Genetically engineering plants for crop improvement.

Dramatic progress has been made in the development of gene transfer systems for higher plants. The ability to introduce foreign genes into plant cells and tissues and to regenerate viable, fertile plants has allowed for explosive expansion of our understanding of plant biology and has provided an unparalleled opportunity to modify and improve crop plants. Genetic engineering of plants offers significant potential for seed, agrichemical, food processing, specialty chemical, and pharmaceutical industries to develop new products and manufacturing processes. The extent to which genetically engineered plants will have an impact on key industries will be determined both by continued technical progress and by issues such as regulatory approval, proprietary protection, and public perception.

Antibody targeting of liposomes: cell specificity obtained by conjugation of F(ab')2 to vesicle surface.

A method devised for conjugating liposomes with protein resulted in the binding of up to 200 micrograms of immunoglobulin G per micromole of lipid. The coupling of antibody to human erythrocyte F(ab')2 in vesicles (140 molecules per vesicle) by this method caused a 200-fold increase in the binding of vesticles to human erythrocytes and resulted in about 80 percent of the vesicle lipid and contents being associated with cells.

Inheritance of functional foreign genes in plants.

Morphologically normal plants were regenerated from Nicotiana plumbaginifolia cells transformed with an Agrobacterium tumefaciens strain containing a tumor-inducing plasmid with a chimeric gene for kanamycin resistance. The presence of the chimeric gene in regenerated plants was demonstrated by Southern hybridization analysis, and its expression in plant tissues was confirmed by the ability of leaf segments to form callus on media containing kanamycin at concentrations that were normally inhibitory. Progeny derived from several transformed plants inherited the foreign gene in a Mendelian manner.

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

Transformation of Arabidopsis thaliana with Agrobacterium tumefaciens.

Transformed Arabidopsis thaliana plants have been produced by a modified leaf disk transformation-regeneration method. Leaf pieces from sterilely grown plants were precultured for 2 days and inoculated with an Agrobacterium tumefaciens strain containing an avirulent Ti (tumor-inducing) plasmid with a chimeric gene encoding hygromycin resistance. After cocultivation for 2 days, the leaf pieces were placed on a medium that selects for hygromycin resistance. Shoots regenerated within 3 months and were excised, rooted, and transferred to soil. Transformation was confirmed by opine production, hygromycin resistance, and DNA blot hybridization of both primary transformants and progeny. This process for producing transgenic Arabidopsis plants should enhance the usefulness of the species for experimental biology.

Engineering herbicide tolerance in transgenic plants.

The herbicide glyphosate is a potent inhibitor of the enzyme 5-enolpyruvylshikimate- 3-phosphate (EPSP) synthase in higher plants. A complementary DNA (cDNA) clone encoding EPSP synthase was isolated from a complementary DNA library of a glyphosate-tolerant Petunia hybrida cell line (MP4-G) that overproduces the enzyme. This cell line was shown to overproduce EPSP synthase messenger RNA as a result of a 20-fold amplification of the gene. A chimeric EPSP synthase gene was constructed with the use of the cauliflower mosaic virus 35S promoter to attain high level expression of EPSP synthase and introduced into petunia cells. Transformed petunia cells as well as regenerated transgenic plants were tolerant to glyphosate.

Isolation of Tissue-Specific cDNAs from Tomato Pistils.

We have used a differential plaque hybridization screening procedure to isolate cDNA clones for genes that show elevated or exclusive expression in tomato pistils. Clones that showed maximal expression in immature pistils (premeiotic to early meiosis) and mature pistils (at anthesis) were isolated. Of nine clones that were characterized, four were found also to express at some stage of anther development. In situ hybridization experiments showed that expression of the genes we have identified is very tightly regulated both spatially and temporally within the pistil. One gene was identified that is expressed in the pistil only in the transmitting tissue of the style. A second gene was found to express exclusively in two to three cell layers of the ovules for a period of less than eight days.

The use of the fluorescent probe alpha-parinaric acid to determine the physical state of the intracytoplasmic membranes of the photosynthetic bacterium, Rhodopseudomonas sphaeroides.

alpha-Parinaric acid has been used to determine the degree of ordering of the hydrocarbon region of purified intracytoplasmic membranes of Rhodopseudomonas sphaeroides. The usefulness of alpha-parinaric acid as a probe of membrane fluidity was established by comparison of its fluorescent properties in phosphatidylcholine vesicles with those of the more commonly used fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene. Both fluorescent probes were shown to monitor similar environments in the phosphatidylcholine vesicles when the phospholipids were maintained at temperatures above their phase transition temperature. The rotational mobility of alpha-parinaric acid in the intracytoplasmic membranes was determined from 0 to 50 degrees C, a region where no phase transitions were detectable. The rotational mobility of alpha-parinaric acid dissolved in vesicles formed from total extracted intracytoplasmic membrane phospholipids, was 2--3-fold greater than that measured in the intact intracytoplasmic membranes; demonstrating that the presence of protein greatly reduces the mobility of the phospholipid acyl chains of the intracytoplasmic membranes. Due to the high protein content of these membranes, the perturbing effect of protein on acyl chain mobility may extend to virtually all the intracytoplasmic membrane phospholipid.

Antibody-directed liposomes. Determination of affinity constants for soluble and liposome-bound antifluorescein.

We have used the binding of liposomes conjugated with antifluorescein antibody specific for fluorescein isothiocyanate-modified erythrocytes as a model for multivalent antigen-antibody interactions. We examined a series of liposome preparations which were conjugated to between 0 and 332 active antibodies per liposome. The antigen binding capacity and mean intrinsic affinity of the soluble and conjugated antibody were determined by fluorescence quenching of carboxyfluorescein. Liposome-cell interaction data were fitted with a Scatchard-type equation. Functional affinity of liposomes for cells was up to 1000-fold greater than the intrinsic affinity of the antibody for soluble ligand. Analysis for binding at high cell concentrations revealed that liposome-induced cell agglutination reduces the number of available binding sites per cell.

Liposome-mediated delivery of tobacco mosaic virus RNA into petunia protoplast : Improved conditions for liposome-protoplast incubations.

Liposome-mediated delivery of TMV RNA into petunia protoplasts and resulting virus antigen production has been used as an assay for determining incubation conditions which favor increased uptake of vesicle contents by plant cells. Vesicle phospholipid composition, incubation buffer divalent metal ion concentration, the type and concentration of polyalcohol used to stimulate vesicle uptake and the RNA content of the liposome preparation were determined to be critical factors influencing the efficiency of delivery. Manipulation of these parameters resulted in a 50-fold improvement in virus antigen production over that obtained with conditions previously used for liposome-protoplast incubations (Proc Natl Acad Sci 79: 1859-1863, 1982). Virus antigen production could be detected following incubation of protoplasts with <0.5 ng of encapsulated TMV RNA, while at higher concentrations of added liposomes, >80% of the protoplasts could be infected. Comparisons with other techniques used to introduce nucleic acids into plant protoplasts indicated that liposome-mediated delivery was 10-to 1 000-fold more efficient than these other methods. The general use of liposomes to introduce RNA and DNA molecules into plant protoplasts is discussed.

Virulence of Agrobacterium tumefaciens Strain A281 on Legumes.

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.

Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer.

Entrapment of pBR322 DNA within liposomes was demonstrated by (i) its comigration with liposomes on Sepharose 4B columns, (ii) resistance of its biological activity to DNase digestion, and (iii) identification of plasmid DNA on agarose gels after lipid extraction. The biological activity of the liposome-entrapped plasmid was determined by transformation assays. The incubation of intact liposomes, containing entrapped pBR322, with competent Escherichia coli cells in the standard transformation mixture resulted in the appearance of tetracycline-resistant colonies at a frequency of 1% of the control frequency. Importantly, this frequency was unaffected by the addition of DNase to the incubation mixture, whereas transformation by free pBR322 DNA was totally eliminated after treatment with DNase.

Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Fate of "old" and "new" membrane.

A nonspecific density labeling technique has been employed to monitor the synthesis of intracytoplasmic membrane in synchronously dividing populations of Rhodopseudomonas sphaeroides. The intracytoplasmic membranes of cells synchronized in D2O-based medium were found to undergo discontinuous decreases in specific density during synchronous cell growth following transfer to H2O-based medium. These abrupt decreases in membrane specific density occurred immediately prior to cell division and were not observed with intracytoplasmic membranes prepared from asynchronously dividing cells (see also Kowakowski, H., and Kaplan, S. (1974) J. Bacteriol. 118, 1144-1157). Discontinuous increases in the net accumulation of cellular phospholipid were also observed during the synchronous growth of R. sphaeroides. This is to be contrasted to the continuous insertion of protein and the photopigment components of the photosynthetic apparatus into the intracytoplasmic membrane during the cell division cycle (Fraley, R.T., Lueking, D.R., and Kaplan, S. (1978) J. Biol. Chem. 253, 458-464; Wraight, C.A., Lueking, D.R., Fraley, R.T., and Kaplan, S. (1978) J. Biol. Chem. 253, 465-471). Further, examination of the protein/phospholipid ratios of purified intracytoplasmic membrane preparations revealed that this ratio undergoes cyclical changes of 35 to 40% during a normal cycle of cell division. In contrast to the results of Ferretti and Gray ((1968) J. Bacteriol, 95, 1400-1406), DNA synthesis was found to occur in a stepwise manner in synchronously dividing cell populations of R. sphaeroides.

Liposome-mediated delivery of tobacco mosaic virus RNA into tobacco protoplasts: A sensitive assay for monitoring liposome-protoplast interactions.

Tobacco mosaic virus (TMV) RNA was encapsulated in large, unilamellar phospholipid vesicles (liposomes), and the encapsulated TMV RNA was shown to be infectious when incubated with tobacco protoplasts under appropriate conditions. Maximal virus production in protoplasts was observed after their incubation with TMV RNA entrapped in phosphatidylserine/cholesterol liposomes. Infection was dependent on the presence of polyalcohols in the incubation mixture. Other parameters, such as the extent of vesicle binding, the cell-induced leakage of vesicle contents, and the degree of liposome toxicity were shown to be important in determining the efficiency of infectivity. Liposome-mediated delivery offers an efficient and reproducible method for introducing RNA into plant protoplasts.

Protein trafficking in plant cells.

The cells of higher plants contain distinct subcellular compartments (organelles) that perform specialized functions such as photosynthesis, carbohydrate and lipid metabolism, and so forth. The majority of the protein constituents of plant organelles are formed as cytosolic precursors with N-terminal extensions that direct transport across one or more membrane bilayers in a post- or co-translational fashion. Since the majority of proteins in plant cells are products of nuclear gene expression, there must be precise sorting mechanisms in the cytoplasm that direct proteins to their correct cellular locations. Based on recent studies of protein targeting to chloroplasts and vacuoles, the details of these intracellular sorting mechanisms are becoming clear. The ability to direct proteins to specific compartments within cells provides new opportunities for improvement of plants by genetic manipulation.

Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn.

The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K(m)app = 1.69 millimolar) than for metolachlor (K(m)app = 8.9 millimolar).

In vitro transcription and translational efficiency of chimeric SP6 messenger RNAs devoid of 5' vector nucleotides.

A plasmid containing the bacteriophage SP6 promoter, designated pHSTO, permits in vitro transcription of RNAs devoid of vector-derived nucleotides. This vector has been characterized for relative transcriptional activity using constructs which alter the conserved nucleotides extending beyond the SP6 transcriptional initiation site. SP6 polymerase efficiently transcribes cDNA inserts which contain a guanosine (G) nucleotide at position +1 relative to the SP6 promoter; however, inserts with an adenosine (A) or pyrimidine at position +1 are not transcribed. Several cellular and viral cDNAs have been transcribed into translatable messenger RNA using this vector; however, SP6 polymerase will not transcribe the A-T rich untranslated leader from alfalfa mosaic virus RNA 4 efficiently unless the viral mRNA cap site is separated from the transcriptional initiation site by twelve base pairs of vector DNA. Chimeric messenger RNAs were created by linking the untranslated leader sequence of several viral mRNAs to the coding region of barley alpha-amylase, and the resultant mRNAs were translated in a wheat germ extract to determine relative translational efficiencies. The untranslated leader sequences of turnip yellow mosaic virus coat protein mRNA and black beetle virus RNA 2 did not increase translational efficiency, while the tobacco mosaic virus leader stimulated translation significantly. The results indicate that substitution of a cognate untranslated leader sequence with a leader derived from a highly efficient mRNA does not necessarily predict enhanced translational efficiency of the chimeric mRNA.

Identification of a cloned cytokinin biosynthetic gene.

A small region of the Ti plasmid (the tmr locus), thought to be involved in phytohormone metabolism in Agrobacterium tumefaciens-transformed plant tissue, was cloned and expressed in Escherichia coli. By enzyme assay, the tmr locus was shown to encode isopentenyltransferase, an enzyme that catalyzes the first step in cytokinin biosynthesis.