Hugo R. Permingeat

A Simple Method for Isolating DNA of High Yield and Quality from Cotton (shape Gossypium hirsutum L.) Leaves

An easy, reproducible and fast procedure to isolate DNA from cotton leaves is described. The addition of 0.5 M glucose in the extraction buffer avoids browning by polyphenolic compounds and improves the quality of DNA for molecular analysis. The DNA yield ranged between 150–400 mg per gram of fresh tissue. The DNA was suitable for digestion by restriction enzymes and amplificatiion by Taq DNA polymerase.

Transient transformation of maize tissues by microparticle bombardment

Abstract Transient transformation of maize coleoptiles with the β-glucuronidase gene was achieved with a particle gun built by us essentially according to the concept of Klein et al. (Nature, 327 (1987) 70–73). Either tungsten or gold particles were used to transform coleoptiles. Transformed blue cells were found in different cell layers and occasionally in as deep as the 8th layer. A transformation efficiency of up to 2% was obtained when the gene was in plasmid pAI 1 GUS driven by the alcohol dehydrogenase promoter plus the intron 1. The 35S promoter of cauliflower mosaic virus (plasmid pBI221) was ten times less efficient. Other maize tissues like immature embryos, leaf basal segments and cell suspensions were also transformed but at lower rates. The time course of the histochemical reaction for the β-glucuronidase activity showed that the number of transformed blue cells detected increased up to 72 h of starting the reaction. It is concluded that transformation of maize coleoptiles with the particle gun is a good method for testing gene expression in transient systems.

Multiple shoot induction and plant regeneration from embryonic axes of cotton

Cytokinins are involved in shoot development of plants. Events of multiple bud formation and shoot development in apical embryonic axes of cotton treated for 2 or 20 days with the cytokinin benzyladenine (BA), were compared with the development of untreated control axes. Meristematic regions (supernumerary vegetative buds) were observed in axes treated for 20 days with BA. An average of 3.4 shoots per embryonary axis was obtained when explants were cultured on medium supplemented with 3 mg l-1 BA. Higher and lower concentrations of the growth regulator yielded fewer shoots per explant. Results shown in this report suggest that BA is directly responsible for re-programming the embryonic apical meristem axes of cotton toward the production of multiple buds and subsequent shoot development.

Stable wheat transformation obtained without selectable markers

Transgenic wheat plants without the selectable marker gene were obtained either in the presence or in the absence of selective pressure during the transformation protocol. When using hygromycin as selective agent in a co-transformation experiment involving a mixture of plasmids pGL2, containing the hpt gene, and pAI1Gus, containing the uidA gene, 3 out of 19 transgenic wheat plants had the uidA gene alone as shown by Southern blots. The gene was transmitted to the progeny following Mendelian rules. Segregation and loss of the selectable marker gene was also found in three out of six events from other experiments where high-molecular-weight glutenin genes were expressed or over-expressed. On the other hand, in 7 experiments where no selective pressure was applied and that involved 1016 bombarded explants, 23 transgenic wheat plants were obtained. The uidA gene was stably integrated as suggested by its transmission to the progeny.

Evaluation of biological pretreatments to increase the efficiency of the saccharification process using Spartina argentinensis as a biomass resource.

Second generation bioethanol obtained from native perennial grasses offers a promising alternative for biofuel production, avoiding land use competition for crops production. Spartina argentinensis is a native perennial C4 grass with high photosynthetic rates, well adapted to halo-hydromorphic soils, though its forage quality (palatability and digestibility) for livestock is quite low due to its high lignin content. Hence, cattle raisers burn these grasslands frequently in order to stimulate the emergence of new leaves with higher digestibility for cattle feeding. Lignin is the main barrier to overcome in order to efficiently hydrolyze the cellulose for bioethanol production. In this work, we evaluate different pretreatments (phosphoric acid, ligninolytic enzymes and fungal supernatants) aimed to remove lignin and improving cellulose hydrolysis efficiency. Results show that pretreatment with Pycnoporus sanguineus supernatant improves fermentable carbohydrates availability, compared with a conventional chemical pretreatment, and that 56.84% of cellulose can be hydrolyzed using this pretreatment.

Bt protein rhizosecreted from transgenic maize does not accumulate in soil

The persistence of CryIAb protein rhizosecreted in soil is important in the assessment of its environmental risk. Here we report that CryIAb protein from transgenic maize does not accumulate at high levels in soils. Levels of CryIAb protein rhizosecreted by three maize transgenic events (BT11, MON810 and 176) were studied in hydroponic cultures and found only in the MON810 and BT11 events but not in event 176 or control plants. Under field conditions, the cryIAb gene and a basal level of CryIAb protein was detected in soils from plots cultivated with transgenic and non-transgenic maize, possibly from Bacillus thuringiensis present in the soils.

Streptomyces thermoautotrophicus does not fix nitrogen.

Streptomyces thermoautotrophicus UBT1 has been described as a moderately thermophilic chemolithoautotroph with a novel nitrogenase enzyme that is oxygen-insensitive. We have cultured the UBT1 strain, and have isolated two new strains (H1 and P1-2) of very similar phenotypic and genetic characters. These strains show minimal growth on ammonium-free media, and fail to incorporate isotopically labeled N2 gas into biomass in multiple independent assays. The sdn genes previously published as the putative nitrogenase of S. thermoautotrophicus have little similarity to anything found in draft genome sequences, published here, for strains H1 and UBT1, but share >99% nucleotide identity with genes from Hydrogenibacillus schlegelii, a draft genome for which is also presented here. H. schlegelii similarly lacks nitrogenase genes and is a non-diazotroph. We propose reclassification of the species containing strains UBT1, H1, and P1-2 as a non-Streptomycete, non-diazotrophic, facultative chemolithoautotroph and conclude that the existence of the previously proposed oxygen-tolerant nitrogenase is extremely unlikely.

Target-site resistance to ALS-inhibiting herbicides in Amaranthus palmeri from Argentina

BACKGROUND Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. RESULTS We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. CONCLUSION This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides.

Target-site resistance to acetolactate synthase (ALS)-inhibiting herbicides in Amaranthus palmeri from Argentina

BACKGROUND Herbicide-resistant weeds are a serious problem worldwide. Recently, two populations of Amaranthus palmeri with suspected cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides (R1 and R2) were found by farmers in two locations in Argentina (Vicuña Mackenna and Totoras, respectively). We conducted studies to confirm and elucidate the mechanism of resistance. RESULTS We performed in vivo dose-response assays, and confirmed that both populations had strong resistance to chlorimuron-ethyl, diclosulam and imazethapyr when compared with a susceptible population (S). In vitro ALS activity inhibition tests only indicated considerable resistance to imazethapyr and chlorimuron-ethyl, indicating that other non-target mechanisms could be involved in diclosulam resistance. Subsequently, molecular analysis of als nucleotide sequences revealed three single base-pair mutations producing substitutions in amino acids previously associated with resistance to ALS inhibitors, A122, W574, and S653. CONCLUSION This is the first report of als resistance alleles in A. palmeri in Argentina. The data support the involvement of a target-site mechanism of resistance to ALS-inhibiting herbicides.

The MAP3K-coding QUI-GON JINN (QGJ) Gene Is Essential To The Formation of Unreduced Embryo Sacs In Paspalum

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.