Efficient delivery of small interfering RNA to plant cells by a nanosecond pulsed laser-induced stress wave for posttranscriptional gene silencing.

Small interfering RNA (siRNA) induced posttranscriptional gene silencing (PTGS) has been an efficient method for genetic and molecular analysis of certain developmental and physiological processes and represented a potential strategy for both controlling virus replication and developing therapeutic products. However, there are limitations for the methods currently used to deliver siRNA into cells. We report here, to our knowledge, the first efficient delivery of siRNA to plant cells by a nanosecond pulsed laser-induced stress wave (LISW) for posttranscriptional gene silencing. Using LISW, we are able to silence gene expression in cell cultures of three different plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.). Gene silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that LISW-mediated siRNA delivery can be a reliable and effective method for inducing PTGS in cultured cells.

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus).

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.

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.

Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos.

We reported establishment of an efficient plant regeneration procedure through direct adventitious shoot (DAS) formation from cotyledons and hypocotyls of eastern white pine (Pinus strobus L.) mature embryos in this investigation. Multiple DASs were initiated from cotyledons of embryos on PS medium containing N6-benzyladenine (BA), thidiazuron (TDZ), or kinetin (KIN). Among different concentrations of casein enzymatic hydrosylate (CH) and glutamine used in this study, 500 mg l(-1) CH or 600 mg l(-1) glutamine induced the highest frequency of DAS formation. Rooting of regenerated shoots was obtained on PS medium supplemented with 0.01-0.1 microM indole-3-acetic acid (IAA) with the highest frequency on medium containing 0.01 muM IAA. No DASs were obtained on medium without TDZ. Measurement of peroxidase (POD) and catalase (CAT) activity during direct shoot induction and differentiation demonstrated that the lowest POD activity appeared in the 5-6th week of culture and lowest CAT activity occurred in the 7-8th week of culture on medium with TDZ. No such a change in POD and CAT activities was observed on medium without TDZ. These results demonstrated that POD and CAT activities were involved in DAS formation induced by TDZ in eastern white pine.

Enhanced tolerance to salt stress in transgenic loblolly pine simultaneously expressing two genes encoding mannitol-1-phosphate dehydrogenase and glucitol-6-phosphate dehydrogenase.

A reproducible approach to improve salt tolerance of conifers has been established by using the technology of plant genetic transformation and using loblolly pine (Pinus taeda L.) as a model plant. Mature zygotic embryos of three genotypes of loblolly pine were infected with Agrobacterium tumefaciens strain LBA 4404 harboring the plasmid pBIGM which carrying two bacterial genes encoding the mannitol-1-phosphate dehydrogenase (Mt1D, EC 1.1.1.17) and glucitol-6-phosphate dehydrogenase (GutD) (EC 1.1.1.140), respectively. Transgenic plantlets were produced on selection medium containing 15 mg l(-1) kanamycin and confirmed by polymerase chain reaction (PCR) and Southern blot analysis of genomic DNA. The Mt1D and GutD genes were expressed and translated into functional enzymes that resulted in the synthesis and accumulation of mannitol and glucitol in transgenic plants. Salt tolerance assays demonstrated that transgenic plantlets producing mannitol and glucitol had an increased ability to tolerate high salinity. These results suggested that an efficient A. tumefaciens-mediated transformation protocol for stable integration of bacterial Mt1D and GutD genes into loblolly pine has been developed and this could be useful for the future studies on engineering breeding of conifers.

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique.

Transgenic cell lines of loblolly pine (Pinus taeda L.) were analyzed by a compact laser-tweezers-Raman-spectroscopy (LTRS) system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.

Post-transcriptional gene silencing induced by short interfering RNAs in cultured transgenic plant cells.

Short interfering RNA (siRNA) is widely used for studying post-transcriptional gene silencing and holds great promise as a tool for both identifying function of novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which were designed against different specific areas of coding region of the same target green fluorescent protein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice (Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Fraser fir [Abies fraseri (Pursh) Poir; AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in the bombarded transgenic cells between two siRNAs, and these results were consistent with the inactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis in tested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be the siRNA specific in different plant species. These results indicate that siRNA is a highly specific tool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could be a reliable approach for large-scale screening of gene function and drug target validation.

Okadaic acid and trifluoperazine enhance Agrobacterium-mediated transformation in eastern white pine.

Mature zygotic embryos of recalcitrant Christmas tree species eastern white pine (Pinus strobus L.) were used as explants for Agrobacterium tumefaciens strain GV3101-mediated transformation using the uidA (beta-Glucuronidase) gene as a reporter. Influence of the time of sonication and the concentrations of protein phosphatase inhibitor (okadaic acid) and kinase inhibitor (trifluoperazine) on Agrobacterium-mediated transformation have been evaluated. A high transformation frequency was obtained after embryos were sonicated for 45-50 s, or treated with 1.5-2.0 microM okadaic acid or treated with 100-200 microM trifluoperazine, respectively. Protein phosphatase and kinase inhibitors enhance Agrobacterium-mediated transformation in eastern white pine. A 2-3.5-fold higher rate of hygromycin-resistant callus was obtained with an addition of 2 microM okadaic acid or 150 microM trifluoperazine or sonicated embryos for 45 s. Stable integration of the uidA gene in the plant genome of eastern white pine was confirmed by polymerase chain reaction (PCR), Southern and northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in eastern white pine and this system would provide an opportunity to transfer economically important genes into this Christmas tree species.

Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine.

An efficient transgenic eastern white pine (Pinus strobus L.) plant regeneration system has been established using Agrobacterium tumefaciens strain GV3850-mediated transformation and the green fluorescent protein (gfp) gene as a reporter in this investigation. Stable integration of transgenes in the plant genome of pine was confirmed by polymerase chain reaction (PCR), Southern blot, and northern blot analyses. Transgene expression was analysed in pine T-DNA transformants carrying different numbers of copies of T-DNA insertions. Post-transcriptional gene silencing (PTGS) was mostly obtained in transgenic lines with more than three copies of T-DNA, but not in transgenic lines with one copy of T-DNA. In situ hybridization chromosome analysis of transgenic lines demonstrated that silenced transgenic lines had two or more T-DNA insertions in the same chromosome. These results suggest that two or more T-DNA insertions in the same chromosome facilitate efficient gene silencing in transgenic pine cells expressing green fluorescent protein. There were no differences in shoot differentiation and development between transgenic lines with multiple T-DNA copies and transgenic lines with one or two T-DNA copies.

Differential gene silencing induced by short interfering RNA in cultured pine cells associates with the cell cycle phase.

The double-stranded short interfering RNA (siRNA) molecules can silence targeted genes through sequence-specific cleavage of the cognate RNA transcript. The rapid adoption of technologies based on this siRNA interference mechanism has been a widely used method to analyze gene function in plants, invertebrates, and mammalian systems. In order to understand the dynamics of siRNA-mediated gene inactivation during cell division, we have investigated the relationship between the cell cycle phase and the post-transcriptional gene silencing mediated by siRNA in gfp transgenic Virginia pine (Pinus virginiana Mill.) cells. Among the different phases of the cell cycle, transgenic cells at the M phase gave 2-3 times lower gfp silencing than those at the G1, S, and G2 phases. The similar results of the siRNA-mediated gfp silencing were obtained in three transgenic cell lines. Differential gfp silencing induced by siRNA has been confirmed by northern blot, laser scanning microscopy, and siRNA analysis. These data suggested that siRNA-mediated gene inactivation is associated with the cell cycle phase in Virginia pine.

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 from callus cultures derived from mature zygotic embryos in white pine (Pinus strobus L.).

Plant regeneration via adventitious shoot organogenesis from callus cultures initiated from mature embryos in white pine (Pinus strobus L.) was achieved in this study. Callus cultures were induced from mature embryos cultured on PS medium supplemented with 2,4-dichlorophenoxyacetic acid, alpha-naphthaleneacetic acid, or indole-3-acetic acid. Adventitious shoot regeneration from callus cultures was induced on medium containing 2 microM indole-3-butyric acid (IBA) and 3-12 microM N(6)-benzylaminopurine, thidiazuron (TDZ), or 6-(gamma,gamma-dimethylallylamino) purine. Sucrose was the most suitable sugar for adventitious shoot organogenesis in white pine. Shoot organogenesis was improved by treatment at 4 degrees C for 6 weeks. The frequency of adventitious shoot formation increased when 0.1 mM putrescine was added to basal medium supplemented with 6 microM TDZ and 2 microM IBA. Putrescine improved adventitious shoot organogenesis by decreasing lipid peroxidation. These findings provide useful information on adventitious shoot organogenesis and may be valuable to genetic transformation in white pine.

Polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine (Pinus virginiana Mill.) plantlets.

Polyamines have been demonstrated to play an important role in adventitious root formation and development in plants. Here, we present a detailed analysis of influence of exogenously added polyamines on adventitious root development and its relationship to cold tolerance in Virginia pine (Pinus virginia Mill.). Our results demonstrated that polyamines putrescine (Put), spermidine (Spd), and spermine (Spm) at 0.001 mM improve rooting frequency and promote root elongation. Put, Spd, and Spm at 0.01-1 mM decrease rooting frequency and reduce root elongation root elongation. Measurements of diamine oxidase (DAO, EC 1.4.3.6) and polyamine oxidase (PAO, EC 1.4.3.4) activities showed that higher DAO and PAO enzyme activities were obtained when high concentrations of polyamines were applied and when plantlets were treated for 5-7 week at 4 degrees C and 16 degrees C. Survival rate of plantlets increased with the treatment of polyamines at low temperature. Polyamines increased mitotic index of cells in root tips of regenerated plantlet cultured on medium containing 0.001 microM Put, Spd, or Spm, but did not increase mitotic index in tissues of needle tips of the same plantlets. These results demonstrated that polyamines promote root elongation and growth by increasing root cell division in regenerated Virginia pine plantlets.

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.

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.

Inducible antisense-mediated post-transcriptional gene silencing in transgenic pine cells using green fluorescent protein as a visual marker.

An inducible post-transcriptional gene silencing (PTGS) system was established in Virginia pine (Pinus virginiana Mill.) cells. This system is based on the activation of an antisense gfp gene construct by a chimeric transcriptional activator GVG (Gal4-binding domain-VP16 activation domain-glucocorticoid receptor fusion) upon application of the inducer to gfp transgenic cell lines. A detailed characterization of the inducible PTGS system in transgenic cell lines demonstrated that this system is stringently controlled. The degree of silencing with this construct could be regulated by the concentration of inducer and the time of treatment. Such transgenic cell lines may provide a useful system to study signaling mechanisms of gene silencing in transgenic pine cells. The inducible system could be a useful tool for functional discovery of novel plant genes.

Regulated gene expression by glucocorticoids in cultured Virginia pine (Pinus virginiana Mill.) cells.

The effects of six glucocorticoids (dexamethasone, hydrocortisone, 6-methylprednisolone, prednisolone, prednisone, and triamcinolone) on inducible gene expression, based on the chimaeric transcriptional activator GVG and carried by the binary expression vector pINDEX3-m-gfp5-ER, were evaluated in transgenic Virginia pine cell cultures. The concentration that activated GVG transcription factor activity, the level of inducible m-gfp5-ER expression, and the kinetics of inducible m-gfp5-ER expression were determined for each glucocorticoid. Transgenic cells produced green fluorescence upon blue light excitation after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone. Green fluorescence was observed at 6-12 h after treatment of all six glucocorticoids at concentrations of 1, 3, 5, and 10 mg l(-1). Differential expression of gfp was confirmed by northern blot analysis and by quantitative fluorescence analyses of confocal images taken by a LSM 510 Laser Scanning Microscope. Fresh and dry weight increases of transgenic cell cultures were not affected by all six glucocorticoids at concentrations of 0.1, 0.5, 1, 3, and 5 mg l(-1). It is shown that triamcinolone had the most potent effect on the GVG system. Different glucocorticoids can therefore be used to regulate the GVG transcriptional activator and to induce gene expression in transgenic plant cells, and this property could be useful in establishing an optimum system of transgene regulation.

Antioxidants enhance in vitro plant regeneration by inhibiting the accumulation of peroxidase in Virginia pine (Pinus virginiana Mill.).

Plant tissue necrosis and subsequent cell death are usually observed during in vitro regeneration in conifers, especially in plant regeneration via somatic organogenesis in pine species. Cell death is correlated with the elevated levels of peroxides. In this investigation, the effects of antioxidants on in vitro regeneration of Virginia pine (Pinus virginiana Mill.) were evaluated. Antioxidants, polyvinylpolypyrrolidone (PVPP) and 1,4-dithio-DL-threitol (DTT), were found to improve callus formation, shoot differentiation and growth, and shoot rooting by inhibiting tissue necrosis during the initiation of cultures and subculture of shoots. These treatments enabled the recovery and regeneration plants at high frequency through somatic organogenesis. Compared to the control, the frequencies of callus formation, shoot growth, and shoot rooting increased 15, 26, and 19%, respectively, by addition of 5 g/l PVPP and 2 g/l DTT. Higher peroxidase activity of tissue cultures during subculture from callus proliferation medium to shoot differentiation medium and to rooting medium was observed. The addition of antioxidants reduces and inhibits browning by reducing the accumulation of peroxidase.

Diurnal changes in water conduction in loblolly pine (Pinus taeda) and Virginia pine (P. virginiana) during soil dehydration.

We studied diurnal changes in water conduction during soil dehydration in 37-month-old seedlings of one Virginia pine (Pinus virginiana Mill.) and two loblolly pine (P. taeda L.) sources, one from North Carolina (NC) and the other from the "Lost Pines" areas of Texas (TX), in an environmentally controlled growth chamber. For seedlings of similar biomass, the TX source had higher values of transpiration, needle conductance, and plant hydraulic conductivity under well-watered conditions than the NC source. Under dry soil conditions, the TX source had lower values of water conduction than the NC source. The Virginia pine source responded similarly to the TX source under both well-watered and dry soil conditions. For all three pine sources, gradients between soil and needle water potentials were greatest when the seedlings were moderately stressed. The TX and Virginia pine sources had higher gradients and lower daytime needle water potentials under moderate stress conditions than the NC source. Predawn needle water potentials did not differ among the pine sources. We conclude that the TX and Virginia pine sources have decreased daytime needle water potentials and increased water potential gradients during the daytime under moderate stress conditions, but with no disruption of recovery at predawn. The greater rates of transpiration and water conduction by the TX source compared with the NC source under well-watered conditions suggest a means by which growth can be maximized prior to the onset of drought, thereby enhancing survival of loblolly pines in drought-prone environments.