WO1987000551A1

WO1987000551A1 - Genetic alteration of plants with retroviruses

Info

Publication number: WO1987000551A1
Application number: PCT/US1986/001493
Authority: WO
Inventors: Inder Mohan Verma; Christopher John Lamb
Original assignee: The Salk Institute For Biological Studies
Priority date: 1985-07-16
Filing date: 1986-07-16
Publication date: 1987-01-29
Also published as: AU6137986A; EP0229174A1

Abstract

Retroviruses are used as vectors for carrying foreign genetic material into cells that serve as precursors for plants. A retroviral vector is provided that includes 5'- and 3'-retroviral LTR sequences including sites for inserting viral sequences into a cell genome, a promoter sequence between the insertion sequences and a sequence, under the promotional control of the promoter sequence, that encodes a protein that imparts a new characteristic to the plant. Precursor cells, such se protoplasts, from which mature plants can regenerated, are prepared. The retroviral vector is introduced into the precursor cells and mature transgenic plants are then regenerated from the cells.

Description

GENETIC ALTERATION OF PLANTS WITH RETROVIRUSES The present invention relates generally to genetically altering plants to impart new characteristics to the plants and more particularly to the use of retroviral vectors to carry genes into plants.

BACKGROUND OF THE I VENTION With rapid advances in genetic engineering, recombinant DNA technology is being used for an increasing variety of purposes. Single cell organisms or tissue cell cultures have been genetically engineered to produce desired protein products. Foreign genes have been introduced into the germ lines of animals to alter their characteristics, e.g., growth hormone gene has been introduced into mice to make them larger. Plants^' important to agriculture are being genetically engineered to be more efficient food producers or to be more resistant to disease or pests.

At the present time, the vehicles generally used for inserting genes into plants are tumor induction plasmids (Ti plasmids) which are transmitted to the plants by means of bacteria. Ti plasmids are useful for introducing genetic material into dicotyledonous flowering plants (hereinafter dicotyledons or dicots) , e.g., tobacco, tomato; however, Ti plasmids have not proven useful to date for introducing genetic material into monocotyledonous flowering plants (hereinafter monocotyledons or monocots) including many major crop plants, such as the cereals rice, maize and wheat.

It would be desirable. to have vectors which are more generally useful for inserting foreign genetic material into plants, including monocotyledons.

SUMMARY OF THE INVENTION Recombinant retroviral vectors are assembled and used to transmit genetic information into the genome of plants, including both dicotyledons and monocotyledons. The vector may take the form of a DNA construct or a packaged retrovirion, but in either case. the vector includes 5¹ and 3' retroviral long terminal repeat (LTR) sequences that enable insertion of retroviral genetic material into a plant cell genome, a promoter sequence and a sequence under the promotional control of the promoter sequence that encodes a protein product of interest.

Protoplasts are isolated from plant tissue, and the retroviral vectors are appropriately introduced into the protoplasts, where the retroviral genome in proviral (double-stranded) DNA form becomes inserted into the genome of tne protoplast. The transformed protoplasts are developed into callus tissue and then regenerated into transgenic plants. The plants derived from the protoplasts and their progeny carry the genetic material of the recombinant retroviral vector in their genomes and express the protein product which alters the characteristics of tne transgenic plant.

IN THE DRAWINGS FIGURE 1.is a schematic representation of the assembly of a DNA construct, pEV-4;

FIGURE 2 is a schematic representation of excision of a nopaline-neomycin phosphotransferase (type II) gene from the bacterial transposon Tn5 clone, pNNeo II. The gene, which imparts resistance to neomycin, G418 and kanamycin antibiotics, is linked to a nopaline promoter. Subsequent insertion of the promoter-gene fragment into the pEV-4 construct of FIGURE 1 provides recombinant vectors, pEV-Neo II-l and pEV-Neo II-2, embodying various features of the present invention.

FIGURE 3a is a schematic representation of the assembly of a helper virus construct, designated pSEM;

FIGURE 3b is a schematic representation of another helper virus construct designated pSAM, derived from pSEM;

DEFINITIONS The following terms are defined below as they are used in this application: Protoplast -- all components of a cell exclusive of the cell wall.

Retrovirus -- a virus having a single-stranded RNA genome and having a life cycle as described in Kornberg, DNA Replication, W.H. Freeman & Co.,

San Francisco, California (1980) pp. 596-601; see also Kornberg, DNA Replication (1982 Supplement), W.H... Freemant & Co., San Francisco, California (1982), pp. S190-S195. Provirus -- the double stranded counterpart of the retroviral genome that comprises an essential stage of the life cycle of a retrovirus.

Virion -- a packaged, infectious viral genome, including retroviral genomes that are packaged with proteins that are not encoded by their own nucleic acid sequences.

Construct -- a recombinant DNA assemblage including pro-retroviral sequences.

Retroviral vector -- a vehicle for introducing genetic material into a cell that includes retroviral sequences; the term is used herein to generally include DNA and RNA material and in packaged or non-packaged form.

Helper virus -- a virion capable of infecting a cell and encoding replication proteins and packaging genomic RNA for further propagation when introduced into a cell.

Helper virus vector -- a helper virus as defined above, including DNA and RNA forms of the virus in packaged or non-packaged form but unable to propagate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, it is found that recombinant retroviral vectors are very useful for introducing foreign genetic material into the genome of a denuded plant cell, i.e., a protoplast. In particular, it is found that the recombinant retroviral vectors can be inserted into prepared protoplasts of a variety of plants, including monocotyledons, dicotyledons, conifers and other lower plants where the genetic information carried by the vector inserts into the protoplast genome. Transgenic protoplasts are developed under appropriate controlled circumstances into fully developed transgenic plants, and these plants carry the inserted genetic material in their germ..and somatic cells and transmit the genetic information to their progeny by usual plant propagation methods. The retroviruses may also be inserted, e.g., by microinjection, into other plant cells from which mature transgenic plants may develop, including but not limited to pollen, pollen tubes and thin-walled somatic cells. Retroviruses are excellent vectors for introducing foreign genetic material into cell genomes Decause as a stage of their life cycle, retroviruses exist in proviral form as double-stranded DNA. .Proviral DNA includes 5'- and 3'-long terminal repeat sequences (LTRs) including sites by which the double-stranded DNA provirus inserts itself into the genome of the infected cell. If a retrovirus is genetically engineered to include a gene of interest, this gene of interest also becomes incorporated into the genome of the cell. If properly promoted, the gene of interest expresses protein product within the cell, imparting a new characteristic to the cell and to the organism of which the cell is a part. Recombinant retroviral vectors according to the invention retain 5' and 3' LTR sequences, including genome insertion sites; include a promoter sequence which may either be a viral promoter associated with the 5¹ LTR sequence or a nonviral promoter and also include the sequence (s) encoding the protein product(s).

Retroviruses are generally infective of animal rather than plant cells, having protein coats adapted for inserting the retroviral genome into particular animal cells. Accordingly, artificial means must generally be used to introduce retroviral vectors into plant cells. To -assure that the genetic information is introduced into the germ line of the plant, and thereby all of the cells of the plant and the progeny of the plant, it is preferred to introduce the retroviral vector into a single cell which serves as a precursor for a mature transgenic plant. The fertilized ovum would serve as such a cell; however, good techniques are not generally available for introducing vectors into fertilized ova of plants. An alternative and preferable pxant precursor cell is a protoplast obtained by denuding (e.g., enzymatically or mechanically removing tne cell wall) cells from plant tissue. Protoplasts are plant cells with their cell walls removed to leave only a thin membrane surrounding their cytoplasm. By a number of techniques, foreign material, such as retroviral vectors, may be introduced into protoplasts through their thin membrane.

Once a retroviral vector is introduced into the protoplast, it functions in the normal manner within the cell, recreating the life cycle of the retrovirus. The insertion of the proviral genome in proviral form into the cell genome occurs irrespective of the form which ^■ the retroviral vector takes. The retroviral vector, for example, may be in the form of a DNA construct in which the retroviral sequences are situated within a plasmid or other cloning vector. In such case, only the viral sequences, from the 5' through the 3' insertion sites, and not the other sequences of the vector, become incorporated into the plant cell genome. Alternatively, the vector may be a complete retrovirion in which an RNA retroviral genome is packaged in a coat of proteins. Generally in the case of plants, retroviral coat proteins do not facilitate introduction of the genome into the cell, i.e., through infection, and thus, packaging of the genome, which involves steps beyond construct assembly, does not in itself facilitate genetic alteration of plant precursor cells relative to the DNA construct itself. However, packaged retrovirion may be obtained in much higher titer than is possible with DNA constructs, and for this reason, the extra effort to package retrovirions is generally considered worthwhile.

In tne normal course of a retroviral life cycle, subsequent to proviral insertion into the infected cell genome, retroviral RNA genome is developed and coat proteins are produced within the cell for packaging the genome as an infectious retrovirion. The retrovirions that are shed from the cell are then capable of infecting other cells. In an animal, this can result in a spreading infection (viremia) . Because retroviruses do not generally infect plant cells, there is little likelihood of the inserted retroviral sequences causing a viremia in the plant itself. However, because the end use of many transgenic plants is for food, including human food, it is preferred to eliminate any likelihood, however small, that the plant could become a "factory" for retrovirion production. Accordingly, recombinant retroviral vectors are engineered according to the invention not only to carry foreign genes of interest, and thereby alter the cnaracteristics of a plant, but also to be incapable of generating recombinant retrovirion which could infect an animal that consumes the plant.

So that transgenic plants do not become virus "factories", retroviral constructs are used for genetic alteration of plants that are defective in sequences needed to package their corresponding RNA genome as complete virions. This may be accomplished, for example, by deleting sequences from a retroviral construct that encode proteins which package the retroviral genome, and in particular the segments that encode the retroviral envelope (env) protein. Preferably, the retroviral constructs encode no viral proteins, as these serve no useful purposes in the plant. In the generally preferred embodiments of the method of the present invention, retroviral vectors are introduced into protoplasts in the form of packaged retrovirions. Because the preferred retroviral constructs are deficient in packaging protein-encoding sequences, their genome must be packaged with proteins from another source. That is, their genome must be pacKaged with proteins encoded by the genome of a second retroviral vector, referred to herein as a helper virus vector. When the genome of a construct is packaged by proteins encoded by a helper virus vector, it is suitable for one-time introduction into a cell and cannot by itself thereafter generate packaged retrovirions within the cell. Retrovirions useful for genetic manipulation of plants are obtained (rescued) from _in vitro cell cultures in which cells contain both the recombinant retroviral construct and the helper virus vector.

It is desirable that rescued retrovirion be free of helper virus. Helper virus genome could, like the recombinant retroviral genome^', insert itself in the genome of the cell, perhaps weakening the plant. If the helper virus is infective of an animal that might consume the plant, there exists the risk of making the plant a "factory" for undesirable retrovirus. Thus, it is preferred that replication-competent helper viral vectors be provided which are also not capable of packaging their corresponding RNA genome as a complete virion. Preferably, helper viral vectors are used for packaging purposes having genomes that encode the requisite coat proteins but are deficient in the sequences that assemble the encoded coat proteins to package the genome. When a replication-competent helper virus that is packaging deficient is used to rescue a retroviral genome that lacks coat protein-encoding sequences from a host cell, the only retrovirions that are shed from the host cell have the retroviral genome of the construct packaged in helper viral proteins. These retrovirions are capable of one-time only introduction into a cell and do not produce any additional retrovirions. Associated with the 5¹ retroviral LTR sequence of retroviruses are powerful promoter sequences, and a gene of interest may be inserted into a retroviral construct under the promotional control of the 5' LTR promoter sequences. However, it may be advantageous to insert a foreign gene under the promotional control of a lin ed, nonviral promoter sequence. If plant cell regulatory mechanisms would inhibit the function of a viral promoter sequence, the gene of interest under its_. promotional control will be expressed in small quantities or may not be expressed at all. On the other hand, a gene that is inserted in a retroviral vector and that is under the promotional control of a suitable linked promoter (i.e., linked operably for transcription under control of the promoter) will not be subject to inhibition by cell regulatory mechanisms. Optionally, a promoter sequence which is regulatable by external stimuli, such as by chemicals that may be applied to the soil in which tne plant grows, may be linked operably for transcription and transcriptional control to the gene of interest.

The gene of interest, that is linked to the promoter and under its promotional control, is selected according to the attribute which is desired to be imparted to the plant. It may be desired, for example, to promote rapid growth of the plant, produce larger fruits or other edible bodies, or have increased resistance to disease or pests. It may even be desirable in some cases to genetically alter plants so that they serve as factories for a particularly desirable gene-encoded protein. An important advantage of retroviruses as gene transfer vectors is that they permit insertion between their bracketing LTR sequences of relatively long foreign genetic sequences, particularly if their coat protein-encoding segments are deleted. Retroviral vectors may be packaged up to a length of about 9 kbp, and by retaining only about 1.5-3 kbp of the viral sequences, the vector can carry up to about 6-8 kbp of foreign genetic material within the region bounded by the 5' and 3' LTR insertion- sequences. Thus, a very long foreign gene may be inserted in a retroviral vector or even several shorter foreign.genes may be inserted.

If the gene of interest is not readily selectaole, it is generally preferred that the construct include, in addition to the gene of interest, a selectable marker for purposes of amplification and purification. Selectable markers may be provided in the plasmid sequences.

The DNA construct in some cases is the vector used to produce transgenic protoplasts. However, where it is desired to have a high titer of vector for more efficient transformation of protoplasts, the DNA constructs are introduced into animal cells, from which they may be rescued in retrovirion form by helper virus vectors. Retrovirions may be produced in a eukaryotic cell line, such as a cultured mammalian tumor cell, into which both the construct and a helper virus vector are introduced. DNA constructs are transfectable into euKaryotic cells by standard techniques, including the well known calcium phosphate precipitation procedure, Granam, F.L. et al. , Virology 52, 456-467 (1973). A cell that is transfected with the construct may express tne products of the genetic sequences carried by the construct, providing the means by which cells containing tne vector may be selected for further culturing.

₇ Titers of up to about 10 units/ml have been obtained. At the present time, the most feasible means of producing transgenic plants with retroviral vectors, either DNA constructs or packaged virions, is by introducing the vectors into prepared protoplasts and tnen culturing the protoplasts so as to eventually produce mature transgenic plants. A variety of protoplast preparation and culturing methods are known for various types of plants, and therefore protoplast isolation and culture will not be described- herein in great detail. Reference is made to Indra Vasil and Vimla Vasil, "Isolation and Culture of Protoplasts", International Review of Cytology, Supplement 11B, Chapter 10, pages 1-19 (1980). Although the invention is generally applicable to the production of transgenic plants, the method is practically limited at this time to species of plants which can be produced from isolated protoplasts or plants which can develop from other precursor cells for which methods of introducing retroviral vectors, such as microinjection, are available.

Generally, in protoplast isolation and culture, plant tissue, such as tissue obtained from leaves or other plant organs, are denuded of their cell walls by enzymatic methods. At this stage, the cells may be manipulated, e.g., fusing protoplasts or introducing foreign genetic material into the cells. The protoplasts are then carefully cultured so that they regenerate their cell wall, undergo cell division and are further cultured to produce callus tissue. By exposing the callus tissue to appropriate treatments, e.g., adjustment of concentration of exogenous auxins . and cytokinins, callus tissue cells can be induced to differentiate and begin the process by which the callus develops into a mature plant.

Manipulation of the protoplasts may be effected by a variety of methods. In some cases, it is possible to introduce retroviral vectors into protoplasts through their membrane by exposing the protoplasts to a titer of vector. Conditions which promote protoplast fusion, as described in Otto Schieder and Indra Vasil, "Protoplast Fusion and Somatic Hybridization", International Review of Cytology, Supplement 11B, Chapter 11, pages 20-46 (1980) , also promote transfer of foreign material, including retroviral vectors, through the protoplast membrane. If introduction into protoplasts is to be effected by direct exposure of protoplasts to vector, it is preferred that the vector be in high titer, such as may be provided by retrovirion rescue. Other methods of introducing foreign genetic material into protoplasts include liposome fusion, Mathews, B., "Liposome-Mediated Delivery of DNA to Plant Protoplasts" in Handbook of Plant Cell Culture, Vol. I (eds. Evans, D.A., et al.), pp. 520-540, MacMillan, New York, N.Y. (1983) , naked bacteria-protoplast fusion (spheroplast fusion) Matsui, C. et al.. Plant Cell Reports 2, 30-32 (1983),

Tanaka, N. et al., Mol. Gen. Genet. 195, 378-380 (1984), and microinjection, Russell, S.H. & Tilton, V.R., "Microinjection of Plant^' Cells I. Instrumentaion and Injection" Plant Physiol. 72, Supplement, Abstract #8 (1983), and Tilton, V.R. & Russell, S.H. ,

"Microinjection of Plant Cells II. Cell Isolation, Preparation and Initial Post-Injection Culture", Plant Physiol. 72, Supplement, Abstract #9 (1983) .

If the retroviral vector provides a marker by which protoplasts that incorporate and express the genetic information may be selected, selection at the protoplast stage or shortly thereafter, e.g., at the callus stage, is preferred. However, selection may occur at any stage at plant development, e.g., by testing plant tissue for the presence of the genetic material or its expressed product, as for example with nucleotide chain hybridization probes and/or immunoassay. Because any plant which develops from the single cell precursor incorporates the genetic materia-1 in the chromosome of all of its cells, it will transmit the genetic information to its progeny in conventional manner through sexual or asexual plant propagation mechanisms. The invention will now be described in greater detail by way of specific examples.

EXAMPLE 1 Assembly of a construct, pEV-4, containing retroviral sequences is schematically illustrated in

FIGURE 1. The recombinant pEV-4 plasmid was constructed using standard techniques as described in Maniatis, T. et al. , Molecular Cloning; A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982) ) . pEV-4 contains 5^f- and 3'-LTR sequences and some sequence therebetween, from a molecular clone in pBR322 of Moloney murine sarcoma proviral DNA (see van Beveren et al. , Cell 2_7, 97-108 (1981)). The insert into pBR322 is at the unique EcoRI site. Furthermore, the PstI site in the amp gene of pBR322 has been eliminated (without altering the protein coded by the gene) so that the entire pEV-4 plasmid construct has a unique Pst I site in the retroviral DNA sequences. The amp gene in the pBR322 sequences serves as a selectable marker (in the presence of a picilin) for intermediate replication and purification in host cell lines. The viral sequences include a genome packaging sequence closely adjacent to the 5' LTR sequences. The 5¹ LTR sequence retains its promoter sequence. The coding regions for viral proteins are either completely removed (gag and pol) or truncated (env) ; thus, no viral proteins can be expressed from the construct.

EXAMPLE 2 An infectious retrovirus carrying the neomycin-resistance gene is constructed for insertion into protoplasts as diagrammed in FIG. 2. A retrovirus construct is assembled from the pEV-4 described in Example 1 by inserting therein a neomycin phosphotransferase II gene (NeoII gene) linked in correct reading frame to a nopaline synthetase promoter (referred to herein as a "nopaline promoter"), the linked nopaline promoter-neomycin phosphotransferase II sequence being cut from a previously described plasmid, pNNeo. The linked promoter and NeoII sequences are bounded by Pst I sites, referred to in the Figure as "PstI Linkers," and although there are internal Pst I sites, the linked promoter and neomycin phosphotransferase II gene may be escised from the plasmid by partial Pst I digestion.

As seen in FIGURE 2, the Pst I promoter-gene segment is directly insertable in the unique Pst I site of pEV-4. Because identical restriction sites exist at each end, approximately half of the nopaline promoter-neomycin phosphotransferase II sequence inserts in parallel with the viral sequences and about half inserts in reverse orientation, giving rise to constructs pEV-Neo-1 (parallel) and pEV-Neo-2 (reverse) . The orientation of the promoter-gene insert is not considered to be consequential because the. neomycin gene is under promotional control of the linked . nopaline synthetase gene promoter and not under the control of a viral promoter sequence.

EXAMPLE 3 Construction of a defective helper virus, pSEM, useful for packaging retroviral genomes, such as the RNA genomes. corresponding to pEV-Neo-1 and pEV-Neo-2 described in Example 2, is diagrammed in FIG. 3a. pMLV-intermediate (pMLV-INT) is described by Berns et al. , J. Virol. 36, pp. 254-263 (1980). pMLV-48 (MO-MLV provirus) is described by Bacheler et al., J^ Virol. 3_2, p. 181-190 (1981)

Briefly, molecularly cloned MO-MLV DNA containing the coding region for gag-pol-env and 3'-LTR sequences is linked to a DNA fragment containing SV40 promoter and enhancer elements as well as splice donor-acceptor sites for viral coat protein. Thus the 5'-LTR insertion and promoter sequences as well as the closely adjacent viral packaging sequences are replaced by SV40 insertion and promoter sequences with no adjacent packaging sequences. Replacement of the 5'-LTR insertion segments with non-retroviral sequences having promoter sequences eliminates the retroviral recombinations that frequently occur when both 5¹- and 3 '-LTR segments are present.

Another useful helper viral construct, pSAM, is assembled from pSEM as diagrammatically illustrated in FIGURE 3b. The construct utilizes segments of amphotropic murine retroviruses that are cut from 407^'OA clone (Chattopadhyay et al., J. Virol. 39, 779-791 (1981).

EXAMPLE 4 pEV-Neo-1 plasmid is amplified in the bacterial cell line DM-1 and this plasmid is cotransfected with pSAM into the TK~ mouse fibroblast cell line NIH/3T3TK-. For transfecting fibroblasts with the constructs, TK~ cells were plated at 5 x 10 cells per 5-cm dish on day 1. ^• On day two, 0.5 ug of each of the uncut recombinant plasmids, pEV-Neo-1 and pSAM along with 8 ug carrier DNA (from NIH 3T3 TK~ cells) were transfected into the cells using the calcium phosphate precipitation procedure of Graham, F.E. Virology 52, 456-476 (1973); Corsaro, CM. et al., Somat-Cell Genet. 7, 603-616 (1981). On day three, the cells were split 1:5 into medium containing G418 drug, and every three days thereafter, the medium was replaced with fresh medium containing G418. Medium from the cells selected in G418 medium was assayed for retrovirion, and retrovirion titers of 10 cfu/ml were obtained.

EXAMPLE 5 A Nicotinia spp. plant is placed in the dark for 24 hours to expand the leaves. Young, fully expanded leaves are sterilized in 8 percent chlorox for 10 minutes and then rinsed three times with sterile distilled water. The leaves are then air-dried under sterile conditions. Lower epidermis is peeled from the leaves with fine disecting forceps, and the peeled leaves are cut into sections.

0.05 grams of leaf tissue is transferred into a 60 x 50 millimeter Petri dish containing 2 ml of a solution that contains cellulase at 0.5 percent (w/v) , macerase at 0.05 percent (w/v), driselase at 0.125 percent (w/v) in 8p medium, pH 5.7 (Kao, K..N. and Michayluk, M.R. Planta 126, 105-110 (1975)). The peeled leaves are incubated in this solution in the dark with occasional shaking until protoplasts are released after 4 to 5 hours.

The protoplast-enzyme mixture is passed through a stainless steel mesh with 67 uM pore size to separate, protoplasts from undigested cells. The enzyme is washed from the protoplasts by centrifuging for four minutes at 40 x g in a sterile conical test tube. The supernatant is discarded with a Pasteur pipette. The protoplasts are gently resuspended in 4 ml of 8p medium. Centrifugation and decantation steps are repeated, and the washed protoplasts are resuspended in 2 ml of

8p medium and droplets of the protoplast suspension are dispersed in a 60 x 15 mm sterile petri dish.

The protoplasts in the Petri dish are overlaid ₄ with 2 ml of the 10 cfu per ml retrovirion titer ^• obtained in Example 4. The plates are sealed with a double seal of Parafilm, and the protoplasts are stored under diffused light in plastic boxes. First division of protoplast occurs within about 2-3 days. At this time, a solution containing 20-200 ug/ml kanamycin, as appropriate, is added for selection of kanamycin-resistant callus. Fresh 8p medium containing appropriate concentrations of kanamycin is added every one to two weeks until rapidly proliferating callus is visible. Shoot regeneration from callus is accomplished by culturing the kanamycin-resistant callus in MS medium containing 6 uM 6BA to the liquid protoplast-derived suspension at two week intervals. Regenerated shoots are rerooted by transferring to half strength MS agar medium containing 0.11 uM 3-aminopyridine. After an extensive root system is formed, the regenerated shoots are transferred to peat pellets for further growth. The humidity is gradually decreased, and the light intensity is gradually increased before the regenerated plant are transferred to greenhouse conditions. The regenerated plants are then grown to maturity. A cell extract is obtained from the leaf cells of the mature plants. These extracts are probed with labeled DNA having a neomycin phosphotransferase II-coding nucleotide sequence. As a control, leaves from a mature, untransformed plants are similarly prooed. Probe hybridization is positive for certain of the presumptively transformed plants. Probe hybridization results are negative for the untransformed plants. This indicates that the neomycin phosphotransferase II gene conferring resistance to kanamycin is incorporated in the genome of those mature transgenic plants.

EXAMPLE 6 Immature embryos of pearl millet (Pennisetum americanum (L.) K. Schum. , var. Gahi 3) are isolated 10-20 days after pollination, and are grown _in_ vitro on Linsmaier and Skoog's medium (Linsmaier, E., Skoog, E., Physiol. Plant 18, 100-127 (1965)., supplemented with 2.5 mg/1 2,4-dichlorophenoxy-acetic acid (2,4-D) and 5% coconut milk, to form a callus tissue. The callus tissue is placed in liquid medium to initiate suspension cultures. Suspension cultures are subcultured every 5-6 days in 35 ml of the medium in 250 ml Erlenmeyer flasks on a Gyrotory shaker at 150 rpm, in the dark at 27°C. Protoplasts are isolated by mixing 10 ml of a

4-5 days old suspenion culture with 60 ml of a filter-sterilized enzyme mixture (2% (w/v) Cellulysin, 1% Macerozyme, 0.5% Driselase, 0.5% Rhozyme, 0.25 M sorbitol, 0.25 M mannitol, 250 mg/1 glucose, 3 mM MES buffer, prepared in the hormone free medium of Linsmaier, E. and Skoog, F. , supra, at pH 5.6). The suspensions are incubated for 1 hr. at room temperature and then incubated for 19 hr. at 14°C in the dark. The protoplast/enzyme mixture is filtered through a layer of Miracloth, and successively through 100 urn and 50 urn stainless steel filters to remove undigested cells and other cellular debris.

Protoplasts are collected and washed three times with fresh nutrient medium by low speed centrifugation (100 x g for 3 min.). At the end of each centrifugation cycle, the protoplasts floating at the top of the nutrient medium and those which have pelleted are collected and mixed. After the final wash, the floating and pelleting protoplasts are mixed and cultured in very thin layers of nutrient medium in

Falcon Petri dishes (35 x 10 mm) , at a density of 10 /ml, and the protoplasts are overlaid with an equal

4 volume of the 10 cfu/ml retrovirion titer obtained in

Example 4. The Petri dishes are sealed with Parafilm and incubated in diffused light in a growth chamber at

27°C, with a 16/8 hr. day/night cycle. 8p nutrient medium is used for the washing and culture of protoplasts, without the free amino acids, nucleic acid oases, riboflavin, and vitamin B,_, b^ut containing 0.4M glucose, 1250 mg/1 sucrose, 2,4-D and 6-benzyl amino purine (6BA) . After 20-celled colonies are formed from the protoplasts, fresh medium with 1% glucose and 2% sucrose is added, and after 4-5 weeks the resulting cell masses are transferred to the same medium solidified with 0.8% agar.

At this time, the cell cultures are resuspended in a like medium but containing 20-200 ug/ml kanamycin, as appropriate. The cultures are incubated for an additional two days in the presence of kanamycin. The cell masses are then transferred from the liquid medium to an agar medium containing kanamycin at an appropriate concentration but without any growth substance, resulting in the growth of cells in tight and discrete groups. Plantlets with shoots and roots form within 4-5 weeks. After an extensive root system is formed, the regenerated shoots are transferred to¹" peat pellets for further growth. The humidity is gradually decreased and the light intensity is gradually increased before the regenerated plants are transferred to greenhouse conditions. The regenerated plants are then grown to maturity.

A cell extract is obtained from the root cells- of the mature plants. These extracts are probed with nick translation labeled DNA corresponding to a restriction fragment of the neomycin-resistance gene (NeoII gene) sequence. As a control, root cell extract from mature, untransformed plants are similarly probed. Probe hybridization results of the presumptively transformed plant are positive and probe hybridization results from the untransformed plants are negative. This indicates that the neomycin phosphotransferase II gene conferring resistance to kanamycin is incorporated in the genome of those mature transgenic plants. EXAMPLE 7

Suspensions of Hemerocallis cv. , a daylilly variety designated "Autumn Blaze", are cultured in nipple flasks containing 220 ml of the basal medium of Murashige and Skoog (B g) , supplemented with coconut water (CW) (10 percent v/v) and 2 mg/1

2,4-dichlorophenoxyacetic acid (2,4-D) at pH 5.7. The flasks are rotated at 1 rotation per minute. At 3 week intervals, sub-cultures are made by passing the contents of the flasks through a no. 30 stainless steel sieve (pore size 592 um) (BioRad Inc., Lebanon, PA), allowing the filtrates to settle for 5 min. , decanting the greater part of the supernatant and adding 4-8 ml of the filtrate (approximtely 1 g f. wt of cells) to 220 ml of fresh medium. Cultures are maintained at- 40 percent relative humidity and continuous darkness at 22°C in walk-in growth rooms. To ensure the use of cells which are in the log phase of their growth, protoplasts are prepared from suspensions which have been sub-cultured 7-12 days earlier. Ten ml are pipetted in 50 ml "

Erlenmeyer flasks containing 15-20 ml of an enzyme mixture comprising 1% Cellulysin TM, cellulolytic enzymes ex. Trichoderma viride (Calbiochem) , 0-5 percent enzyme (pectmase) mixture Macerase R T,M_n ex.

Rhizopus (Calbiochem, San Diego, California, U.S.A.),

4-5 mM CaCl₂, 0.3 M each of sorbitol and mannitol, all ^' in BM.,Sr, buffered with 3 mM MES [2- (N-morpholine) ethanesulfonic acid] at pH 5.7. The flasks are incubated at 28°C in the light on a rotary snaker at 60 rotations per minute.

Protoplasts are separated from undigested cells after 5-6 hr. by a two-step filtering process. A first filtration is through two layers of Miracloth (Chicopee Mills, Inc., Milltown, NJ) supported on a 65 mm diameter funnel with its stem (63 mm) directly placed in a 12 ml glass centrifuge tube. A second filtration through a no. 500 stainless steel sieve (pore size 25 urn) removes the remaining undigested fragments and debris. The final filtrate is centrifuged in the tube with its metal closure in place at 100 x g for 10 min. The pellet is gently rinsed six times by dribbling about 4-5 ml of wash medium down the sides of the tube with a Pasteur pipette. The rinse medium is identical to the digestion mixture but lacks the enzymes.

The pellet is suspended in 1-2 ml of the regeneration medium-B salts and vitamins

MS supplemented with 4.5 mM CaCl₂, 1-6 mM xylose, 0.555 mM inositol, 0.3 M each of mannitol and sorbitol, 55.5 mM glucose, 2 percent coconut water, 0.5 mg/1 kinetin and 200 ml/1 casein hydrolysate (enzymatic digest) . Counts are made with a Levy counting chamber with a Fuchs-Rosenthal Double Ruling grid. The¹ concentration of suspended protoplasts is adjusted so that when 1 ml of suspended protoplasts is added to each 250 ml (75 cm ) plastic tissue culture flask (Falcon no. 3024) containing 8 ml of regeneration medium, the

4 final concentration is approximately 5 x 10 protoplasts/ml or higher.

To each flask is added 1 ml of the 10 cfu/ml retrovirion titer obtained in Example 4. The protoplasts are incubated with the retrovirion at 24°C on a 14:10 light:dark cycle for 72 hours. The protoplasts are centrifuged in the flask at 100 x g for- 10 min. , and the supernatant removed. The protoplasts are washed two times in 8 ml of regeneration medium per flask, and the protoplasts are resuspended in 8 ml of regeneration medium.

After 2-4 days, the cultures are resuspended in a like medium but containing 20-200 ug/ml kanamycin, as appropriate.

Flasks are maintained at 24°C on a 14:10 light: dark cycle. After 7-12 days, 5 ml of fresh regeneration medium at a reduced osmotic level is added to each of the flasks and each subsequent week the osmotic level is further reduced by adding additional medium. Then the entire contents of individual plastic culture flasks with surviving cultures are transferred into individual nipple culture flasks containing 220 ml of B_MS supplemented with coconut water 10 percent (v/v) and 2 mg/1 2,4-D together with kanamycin at an appropriate concentration. The suspension from this point onwards is maintained and grown as already described.

Plantlets are produced from these suspensions using the sequence of media changes described by Krikorian, A.D. and Kann, R.P. (1980) Hemerocallis

J. 34, 35-38. This involves transfer from the medium containing 2,4-D and coconut water to a B_MS with coconut water but lacking auxin; and then to another basal medium (Schenk, R.U. and Hildebrandt, A.C. , (1972) Can. J. Bot. 50, 190-204) at approximately 30-day intervals. Plantlets are finally removed from liquid and transferred to an agar medium until they are at a size sufficient to ensure their successful growth in soil in the greenhouse.

By assaying tissue from mature plants with DNA probes for the neomycin-resistance gene, as described in Example 5 above, it is demonstrated that certain of the transformed plants contain the neomycin gene. Hybridization results are negative for untransformed plants.

The above examples demonstrate the usefulness of recombinant retroviral vectors for producing transgenic plants, including dicotyledons (Example 5) and monocotyledons (Example 6) . These represent the two classes of flowering plants; however, there is no reason why retroviral vectors would not similarly work for other seed bearing plants, i.e., the Gymnosperms (conifers) , and lower plants.

It is very advantageous that retroviral vectors can transform monocotyledons, which so far have not been transformed with Ti-plasmids or otherwise. In expanding the applicability of genetic engineering techniques to monocotyledons, the means are available for improving the output and/or sturdiness of a wide variety of plants which currently serve as food sources.

Furthermore, it is useful to have alternatives to Ti-plasmids as transforming vehicles. Retroviral vectors inherently have several advantageous attributes, including the capability of accepting large nucleotide chain inserts and wide applicability to transforming species of plants. It is not intended that the invention be limited to a particular method of introducing retroviral vectors into cells. Although the examples describe introducing retroviral vectors into protoplasts by exposing the protoplasts to high titers of retroviral vectors, other method of introducing the vectors, such as microinjection, liposomes, bacterial spheroplasts etc., are also considered to be within the scope of the invention. Genetic information imparted to plants by retroviral vectors is carried through their germ lines and, as such, transmissable to their progeny. It may be noted that whereas in the above-examples, kanamycin was the antibiotic used to exert selective pressure on cells or protoplasts transformed with a retroviral vector containing the neomycin phosphotransferase II gene, other antibiotics,- such as neomycin and G418 would also apply selective pressure, and it may be desirable to use either of these for selecting cells or protoplasts derived from plants that naturally are quite highly resistant to kanamycin.

While the invention has been described primarily in terms of transforming protoplasts, retroviral vectors may be introduced into other cells which serve as precursors for plants. For example, retroviral vectors may be microinjected into pollen, and pollen tubes and directly into thin-walled embyrionic cells. The retroviral vectors may also be microinjected into thin-walled somatic cells, thereby producing mosaic plants. Some of the progeny of such mosaic plants would be expected to be transgenic, carrying the genetic information of the retroviral vector in their genome.

Various features of the invention are set forth in the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a transgenic plant in which a foreign protein is expressed, giving the plant a new characteristic, the method comprising: isolating a ^• precursor cell; providing a retroviral vector including

5 5' and 3' retroviral LTR sequences, each including sites for inserting viral sequences into a cell genome, a promoter sequence between said insertion sequences and a sequence under the promotional control of said promoter sequence that encodes said foreign protein; introducing 0 said retroviral vector into said precursor cell; and developing a mature plant from said precursor plant.

2. A method according to Claim 1 wherein said vector is provided in double-stranded DNA form.

3. A method according to Claim 2 wherein said 5 vector is a plasmid.

4. A method according to Claim 1 wherein said vector is a packaged retrovirion.

5. A method according to Claim 4 wherein said genome is deficient in sequences encoding the coat 0 proteins required to repackage its genome as a retrovirion.

6. A method according to Claim 5 wherein said retrovirion is provided free of helper virus.

7. A transgenic plant produced by a method of ⁵ Claims 1-6.

8. A transgenic plant according to Claim 7, which plant is monocotyledenous.

9. A transgenic plant according to Claim 7, which plant is dicotyledenous.

~ ~ 10. A retroviral vector for introduction into a precursor plant cell for the purpose of producing a transgenic plant, the retroviral vector including 5' and 3' retroviral LTR sequences each including sites for inserting viral sequences into a cell genome, a promoter

35 sequence between said insertion sites and a sequence under the promotional control of said promoter sequence that encodes a protein product that imparts a new characteristic to a plant.

11. A retroviral vector according to Claim 10 wherein said vector is in double-stranded DNA form.

12. A retroviral vector according to Claim 10 wherein said vector is a plasmid.

13. A retroviral vector according to Claim 10 also including a packaging sequence for assemblying coat proteins around the vector in RNA genome form.

14. A retroviral vector according to Claim 13 being deficient in sequences encoding the coat proteins required for packaging itself in RNA genome form.

15. A retroviral vector according to Claim 14 encoding no coat proteins.

16. In combination, a retroviral vector according to Claim 14 and a helper virus vector which encodes the proteins required for packaging said RNA genome.

17. A combination in accordance with Claim 16 wherein said helper virus vector is deficient in pacKaging sequences for assemblying coat proteins around its own genome.

18. A retroviral vector according to Claim 10 wherein said vector is a retrovirion including an RNA genome and a coat of proteins packaging said RNA genome.

19. A retrovirion according to Claim 18 wnerein said genome is deficient in sequences encoding the coat proteins required to repackage its genome as a retrovirion.

20. A composition comprising retrovirion according to Claim 19 and free of helper virus.