Yongqin Chen

The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters

Here we report the effect of the 35S promoter sequence on activities of the tissue- and organ-specific gene promoters in tobacco plants. In the absence of the 35S promoter sequence the AAP2 promoter is active only in vascular tissues as indicated by expression of the AAP2:GUS gene. With the 35S promoter sequence in the same T-plasmid, transgenic plants exhibit twofold to fivefold increase in AAP2 promoter activity and the promoter becomes active in all tissue types. Transgenic plants hosting the ovary-specific AGL5:iaaM gene (iaaM coding an auxin biosynthetic gene) showed a wild-type phenotype except production of seedless fruits, whereas plants hosting the AGL5:iaaM gene along with the 35S promoter sequence showed drastic morphological alterations. RT-PCR analysis confirms that the phenotype was caused by activation of the AGL5:iaaM gene in non-ovary organs including roots, stems and flowers. When the pollen-, ovule- and early embryo-specific PAB5:barnase gene (barnase coding a RNase gene) was transformed, the presence of 35S promoter sequence drastically reduced transformation efficiencies. However, the transformation efficiencies were restored in the absence of 35S promoter, indicating that the 35S promoter might activate the expression of PAB5:barnase in non-reproductive organs such as calli and shoot primordia. Furthermore, if the 35S promoter sequence was replaced with the NOS promoter sequence, no alteration in AAP2, AGL5 or PAB5 promoter activities was observed. Our results demonstrate that the 35S promoter sequence can convert an adjacent tissue- and organ-specific gene promoter into a globally active promoter.

Invasive Ornamental Plants: Problems, Challenges, and Molecular Tools to Neutralize Their Invasiveness

The spread of invasive plants is one of the most challenging ecological problems in the 21st Century, causing a

Rapid clonal propagation of Dioscorea zingiberensis

35 billion loss per year to the economy in the United States alone. More than 50% of all invasive plants and 85% of invasive woody species were introduced originally for ornamental and landscape use. Because many nonnative plants are commercially important and widely utilized for various purposes, completely banning their use and prohibiting their imports are unpractical solutions for control. On the other hand, the methods currently used to control the spread of nonnative plants are ineffective, expensive, or environmentally problematic. Recent advances in plant biotechnology may enable us to create sterile cultivars of these nonnative ornamental crops of commercial value. The use of sterile cultivars should reduce or eliminate the undesirable spread of some nonnative invasive plants into natural areas.

The maize Knotted1 gene is an effective positive selectable marker gene for Agrobacterium-mediated tobacco transformation

A protocol was developed for rapid in vitro propagation of Dioscorea zingiberensis Wright using stems as explants. MS medium with the macroelements at half strength and supplemented with 20.0 g l−1 sucrose and 8.0 g l−1 agar was used as basal medium. Lateral buds on nodal cuttings grew into shoots within 20 days after culture on basal medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 1.1 μM α-naphthalene acetic acid (NAA). The shoots were cut into segments and cultured on medium with 8.9 μM BA and 5.4 μM NAA for 30 days for callus formation. The callus was cut into pieces and cultured on medium containing 22.2 μM BAP and 1.1 μM NAA, on which 87.5% of the callus pieces regenerated multiple shoots within 50 days. The shoots were rooted on medium containing 4.9 μM indole-3-butyric acid (IBA) for 20 days. Adventitious buds and shoots could be repeatedly formed after the calli were cut into pieces and cultured on the medium containing 8.9 μM BAP plus 1.1 μM NAA. More than 85% of the regenerated plantlets survived and grew vigorously 1 month after they were transplanted in vermiculite and each plant formed 2–4 microtubers 3 months of transplanting.

In vitro plant regeneration from the immature seeds of Cymbidium faberi

We have assessed the use of a homeobox gene knotted1 (kn1) from maize as a selectable marker gene for plant transformation. The kn1 gene under the control of cauliflower mosaic virus 35S promoter (35S::kn1) was introduced into Nicotiana tabacum cv. Xanthi via Agrobacterium-mediated transformation. Under nonselective conditions (without antibiotic selection) on a hormone-free medium (MS), a large number of transgenic calli and shoots were obtained from explants that were infected with Agrobacterium tumefaciens LBA4404 harboring the 35S::kn1 gene. On the other hand, no calli or shoots were produced from explants that were infected with an Agrobacterium strain harboring pBI121 (nptII selection) or from uninfected controls cultured under identical conditions. Relative to kanamycin selection conferred by nptII, the use of kn1 resulted in a 3-fold increase in transformation efficiency. The transgenic status of shoots obtained was confirmed by both histochemical detection of GUS activity and molecular analysis. The results presented here suggest that kn1 gene could be used as an effective alternative selection marker with a potential to enhance plant transformation efficiency in many plant species. With kn1 gene as a selection marker gene, no antibiotic-resistance or herbicide-resistance genes are needed so that potential risks associated with the use of these traditional selection marker genes can be eliminated.

Isolation of prostrate turfgrass mutants via screening of dwarf phenotype and characterization of a perennial ryegrass prostrate mutant

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l−1α-naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l−1 NAA plus 2.0 or 5.0 mg l−1N6-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l−1 activated charcoal for 50 days and the plantlets grew normally after transplanting.

Problems and Challenges of Invasive Ornamental Plants and Molecular Tools to Control Their Spread

Prostrate turf varieties are desirable because of their increased low mowing tolerance, heat resistance, traffic resistance and ground coverage compared with upright varieties. Mutation breeding may provide a powerful tool to create prostrate varieties, but there are no simple, straightforward methods to screen for such mutants. Elucidation of the molecular basis of the major ‘green revolution’ traits, dwarfism and semi-dwarfism, guided us to design a simple strategy for isolating dwarf mutants of perennial ryegrass (Lolium perenne L.). We have shown that gamma-ray-mediated dominant dwarf mutants can be easily screened for at the three-leaf stage. About 10% of dwarf mutant lines also displayed a prostrate phenotype at mature stages (>10 tillers). One prostrate line, Lowboy I, has been characterized in detail. Lowboy I had significantly shorter canopy, leaf blade and internode lengths compared with wild type. Lowboy I also exhibited greater tolerance to low mowing stress than wild type. Exogenous gibberellic acid (GA) restored Lowboy I to a wild-type phenotype, indicating that the dwarf and prostrate phenotypes were both due to GA deficiency. We further showed that phenotypes of Lowboy I were dominant and stably inherited through sexual reproduction. Prostrate turfgrass mutants are difficult to screen for because the phenotype is not observed at young seedling stages, therefore our method represents a simple strategy for easily isolating prostrate mutants. Furthermore, Lowboy I may provide an outstanding germplasm for breeding novel prostrate perennial ryegrass cultivars.

‘GM‐gene‐deletor’: fused loxP‐FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants

Abstract Invasive plants, one of the most devastating ecological problems in the 21st century, cause an estimated

In vitro regeneration and Agrobacterium-mediated genetic transformation of Euonymus alatus

35 billion loss per year to the economy in the United States alone. More than 50% of all invasive plant species and 85% of invasive woody plant species were introduced originally for ornamental and landscape use. Because many non-native ornamentals are commercially important and widely utilized for various purposes, completely banning their use and prohibiting their import are unpractical solutions. On the other hand, currently used methods to control the spread of non-native plants are ineffective, expensive, or environmentally problematic. Recent advances in plant molecular biology and plant genetic transformation may enable us to create sterile cultivars of these non-native ornamental crops of high commercial value. The use of sterile cultivars should reduce or eliminate the undesirable spread of some non-native invasive plants into natural areas.