used in the transformation process. The effect of kanamycin and hygromycin was studied on barley microspores at different stages of development. The resul showed that barley microspores have a high tolerance for kanamycin, whereas hygromycin completely inhibits cell- division in the microspores. When developing the trans¬ formation method, use was therefore made of a gene con¬ struction based on hygromycin resistance, which has been introduced into Agrobacterium tumefaciens. The Agrobacterium tumefaciens used includes the vec¬ tor pCVHPT GUS (Fig. 2) which results in hygromycin resis tance. The vector pCVHPT GUS is based on another vector, pPCV 002 (Fig. 1) (Walden et al 1990).

In pCVHPT GUS, a gene for hygromycin resistance was inserted, viz. HPT 2030 bp, at the EcoRI-Sall-site in the polylinker molecule from pPCV 002. The reporter gene for beta glucuronidase (GUS) (Jefferson et al 1986) replaced the NPT II gene in pCVHPT GUS between pNOS and pAccs from pPCV 002. Co-cultivation of Agrobacterium and Microspore Culture

Agrobacterium tumefaciens with the vector pCVHPT GUS propagated at 28^CC for 2 days in LB medium (Maniatis et a 1982) mixed with ampicillin (50 μg/ml) and carbenicillin (100 μg/ml) was used in co-cultivation with barley micro- spores.

Barley microspores at a late single nucleus to early double nuclei stage are isolated from ear which has been refrigerated as +4°C for 4 weeks. The microspores are cul tivated in 7 cm Petri dishes in the dark at 25°C in N6 medium (Chu 1978) mixed with 1.75 mg/1 2,4-D 0.25 mg/1 kinetin 63 mg/1 maltose.

At different time intervals after isolation of the microspores, the culture medium is replaced by a new N6 medium mixed with 0.5 % MES

50 μl bacteria suspension/7 ml medium.

After adding the bacteria, the material is placed in the dark where it is first agitated at 30-40 rpm for 20 min. and is then allowed to stand for 3 days. Selection and Regeneration of Transgenic Barley Plants After the period of 3 days, the culture medium is replaced by selective N6 medium mixed with 10 mg/1 hygromycin 200 mg/1 Claforan.

The function of Claforan is to stop the growth of the excess of Agrobacterium cells, while the hygromycin is added for selection of transformed tissue only, to which the hygromycin gene has been transferred.

4-5 weeks after the microspore isolation, the embryo material is transferred to a regeneration medium consist¬ ing of J-25-8-medium (Jensen 1983) mixed with 0.5 mg/1 IAA 1 mg/1 kinetin

10 mg/1 hygromycin 200 mg/1 Claforan 7 g/1 agar.

About 8 weeks after the initial microspore isolation, barley plants have been regenerated. They are transferred to soil and placed in a greenhouse.

Control and Analysis of the Expression of the Genetic Mate¬ rial

The success of the transformation and the genes from pCVHPT GUS being incorporated into the barley genome are checked by two different methods. The presence and the number of copies of the hygromycin gene are analysed by Southern blot, while the expression of the GUS gene is judged by a fluorometric method (Jefferson 1988). Further control analyses, e.g. determination of the chromosome number, are performed but on the plants giving a positive signal by Southern and/or GUS analysis. Southern analysis:

Small regenerated barley plants are cut into pieces and freeze-dried. Total DNA is extracted according to the CTAB metod (Murray et al 1980) from 10-25 mg of freeze- dried tissue and is restriction-cleaved by the enzyme Hin III. The presence of the hygromycin gene (= the HPT frag¬ ment, 2030 bp) in the barley genome was analysed by Southern blot, using the entire HPT fragment as the probe (Fig. 3). The membrane containing samples with positive signal for the hygromycin gene are again probed by Southern blot After the first probe, the HPT fragment, has been washed away, a new probe is used. This consists of a 500 bp vec¬ tor sequence from pCVHPT GUS which is not integrated between the T-DNA boundary sequences and which thus canno be transferred to the barley genome. This additional con¬ trol is performed in order to determine that the initial positive signals do not derive from surviving Agrobac¬ terium cells. The result obtained therefore is a complete ly blank membrane without any signals (Fig. 4).

To make sure that the barley DNA is not washed away between the hybridisations (the Southern analyses), a third Southern analysis is performed on the same membrane and in this case with the original probe, the HPT frag- ment. The result of this analysis then corresponds to the original result, i.e. positive signals for the presence o the hygromycin gene in the barley genome are obtained (Fig. 5). GUS analysis: The presence of the GUS gene (the gene for beta glu- curonidase from E. coli) is analysed in nonfreeze-dried plant material. The activity of beta glucuronidase is analysed on leaf extracts from the regenerated barley plants by visual examination. Determination of the chromosome number:

Since the chromosome number is sometimes spontaneous- ly doubled in plants from microspores with n=7, it is necessary that the chromosome number is determined so as to distinguish the haploid plants from the diploid. As it is necessary to obtain diploid plants, the transformed haploid plants are treated with colchicine in order to obtain a chromosome number duplication to 2n « 14. The spontaneously diploid plants continue to grow without any form of treatment.

Results

Transformed, regenerated barley plants have been obtained from barley microspores treated according to the method described above. A total of 150 barley plants have been regenerated, 10% of which are of albino type.

Total DNA from all plants was analysed by Southern blot for checking the presence of the hygromycin gene in the barley genome. A positive result was obtained from 23 plants, but only 9 thereof survived. Both a negative and a further positive control by Southern analysis of these 9 barley plants showed that the positive signals do not derive from surviving Agrobacterium cells. GUS analysis of the 9 surviving barley plants which are positive by Southern blot, resulted in 5 positive and 4 negative judgements. Of the 5 double positive barley plants, one has so far been established to be a sponta¬ neously chromosome-doubled fertile plant. The remaining 4 double positive plants have not yet formed ears, but two of them are probably haploid. These will be chromosome- doubled by colchicine.

Of the 4 surviving barley plants which are Southern- positive but GUS-negative, 3 are fertile, having a doubled chromosome number. The fourth plant is haploid and has therefore been treated with colchicine. Both Southern and GUS analyses are repeated for this different material. Fig. 6 shows a photo of a regenerated, fertile transgenic plant which has been obtained by the method according to the invention. Conclusion By the method described above for co-cultivation of barley microspores with Agrobacterium tumefaciens cells, it has been proved that it is possible to transform tis¬ sues from e.g. barley, so that regenerated, fertile trans genic plants are obtained. It has further been proved tha the Agrobacterium system functions also for monocotyledo¬ nous species of plants, such as barley, in that the gene¬ tic material transferred with Agrobacterium is incorpo¬ rated into the plant genome, in this case barley. Bibliography - Chu, C. C. 1978, Proc. Symposium on Plant Tissue

Culture, Science, Press, Beijing, Kina, 1978, pp 43-50.

- Freiberg, B. 1990. Ag. Biotechnology News March/April:2

- Jefferson, R. A. 1988. In Genetic Engineering/Principle and Methods. Vol. 10: 247-263. - Jefferson, R. A. et al. 1986. PNAS 83: 8447-8451.

- Jensen, C. J. 1983. In Cell and Tissue Culture Techni¬ ques for Cereal Crop Improvement. Science Press, Beijing, Kina, pp 55-79.

- Lorz, H. et al. 1988. Plant Breeding 100: 1-25. - Murray, M. G. and Thompson, W. F. 1980. Nucleic Acids Res. 8(19): 4321-4325.

- Paszkowski, J. and Saul, M. W. 1986. In Methods in Enzy mology. Vol. 118: 668-684.

- Potrykus, I. 1990. Biotechnology 8(6): 535-543. - Walden, R. et al. 1990. MMCB 1(5/6): 175-194.

- Zambryski, P. et al. 1984. In Genetic Engineering/Prin¬ ciples and Methods. Vol. 6: 253-278.

Molecular biological methods which are generally used are described in the following manuals: - Draper, J., Scott, R., Armitage, P., Walden, R. 1988. Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell. Scientific Publications. - Maniatis, T., Fritsch, E. F., Sambrook, J. 1982. Mole¬ cular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory. Figure Legends: Fig. 1: pPCV002: LB and RB are the right and left T-DNA boundary; oriV and oriT, replication start functioning in Agrobacterium; ori, bom, ColEl, replication start and mobilising sequence functioning in E. coll; Amp, ampicillin resistance gene: pNos, nopaline syntase promotor; NPTII, neomycin phosphotransferase gene; pAcos, sequence for oσtopine syntase polyA. Fig. 2: pCVHPT GUS: LB and RB are the right and left T-DNA boundary; oriV and oriT, replication start functioning in AgroJbacterium; ori, bom, ColEl, replication start and mobilising sequence functioning in E. coli Amp, ampicillin resistance gene: pNos, nopaline syntase promotor; GUS, β-Gucuronidase gene; pAocs, sequence for octopine syntase polyA; HPT, hygromycin resistance gene. pCVHPT GUS is based on pPCV002.

Figs. 3 A-C. Southern blot with

A. HPT fragment,

B. 500 bp vector sequence,

C. HPT fragment as a probe. Line 1 is DNA from transformed barley plan . Lines 2 and 3 are HPT fragments from pCVHPT GUS. Fig. 4 Regenerated transformed barley plant.

SUBSTITUTESHEET

Claims

1. Method for genetic transformation of tissues from a monocotyledonous plant, c h a r a c t e r i s e d in that genetic material is inserted into a vector in a bacterium, whereupon the bacterium is co-cultivated with a microspore culture of the plant to be transformed.

2. The method as claimed in claim 1, c h a r a c - t e r i s e d in that a selectable gene is inserted into the bacterium vector, before the bacterium is co-culti¬ vated with the microspore culture.

3. The method as claimed in claim 1 or 2, c h a ¬ r a c t e r i s e d in that the monocotyledonous plant is a cereal species.

4. The method as claimed in claim 3, c h a r a c ¬ t e r i s e d in that the cereal species is barley.

5. The method as claimed in one or more of claims 1-4, c h a r a c t e r i s e d in that the bacterium is an Agrobacterium.

6. The method as claimed in claim 5, c h a r a c ¬ t e r i s e d in that the bacterium is Agrobacterium tumefaciens.

7. The method as claimed in one or more of the pre- ceding claims, c h a r a c t e r i s e d in that the vector is pCVHPT GUS.

8. Genetically transformed tissues from a monocotyle¬ donous plant, c h a r a c t e r i s e d in that they have been transformed by inserting genetic material into a vector in a bacterium which is subsequently co-cultivated with a microspore culture of said monocotyledonous plant.

9. Tissues as claimed in claim 8, c h a r a c ¬ t e r i s e d in that they derive from cereals.

10. Tissues as claimed in claim 9, c h a r a c - t e r i s e d in that they derive from barley.