ABSTRACT
A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.
Subject(s)
Gene Expression Regulation, Plant , Genes, Plant/genetics , Glutamic Acid/metabolism , Manihot/growth & development , Manihot/genetics , Plant Proteins/genetics , Promoter Regions, Genetic/genetics , Agriculture , Base Sequence , Cloning, Molecular , DNA, Bacterial/genetics , Daucus carota/genetics , Gene Expression Profiling , Glucuronidase/metabolism , Immunohistochemistry , Molecular Sequence Data , Organ Specificity , Plant Proteins/metabolism , Plant Roots/enzymology , Plant Roots/genetics , Plants, Genetically Modified , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Fusion Proteins/metabolism , Transformation, GeneticABSTRACT
Transgenic plants of cassava (Manihot esculenta) resistant to the herbicide Basta were obtained through Agrobacterium-mediated transformation. The plants also expressed the uidA gene and two were positive for PCR- and/or Southern-based detection of the nptII gene. Somatic-embryo-derived cotyledons were used as source of explants. A non-disarmed Agrobacterium strain (CIAT 1182) was used to transfer the genes of interest into cassava cultivar MPer183. Greenhouse tests of resistance to Basta (Hoechst) showed three plant lines with different levels of tolerance to the herbicide. Based on Southern tests of transgenesis, the transformation efficiency was 1%. The results constitute the first report of the bar gene conferring herbicide resistance to cassava plants.
ABSTRACT
We developed genetic markers for three microsatellite loci in the tropical tree Symphonia globulifera and used them to examine the demographic genetic consequences of forest fragmentation. High levels of genetic variation were revealed in samples of adults, saplings, and seedlings. The more-variable loci exhibited less stability in allelic composition across sites and stages. The number of alleles per hectare (ha) of forest was similar when continuous forest plots were compared to plots from fragmented forest for all three stages. This pattern also held for the number of unique multilocus adult and sapling genotypes, but the number of unique seedling genotypes per ha of fragmented forest greatly exceeded expectations based on continuous forest data, probably due to the concentration of seeds into remnant forest patches by foraging bats. Significant inbreeding and genetic differentiation were most often associated with the fragmented forest and the seedlings. Finally, principal component analysis reaffirmed that a bottleneck, acting in concert with pre-existing genetic structure in the adults, had led to enhanced and rapid divergence in the seedlings following deforestation, a result that is of central interest for landscape management.
Subject(s)
Demography , Dinucleotide Repeats/genetics , Ecology , Genetic Variation/genetics , Trees/genetics , Age Factors , Alleles , Animals , Base Sequence , Blotting, Southern , Chiroptera , Chromatography, Gel , Costa Rica , DNA Primers/chemistry , DNA, Plant/chemistry , Molecular Sequence Data , Plant Leaves/chemistry , Polymerase Chain Reaction , Trees/physiologyABSTRACT
Methods for improving the efficiency of plant recovery from somatic embryos of cassava (Manihot esculenta Crantz) were investigated by optimizing the maturation regime and incorporating a desiccation stage prior to inducing germination. Somatic embryos were induced from young leaf lobes of in vitro grown shoots of cassava on Murashige and Skoog medium with 2,4-dichlorophenoxy acetic acid. After 15 to 20 days of culture on induction medium, the somatic embryos were transferred to a hormone free medium supplemented with activated charcoal. In another 18 days mature somatic embryos became distinctly bipolar and easily separable as individual units and were cultured on half MS medium for further development. Subsequent desiccation of bipolar somatic embryos resulted in 92% germination and 83% complete plant regeneration. The plants were characterized by synchronized development of shoot and root axes. Of the non-desiccated somatic embryos, only 10% germinated and 2% regenerated plants. Starting from leaf lobes, transplantable plantlets were derived from primary somatic embryos within 70 to 80 days.