CA2253292A1 - Transgenic plants with enhanced sulfur amino acid content - Google Patents

Abstract

An enhanced seed content of sulfur-containing amino acids is achieved, without significant allergenicity, in transgenic plants, such as soybean plants, that contain a heterologous DNA segment embodying one or more copies of a gene that codes for a 10 kDa or 16 kDa rice prolamin or sunflower 2S albumin protein.

Description

CA 022~3292 1998-10-29 TRANSGENIC PLANTS WITH ENHANCED
SULFUR AMINO ACID CGl.l~i FIELD OF THE I~V~;N110N
The present invention relates to transgenic plants 5that have elevated levels of sulfur-containing amino acids. More specifically, the invention relates to the production of transgenic plants that contain a DNA
segment with a coding sequence corresponding to that of a gene for a sulfur-rich rice prolamin or sunflower 2S
albumin protein.
BACKGROUND OF THE INVENTION
Many animal feed formulations are based on soybean or corn, which provides a concentrated source of protein.
Soybean meal in particular lacks the essential sulfur-containing amino acids, methionine and cysteine, because the most abundant proteins accumulated in soybean seeds are deficient in these amino acids. Supplementation of animal feeds based on soybean or corn to bring the levels of essential sulfur-containing amino acids up to that required is a significant added expense.
Sulfur content of animal feed formulations based on soybeans has been increased by using soybeans transformed to express Brazil nut 2S albumin gene. The Brazil nut 2S
albumin protein is a seed storage protein that is high in sulfur-containing amino acids, containing 18 mol per cent methionine and eight mol per cent cysteine. Since the Brazil nut 2S albumin is largely responsible for Brazil nut allergenicity, however, the use of seed from soybean plants transformed to express the Brazil nut 2S albumin gene is an unacceptable method of raising the sulfur content of animal feeds based on soybeans.
Brazil nut 2S albumin is a member of a superfamily of homologous proteins hypothesized to have a common ancestral qene(s). Kreis et al ., J. Mol . Biol . 183: 499 (1985). Many other seeds that have 2S albumin storage proteins are known to contain~potent allergens. Melo et al., Food & Agricultural I~r1munology 6: 185 (1994) . This CA 022~3292 1998-10-29 W O 97/41239 PCTrUS97/06180 is not surprlsing, given the degree of homology of 2S
albumin storage proteins.
Monocot plants also contain seed storage proteins.
The predominant storage proteins in most of the agronomically important cereals are the prolamins. Rice and oats are unusual cereal crops, in that the major storage protein is an llS-type globulin, and only small amounts of prolamin proteins are found. Fewer monocot genes havo been introduced successfully into dicot plants, for several reasons. There are definite differences in seed composition, gene regulation and post transciptional processing of storage proteins in dicots and monocots. In particular, monocot promoters typically do not function well in dicots. Other concerns relate to the stability of the sulfur-rich gene in the seed, and possible affects of the introduced gene on normal development of the plant. Altenbach et al., TIBTECH 8:
156 (1990).
SU ~ RY OF THE lNV ~:NllON
It is therefore an object of the present invention to provide plants that express an exogenous DNA sequence encoding a protein which is high in sulfur-containing amino acids but which does not cause an allergenic reaction in humans or animals.
It also is an object of the present invention to provide a method of increasing the content of sulfur-containing amino acids in an animal feed without supplementation.
It also is an object of the present invention to provide a~ approach for altering a plant so that it produces seeds characterized by levels of sulfur-containing amino acids that are higher than those of plants that have not been so altered.
In accomplishing these and other objectives, there has been provided, in accordance with one aspect of the present invention, a transgenic plant that contains a DNA
molecule comprised of (A) a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or CA 022~3292 1998-10-29 W O 97/41239 PCT~US97/06180 sulfur-rich sunflower 2S albumin gene and (B) a promoter, operably linked to the nucleotide sequence, to effect expression thereof by the transgenic plant. The transgenic plant preferably is a corn or soybean plant.
A preferred rice prolamin gene is a sulfur-rich 10 kDa rice prolamin gene, while a preferred sunflower 2S
albumin gene is SFA-7 and SFA-8, preferably SFA-8. The invention also provides seeds produced by the transgenic plants.
A method of altering a plant to produce seed that contains levels of sulfur-containing amino acids that are higher than those of an unaltered plant is provided, comprising steps of (i) providing cells or tissues of a plant tran~formed with an expression vector that contains a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene;
(ii) propagating plants from the transformed cells or tissue; and (iv) selecting for further propagation those plants that produce sulfur-rich rice prolamin or sulfur-rich sunfl~wer 2S albumin.
A method also is provided for increasing the content of sulfur-containing amino acids in an animal feed, preferably without supplementation, which comprises steps of (i) providing seeds from a plurality of plants, at least some of which plants contain a DNA molecule comprised of a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene, operably linked to a promoter to effect expressior. of the nucleotide sequence by the plants; and (ii) processing the seeds into animal feed. The method results in a feed product that has not been supplemented by addition of sulfur-containing amino acids and that contains soybean or corn meal obtained from seed of transgenic plants.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, CA 022~3292 1998-10-29 while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invent on will become apparent to th~se skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the nucleotide and deduced amino acid sequence of sunflower 2S albumin 8 (SFA-8).
Figure 2 is the nucleotide and deduced amino acid sequence of the 10 kDa rice prolamin clone.
DETAILED DESCRIPTION OF THE PREFERRED EMBODl~l~NlS
The present invention allows for the production of transgenic plants that bear seeds characterized hy a higher content of sulfur-containing amino acids, relative to plants that lack the heterologous DNA in question.
Thus, an enhanced sulfur content in soybean (Glycine max) or corn (Zea mays), for example, is achieved, without significant allergenicity problems, by transforming soybean Ol corn material with a heterologous DNA segment that has a nucleotide sequence corresponding to one or more copies of a gene for a rice (Oryza sativa L) 10 kDa or 16 kDa prolamin or a sunflower (Helianthus annus L) 2S
albumin protein, which are rich in sulfur.
In view of the similarity between the Brazil nut 2S
albumin p-otein and its sunflower homolog, it is surprising that the sulfur-rich sunflower 2S albumin protein does not cause allergenicity problems. It also is surprising that a prolamin gene from rice, a monocot, can be successfully expressed in soybean, a dicot, without stability problems.
Sulfur-rich sunflower 2S albumin proteins are described, by example, by Lilley et al., "Isolation and Primary Structure for a Novel Methionine-rich Protein from Sunflowerseeds (Helianthusannus.L)," in PROCEEDINGS
OF THE WORLD CONGRESS ON VEGETABLE PROTEIN UTILIZATION IN HUMAN
FOODS AND ANIMAL FEEDSTUFFS 497-502 (1989), andby Kortt et al., Phytochemistry 29: 2805 (1990), the respective contents of which are incorporated herein by reference.

CA 022~3292 l998-l0-29 W O 97/41239 PCTrUS97/06180 Eight proteins, denoted sunflower albumins (SFA) 1 to 8, are identified in the 2S albumin fraction. Two of these, SFA-7 and SFA-8, are sulfur-rich. They contain about eight mol per cent cysteine and 16 mol per cent methionine. SFA-8 is present in greater amounts than SFA-7, and for this reason is preferred. The nucleotide sequence of SFA-8 is shown in Figure 1.
Matsumura et al., Plant Molec. Biol. 12: 123 (1989), describe three prolamin polypeptides isolated from rice seeds, denoted "10 kDa," "13 kDa" and "16 kDa." The 10 kDa and 16 kDa proteins are said to be sulfur-rich.
A full-length clone for the 10 kDa prolamin also is described, and this embodiment is preferred for use according to the present invention. The nucleotide sequence of the 10 kDa clone is shown in Figure 2.
In accordance with the present invention, a DNA
molecule comprising a transformation/expression vector is engineered to include a sequence from one of the sulfur-rich sunflower 2S albumin genes or from the 10 kDa or 16 kDa sulfur-rich rice prolamin gene. Isolation and cloning of these genes by standard methodology is described in Lilley et al., Kortt et al. and Matsumura et al ., supra . The rice prolamin genes first may be modified 'o reflect preferred codon usage in dicots, prior to introduction into soybean.
The sulfur-rich sunflower 2S albumin genes and the 10 kDa or 16 kDa sulfur-rich rice prolamin gene of the present invention can be isolated from a natural source or synthesized from a known sequence. The sequence may be obtained by making a cDNA from mRNA. The sequence may also be derived from a genomic DNA sequence. The sequence may be subcloned in a vector of choice. When isolating the sulfur-rich sunflower 2S albumin or sulfur-rich 10 kDa or 16 kDa rice prolamin gene from the genome, or subcloning these genes, the methodologies used would include identification of the gene by hybridization with probes, PCR, probe/primer/synthetic gene synthesis, CA 022~3292 1998-10-29 W O 97/41239 PCT~US97/06180 sequencing, molecular cloning and other techniques which are well-known to those skilled in molecular biology.
Once the gene of choice has been isolated, a copy of its sequence is placed into an expression vector by standard methods. For a general description of plant expression vectors, see Gruber et al ., "Vectors for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY 89-119 (CRC Press, 1993). It is preferred to insert multiple copies of an expression cassette containing the gene to be introduced into an expression vector, preferably at least four copies. The selection of an appropriate expression vector will depend upon the method of introducing the expression vector into host cell~.
A typical expression vector contains: prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the growth and selection of the expression vector in the bacterial host; a cloning site for insertion of the exogenous DNA sequencei eukaryotic DNA elements that control initiation of transcription of the exogenous DNA
sequence, such as a promoter and an optional enhancer;
and DNA elements that control the processing of transcripts, such as a transcription termination-polyadenylation sequence. The vector also could containadditional sequences that are necessary to allow for the eventual integration of the vector into a chromosome.
Expression of the gene sequence is under the control of a promoter. Examples of suitable promoters are the promotor for the small subunit of ribulose-1,5-bis-phosphate carboxylase, promoters from tumor-inducing plasmids of Agrobacterium tumefaciens, such as the nopaline synthase and octopine synthase promoters, and viral promoters such as the cauliflower mosaic virus (CaMV) l9S and 35S promoters or the figwort mosaic virus 35S promoter. The promoter can be constitutive or inducible.

CA 022~3292 l998-l0-29 W O 97/41239 PCTrUS97/06180 Especially preferred is a "seed tissue-preferred" or ~seed tissue-specific" promoters, that is, promoters that drive high expression of the heterologous DNA segment in seed tissue where control of genes that are involved in seed metabolism is desired, and little or no expression in other parts of the plant. Manufacture of sulfur-rich proteins in other parts of the plant needlessly expends the plant's energy. Examples of known seed tissue-preferred or seed tissue-specific promoters include the soybean prcmoter of ~3-conglycinin, also known as the 7S
protein, which drives seed-directed transcription (Bray, Planta 172: 364 (1987)) and seed-directed promoters from the zein genes of maize endosperm (Pedersen et al., Cell 29: 1015 (1982)). Dicot promoters are preferred for use in soybeans, and a particularly preferred promoter is the bean phaseolin promoter.
In addition to a suitable promoter, one or more enhancers are useful in the invention to increase transcrip~_on of the introduced DNA segment. The enhancer or enhancer-like element can be inserted into the promoter to provide higher levels of transcription.
Examples of such enhancers include inter alia, viral enhancers like those within the 35S promoter, as shown by Odell et al., Plant Mol. Biol. 10: 263-72 (1988), and an enhancer f:.om an opine gene as described by Fromm et al ., Plant Cell 1: 977 (1989).
Selec.able marker genes, in physical proximity to the introduced DNA segment, are used to allow transformed cells to be recovered by either positive genetic selection or screening. The selectable marker genes also allow for maintaining selection pressure on a transgenic plant population, to ensure that the introduced DNA
segment, and its controlling promoters and enhancers, are retained by the transgenic plant.
Many of the commonly used positive selectable marker genes for plant transformation have been isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or CA 022~3292 l998-l0-29 WO 97/41239 PCTnUS97/06180 a herbicide. Other positive selection marker genes encode an altered target which is insensitive to the inhibitor.
A preferred selection marker gene for plant transformation is the neomycin phosphotransferase II
(nptII) gene, isolated from Tn5, which confers resistance to kanamycin when placed under the control of plant regulatory signals. Fraley et al., Proc. Nat'l Acad.
Sci. USA 80: 4803 (1983). Another useful selectable marker is the hygro~ycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plan Mol. Biol. 5: 299 (1985). Additional positive selectable markers genes of bacterial origin that confer resistance to antibiotics include gentamicin acetyl ttansferase, streptomycin phosphotransferase, aminoglycoside-3'-adenyl transferase and the bleomycin resistance determinant. Hayford et al., Plant Physiol.
86: 1216 (1988); Jones et al ., Mol . Gen. Genet. 210: 86 (1987); Svab et al., Plant Mol . Biol . 14 : 197 (1990);
Hille et al., loc. cit. 7: 171 (1986).
Other positive selectable marker genes for plant transformation are not of bacterial origin. These genes include mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67 (1987); Shah et al., Science 233: 478 (1986); Charest et al ., Plant Cell Rep. 8 : 643 (1990).
Another class of useful marker genes for plant transformation with the DNA sequence requires screening of presum~tively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic.
These genes are particularly useful to quantitate or visualize the spatial pattern of expression of the DNA
sequence in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening CA 022~3292 1998-10-29 W O 97141239 PCTrUS97/06180 g presumptively transformed cells include ~-glucuronidase (GUS), ~-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, Plant Mol. Biol. Rep. 5:
387 (1987); Teeri et al., EMBO J. 8: 343 (1989); Koncz et 5 al., Proc. Nat'l Acad. Sci. USA 84: 131 (1987); De Block et al., EMBO J. 3: 1681 (1984). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., Science 247: 449 ( 1990 ) .
In order to create an expression vector containing the gene or genes of interest, an expression cassette first is made by inserting a cloned sunflower 2S albumin or rice prolamin gene into a plasmid under the control of a regulatory sequence. The resulting expression cassette can be ligated back to itself to produce an expression cassette with a tandem repeat of the cloned gene. A
further ligation can be performed to generate a construct that contains four tandem copies of the gene. ~ne or more copies of the expression cassette containing the introduced DNA segment corresponding to the sulfur-rich 2S albumin sunflower gene or sulfur-rich 10 kDa or 16 kDa rice prolamin gene then is transferred to an expression vector. In a preferred embodiment, the vector also contains a gene encoding a selection marker which is functionally linked to promoters that control transcription initiation. Preferably, a disarmed Ti-plasmid is used as a vector for foreign DNA sequences.
To create a transgenic plant, an expression vector containing a sulfur-rich 2S albumin sunflower gene or sulfur-rich 10 kDa or 16 kDa rice prolamin gene can be introduced into protoplasts; into intact tissues, such as immature embryos and meristems; into callus cultures or into isola~ed cells. Preferably, expression vectors are inserted into intact tissues, particularly explants derived from hypocotyl or cotyledonary nodes of a germinated seed. In this regard, an explant is a piece CA 022~3292 1998-10-29 WO97/41239 PCT~S97/06180 of tissue that is taken from a donor plant and is capable of producing callus in culture. Hypocotyl tissue is that portion of the stem of a plant embryo or seedling below the cotyledons and above the root. A cotyledon is an embryonic leaf, and a cotyledonary node is that part of the seedling between the embryonic axis and the cotyledons which botanically defines the division of the hypocotyl and the epicotyl, or embryonic shoot. General methods of culturing plant tissues are provided, for example, by Miki et al., "Procedures for Introducing Foreign DNA into Plants," in METHODS IN P~ANT MOLECULAR
BIOLOGY AND BIOTECHNOLOGY 67-88 (CRC Press 1993).
A preferred method for introducing an expression vector into plant tissue is direct infection or co-cultivation of plant tissue with A. tumefaciens thatcontains an expression vector with the gene of interest and associated regulatory elements. Horsch et al., Science 227: 1229 (1985). A preferred expression vector is the veccor pARC12 tpl830), a plasmid which is part of a binary Ti plasmid system of A. tumefaciens and which containsnopaline synthase/neomycin phosphotransferaseII
(NPTII) as a promoter and selectable marker for transformed plant cells.
The expression vector preferably is transformed into A. tumefaciens using a freeze-thaw technique, as described in PLANT MO~ECULAR BIOLOGY (Gelvin and Schilperoort, eds., Kluwer Academic Publishers, 1988).
After an A. tumefaciens strain is confirmed to contain a complete copy of the construct, the construct can be transferred to the target plant. A suitable model for demonstrating transformation of a plant by the sulfur-rich sunflower 2 S albumin and sulfur-rich rice prolamin genes is Agrobacterium-mediated transformation of the tobacco plant, Nicotiana tabacum. In this model, an Agrobacterium strain confirmed to contain the construct is used to inoculate wounded tobacco plants to generate the transgenic events. Spielmann et al. Mol.
Gen. Genet. 205: 34 (1986).

CA 022~3292 1998-10-29 When Agrobacterium is used to transfer an expression vector to soybean, induction of the virulence (vir) genes in Agrobacterium leads to enhanced transformation.
Cultured soybean cells lack or have a limiting amount of the necessary signal molecules to initiate the transformation process, and induction of the vir genes is necessary to achieving successful transformation.
Various physical parameters can be used to induce the vir genes. Various compounds can be used, individually or in combination, to induce the vir gene. Exemplary of such compounds are phenolic compounds such as acetosyringone, ~-hydroxyacetosyringone, acetovanillone, syringaldehyde, syringic acid and sinapinic acid.
Low temperatures and low pH during co-cultivation also lead to improved transformation, presumably as a result of an effect on the vir genes. Temperatures below 26~ result in more effective transformation, and a temperature of about 20~ is preferred. Buffering at a pH
below 6.0, preferably a pH of about 5.5, also improves transformation efficiency.
Transformation of soybean by Agroba c t eri um also is dependent upon concentration of bacteria in the inoculum.
In general, higher numbers of bacteria result in more transformation events. Preferably an inoculation period of at least 30-minutes, and a concentration of bacteria of at least 3X108 viable cells/ml are used.
For transformation of corn, techniques other than Agrobacterium-mediated transformation are preferred. An especially preferred methodology in this regard entails subjecting an exposed corn meristem to bio~istic bombardment, in order to target non-differentiated meristem cells for transformation, as described in international application PCT 96/04392. According to this approach, meristem tissue is manipulated following bombardment in order to enlarge transgenic sectors, either through selection and/or through effecting a proliferation from the tissue of shoots or multiple meristems per se. The shoot population thus obtained , .

CA 022~3292 l998-l0-29 W O 97/41239 PCT~US97tO6180 then is screened, by means of a nonlethal enrichment assay, to identify either chimeric sectors that will contribute to germline transmission, or non-sectored, periclinal chimeras that will by definition transmit to progeny. Increased time in culture, under selection, enhances the prospects for sectoral-to-periclinal conversions, and also selects for L1-to-L2 conversions which, through a shift in position, ultimately contribute to the germline.
After screening, transgenic plants are established from transformed explants by conventional techniques known to the skilled artisan. A preferred technique is cultivation of transformed explants in liquid counterselection medium, followed by transfer to solidified selection medium. The process is repeated as described in the art for identification by transgenic markers such as ~-glucuronidase (gus). McCabe e~ al., Bio/Technology 6: 922 (1988). Shoots are induced in transgenic explants by known methods. Wright et al., Plant Cell Rep. 5: 150 (1986); Barwale et al., Planta 167: 473 ~1986). The shoots are excised and, by the addition of pyroglutamic acid to a hormone-enriched growth medium, roots readily are induced. Whole mature, reproductive plants are produced after transfer to greenhouse culture in soil.
Seed from transgenic plants according to the invention contain significant levels of sulfur-containing amino acids, particularly methionine. Expression of the sulfur-rich genes was accompanied by a concomitant decrease in trypsin and chymotrypsin inhibitor activities. These endogenous inhibitors are relatively rich in sulfur-containing amino acids, and the overexpression of the introduced genes may be depleting the normal free sulfur pools in the seed, with the result that sulfur is being scavenged from endogenous proteins.
The transgenic plants appear to accumulate the heterologous proteins at the expense of certain endogenous proteins to maintain a homeostatic condition.

CA 022~3292 1998-10-29 W O 97/41239 PCT~US97/06180 The changes produce no apparent effect on the viability of the transgenic plant or on the total seed protein.
Analysis of seed proteins from transgenic plants reveals no larger precursor proteins, suggesting that the transgenic protein is being processed completely, as it would be in the plant of origin. Amino acid analysis of seeds from transgenic plants shows no significant changes as compared to controls, except for increased levels of methionine.
While the present invention has been described in detail with respect to particular preferred embodiments, it should be understood that such description is presented by way of illustration and not limitation.
Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modifications. Thus, while A. tumefaciens is a preferred vector, other types of vectors can be used for transformation by procedures such as direct gene transfer, as described, for example, in PCT application WO 85/01856 and in European application 0 275 069; in vitro protoplast transformation, which is the subject of U.S. patent No. 4,684,611, for instance; plant virus-mediated transformation, illustrated in European application 0 67 553 and U.S. patent No. 4,407,956; and liposome-mediated transformation according to U.S. patent No. 4,536,475, among other disclosures. Direct transfer methods also may be employed, such as microprojectile-mediated delivery, DNA injection, and electroporation.
See, for example, Gruber et al. and Miki et al., both cited above, and Klein et al., Bio/Technology 10: 268 (1992).
The present invention is further described by - reference to the following, illustrative example.

CA 022~3292 l998-l0-29 W O 97/41239 PCTrUS97/06180 EXAMPLE~ ,ROBACTERIUM-MEDIATED TRANSFORMATION OF TOBACCO
OR SOYBEAN TO PRODUCE A TRANSGENIC PLANT THAT HAS A HIGH
CON~N-l~ATION OF SULFUR-CONTAINING AMINO ACIDS
A. Creation of expreRsion vector that contains sulfur-rich sunflower 2S
albumin and rice prolamin genes Full-length cDNA clones for the 10 kDa rice prolamin and sunflower SFA-8 proteins were obtained using RT-PCR
with first strand cDNA as template and gene-specific primers designed against published sequences. See Matsumura et al. and Lilley et al., supra. The resulting PCR products were subcloned into pBluescript SKII and confirmed by sequence analysis. The genes were transferred into p4752, which contains the 5' and 3' regulatory sequences from phaseolin. Sengupta-Gopalan et al., Proc. Nat'l Acad. Sci. USA 82: 3320 (1985). The resulting expression cassettes contained SFA-8 (p6445) and 10 kDa rice prolamin (p6465) under control of the phaseolin regulatory sequences. p6445 and p6465 were then ligated back to themselves to produce p6668 and p6670, respectively, which contained tandem repeats of the expression cassettes. A final set of ligations was performed with p6668 and p6670 to generate p7518 and p7519, respectively, constructs which contain four tandem copies of the expression cassettes.
The constructs containing the four tandem copies were then transferred to the binary vector pARC12 (pl830), a vector which contains the NPTII selectable marker for plant selection. The resulting binary vectors for 4X SFA-8 and 4X 10 kDa rice prolamin are denoted p6704 and p7499, respectively. These vectors were transformed into Agrobacterium tumefaciens strain LBA4404 by a freeze-thaw method (PLANT MOLECULAR BIOLOGY, op cit.), and presence of a complete copy of the 4X
construct was confirmed.

CA 022~3292 1998-10-29 B. Explant preparation, transformation and transgenic plant recovery Tobacco Tobacco plants (Nicotiana tabacum) var. Xanthii, were cultured from 1 cm apical or axillary explants on OMS, according to Murashige et al., Physiologia Plantarum, 15:473 (1962), in a Magenta Box (Magenta Corp., Chicago, Ill.) at 27~C with a 16 hour light/8 hour dark photoperiod. The first four fully expanded leaves below the apex (leaves 3, 4, 5 or 6) were removed.
Leaves were placed, one at a time, in a 100 mm petri dish with several Whatman #1 filter discs, 7cm, soaked with liquid medium. A sterile, sharp, #2 cork bore (0.5 cm i.d.) was used to punch leaf discs, avoiding the midrib and veins. Discs were held on filter paper at 100~
humidity in a separate petri dish with Whatman filters and liquid medium.
Overnight cultures of A. tumefaciens LBA4404 harboring p6704 and p7499, respectively, were grown to log phase in Minimal A medium containing tetracycline, 1.0 ~g/ml. Cultures were pooled and an optical density at 550 nm was measured. Culture density was adjusted by dilution with liquid medium to about 5 x 105 bacteria/ml.
Leaf discs were inoculated by dipping them individually in a suspension of Agrobacterium harboring the 4X construct. Each disc was held submerged in the suspension for 0.5 -1.0 second, to ensure that all edges were infected. Each disc then was placed on a filter on cocultiva~ion medium the induces regener~tion of shoots in a petri dish. The medium was supplemented with phytohormones and included major salts and MS
micronutrients (Murashiga, op. cit.) supplemented with sucrose, 3.0~ w/v; naphthalene acetic acid (NAA), 0.1 mg/L and 6-benzylaminopurine (BAP), 1.0 mg/L. The medium was buffered to pH 5.7. The filter was dry enough to wick away excess inoculum. Ten to twelve discs per 7 cm filter per 100 mm plate were plated. The petri dishes CA 022~3292 1998-10-29 WO97/41239 PCT~S97/06180 were covered with parafilm and kept in low light at 28~C
for two days.
After two days the explants were moved to a liquid counterselection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of cefotaxime, 500 ~g/ml and vancomycin, 100 ~g/ml. The discs were washed with gentle continuous gyratory shaking for 3-6 hours with at least one change of liquid medium.
The discs then were placed on solid medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ~g/ml and carbenicillin, 500 ~/ml. Excess liquid was evaporated before plating ten to twelve discs per 100 mm plate. The explants were cultures for 3 days at 28~C in low light.
Discs were transferred to solid selection medium, a medium having the same basic composition as that used during cocultivation, but with the addition of vancomycin, 100 ~g/ml; carbenicillin, .00 ~g/ml; and kanamycin, 100 ~g/ml. The discs were cultured at 26~C in high light with 16 hour light/8 hour dark photoperiod.
Transformed sectors became visible approximately two weeks later. Shoots appeared soon after callus and these were removed from the callus and place on hormone-free medium fo~ rooting.
Soybean Seeds of soybean (Glycine max), var. PHI9341, were surface sterilized by exposure to chlorine gas evolved in a glass bell jar. Gas was produced by adding 3.5 ml hydrochloric acid (34-37% w/w) to 100 ml to sodium hypochlorite (5.25% w/w). Exposure was for 16-20 hours in a container approximately one cubic foot in volume.
Surface sterilized seed was stored in petri dishes at room temperature. Seed was germinated by plating of 1/10 strength agar solidified medium according to Gamborg (B5 basal medium with minimal organics, Sigma Chemical Co., cat. no. G5893, 0.32 gm/L; sucrose, 0.2% w/v and CA 022~3292 1998-10-29 W O 97/41239 rCT~US97106180 2-[N-morpholino]ethanesulfonic acid] (MES), 3.0 mM
without plant growth regulators and culturing at 28~C
with a 16 hour day length and cool white fluorescent illumination of approximately 20 ~Em2S~. After three or four days, seed was prepared for co-cultivation. The seed coat was removed and the elongating radicle was removed 3-4 mm below the cotyledons. Ten prepared seed were held in each of several petri dishes.
Overnight cultures of A. tumefaciens LBA4404 harboring p6704 and p7499, respectively, were grown to log phase in Minimal A medium containing tetracycline, 1.0 ~g/ml. Cultures were pooled and an optical density at 550 nm was measured. An amount of culture sufficient to collect upon sedimentation between 1.0 and 2.0 x 101~
cells, where O.D. 550 1.0 = 1.4 x 109 cells/ml, was placed in a 15 ml conical centrifuge tube, and spun down at 6000 g for 10 minutes. After centrifugation the supernatant was decanted and the tubes were held at room temperature until inoculum was needed, but not longer than one hour.
Inoculations were conducted in batches such that each plate of seed was treated with a newly resuspended pellet of A. tumefaciens harboring the 4X construct. One at a time the pellets were resuspended in 20 ml inoculation medium. Inoculation medium consisted of B5 salts (B5893), 3.2 gm/L; sucrose, 2.0~ w/v; BAP, 44 ~M;
and indolebutyric acid (IBA), 0.5 ~M. Acetosyringone (AS), 100 ~M was added and the medium was buffered to pH 5.S with MES, 10 mM.
The mixture was resuspended by vortexing and the inoculum was poured into a petri dish containing prepared seed and the cotyledonary nodes were macerated with a surgical blade. This was accomplished by dividing seed in half by longitudinal section through the shoot apex, preserving the two whole cotyledons. The two halves of the shoot apex were broken off at their respective cotyledons by prying them away with a surgical blade.
The cotyledonary node was then macerated with the CA 022~3292 1998-10-29 WO97141239 PCT~S97/06180 surgical blade by repeated scoring along the axis of symmetry. Care was taken not to cut entirely through the explant to the abaxial side. Twenty explants were prepared in roughly five minutes and then incubated for minutes at room temperature without agitation.
Additional plates were prepared during this time. After 30 minutes the explants were transferred to plates of the same medium solidified with Gelrite (Merck & Co., Inc.), 0.2~ w/v. Explants were embedded with the adaxial side up and level with the surface of the medium and cultured at 22~C for three days under cool white fluorescent light, approximately 20 ~Em2S~.
After three days the explants were moved to liquid counterselection medium. Counterselection medium consisted of B4 salts (G5893), 3.2 gm/L; sucrose, 2.0~ w/v; BAP, 5.0 ~M; IBA, 0.5 ~M; vancomycin, 200 ~/ml;
cefotaxime, 500 ~g/ml and was buffered to pH 5.7 with MES, 3 mM. Ten explants were washed in each petri dish with constant, slow gyratory agitation at room temperature for four days. Counterselection medium was replaced four times.
The explants were picked to agarose solidified selection medium. Selection medium consisted of B5 salts (G5893), 3.2 gm/Li sucrose, 2.0~ w/v; BAP, 5.0 ~M; IBA, 0.5 ~M; kanamycin sulfate, 50 ~g/ml; vancomycin, lO0 ~g/ml; cefotaxime, 30 ~g/ml; timentin, 30 ~g/ml and was buffered to pH 5.7 with MES, 3.0 mM. Selection medium was solidified with SeaKem agarose, 0.3~ w/v. The explants ~ere embedded in the medium, adaxial side down and cultured at 28~C with a 16 hour day length and cool white fluorescent light, of 60-80 ~Em2S~.
After two weeks explants were washed with liquid medium on the gyratory shaker. This time the wash was conducted overnight in counterselection medium containing kanamycin sulfate, 50 ~g/ml. The following day explants were picked to agarose solidified selection medium.
Again they were embedded in the medium, adaxial side down, and cultured as before for another two weeks.

CA 022~3292 1998-10-29 W O 97/41239 PCTnUS97/06180 After one month on selective media transformed tissue became visible as green sectors of regenerating tissue against a background of bleached, less healthy tissue. Explants without green sectors were discarded, explants with green sectors were transferred to elongation medium. Elongation medium consisted of B5 salts (G5893), 3.2 gm/L; sucrose, 2.0~ w/v; IBA, 3.3 ~M;
gibberellic acid, 1.7 ~M; vancomycin, 100 ~g/ml;
cefotaxime, 30 ~g/ml; timentin, 30 ~g/ml and was buffered to pH 5., with MES, 3.0 mM. Elongation medium was solidified with gelrite, 0.2~ w/v. Explants were embedded adaxial side up and culture as before. Culture was continued on this medium with transfers to fresh plates every two weeks.
When shoots became 0.5 cm in length they were excised at the base and placed in rooting medium in 13 x 100 mm test tubes. Rooting medium consisted of B5 salts (G5893), 3.2 gm/L; sucrose, 15 gm/L; nicotinic acid, 20 ~M; pyroglutamic acid (PGA), 900 mg/L and IBA, 10 ~M. The medium was buffered to pH 5.7 with MES, 3.0 mM and solidified with GelRite, 0.2~ w/v. After ten days the shoots were transferred to the same medium without IBA or PGA. Shoots were rooted and held in these tubes under the same environmental conditions as before.
Once a root system was well established, the plantlet was transferred to sterile soil mix in plant cons (ICN Biomedicals, Inc.; catalogue Nos. 26-720 and 1-02). Temperature, photoperiod and light intensity remained the same as before. Under these conditions the regenerants became vigorous, somewhat small, but mostly normal plants. When their root systems became well established, a corner of the plant con was cut off and the plants were gradually hardened off in an environmental chamber or greenhouse. Finally they were potted in soil mix and grown to maturity, bearing seed, in a greenhouse.
C. Analysis of seeds produced by transgenic plants CA 022~3292 l998-l0-29 W O 97/41239 PCTrUS97/06180 Mature seed from several independent trans~enic lines containing either the SFA-8 or 10 kDa rice prolamin genes were extracted in 2X Laemmli buffer. Proteins were quantitated using a modified Lowry kit from Bio-Rad using bovine serum albumin as a standard. Equal amounts of protein were loaded onto 4-20% Tris-glycine polyacrylamide gels. Molecular weight standards in the range of 3-116 kD were obtained from Novex and run alongside the protein samples. Gels were run in duplicate with one being electrophoretically transferred to Immobilon PVDF membrane for western blotting and the other being used for Coomassie staining. Western blots were performed using affinity purified antibodies against either the sunflower 2S albumin or rice prolamin and the signal was detected by the Western-Light Chemiluminescent Detection System from Tropix, Inc. (Bedford, MA) as per the manufacturer's instructions.
Total amino acid composition can be determined by acid hydrolysis of tobacco or soybean meal by standard protocols. Seed proteins also can be analyzed for trypsin inhibitory activity according to the previously described protocols of Kollipara et al., J. Agricul. Food Chem. 40:2356 (1992). Similarly chymotrypsin inhibitory assays can be done according to Geiger, Chymotrypsin. In "Methods of Enzymatic Analysis," pp. 99-109 (1984).
Analysis of seed proteins from 6 independent transgenic tobacco lines that contain the gene for SFA-8 revealed a band that comigrated with purified SFA-8. No signal was detected in protein samples from non-transformed tobacco. By comparing the intensity of the immunodetected band to a dilution series of purified SFA-8, the transgenic SFA-8 protein accumulated as a percentage of total seed protein was calculated to be 5-10%. No larger precursor proteins were detected, indicating that the transgenic is processed as it normally is in sunflower.
Similar results were obtain for transgenic tobacco plants expressing the rice prolamin gene. A band was CA 022~3292 1998-10-29 WO97/41239 PCT~S97/06180 detected that comigrated with the purified rice prolamin in all of the transgenic tobacco lines. No signal was detected in protein samples from non-transformed tobacco.
Using a dilution series of purified rice prolamin as a standard, the transgenic rice prolamln protein accumulated as a percentage of total seed protein was calculateà to be 1-5~. Surprisingly, no larger precursor proteins were detected, indicating that the transgenic tobacco plant, a dicot, processed the protein in the same way as rice, a monocot.

Claims (15)

What Is Claimed Is:

1. A transgenic plant that contains a DNA molecule comprised of (A) a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene and (B) a promoter, operably linked to said nucleotide sequence, to effect expression thereof by said transgenic plant.

2. A transgenic plant according to claim 1, wherein said plant is a soybean plant.

3. A transgenic plant according to claim 2, wherein said nucleotide sequence corresponds to a sulfurich 10 kDa rice prolamin gene.

4. A transgenic plant according to claim 3, wherein said nucleotide sequence is a sulfur-rich 10 kDa rice prolamin gene as shown in Figure 2.

5. A transgenic plant according to claim 2, wherein said nucleotide sequence corresponds to one of SFA-7 and SFA-8.

6. A transgenic plant according to claim 5, wherein said nucleotide sequence is SFA-8.

7. A transgenic plant according to claim 1, wherein said plant is a corn plant.

8. A seed produced by a plant as claimed in claim 1.

9. A method of increasing the content of sulfur-containing amino acids in an animal feed, comprising the steps of (i) providing seeds from a plurality of plants, at least some of which plants contain a DNA molecule comprised of a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S albumin gene, operably linked to a promoter to effect expression of said nucleotide sequence by said plants;
and (ii) processing said seeds into animal feed.

10. A method according to claim 8, wherein said plants are soybean plants.

11. A method according to claim 10, wherein said nucleotide sequence corresponds to a sulfur-rich 10 kDa rice prolamin gene.

12. A method according to claim 9, wherein said nucleotide sequence corresponds to a sulfur-rich sunflower 2S albumin gene.

13. A feed product that contains soybean meal obtained from seed of a transgenic plant according to claim 1.

14. A feed product according to claim 13, wherein said feed product has not been supplemented by addition of sulfur-containing amino acids.

15. A method of altering a plant to produce seed that contains levels of sulfur-containing amino acids that are higher than those of an unaltered plant, comprising the steps of (i) providing cells or tissues of a plant transformed with an expression vector that contains a nucleotide sequence corresponding to a sulfur-rich rice prolamin gene or sulfur-rich sunflower 2S
albumin gene; (ii) propagating plants from said transformed cells or tissue; and (iv) selecting for further propagation those plants that produce sulfur-rich rice prolamin or sulfur-rich sunflower 2S albumin.