Plant Pathology Commons^™

Certification And The American Phytopathological Society, L. J. Stowell, J. Amador, O.W. Barnett, R. J. Cook, D. E. Mathre, A. K. Vidaver, S. A. Tolin

Department of Plant Pathology: Faculty Publications

The profession of plant pathology strives to improve the health and safety of the public through improved plant health systems. This includes the benefits afforded by well-managed urban landscapes and abundant food and fiber. However, the proud glow of the profession of plant pathology has dimmed over the past 20 years to the point that some members of the American Phytopathological Society (APS), the principal professional society of plant pathologists in the United States, are not sure what a plant pathologist should know or what a plant pathologist is responsible for (Phytopathology News 30:162). Without a unified, positive direction for …

Phylogenetic Relationships Within The Family Potyviridae: Wheat Streak Mosaic Virus And Brome Streak Mosaic Virus Are Not Members Of The Genus Rymovirus, Drake C. Stenger, Jeffrey S. Hall, Il-Ryong Choi, Roy C. French

Department of Plant Pathology: Faculty Publications

The complete nucleotide sequence of wheat streak mosaic virus (WSMV) has been determined based on complementary DNA clones derived from the 9,384-nucleotide (nt) RNA of the virus. The genome of WSMV has a 130-nt 5&#;&#;leader and 149-nt 3&#;-untranslated region and is polyadenylated at the 3&#;&#;end. WSMV RNA encodes a single polyprotein of 3,035 amino acid residues and has a deduced genome organization typical for a member of the family Potyviridae (5&#;-P1/HC-Pro/P3/6K1/CI/6K2/VPg-NIa/NIb/CP-3&#;). Because WSMV shares with ryegrass mosaic virus (RGMV) the biological property of transmission by eriophyid mites, WSMV has been assigned to the genus Rymovirus, of which RGMV is …

Expression Of Human Lactoferrin Cdna Confers Resistance To Ralstonia Solanacearum In Transgenic Tobacco Plants, Zhanyuan Zhang, Dermot P. Coyne, Anne K. Vidaver, Amitava Mitra

Department of Plant Pathology: Faculty Publications

A construct containing a human lactoferrin cDNA was used to transform tobacco (Nicotiana tabacum) using an Agrobacterium-mediated DNA-transfer system to express this human protein in transgenic plants. Transformants were analyzed by Southern, Northern, and Western blots to determine integration of the cDNA into the plant genome and lactoferrin gene expression levels. Most transgenic plants demonstrated significant delays of bacterial wilt symptoms when inoculated with the bacterial pathogen Ralstonia solanacearum. Quantification of the expressed lactoferrin protein by enzyme-linked immunosorbent assay in transgenic plants indicated a significant positive relationship between lactoferrin gene expression levels and levels of disease …

Physiologic Specialization Of Puccinia Recondita F. Sp. Tritici In Nebraska During 1995 And 1996, J. E. Watkins, S. S. Rutledge, P. Stephen Baenziger, W. Youngquist

Department of Plant Pathology: Faculty Publications

Field samples of Puccinia recondita f. sp. tritici, collected from four wheat-growing regions in Nebraska in 1995 and from three in 1996, were characterized for virulence. Twenty virulence phenotypes were identified in 1995 and 18 in 1996. Virulence phenotypes MBR-10,18 (virulent on Lr genes, 1, 3, 3ka, 10, 11, 18, and 30) and MDR-10,18 (virulent on Lr genes 1, 3, 3ka, 10, 11, 18, 24, and 30) were the most prevalent, with each phenotype comprising 21.6% of the isolates characterized in 1995. Of the 1995 isolates, 24% were virulent on 10 or more host genes. No virulence to Lr16 and …

Polymerase Chain Reaction Detection And Phylogenetic Characterization Of An Agent Associated With Yellow Vine Disease Of Cucurbits, Francisco J. Avila, Benny D. Bruton, Jacqueline Fletcher, J. L. Sherwood, Sam D. Pair, Ulrich Melcher

Department of Plant Pathology: Faculty Publications

Diagnosis of yellow vine disease (YVD) in cucurbits, an important disease in the south-central United States, relies on external symptom appearance, phloem discoloration, and the presence of bacterium-like organisms (BLOs) in phloem. Polymerase chain reaction (PCR) amplification of BLO nucleotide sequences was explored as a means to improve diagnostic techniques. PCR, using a primer pair based on sequences of the citrus-greening BLO, amplified a 0.15-kilobase (kb) fragment from the DNA of symptomatic plants, but not from that of asymptomatic plants. Its nucleotide sequence suggested that the DNA amplified was of prokaryotic origin. A primer pair, designed to amplify nonspecific prokaryotic …

Lindsay M. Black, 1907 To 1997, Myron K. Brakke, D. V. R. Reddy

Department of Plant Pathology: Faculty Publications

Plant pathology and virology lost a pioneer when Lindsay MacLeod Black died on 23 December 1997. We lost a friend and mentor. Black was noted for proving conclusively that a plant virus can multiply in both leafhopper vectors and plants; characterizing unstable, leafhopper-transmitted viruses; and developing insect tissue cultures for studying plant viruses.

Signaling Via Camp In Fungi: Interconnections With Mitogen-Activated Protein Kinase Pathways, James Kronstad, Adriana De Maria, Deanna L. Funnell, R. David Laidlaw, Nancy Lee, Mário Moniz De Sá, Marilee Ramesh

Department of Plant Pathology: Faculty Publications

The cAMP signal transduction pathway controls a wide variety of processes in fungi. For example, considerable progress has been made in describing the involvement of cAMP pathway components in the control of morphogenesis in Saccharomyces cerevisiae, Ustilago maydis, and Magnaporthe grisea. These morphological processes include the establishment of filamentous growth in S. cerevisiae and U. maydis, and the differentiation of an appressorial infection structure in M. grisea. The discovery that appressorium formation requires cAMP signaling provides an immediate connection to fungal virulence. This connection may have broader implications among fungal pathogens because recent work indicates that …

Management Of Red Squirrel Feeding Damage To Lodgepole Pine By Stand Density Manipulation And Diversionary Food, Thomas P. Sullivan

Vertebrate Pest Conference Proceedings: 18th (1998)

The red squirrel (Tamiasciurus hudsonicus) feeds on the vascular tissues of sapling lodgepole pine (Pinus contorta) during spring periods in forests of interior British Columbia and Alberta, Canada. This damage may lead to mortality and reduced growth of crop trees in managed stands. Manipulation of stand density by pre-commercial thinning to densities < 1,000 stems/ha is an effective method to lower squirrel populations and feeding damage. Lowering stand density enhances the growth of crop trees, and understory herbs and shrubs as wildlife habitat, while protecting trees from squirrel feeding. This approach has been successful in several forest ecological zones. An alternative management tool is provision of diversionary food (sunflower seed) for those stands susceptible to feeding damage, and where stand thinning has already been completed. Diversionary food can be applied aerially and is very cost effective for protecting managed stands. These techniques may be used to maintain or even enhance species diversity of small mammal communities in those forest stands requiring protection.

Characterization Of The Large (L) Rna Of Peanut Bud Necrosis Tospovirus, S. Gowda, Satyanarayana Tatineni, R. A. Naidu, A. Mushegian, W. O. Dawson, D. V. R. Reddy

Department of Plant Pathology: Faculty Publications

The nucleocapsids purified from peanut plants systemically infected with peanut bud necrosis virus (PBNV), a member of the genus Tospovirus, contained both viral(v) and viral complementary(vc) sense L RNAs. Defective forms of L RNA containing ‘core polymerase region’ were observed. The full length L RNA of PBNV was sequenced using overlapping cDNA clones. The 8911 nucleotide L RNA contains a single open reading frame (ORF) in the vc strand, and encodes a protein of 330 kDa. At the 50 and 30 termini of the v sense RNA there were 247 and 32 nt untranslated regions, respectively, containing an 18 …

A Phylogenetic Analysis Of Heterorhabditis (Nemata: Rhabditidae) Based On Internal Transcribed Spacer 1 Dna Sequence Data, B. J. Adams, A. M. Burnell, Thomas O. Powers

Department of Plant Pathology: Faculty Publications

Internal transcribed spacer 1 sequences were used to infer phylogenetic relationships among 8 of the 9 described species and one putative species of the entomopathogenic nematode genus Heterorhabditis. Sequences were aligned and optimized based on pairwise genetic distance and parsimony criteria and subjected to a variety of sequence alignment parameters. Phylogenetic trees were constructed with maximum parsimony, cladistic, distance, and maximum likelihood algorithms. Our results gave strong support for four pairs of sister species, while relationships between these pairs also were resolved but less well supported. The ITS1 region of the nuclear ribosomal repeat was a reliable source of …

Peanut Yellow Spot Virus Is A Member Of A New Serogroup Of Tospovirus Genus Based On Small (S) Rna Sequence And Organization, Satyanarayana Tatineni, S. Gowda, K. Lakshminarayana Reddy, S. E. Mitchell, W. O. Dawson, D. V. R. Reddy

Department of Plant Pathology: Faculty Publications

Peanut yellow spot virus (PYSV) represents a distinct tospovirus species based on serology and nucleic acid hybridization. The sequence of the S RNA was 2 970 nucleotides with 22 nucleotide long inverted repeats (with three mismatches) at the termini. The coding was ambisense with a long open reading frame (ORF) in each strand. The 5'-large ORF (1 440 nucleotides in the viral sense (v) strand) encoded a protein with a predicted size of 53.2 kDa that was identified as the nonstructural (NSs) protein based on 16–21% sequence identity and 42– 48% sequence similarity with other tospoviruses. A 3' ORF (741 …

Hyaluronan Synthase Of Chlorella Virus Pbcv-1, Paul L. Deangelis, Wei Jing, Michael V. Graves, Dwight E. Burbank, James L. Van Etten

Department of Plant Pathology: Faculty Publications

Sequence analysis of the 330-kilobase genome of the virus PBCV-1 that infects a chlorella-like green algae revealed an open reading frame, A98R, with similarity to several hyaluronan synthases. Hyaluronan is an essential polysaccharide found in higher animals as well as in a few pathogenic bacteria. Expression of the A98R gene product in Escherichia coli indicated that the recombinant protein is an authentic hyaluronan synthase. A98R is expressed early in PBCV-1 infection and hyaluronan is produced in infected algae. These results demonstrate that a virus can encode an enzyme capable of synthesizing a carbohydrate polymer and that hyaluronan exists outside of …

Identification Of The Wheat Curl Mite As The Vector Of The High Plains Virus Of Corn And Wheat, Dallas L. Seifers, Tom L. Harvey, T. J. Martin, Stanley G. Jensen

Department of Plant Pathology: Faculty Publications

Wheat with virus-like symptoms (extracts containing a 33-kDa protein in sodium dodecyl sulfate- polyacrylamide gel electrophoresis, negative in enzyme-linked immunosorbent assay to wheat streak mosaic virus, and not infectious in a backassay to other wheat) reacted positively to antiserum made against a protein purified from symptomatic corn infected with the High Plains virus (HPV), indicating a serological relationship between the corn and wheat pathogens. The wheat curl mite (WCM, Aceria tosichella Keifer) was identified as the vector of the virus and caused persistent infection of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) in greenhouse experiments. The …

Characteristics Of Beet Soilborne Mosaic Virus, A Furo-Like Virus Infecting Sugar Beet, G.B. Heidel, C.M. Rush, T.L. Kendall, S.A. Lommel, Roy C. French

Department of Plant Pathology: Faculty Publications

Beet soilborne mosaic virus (BSBMV) is a rigid rod-shaped virus transmitted by Polymyxa betae. Particles were 19 nm wide and ranged from 50 to over 400 nm, but no consistent modal lengths could be determined. Nucleic acids extracted from virions were polyadenylated and typically separated into three or four discrete bands of variable size by agarose-formaldehyde gel electrophoresis. RNA 1 and 2, the largest of the RNAs, consistently averaged 6.7 and 4.6 kb, respectively. The sizes and number of smaller RNA species were variable. The molecular mass of the capsid protein of BSBMV was estimated to be 22.5 kDa. …

The Aspergillus Nidulans Sepa Gene Encodes An Fh1/2 Protein Involved In Cytokinesis And The Maintenance Of Cellular Polarity, Steven D. Harris, Lisbeth Hamer, Kathryn E. Sharpless, John E. Hamer

Department of Plant Pathology: Faculty Publications

Cytokinesis (septation) in the fungus Aspergillus nidulans occurs through the formation of a transient actin ring at the incipient division site. Temperature-sensitive mutations in the sepA gene prevent septation and cause defects in the maintenance of cellular polarity, without affecting growth and nuclear division. The sepA gene encodes a member of the growing family of FH1/2 proteins, which appear to have roles in morphogenesis and cytokinesis in organisms such as yeast and Drosophila Results from temperature shift and immunofluorescence microscopy experiments strongly suggest that sepA function requires a preceding mitosis and that sepA acts prior to actin ring formation. Deletion …

United States Patent, Number: 5,633,447: Plant Tissue Comprising A Subgenomic Promoter, Paul G. Ahlquist, Roy C. French

Department of Plant Pathology: Faculty Publications

A subgenomic promoter of a positive strand RNA virus is disclosed which directs the amplified expression of a structural gene in plant tissue. The core region and an upstream activating domain of the subgenomic promoter are identified. This promoter can be utilized in a modified virus. or in an appropriate engineered recombinant DNA derivative which may be chromosomally integrated or maintained as an episome in transformed cells.

Comparison Of Avrd Alleles From Pseudomonas Syringae Pv. Glycinea, Lisa Wolfson Keith, Carol Boyd, Noel T. Keen, J. E. Patridge

Department of Plant Pathology: Faculty Publications

Avirulence gene D alleles resided on indigenous plasmids in races 0, 2, 3, 4, 5, and 6 of Pseudomonas syringae pv. glycinea (Psg), but the allele in race 1 appeared to be chromosomal. These were all nonfunctional avirulence genes because they neither induced the avirulence phenotype on Rpg4 soybean cultivars nor directed the production of syringolide elicitors when expressed in Escherichia coli cells. The predicted proteins encoded by the seven Psg avrD genes were very similar to that of a functional class II allele from P. syringae pv. phaseolicola G50 race 2, but contained mutations collectively …

Chlorella Virus Pbcv-1 Encodes A Homolog Of The Bacteriophage T4 Uv Damage Repair Gene Denv, Masakazu Furuta, John Schrader, Holly Schrader, Tyler Kokjohn, Simon Nyaga, Amanda Mccullough, R. Stephen Lloyd, Dwight Burbank, Dorit Landstein, Leslie C. Lane, James L. Van Etten

Department of Plant Pathology: Faculty Publications

The bacteriophage T4 denV gene encodes a well-characterized DNA repair enzyme involved in pyrimidine photodimer excision. We have discovered the first homologs of the denV gene in chlorella viruses, which are common in fresh water. This gene functions in vivo and also when cloned in Escherichia coli. Photo-damaged virus DNA can also be photoreactivated by the host chlorella. Since the chlorella viruses are continually exposed to solar radiation in their native environments, two separate DNA repair systems, one that functions in the dark and one that functions in the light, significantly enhance their survival.

Proceedings Of The 24th Annual Meeting, Southern Soybean Disease Workers (March 15-16, 1997, Fort Walton Beach, Florida), Robert P. Mulrooney, Gabe Sciumbato, Steve Koenning, John Russin, Glenn G. Hammes

Southern Soybean Disease Workers: Conference Proceedings

Contents

Business session

Soybean Disease Loss Estimate for the Southern United States during 1996. Compiled by PW Pratt

Treasurer report. GG Hammes

SSDW Committee chairmen for 1996-1997

Graduate student papers

Characterization of Races of Phytophthora sojae in Arkansas and Their Effects on Commonly Grown Cultivars. TA Jackson, TL Kirkpatrick, and JC Rupe

Induction of Defense Related Proteins During Compatible and Incompatible Soybean-Cercospora sojina Interactions. WA Baker, J Qiu, CB Lawrence, S Tuzun, and DB Weaver

Contributed papers

Performance and Cyst Nematode Resistance of Selected Soybean Cultivars in North Carolina Production Fields. SR Koenning and KR Barker

Distribution and Diversity …

Characterization Of An Atp-Dependent Dna Ligase Encoded By Chlorella Virus Pbcv-1, C. Kiong Ho, James L. Van Etten, Stewart Shuman

Department of Plant Pathology: Faculty Publications

We report that Chlorella virus PBCV-1 encodes a 298-amino-acid ATP-dependent DNA ligase. The PBCV-1 enzyme is the smallest member of the covalent nucleotidyl transferase superfamily, which includes the ATP-dependent polynucleotide ligases and the GTP-dependent RNA capping enzymes. The specificity of PBCV-1 DNA ligase was investigated by using purified recombinant protein. The enzyme catalyzed efficient strand joining on a singly nicked DNA in the presence of magnesium and ATP (K_m, 75 μM). Other nucleoside triphosphates or deoxynucleoside triphosphates could not substitute for ATP. PBCV-1 ligase was unable to ligate across a 2-nucleotide gap and ligated poorly across a 1-nucleotide …

United States Patent , Number: 5,602,242: Hybrid Rna Virus, Paul G. Ahlquist, Roy C. French, Robert F. Sacher

Department of Plant Pathology: Faculty Publications

A recombinant RNA virus is provided allowing encapsidation of genetically engineered viral sequences in heterologous, preferably rod-shaped coat, protein capsids. Since icosahedral viruses are limited in the amount of RNA they can carry, and rod-shaped viruses are expansible, this invention allows the size of recombinant virus RNA components to be increased (or decreased). Methods of making and using such recombinant viruses are also provided, specifically with respect to the transfection of plants to bring about genotypic and phenotypic changes.

Genetic Variability Among Maize Inbred Lines For Resistance To The High Plains Virus–Wheat Streak Mosaic Virus Complex, A. Marçon, S. M. Kaeppler, S. G. Jensen

Department of Plant Pathology: Faculty Publications

High plains virus (HPV) is a pathogen that causes a severe disease, especially in susceptible corn genotypes. The virus is transmitted by the eriophyid mite Aceria tosichella, which also transmits wheat streak mosaic virus (WSMV). This often results in a mixed infection by these two viruses. Genetic variability for resistance to the HPV exists among maize inbred lines but has not been characterized. The disease reaction of 30 maize inbred lines to the mixed infection and to WSMV alone was characterized in this study. Evaluation was based on symptom expression and virus titer (HPV and WSMV), as measured by …

Seasonal Occurrence Of Rice Yellow Mottle Virus In Lowland Rice In Côte D’Ivoire, E. A. Heinrichs, A. A. Sy, S. K. Akator, I. Oyediran

Department of Entomology: Faculty Publications

Monthly plantings of the rice variety Bouaké 189 were made under lowland irrigated conditions, to obtain information on the phenological and seasonal occurrence of pests and diseases on the West African Rice Development Association (WARDA) research farm near Bouaké, Côte d’Ivoire. Regular sampling of insect pests and observations on rice yellow mottle virus (RYMV) disease infection throughout the year provided information on the occurrence of RYMV and potential insect vectors. RYMV incidence and grain yields varied depending on planting date, and for a given planting date, varied from one year to another. There was no evidence that RYMV incidence increases …

The Internal Transcribed Spacer Region Of Belonolaimus (Nemata: Belonolaimidae), T. Cherry, A. L. Szalanski, T. C. Todd, Thomas O. Powers

Department of Plant Pathology: Faculty Publications

Belonolaimus isolates from six U.S. states were compared by restriction endonuclease digestion of amplified first internal transcribed spacer region (ITS1) of the nuclear ribosomal genes. Seven restriction enzymes were selected for evaluation based on restriction sites inferred from the nucleotide sequence of a South Carolina Belonolaimus isolate. Amplified product size from individuals of each isolate was approximately 700 bp. All Midwestern isolates gave distinct restriction digestion patterns. Isolates identified morphologically as Belonolaimus longicaudatus from Florida, South Carolina, and Palm Springs, California, were identical for ITS1 restriction patterns. The correlation between ITS1 restriction patterns and the distribution of B. longicaudatus isolates …

Identification Of Cyst Nematodes Of Agronomic And Regulatory Concern With Pcr-Rflp Of Its1, Allen L. Szalanski, Dezhi D. Sui, T. S. Harris, Thomas O. Powers

Department of Plant Pathology: Faculty Publications

The first internally transcribed spacer region (ITS1) from cyst nematode species (Heteroderidae) was compared by nuc.leotide sequencing and PCR-RFLP. European, Asian, and North American isolates of five heterodefid species were examined to assess intraspecific variation. PCR-RFLP patterns of amplified ITS1 DNA from pea cyst nematode, Heterodera goettingiana, from Northern Ireland were identical with patterns from Washington State. Sequencing demonstrated that ITS1 heterogeneity existed within individuals and between isolates, but did not result in different restriction patterns. Three Indian and two U.S. isolates of the corn cyst nematode, Heterodera zeae, were compared. Sequencing detected variation among ITS1 clones from …

The Rdna Internal Transcribed Spacer Region As A Taxonomic Marker For Nematodes, Thomas O. Powers, T. C. Todd, A. M. Burnell, P. C. B. Murray, C. C. Flemming, Allen L. Szalanski, B. A. Adams, T. S. Harris

Department of Plant Pathology: Faculty Publications

The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group membership, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchus had species with large differences in size. ITS heterogeneity in individuals and populations was identified in several nematode taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that …

Cross-Polarized Magic-Angle Spinning 13c Nuclear Magnetic Resonance Spectroscopic Characterization Of Soil Organic Matter Relative To Culturable Bacterial Species Composition And Sustained Biological Control Of Pythium Root Rot, Michael J. Boehm, Tianyun Wu, Alex G. Stone, Bart Kraakman, Donna A. Iannotti, G. Edwin Wilson, Laurence V. Madden, Harry A.J. Hoitink

Department of Plant Pathology: Faculty Publications

We report the use of a model system that examines the dynamics of biological energy availability in organic matter in a sphagnum peat potting mix critical to sustenance of microorganism-mediated biological control of pythium root rot, a soilborne plant disease caused by Pythium ultimum. The concentration of readily degradable carbohydrate in the peat, mostly present as cellulose, was characterized by cross-polarized magic-angle spinning 13C nuclear magnetic resonance spectroscopy. A decrease in the carbohydrate concentration in the mix was observed during the initial 10 weeks after potting as the rate of hydrolysis of fluorescein diacetate declined below a critical threshold level …

Promoters From Chlorellavirus Genes Providing Expression Of Genes In Prokaryotic And Eukaryotic Hosts, Amitava Mitra, James L. Van Etten

Department of Plant Pathology: Faculty Publications

The invention is directed to novel promoters or mutants thereof from Chlorella virus DNA methyltransferase genes. A Chlorella virus gene promoter is operably linked to a first and/or second DNA sequence encoding a gene that is different from the Chlorella virus gene to form an expression cassette. An expression cassette can be introduced into prokaryotic and/or eukaryotic cells and can provide for a high level of expression of the gene encoded by the first and/or second DNA sequence. The invention also provides a method for screening other Chlorella virus genes for promoters that can function to express a heterologous gene …

Expression And Characterization Of An Rna Capping Enzyme Encoded By Chlorella Virus Pbcv-1, C. Kiong Ho, James L. Van Etten, Stewart Shuman

Department of Plant Pathology: Faculty Publications

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5’ diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant A103R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is …

United States Patent 5,500,360: Rna Transformation Vector, Paul G. Ahlquist, Roy C. French

Department of Plant Pathology: Faculty Publications

A + strand RNA viral transformation of host organisms with foreign RNA, and expression of said foreign RNA. The foreign RNA is inserted into an infective RNA viral segment containing replication elements, and allowed to infect the host organism. The invention is exemplified utilizing brome mosaic RNA modified to contain a gene coding for chloramphenicol acetyl transferase (CAT) in the transformation of barley protoplasts.