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Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high-yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence-derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock.

RUBY

Data for Miscanthus giganteus Biolistic Transformation Using the Visible RUBY Red Marker Gene to Monitor Transformation Efficiency

Miscanthus × giganteus ( M × g ) is a high- yielding perennial C4 bioenergy crop, but genetic improvement by breeding is constrained by triploid sterility and clonal propagation. Improving genetic transformation methods for M × g would provide opportunities for advantageous trait introgression. Use of an easy to phenotype reporter gene is a promising strategy to improve transformation processes and efficiency. This study presents an efficient novel method for biolistic transformation of inflorescence- derived callus in M × g and demonstrates its efficacy using RUBY, a betalain-based noninvasive reporter that is visible throughout the transformation process. RUBY expression ( Zea mays codon optimized) was visible from callus stage through plantlet development into maturity. RUBY expressing independently transformed plants were confirmed by hygromycin phosphotransferase ELISA and by genomic PCR demonstrating that the RUBY phenotype is sufficient for screening transformants. The Zea mays codon optimized hygromycin selection marker was driven by previously established promoters for Miscanthus, ZmUBI and 2×35S, while the RUBY gene expression was controlled by a known Zea mays C4 promoter, Brachypodium UBI10, newly employed in Miscanthus. The construct containing the 2×35S promoter for hygromycin had a 15.1% transformation efficiency while the ZmUBI promoter had a 20.5% transformation efficiency. This study provides a novel, highly efficient protocol for successful biolistic transformation of M × g for stable expression. This study also demonstrates that RUBY expression can be used as a convenient and powerful monitor of transformation in ongoing and future work to engineer M × g into an improved bioproduct feedstock. **NOTE: in "TableS2_ProtocolComparison.csv", the data from row 665 to 971 should be removed.

Gene Editing

Bistable direction switching in an off-axis pumped continuous wave ruby laser

A report is presented of the observation of hysteretic bistable direction switching in a single-mode CW ruby laser system. This effect is only observed when the pump beam which is focused into the ruby rod is misaligned with respect to the rod end faces. At low pump powers, the ruby lases in a mode nearly collinear with the pump axis. At a higher pump power the ruby switches to a mode that is collinear with the rod end faces and preserves the original polarization. The effect is large enough to switch the beam by an angle equal to twice the diffraction angle. The observations show that under steady-state pumping, a CW ruby laser can exhibit bistable operation in its output direction and power. A calculation using the heat equation with two concentric cylinders with one as a heat source (pump laser) and the outer wall of the other held at 77 K, gives an increase in core temperature of about 0.01 K. Therefore, the increase in temperature is not large enough to change the index of refraction to account for such large macroscopic effects.

Afzal, R. Sohrab

Spin-lattice relaxation and the calculation of gain, pump power, and noise temperature in ruby

The use of a quantitative analysis of the dominant source of relaxation in ruby spin systems to make predictions of key maser amplifier parameters is described. The spin-lattice Hamiltonian which describes the interaction of the electron spins with the thermal vibrations of the surrounding lattice is obtained from the literature. Taking into account the vibrational anisotropy of ruby, Fermi's rule is used to calculate the spin transition rates between the maser energy levels. The spin population rate equations are solved for the spin transition relaxation times, and a comparison with previous calculations is made. Predictions of ruby gain, inversion ratio, and noise temperature as a function of physical temperature are made for 8.4-GHz and 32-GHz maser pumping schemes. The theory predicts that ruby oriented at 90 deg will have approximately 50 percent higher gain in dB and slightly lower noise temperature than a 54.7-deg ruby at 32 GHz (assuming pump saturation). A specific calculation relating pump power to inversion ratio is given for a single channel of the 32-GHz reflected wave maser.

Lyons, J. R.

The superconducting cavity stability ruby maser oscillator

Analysis of an application of the rudy maser to a superconducting Cavity Stabilized oscillator shows many attractive features. These derive from the mechancial stability inherent in an all-cryogenic design and from the properties of the ruby maser itself. A multiple-cavity design has been developed to allow physical separation of the high-Q superconducting cavity and the ruby element with its requried applied magnetic field. Mode selection is accomplished in this design by tuning the ruby by means of the applied field. We conclude that such an oscillator would perform well, even with cavity Q's as low as 10 to the 8th power allowing the use of a superconductor-on-sapphire resonator with its greater rigidity and lower thermal expansion. A first test of the Superconducting Cavity Stabilized Maser Oscillator (SCSMO) confirms the efficacy of the multiple-cavity design and the applicability of the ruby maser. Frequency variation less than 4x10 to the minus 11th power was measured in the stabilized mode and is attributed to the reference oscillator and to instabilities in the pump source. Variation of 10 to the minus 10th power was observed in the low-Q unstabilized mode, again attributable to pump fluctuations. Even so, direct scaling to a Q of 10 the 9th power predicts a stability better than 10 to the minus 15th power. Together with results showing the lowest losses to date in sapphire at microwave frequencies, and preliminary experiments on superconductor-on-sapphire resonators, frequency stability, levels as low as 10 to the minus 17th power are indicated.

Dick, G. J.

High-pressure ruby and diamond fluorescence - Observations at 0.21 to 0.55 terapascal

A diamond-anvil, high-pressure apparatus was used to extend the upper pressure limit of static laboratory experiments. Shifts of the R1 strong fluorescent line of ruby were observed that correspond to static pressures of 0.21 to 0.55 terapascal (2.1 to 5.5 megabars) at 25 C. Sensitive spectroscopic techniques in the pressure range 0.15 to 0.28 terapascal were used to observe ruby and diamond fluorescence separately; these two fluorescent emissions overlap strongly at high pressures. At pressures greater than approximately 0.28 terapascal, the diamond fluorescence diminished and the ruby fluorescence reappeared strongly. Pressure was determined by extrapolation of the calibrated shift of the ruby R1 line.

Xu, J. A.

Experimental study of the dynamics of a ruby laser pumped by a CW argon-ion laser

A study of the dynamics of a ruby laser pumped by a CW argon-ion laser is presented. The ruby laser is predominantly stable but has two accessible unstable states. One state exhibits chaotic output, while the other results in regular self-pulsing. The conditions needed for instability are discussed and homodyne spectra and temporal maps of the phase-space attractors are obtained. In addition, a numerical simulation of nonlinear beam propagation in ruby is presented that shows that strong deviations from plane-wave behavior exist, and that transverse effects must be incorporated into theoretical models of the instability.

Afzal, R. S.

Experimental investigation of self-induced transparency and pulse delay in ruby.

We have investigated the self-induced transparency effect in ruby over a range of input energies which range from linear absorption to full transparency. The transmission, pulse delay, and pulse broadening were studied as a function of input energy. The transition region is narrower than that found in similar studies of the CO2/SF6 system; this is consistent with predictions based on ensembles of two-level systems. Included are the first pulse-delay and pulse-broadening curves to be obtained for the ruby system.

Asher, I. M.

Ionizing mechanisms in a cesium plasma irradiated with a ruby laser

A cesium filled diode--laser plasmadynamic converter was built to investigate the feasibility of converting laser energy to electrical energy at large power levels. Experiments were performed with a pulsed ruby laser to determine the quantity of electrons and cesium ions generated per pulse of laser beam and to determine the output voltage. A current density as high as 200 amp/sq cm from a spot of approximately 1 sq mm area and an open circuit voltage as high as 1.5 volts were recorded. A qualitative theory was developed to explain these results. In the operation of the device, the laser beam evaporates some of the cesium and ionizes the cesium gas. A dense cesium plasma is formed to absorb further the laser energy. Results suggest that the simultaneous absorption of two ruby laser photons by the cesium atoms plays an important role in the initial ionization of cesium. Inverse bremsstrahlung absorption appears to be the dominant mechanism in subsequent processes. Recombinations of electrons and cesium ions appear to compete favorably with the simultaneous absorption of two photons.

Shimada, K.

Far-infrared lasing in ruby

The feasibility of submillimeter wavelength (870 GHz) lasing in Al2O3 is discussed. The proposed scheme is based on the pumping of the 2A level of ruby via a ruby laser operating on its R2 line. Lasing is expected on the 2A to E transition of the split E-2 level.

Lawandy, N. M.

Experimental evaluation of a ruby maser at 43 GHz

Inversion ratio measurements were conducted at several frequencies between 27 and 43 GHz for a pink ruby material (0.05% Cr/3+/ in Al2O3) at the push-pull pump angle of 54.7 degrees in order to determine the upper frequency limit where pink ruby could be expected to operate as a practical maser amplifier. Based on these measurements, a single-stage maser was developed which yielded 8 + or - 1 dB net gain and a 3 dB bandwidth of 180 MHz at a center frequency of 42.5 GHz. It is concluded that a multistage reflected wave maser could achieve bandwidths exceeding 1 GHz with 30 dB net gain at center frequencies near 40 GHz.

Moore, C. R.

Evolution of basin and range structure in the Ruby Mountains and vicinity, Nevada

Results from various age dating techniques, seismic reflection profiling hydrocarbon maturation studies, and structural analysis were used to evaluate the Cenozoic deformation in the Ruby Mountains and adjoining ranges (pinyon Range and Cortez Range) in Elko and Eureka Counties, Nevada. Age dating techniques used include potassium-argon ages of biotites from granites published by Kistler et al. (1981) and fission track ages from apatite and zircon. Fission track ages from apatite reflect a closing temperature of 100 plus or minus 20 deg C. Zircon fission track ages reflect a closing temperature of 175 plus or minus 25 deg C and potassium-argon ages from brotite reflect a closing temperature of 250 plus or minus 30 deg C. Thus these results allow a reasonably precise tracking of the evolution of the ranges during the Cenozoic. Seismic reflection data are available from Huntington Valley. Access to seismic reflection data directly to the west of the Harrison Pass Pluton in the central Ruby Mountains was obtained. In addition results are available from several deep exploration holes in Huntington Valley.

Blackwell, D. D.

Alexandrite as a high-temperature pressure calibrant, and implications for the ruby-fluorescence scale

The wavelength shifts of the R1 and R2 fluorescence lines of alexandrite (BeAl2O4:Cr(+3)) have been experimentally calibrated against the ruby-fluorescence scale as a function of both hydrostatic and nonhydrostatic pressures between 0 and 50 GPa, and simultaneously as a function of temperatures between 290 and 550 K. It is found that the pressure-temperature cross derivative of the fluorescence wavelength shifts are negligible for both ruby and alexandrite.

Jahren, A. H.

Ruby on Rails Applications

Ruby on Rails is an open source web application framework for the Ruby programming language. The first application I built was a web application to manage and authenticate other applications. One of the main requirements for this application was a single sign-on service. This allowed authentication to be built in one location and be implemented in many different applications. For example, users would be able to login using their existing credentials, and be able to access other NASA applications without authenticating again. The second application I worked on was an internal qualification plan app. Previously, the viewing of employee qualifications was managed through Excel spread sheets. I built a database driven application to streamline the process of managing qualifications. Employees would be able to login securely to view, edit and update their personal qualifications.

Hochstadt, Jake

Spin-lattice relaxation in ruby.

Spin-lattice relaxation times for ruby of very low chromium concentration at low temperatures for one-phonon kronig-van vleck process

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