William E. Dyer

Improved biomass productivity and water use efficiency under water deficit conditions in transgenic wheat constitutively expressing the barley HVA1 gene.

The ABA-responsive barley gene HVA1, a member of group 3 late embryogenesis abundant (LEA) protein genes, was introduced into spring wheat (Triticum aestivum L.) cv. Hi-Line using the biolistic bombardment method. High levels of expression of the HVA1 gene, regulated by the maize ubi1 promoter, were observed in leaves and roots of independent transgenic wheat plants and were inherited by offspring generations. T(3) progenies of four selected transgenic wheat lines were tested under greenhouse conditions for tolerance of soil water deficit. Potted plants were grown under moderate water deficit and well-watered conditions, respectively. Two homozygous and one heterozygous transgenic lines expressing the HVA1 gene had significantly (P<0.01) higher water use efficiency values, 0.66-0.68 g kg(-1), as compared to 0.57 and 0.53 g kg(-1), respectively, for the non-expressing transgenic and non-transgenic controls under moderate water deficit conditions. The two homozygous transgenic plant lines also had significantly greater total dry mass, root fresh and dry weights, and shoot dry weight compared to the two controls under soil water deficit conditions. Results of this study indicate that growth characteristics were improved in transgenic wheat plants constitutively expressing the barley HVA1 gene in response to soil water deficit.

Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses.

The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of imbibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA3 treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.

Dicamba resistance in kochia

Abstract Kochia plants resistant (R) to field rates of dicamba were characterized for their frequency of occurrence and levels of resistance and for the physiological fate of applied 14C-dicamba. Of 167 randomly sampled fields and seven fields identified by producers to contain R kochia, 19 contained plants that produced 1% or more R progeny. The maximum percentage of R progeny produced by parental plants from any field was 13%. An inbred R line derived from a field collection was 4.6-fold more resistant to dicamba than an inbred susceptible (S) line. Rates of 14C-dicamba uptake and translocation were similar in R and susceptible (S) plants up to 168 h after treatment (HAT). Concentrations of the primary metabolite, 5-hydroxy dicamba, were similar in R and S tissues up to 60 HAT, although amounts were significantly greater in R treated leaves by 96 and 168 HAT. However, because there were negligible levels of dicamba metabolites in R shoots and because the rate of metabolism was relatively slow, the observed changes were inadequate to account for observed resistance levels. Thus, dicamba resistance in kochia cannot be attributed to differential herbicide absorption, translocation, or metabolism. These findings, together with our field observations on the unusually slow spread of resistance within and among fields may indicate that dicamba resistance is a quantitative trait. Nomenclature: Dicamba; kochia, Kochia scoparia (L.) Schrad KCHSC.

Resistance to wheat streak mosaic virus in transgenic wheat engineered with the viral coat protein gene.

Wheat (Triticum aestivum) plants were stably transformed with the coat protein (CP) gene of wheat streak mosaic virus (WSMV) by the biolistic method. Eleven independently transformed plant lines were obtained and five were analyzed for gene expression and resistance to WSMV. One line showed high resistance to inoculations of two WSMV strains. This line had milder symptoms and lower virus titer than control plants after inoculation. After infection, new growth did not show symptoms. The observed resistance was similar to the ‘recovery’ type resistance described previously using WSMV NIb transgene and in other systems. This line looked morphologically normal but had an unusually high transgene copy number (approximately 90 copies per 2C homozygous genome). Northern hybridization analysis indicated a high level of degraded CP mRNA expression. However, no coat protein expression was detected.

Resistance to wheat streak mosaic virus in transgenic wheat expressing the viral replicase (NIb) gene

Wheat (Triticum aestivum L. cv. Hi-Line) immature embryos were transformed with the replicase gene (NIb) of wheat streak mosaic virus (WSMV) by the biolistic method. Six independent transgenic plant lines were analyzed for transgene expression and for resistance to mechanical inoculation of WSMV at R3 or R4 generation. Four out of the six lines showed various degree of resistance to WSMV. These lines had initially milder symptoms than controls, and the new growth ranged from milder symptoms, a substantial delay in symptom development, or asymptomatic. Two lines displayed higher resistance with very mild virus symptoms after inoculation and the new growth of 72% and 32% plants from these lines were asymptomatic and had no detectable virus through the plant life cycle. Interestingly, five out of the six transgenic lines had no detectable transgene mRNA expression by RNA gel blot hybridization. The only line that had detectable transgene mRNA did not show delay in the symptom development but had overall milder symptom to the virus.

In vitro regeneration and multiplication of safflower (Carthamus tinctorius L.)

Abstract We have developed a useful system for direct shoot regeneration from primary seedling explants and immature embryos of the American safflower ( Carthamus tinctorius L.) cultivar. Centennial. Direct shoot regeneration from primary explants was optimal on Murashige and Skoog [1] (MS) basal salts medium containing 0.1 mg/1 1-naphthaleneacetic acid (NAA) and 0.5 mg/1 6-benzylaminopurine (BAP) or 0.1 mg/1 thidiazuron (TDZ). Numbers of regenerated shoots were comparable on media containing BAP or TDZ, althoughTDZ medium was superior in reducing shoot hyperhydricity and permitting multiple harvests of regenerated shoots from primary explants. Shoot regeneration from immature embryos was observed on MS medium containing NAA and TDZ. For shoot multiplication, numerous axillrry shoots were obtained from 1 cm long internodal seedling shoot sections on MS medium containing 1 mg/1 2-isopentenyladenine (2iP). Shoots regenerated from primary Montola and Centennial seedling explants were rooted on 0.5X MS medium containing 1 mg/1 NAA and successfully transferred to the greenhouse.

Factors influencing Agrobacterium tumefaciens-mediated transformation and regeneration of the safflower cultivar ‘centennial’

The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. ‘Centennial’ were examined. Agrobacterium tumefaciens strain EHA105/p35SGUSInt was more infective than LBA4404/pBI121 as determined by numbers of sectors expressing β-glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by addition of acetosyringone, indicating that a hypersensitive response to bacterial infection may reduce organogenetic potential. Likewise exposure of co-cultivated explants to kanamycin or geneticin in selective media reduced regeneration efficiency. Addition of 500 mg l-1 carbenicillin slightly increased numbers of regenerating shoots. Tranfformed shoots were obtained only when kanamycin selection was initiated 1 or 2 days after co-cultivation. Presence of transgenes in geneticin-resistant shoots was confirmed using polymerase chain reaction and Southern hybridization assays.

Physiological characterization of auxinic herbicide-resistant biotypes of kochia (Kochia scoparia)

Abstract Auxin-mediated responses of kochia biotypes resistant to dicamba (HRd) or resistant to dicamba and fluroxypyr (HRdf) were compared with those of two susceptible biotypes. Rates of shoot and root gravitropic response and patterns of apical dominance, as determined by lateral bud sprouting after decapitation, were determined in the absence of herbicide treatment. Shoots of susceptible plants reoriented toward vertical at a rate of 23.4° h−1, whereas the rates of HRd and HRdf shoot reorientation were significantly slower at 7.2° and 14.4° h−1, respectively. Root gravitropic responses were not different between resistant and susceptible biotypes. In contrast to susceptible biotypes, both apical and basal lateral buds on HRd plants elongated after decapitation, although differences between HRd and susceptible biotypes became smaller during succeeding weeks. The elongation pattern of HRdf lateral buds was intermediate to that of susceptible and HRd plants. Inhibition assays of root growth by natural and synthetic auxins showed that HRd root growth was less sensitive to dicamba, 2,4-D, naphthalene-1-acetic acid, and indole-3-acetic acid than was root growth of HRdf or the susceptible biotypes. Collectively, results support the hypothesis that auxin binding or signal transduction pathways are impaired in resistant biotypes and that HRd may contain different lesions than does HRdf. Nomenclature: Dicamba; fluroxypyr; 2,4-D; kochia, Kochia scoparia L. Schrad KCHSC.

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar ‘Centennial’ on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. ‘Centennial’ expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.

Dicamba-responsive genes in herbicide-resistant and susceptible biotypes of kochia (Kochia scoparia)

Abstract The herbicide resistance to dicamba (HRd) biotype of kochia is resistant to several auxinic herbicides and is impaired in shoot gravitropism and other auxin-mediated responses. To better characterize the biotype and investigate its mechanism of resistance, we used messenger RNA (mRNA) differential display to compare patterns of dicamba-induced gene expression in HRd and susceptible (S1) plants. More than 60,000 complementary DNA fragments were generated and examined, 106 of which were isolated and used as probes on Northern blots to confirm gene expression levels. Steady-state levels of > 90% of mRNAs did not change after dicamba application. However, several mRNAs were detected whose levels were decreased, increased, or differentially regulated between the biotypes within minutes of dicamba treatment. The abundance of three mRNAs decreased after treatment, two of which had significant sequence similarity to choline monooxygenase and 5,10-methylenetetrahydrofolate reductase, respectively. Conversely, increased expression levels were observed for a putative chloride channel protein, 1-aminocyclopropane-1-carboxylate synthase, and an unknown gene. Genes differentially expressed between HRd and S1 plants included those similar to a putative translation initiation factor, xyloglucan endotransglycosylase, and a hypothetical protein cloned from several organisms. The results demonstrate that mRNA differential display is a useful technique for discovering genes that are rapidly regulated as part of a physiological response, and that this approach may provide insight into the mechanism of auxinic herbicide resistance in kochia. Nomenclature: Dicamba; kochia, Kochia scoparia L. Schrad. KCHSC.