Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
  • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
  • C12N15/8205—Agrobacterium mediated transformation

Definitions

• Dicotyledonous plants are susceptible to infection from two pathogenic species of Gram-negative soil bacteria, Agrobacterium tumefaciens and Agrobacterium rhizogenes. see review by Bevan and Chilton, 1982, Ann. Rev. Genet., 16:357-384 which results in the diseases known as Crown Gall and Hairy Root, respectively.
• the causative agent has been identified as resulting from the transfer and integration of a Segment of the large pTi or pRi DNA plasmid, known as the T-DNA regions Chilton et al., 1977, Cell 11:263-271; Chilton et al., 1982, Nature, 295:432-434.
• Such plasmids contain, the Agrobacterium-derived pTi or pRi DNA signals necessary for the transfer of an engineered T-DNA region. These plasmids are referred to as binary plasmids, Bevan, 1984, Nucl. Acids. Res., 12:8711-8721; An et al., 1985).
• Plant means a plant sufficiently developed to have a shoot and a root that is asexually reproduced by cell culture.
• “Explaht” means a section or a piece of tissue from any part of a plant or plantlet for culturing, whether wild or cultivated, hybrid (somatic or sexual), or genetically variant or transformed.
• Hetere means a plant growth regulator that affects the growth or differentiation of plants, and is exogenous as used in reference to the various media herein.
• Embryoid means a structure similar in appearance to plant zygotic embryo
• “Medium” means culturing media known in the art to be useful for culturing plant tissue
• Ketin is a known hormone
• This invention provides:
• a method of generation of Cucumis sativus embryoids, from Cucumis sativus explants obtained from cotyledon or hypocotyl tissue which comprises culturing the explants in an induction medium consisting of MS medium and exogenous hormones 2,4-D and kinetin for four to five weeks sufficient to induce competent embryoids.
• the embryoids provided by this method may be either transgenic or non- transgenic.
• Transgenic embryoids can be prepared by introducing foreign DNA into the plant material by either using agrobacterium as a vector or by microprojectile bombardment.
• a method for producing Cucumis sativus transgenic plants which comprises subjecting embryoids generated by the method of claim 18 to conversion to obtain whole tranformed plants.
• a composition consisting of Cucumis sativus plant tissue, an induction media, 2,4-D and kinetin.
• Seeds of cucumber (Cucumis sativus L cv. Poinsett 76, Asgrow Seed Co.) were soaked in tap water for approximately 15 minutes. The seed coats were removed manually. The decoated seeds were surface sterilized with 70% ethyl alcohol for one minute. A twenty-five minute treatment with 25% (v/v) solution of commercial bleach (5.25% sodium hypochlorite) followed. The seeds were then rinsed four times with sterile distilled water. Sterilized seeds were germinated at 28oC on 0.8% water agar (Difco Laboratories) in darkness. Unlsss otherwise stated, all media were supplemented with 3% sucrose and solidified with 0.8 % phytagar (Gibco). The pH of all media was adjusted to 5.8 before autoclaving. All media were autoclaved at 121oC for 20 minutes.
• the cultures were then transferred to MS medium + 1.0 mg/1 NAA + 0.5 mg/1 kinetin. These cultures were incubated for an additional two weeks at 26oC under diffuse cool white fluorescent lamps (4K1x) with a 16-hour photoperiod.
• the tissues were then transferred to MS medium with no growth regulators.
• the concentration of 2,4-D added to the induction media range from 1.0 mg/1 to 2.0 mg/1 when explants from cotyledons and hypocotyl are used.
• the concentration of kinetin added is about 0.5 mg/1 for explants from cotyledons and hypotocol.
• Example 3 The Use of Microprojectiles for Transformation Cucumber somatic embryos derived using the tissue sources described in the embodiment of the invention are placed on MS medium containing 2.0 mg/1 of 2,4,D and 0.5 mg/1 kinetin. These tissues were bombarded with microprojectiles which have been coated with DNA containing a plasmid encoding a beneficial gene(s), as described by the manufacturer. After bombardment these tissues were transferred to plates containing fresh media. If the DNA construction used contained the plant expressible NPT II gene, the selection drug, kanamycin 100 to 200 mg/ml was added to the fresh plates. Cucumber tissues bombarded with microprojectiles were regenerated using the procedure described above in the embodiment of the invention.
• transformation with microprojectiles can also be achieved by bombarding the embryogenlc callus.
• Regeneration of potentially transformed cucumber callus tissues was done as follows: The cotyledon pieces from 3-6 days old germinating seedlings were cultured on MS basal medium supplemented with 2.0 mg/1 2.4D and 0.5 mg/1 kinetin. After four days, the Agrobacterium-infected cucumber cotyledon pieces were transferred to the same medium supplemented with 500 mg/ml carbinicillin and 100-200 mg/ml kanamycin and cultured for six additional weeks in the dark at 26oC.
• Example 5 The transfer of virus coat protein genes
• the purpose of this example is to generate a construction for the expression of a plant virus coat protein gene which, when expressed in a plant, results in reduced symptoms or resistance to later infections by that virus.
• a coat protein gene after the identification of a coat protein gene by nucleotide sequencing, its sequences can be modified by using specific oligomers and the technique referred to as polymerase chain reaction (PCR), to attach specific restriction enzyme sites to any coat protein gene.
• PCR polymerase chain reaction
• These restriction enzyme sites can be used to clone the coat protein gene into a plant expression vector which contains the necessary gene regulatory elements needed for controlling expression of the gene after transfer into the genome of various plants.
• This example illustrates how to generate plant expressible genes which allow a plant to be resistant to specific classes of herbicides.
• Such plants are useful for several reasons, (1) herbicides normally lethal can be used, and (2) different crops can be used in close rotations on soil which may contain residual amounts of a previously used herbicide that is normally lethal to the second crop.
• ALS acetolactate synthase
• EPSPS enolpyruvylshikimate-3-phosphate synthase
• a gene which encodes an important enzyme which is either resistant to or detoxifies a specific herbicide is cloned downstream from a plant promoter, such as CaMV 35S (Pietrzak et al, 1986), ribulose-1,5-biphosphate carboxylase small subunit gene (Mazur and Chui, 1985) or other strong plant gene promoter and upstream from a plant gene poly (A) signal sequence (see Chart 3).
• a plant promoter such as CaMV 35S (Pietrzak et al, 1986), ribulose-1,5-biphosphate carboxylase small subunit gene (Mazur and Chui, 1985) or other strong plant gene promoter and upstream from a plant gene poly (A) signal sequence (see Chart 3).
• This gene is then cloned into an Agrobacterium-derived vector (either binary or cis) and using the above described plant transformation methods this genetic material can be transferred and integrated into the genome (Chart 3).
• This gene can also be cloned into a vector containing a plant expressible marker gene (Chart 3) and transferred into cucumber plants using the microprojectile- mediated gene transfer method (see Example 3).
• Promoters for the expression of these antimicrobial polypeptide genes can include the constitutive type or others which have tissue specificity.
• tissue specific promoters, or wound inducible promoters (Sanchez-Serrano et al, 1987) would be useful so as to produce the antimicrobial peptide only when it is needed or in tissues which are more vulnerable to attack by bacterial or fungi, or both pests.
• the engineering of genes encoding antimicrobial polypeptide genes combined with the plant transformation and regeneration schemes described in and Examples 2 and 3 would allow for the transfer of a useful trait to squash plants.
• Chart 5 summarizes the construction of plasmids which could be used with the Agrobacterium-mediated and microprojectile-mediated gene transfer systems.
• the EcoRI fragments from lambda clone WL3Z8 were transferred to the plasmid vector, pUC19 (available from Bethesda Research, P.O. Box 6009, Gaithersburg, Md 20877), using standard methods to obtain clone pWL3Z8.1.
• the EcoRI cloned fragments in pUC19 were then sequenced by the technique described by Maxam and Gilbert (Methods in Enzvmologv 65:499, 1980). Based on this information the complete sequence of the CMV-WL coat protein gene was determined and this is shown in Chart 1.
• clone pWL3Z8.1 contains all but the 5'193 bp of the CMV-WL RNA3 molecule, as determined by comparison with the complete sequence of CMV-Q RNA3 (Davis and Symons, 1988, Virology 164: In press).
• the nucleotide and amino acid sequence of GMV-WL and CMV-C differ by 22.7% (Chart 2A) and 16% (Chart 3B), respectively.
• Example 4 Construction of a micro T-DNA plasmid containing a plant- expressible CMV-WL coat protein gene with the CaMV 35S polyadenylation signal
• the plant expressible coat protein gene was then moved into a vector suitable for Agrobacterium-mediated gene transfer. Following partial digestion with EcoRI, the EcoRI to EcoRI fragment of about 1.9kb was removed from pDH51/CPWL and placed into the EcoRI site of the plasmid, pUC1813 (available from Robert Kay, Dept. of Chemistry, Washington State University, Pullman, Washington), creating the plasmid pUC1813/CPWL. A 1.9 kb fragment containing this plant expressible CMV-WL coat protein gene was removed by partial Hindlll digestion and ligated into the Hindlll site of the vector, pGA482 (An, 1986) (available from Gynehung An, Institute of Biological Chemistry. Washington State University).
• the plasmid pGA482 was previously modified to contain the plant expressible ⁇ -glucuronide gene as described in WO 89/05858, incorporated above, and the modified plasmid is referred to as pGA482/G. After cloning the expression cassette the plasmid was designated pGA482/CPWL/G (see Chart 5A).
• the following expression cassette was constructed to provide the necessary plant regulatory signals (which include the addition of a promoter, 5' untranslated region, translation initiation codon, and polyadenylation signal) to the gene inserts in order to achieve high level expression of the inserts.
• the expression cassette may be used to express any genes inserted therein. Accordingly, the applicability of the expression cassette goes beyond its use in expressing coat protein genes. Rather, the expression cassette may be used to express any desired protein in transgenic plants.
• the expression cassette is the preferred expression system for expressing viral coat protein genes in plants.
• the expression cassette of the preferred embodiment contains: a constitutive promoter; a 5' untranslated region which enhances gene expression; an initiation codon which comprise Kozak's element; a cloning site where the gene to be expressed may be inserted to produce a functional expression unit; and a 3' untranslated region which comprises a poly(A) addition signal and untranslated flanking regions which result in a higher level of expression.
• the expression cassette which is the preferred embodiment of the present invention consists of the cauliflower mosaic virus (CaMV) 35S transcript promoter, the 5'- untranslated region of cucumber mosaic virus (CMV), the CMV translation initiation codon, and the CaMV polyadenylation signal.
• the construction of this expression cassette utilized the Polymerase Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition of a coat protein gene and the excision of the completed cassette so it can be transferred to other plasmids.
• PCR Polymerase Chain Reaction
• 5'-GAAGCTTCCGGAAACCTCCTCGGATTCC-3' contains a Hindlll site at its 5' -end and contains 21 bases which are identical to 21 bases in the 5'-flanking region of CaMV.
• 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3' contains a Ncol site at its 5' -end which contains a translation initiation codon which conforms to Kozak's rules and has 21 bases which are identical to 21 bases in the antisense strand of the CMV 5' -untranslated region.
• 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3' contains a Ncol site at its 5'-end (which contains the same translation initiation codon as oligomer 2) and has 20 bases which are identical to 20 bases in the 3'-untranslated region of CaMV.
• 5'-GAAGCTTGGTACCACTGGATTTTGGTT-3' contains a Hindlll site at its 3' -end and has a 20 base match with the flanking DNA region 3' of the CaMV polyadenylation site (on the antisense strand).
• oligomers were used to amplify sequences contained within the CMV expression clone referred to as pUC1813/CP19, shown in Chart 6B, and referred to above. As depicted in Chart 8, the PCR technique was used to amplify the gene regulatory regions in pUC1813/CP19.
• AAXXATGG resultsed in a fragment of about 400 base pairs in length and amplification of the CaMV 3-untranslated and flanking regions resulted in a fragment of about 200 base pairs in length.
• These fragments were digested with Ncol and Hindlll, isolated from a polyacrylamlde gel, and then ligated into Hindlll digested and phosphatase treated pUC18. The resulting clone is referred to as p18CaMV/CMV-exp and is shown in Chart 8B.
• the length of the WMVII coat protein gene coding region (281 amino acids) is consistent with a gene encoding a protein of about 33 kD.
• the sequences of this WMVII coat protein gene and protein are shown in Chart 2B.
• comparison of this sequence with that obtained from the related virus Soybean Mosaic Virus (SMV) strain N described by Eggeriberger et al. shows that they share overall about 88% identity and excluding the N- terminal length differences they share about 92.5% identity, see Chart 5B. Because these two virus coat proteins share extensive amino acid identities, expression of the coat protein gene from WMVII is expected to yield plants resistant to WMVII infection and could yield plants resistant to SMV infection.
• SMV Soybean Mosaic Virus
• Example 10 Construction of a Plant-expressible WMVII Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG.
• pl8WMVII-exp a plant expressible WMVII gene which, following transcription and translation, will generate a WMVII coat protein which is identical to that derived from the WMVII coat protein gene nucleotide sequence, see Chart 2B.
• this coat protein will differ, because of necessary genetic engineering to add the ATG initiation codon and by including the last four amino acids of the 54 kD nuclear inclusion protein (which is adjacent to the Glu-Ser protease cleavage site); the amino acids added are Val-Ser-Leu-Glu-N-ter WMVII.
• the plant expression cassette for the WMVII coat protein gene was transferred into a suitable micro-T-DNA vector which contains the necessary Agrobacterium T-DNA transfer signals (to mediated transfer from an Agrobacterium and integration into a plant genome) and wide-host range origin of replication (for replication in Agrobacterium) to form plasmid pGA482/G/CPWMVII-exp.
• plasmid pl8WMVII-exp was digested with Hind III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and an 1.8 kb fragment containing the plant-expressible cassette was removed and ligated into the Hind III site of the modified Agrobacterium-derived microvector pGA482 (modification included the addition of the ⁇ - glucuronidase gene).
• the micro T-DNA vector, pGA482 is shown in Chart 7B and available from G. An, Institute of Biological Chemistry, Washington State University, Pullman, WA.
• the resulting plasmid was designated, pGA482/G/CPWMVII-exp is shown in Chart 10B.
• This plasmid (or derivatives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumefaciens or rhizogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b), and others.
• Strains A208 C58, LBA4404, and A4RS are available from American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD.
• Bacteria A4RS(pRiB278b)) is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Paris, France.
• Bacteria C58Z707 is available from Dr. A.G.Hepburn, Dept. of Agronomy, University of Illinois, Urbana, IL.
• these Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible WMVII coat protein gene contained within its T-DNA region. This transfer can be accomplished vising the standard methods for T-DNA transfers which are knoVm to those skilled in the art, or this transfer can be accomplished using the methods described in U.S. Patent application SN 07/135,655 filed December 21, 1987 entitled "Agrobacterium Mediated Transformation of Germinating Plant Seeds". In addition, it has recently been shown that such Agrobacteria are capable of transferring and integrating their T-DNA regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible WMVII coat protein gene into the genome of soybean to develop a soybean plant line which is resistant to infection from soybean mosaic virus strains.
• Plasmid pUC18cpZYMV (Chart 12B) was digested with Hind III (which cuts within the polycloning sites of pUC18, well outside of the expression cassette), and a 1.6 kb fragment containing the plant-expressible cassette v ⁇ s removed and ligated into the Hind III site of the microvector pGA482 (Chart 7B). The resulting plasmid was designated, pGA482GG/cpZYMV is shown in Chart 13B.
• the Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible ZYMV coat protein gene contained within its T-DNA region.

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