Larry Erickson

PO149, a new member of pollen pectate lyase-like gene family from alfalfa

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5′-and 3′-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.

Compostability and biodegradation study of PLA-wheat straw and PLA-soy straw based green composites in simulated composting bioreactor.

A laboratory scale simulated composting facility (as per ASTM D 5338) was designed and utilized to determine and evaluate the extent of degradation of polylactic acid (PLA), untreated wheat and soy straw and injection moulded composites of PLA-wheat straw (70:30) and PLA-soy straw (70:30). The outcomes of the study revealed the suitability of the test protocol, validity of the test system and defined the compostability of the composites of PLA with unmodified natural substrate. The study would help to design composites using modified soy straw and wheat straw as reinforcement/filler to satisfy ASTM D 6400 specifications.

Stimulation of in vitro shoot proliferation from nodal explants of cassava by thidiazuron, benzyladenine and gibberellic acid

Multiple shoots were produced from nodal explants of cassava (Manihot esculenta Crantz) by a two-step procedure: a 6- to 8-day exposure to 0.11–0.22 µM thidiazuron (TDZ) in liquid Murashige and Skoog (MS) medium followed by culture on agar-solidified MS medium supplemented with 2.2 µM 6-benzyladenine (BA) and 1.6 μM gibberellic acid (GA3). TDZ caused the nodal explants to expand and this expansion (growth) continued during culture with BA and GA3. From this expanded explant, clusters of buds and fasciated stems developed continuously and these gave rise to shoots. The shoot proliferation process was open-ended, yielding an average of 31.5 shoots per nodal explant after 10 weeks of culture with genotype CG 1–56. A positive response was also obtained from seven other genotypes evaluated with this protocol.

Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens

The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR.

Stable transformation of alfalfa (Medicago sativa L.) by particle bombardment

SummaryStable transformation of alfalfa (Medicago sativa L.) was obtained by particle bombardment of calli (derived from petiole and stem sections cultured for three weeks on SH medium), followed by delayed selection with 50 mg/l kanamycin on BOi2Y medium. Selection at a lower level of kanamycin (25 mg/l) in late stages of culture resulted only in escapes. Analysis of seven transgenic plants revealed that all were derived from one transformation event. Segregation of the neomycin phosphotransferase gene in test cross progeny followed a 1∶1 Mendelian ratio for a single locus insertion in a heterozygous state.

A comparison of stilbene and chalcone synthases including a new stilbene synthase gene from Vitis riparia cv. Gloire de Montpellier

A stilbene synthase gene was cloned from Vitis riparia cv. Gloire de Montpellier after PCR amplification with primers designed to include the start and stop codons of stilbene synthase genes of V. vinifera. The exon was very similar to that of other stilbene synthases, particularly those from V. vinifera (99% nucleotide identity). An intron was found which interrupted the predicted codon for cysteine in the same location as in other stilbene and chalcone synthase genes. The intron showed high nucleotide identity (86%) with an intron from a stilbene synthase gene of V. vinifera. The V. riparia sequence was used in an evaluation of the relatedness of stilbene and chalcone synthases of plants. Five procedures involving distance, parsimony and maximum likelihood methods were used for constructing phylogenetic trees, and they yielded slightly to considerably different results. However, none of the trees were consistent with a previous hypothesis that stilbene and chalcone synthases cluster solely based on the genetic relatedness of the species, implying that stilbene synthase genes arose independently in plant families. In our analyses, stilbene and chalcone synthases of Vitis always clustered separately. The relatedness and origin of stilbene and chalcone synthases appears to be more complex than originally believed.

A new arabinogalactan protein-like gene expressed in the pollen of alfalfa☆

Abstract Differential screening of an alfalfa microspore/pollen cDNA library led to the isolation of a cDNA clone ( PO2 ) which contains an open reading frame coding for a polypeptide with features characteristic of arabinogalactan proteins. This deduced polypeptide is composed of 259 amino acids and has three distinct domains: an N-terminal signal peptide, a less hydrophobic central domain with several amino acid repeats, and a C-terminal hydrophobic domain. The predicted mature protein is rich in Ala, Ser and Pro, has a molecular mass of ca. 24 kD, a pI of 4.37 and no N -glycosylation sites, PO2 represents a low-copy-number gene in the alfalfa genome and is transcribed during the late stage of pollen development. The corresponding genomic sequence of PO2 has no introns and the promoter region contains no sequence in common with other eukaryotic promoters including plant pollen-expressed promoters.

The Role of a Phenylalanine Ammonia Lyase Gene during Cassava Bacterial Blight and Cassava Bacterial Necrosis

Manihot esculenta). PAL enzyme activity and MEPAL mRNA levels were measured in leaves of cultivar MCOL 22 following mechanical wounding or inoculation with 2 bacterial pathogens. MCOL 22 is resistant to Xanthomonas cassavae, which causes cassava bacterial necrosis and susceptible to X. axonopodis pv. manihotis, which causes cassava bacterial blight. PAL enzyme activity in the resistant interaction was significantly higher than in the susceptible interaction or the control. Similarly, MEPAL transcripts were detected in leaves during the resistant interaction, but not during the susceptible reaction. RFLP analysis with MEPAL revealed a divergence between South American and African/Asian cultivars and showed promise for MEPAL as a marker for resistance to Xanthomonads.

Cloning of a peroxidase gene from cassava with potential as a molecular marker for resistance to bacterial blight

Cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis, is considered one of the most important bacterial diseases of cassava (Manihot esculenta Crantz). In order to characterize the cassava genes involved in resistance to this disease, a genomic clone of a cationic peroxidase gene, MEPX1, was isolated by PCR from cassava cultivar MCOL 22. The DNA sequence of MEPX1 showed high homology with other plant peroxidase genes and contained a large intron typical of peroxidase genes. The predicted translation product showed a heme-ligand motif, also a characteristic of peroxidases, as well as phosphorylation, myristoylation and glycosylation sites. The amino acid sequence had 75 % homology with two Arabidopsis thaliana peroxidases. A Southern blot of 17 cassava cultivars, probed with MEPX1, showed multiple hybridization bands. Polymorphisms between cultivars generally reflected geographic origin, but there was also an association with resistance to CBB, indicating that MEPX1 could be a potentially useful marker for this trait.

Differential expression of eight defensin genes of N. benthamiana following biotic stress, wounding, ethylene, and benzothiadiazole treatments

Eight Nicotiana benthamiana defensin genes were identified that could be divided into two classes with class II defensins being longer than class I defensins due to an additional acidic C-terminal domain. Class I defensins were NbDef1.1, NbDef1.2, NbDef1.3, NbDef1.4, NbDef1.5, and NbDef1.6, and class II were Nbdef2.1 and NbDef2.2. Relative RT-PCR showed that NbDef1.1, NbDef1.2, and NbDef1.4 had relatively similar expression levels in healthy leaves, stems, roots, flowers, and seeds. However, Nbdef1.3, NbDef1.5, and NbDef1.6 had varying degrees of tissue specific expression, and Nbdef2.1 and NbDef2.2 had strictly flower-specific expression. None of the defensins were significantly induced by infection by Colletotrichum destructivum or C. orbiculare. However, infection by Pseudomonas syringae pv. tabaci resulted in increased expression of Nbdef1.2 and Nbdef2.2, and decreased expression of NbDef1.1, NbDef1.4, and NbDef1.6. In the hypersensitive response of N. benthamiana containing Pto with P. syringae pv. tabaci containing AvrPto, only NbDef2.2 was significantly up-regulated. Expression of the genes was also affected by abiotic treatments. Both wounding and ethylene treatments resulted in a strong induction of NbDef2.2 and a moderate to weak induction of NbDef1.1, NbDef1.2, and NbDef1.4. Only weak or no induction was observed with treatment with benzothiadiazole. The expression of these eight defensin genes demonstrates that only a small fraction of the members of a defensin gene family will respond to a particular hemibiotrophic pathogen as well as to abiotic stress or signaling molecules.