Expression of a transcription factor from Capsicum annuum in pine calli counteracts the inhibitory effects of salt stress on adventitious shoot formation.

Transcription factors regulating the stress-responsive gene expression play an important role in plant stress adaptation. In this study, we examined the salt stress tolerance of transgenic Virginia pine (Pinus virginiana Mill.) overexpressing a Capsicum annuum ERF/AP2-type transcription factor (CaPF1), which may enhance the ability of transgenic plants to tolerate various kinds of stresses during vegetative growth. CaPF1 transgene increased the salt and oxidative stress tolerances of pine tissues and counteracted the inhibitory effects of salt stress on the growth of transgenic Virginia pine calli, shoots, and plants. To our surprise, the ability of shoot formation was enhanced in three CaPF1 transgenic Virginia pine cell lines under stress of different NaCl concentrations. NaCl at 200 mM significantly increased the frequency of adventitious shoot formation and the number of shoots per gram calli. Measurement of plant hormone demonstrated that the levels of cytokinin was altered in CaPF1-overexpressed Virginia pine calli, compared to the control. Based on our results, we speculate that the altered level of cytokinin may result in enhancing adventitious shoot formation of transgenic calli exposed to salt for 1 week via an unknown mechanism.

Plant regeneration through multiple adventitious shoot differentiation from callus cultures of slash pine (Pinus elliottii).

A plant regeneration system through multiple adventitious shoot differentiation from callus cultures has been established in slash pine (Pinus elliottii). Influences of seven different basal media on callus induction, adventitious shoot formation, and rooting were investigated. Among the different basal media, B5, SH, and TE proved to be suitable for callus induction and plantlet regeneration. Multiple adventitious shoot formation was obtained from callus cultures of slash pine on B5, SH, and TE media containing indole-3-butyric acid, N6-benzyladenine, and thidiazuron. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. These results indicate that an efficient plant regeneration protocol for micropropagation of slash pine had been established. This protocol could be most useful for future studies on genetic transformation of slash pine.

Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth.

Transcription factors play an important role in regulating gene expression in response to stress and pathogen tolerance. We describe here that overexpression of an ERF/AP2 pepper transcription factor (CaPF1) in transgenic Virginia pine (Pinus virginiana Mill.) confers tolerance to heavy metals Cadmium, Copper, and Zinc, to heat, and to pathogens Bacillus thuringiensis and Staphylococcus epidermidis, as by the survival rate of transgenic plants and the number of decreasing pathogen cells in transgenic tissues. Measurement of antioxidant enzymes ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD) activities demonstrated that the level of the enzyme activities was higher in transgenic Virginia pine plants overexpressing the CaPF1 gene, which may protect cells from the oxidative damage caused by stresses, compared to the controls. Constitutive overexpression of CaPF1 gene enhanced organ growth by increasing organ size and cell numbers in transgenic Virginia pine plants over those in control plants.

High efficiency inducible gene expression system based on activation of a chimeric transcription factor in transgenic pine.

Inducible gene expression systems are needed in functional genomics of tree species. A glucocorticoid-inducible gene expression system was established in a gymnosperm species Virginia pine (Pinus virginiana Mill.) through Agrobacterium tumefaciens-mediated genetic transformation. The results demonstrate that expression of the m-gfp5-ER reporter gene was tightly controlled and 0.1 microM of the glucocorticoid hormone triamcinolone was able to induce m-gfp5-ER expression in transgenic cells. Differential expression of gfp in transgenic cells induced by different concentrations of triamcinolone was observed and confirmed by Northern Blot analysis and by quantitative green fluorescence analyses with Laser Scanning Microscopy. In transgenic plantlets, triamcinolone was taken up efficiently by roots. Triamcinolone was able to induce m-gfp5-ER activity throughout the whole plant. The phenotype of transgenic plantlets was not affected 6 weeks after treatment with 0.1-10 microM triamcinolone. However, 6-week inductions with 100 microM triamcinolone caused growth retardation and developmental defects, as well as inhibition of root formation and elongation. With careful selection of transgenic lines, the inducible gene expression presented in this study could be a very valuable alternative for functional identification of novel genes in plants, especially in pine.