DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73.

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.

Effect of Video Camera Angle on the Detection of Compensatory Movements during Motion Observation.

When exercise instructions are provided over the Internet, such as in online personal training, an instructor checks the user's form by watching their motion video recorded using a single camera device. However, fixed shooting angles may affect the detection of incorrect forms, including compensatory movements. This study aimed to verify whether differences in the shooting direction could influence compensatory movement detection by conducting motion observation using training motion videos shot from two angles. Videos of four training movements, including compensatory movements, were simultaneously captured from the front and side. Ten university students studying physical therapy watched the videos from each angle to detect compensatory movements. This study revealed significant differences between the plane of motion in which the compensatory action occurred and the direction of shooting for the false responses in the compensatory action detection for the three movements (p < 0.05). The results indicated that the shooting direction and the plane of motion in which the compensatory action occurred affected the detection of compensatory movements, which was attributable to differences in information on the amount of joint change depending on the direction of joint motion observation and to a lack of binocular visual information necessary for depth motion detection.

Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos.

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., 'Nipponbare') regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The natural mutation rate of 1.2 × 10-8 calculated using the variants in the seed-propagated plants was consistent with the values reported previously. The ratio of single nucleotide variants (SNVs) among the variants in the seed-propagated plants was 91.1%, which is higher than 56.1% previously reported, and not significantly different from those in the regenerants. Overall, the ratio of transitions to transversions of SNVs was lower in the regenerants as shown previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

Comparison of Smoothness, Movement Speed and Trajectory during Reaching Movements in Real and Virtual Spaces Using a Head-Mounted Display.

Virtual reality is used in rehabilitation and training simulators. However, whether movements in real and virtual spaces are similar is yet to be elucidated. The study aimed to examine the smoothness, trajectory, and velocity of participants' movements during task performance in real and virtual space. Ten participants performed the same motor task in these two spaces, reaching for targets placed at six distinct positions. A head-mounted display (HMD) presented the virtual space, which simulated the real space environment. The smoothness of movements during the task was quantified and analysed using normalised jerk cost. Trajectories were analysed using the actual trajectory length normalised by the shortest distance to the target, and velocity was analysed using the time of peak velocity. The analysis results showed no significant differences in smoothness and peak velocity time between the two spaces. No significant differences were found in the placement of the six targets between the two spaces. Conversely, significant differences were observed in trajectory length ratio and peak velocity time, albeit with small effect sizes. This outcome can potentially be attributed to the fact that the virtual space was presented from a first-person perspective using an HMD capable of presenting stereoscopic images through binocular parallax. Participants were able to obtain physiological depth information and directly perceive the distance between the target and the effector, such as a hand or a controller, in virtual space, similar to real space. The results suggest that training in virtual space using HMDs with binocular disparity may be a useful tool, as it allows the simulation of a variety of different environments.

Visual feedback in the lower visual field affects postural control during static standing.

BACKGROUND: The dorsal parietal visual system plays an important role in self-motion perception and spatial cognition. It also strongly responds to visual inputs from the lower visual field. Postural control is modified in a process called sensory reweighting based on the reliability of available sensory sources. The question of whether visual stimuli presented to either the lower or upper visual field affect postural control and sensory reweighting has not been resolved. RESEARCH QUESTION: Do visual stimuli presented to the lower and upper visual fields affect postural control and sensory reweighting? METHODS: Twenty-nine healthy young adults participated in the study. Four conditions (full visual field, upper visual field, lower visual field, and no optic flow condition) were simulated in a VR environment using a head-mounted display. The optic flow stimuli used were swarms of small white spheres originating from the central point of the visual field, moving radially towards the periphery, and expanding across the scene. Participants were instructed to stand quietly for 50 s under each visual condition. Using force plate signals, we measured the center of pressure (COP) signal in the horizontal plane and calculated its 95 % ellipse area, root mean square (RMS) deviations, the mean velocity, and power spectral density (PSD). RESULTS: Optic flow in the full and lower visual fields produced significantly smaller 95 % ellipse area and RMS of COP in the anterior-posterior direction compared to optic flow in the upper visual field. Furthermore, the PSD of the lower frequency band (0-0.3 Hz) was decreased and that of higher frequency bands (0.3-1 Hz and 1-3 Hz) was increased for the lower compared to the upper visual field. SIGNIFICANCE: Visual feedback affects static postural control more when presented in the lower visual field compared to the upper visual field.

Isolation of gametes and zygotes from Setaria viridis.

Setaria viridis, the wild ancestor of foxtail millet (Setaria italica), is an effective model plant for larger C4 crops because S. viridis has several desirable traits, such as short generation time, prolific seed production and a small genome size. These advantages are well suited for investigating molecular mechanisms in angiosperms, especially C4 crop species. Here, we report a procedure for isolating gametes and zygotes from S. viridis flowers. To isolate egg cells, ovaries were harvested from unpollinated mature flowers and cut transversely, which allowed direct access to the embryo sac. Thereafter, an egg cell was released from the cut end of the basal portion of the dissected ovary. To isolate sperm cells, pollen grains released from anthers were immersed in a mannitol solution, resulting in pollen-grain bursting, which released sperm cells. Additionally, S. viridis zygotes were successfully isolated from freshly pollinated flowers. Isolated zygotes cultured in a liquid medium developed into globular-like embryos and cell masses. Thus, isolated S. viridis gametes, zygotes and embryos are attainable for detailed observations and investigations of fertilization and developmental events in angiosperms.

In Vitro Fertilization System Using Wheat Gametes by Electric Fusion.

In vitro fertilization (IVF) systems using isolated gametes have been used to dissect post-fertilization events in angiosperms, as female plant gametophytes are deeply embedded within the ovaries. In addition, hybrid and polyploid zygotes can be produced by using IVF systems. Complete IVF systems of maize and rice, two out of three major energy-providing crops, have been established in order to acquire detailed knowledge of mechanisms of fertilization and early embryogenesis. Following in the footsteps of previous success, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a modified electrofusion method, and the zygote formed a cell wall and two nucleoli. The zygotes divided into symmetric two-celled embryos, globular-like embryos and multicellular club-shaped embryos which are mostly consistent with those in the embryos in planta. IVF-produced club-shaped embryos developed into compact embryonic calli and subsequently regenerated into fertile plants. In this chapter, we provide a detailed description of wheat IVF system that might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for investigating fertilization-induced events in wheat.

Genome editing approaches using reproductive cells/tissues in flowering plants.

Targeted mutagenesis via programmable nucleases including the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been broadly utilized to generate genome-edited organisms including flowering plants. To date, specific expression of Cas9 protein and guide RNA (gRNA) in reproductive cells or tissues is considered one of the most effective genome-editing approaches for heritable targeted mutagenesis. In this report, we review recent advances in genome editing methods for reproductive cells or tissues, which have roles in transmitting genetic material to the next-generation, such as egg cells, pollen grains, zygotes, immature zygotic embryos, and shoot apical meristems (SAMs). Specific expression of Cas9 proteins in initiating cells efficiently induces targeted mutagenesis via Agrobacterium-mediated in planta transformation. In addition, genome editing by direct delivery of CRISPR/Cas9 components into pollen grains, zygotes, cells of embryos and SAMs has been successfully established to generate genome-edited plant lines. Notably, DNA-free genome editing by the delivery of Cas9-gRNA ribonucleoproteins (RNPs) is not associated with any legislative concerns about genetically modified organisms. In summary, the genome editing methods for reproductive cells or tissues have enormous potential for not only basic studies for plant reproduction but also applied sciences toward molecular plant breeding.

Effects of simulated peripheral visual field loss on the static postural control in young healthy adults.

BACKGROUND: Integration of visual, vestibular, and proprioceptive sensations contributes to postural control. People with peripheral visual field loss have serious postural instability. However, the directional specificity of postural stability and sensory reweighting caused by gradual peripheral visual field loss remain unclear. RESEARCH QUESTION: What are the effects of peripheral visual field loss on static postural control? METHODS: Fifteen healthy young adults participated in this study. The participants were asked to stand quietly on a foam surface. Three conditions of virtual visual field loss (90°, 45°, and 15°) were provided by a head-mounted display, and ground reaction forces were collected using a force plate to calculate the displacements of the center of pressure (COP). RESULTS: The root mean square (RMS), mean velocity, and 95% ellipse area of COP displacements in the horizontal plane increased, and RMS in the anteroposterior (AP) direction was unchanged under the smallest visual field condition compared to the largest one. The power spectrum density of COP displacements in the low-frequency band was decreased and that in the medium-frequency band was increased in the AP direction. SIGNIFICANCE: During quiet standing of young healthy adults with peripheral visual field loss, increased peripheral visual field loss resulted in lower postural stability. Postural stability in the AP direction was maintained contrary to the functional sensitivity hypothesis. Peripheral visual field loss reduced the weighting of the visual input and increased that of the vestibular input in the AP direction to maintain equilibrium.

High-throughput phenotypic screening of random genomic fragments in transgenic rice identified novel drought tolerance genes.

KEY MESSAGE: Novel drought tolerance genes were identified by screening thousands of random genomic fragments from grass species in transgenic rice. Identification of agronomically important genes is a critical step for crop breeding through biotechnology. Multiple approaches have been employed to identify new gene targets, including comprehensive screening platforms for gene discovery such as the over-expression of libraries of cDNA clones. In this study, random genomic fragments from plants were introduced into rice and screened for drought tolerance in a high-throughput manner with the aim of finding novel genetic elements not exclusively limited to coding sequences. To illustrate the power of this approach, genomic libraries were constructed from four grass species, and screening a total of 50,825 transgenic rice lines for drought tolerance resulted in the identification of 12 reproducibly efficacious fragments. Of the twelve, two were from the mitochondrial genome of signal grass and ten were from the nuclear genome of buffalo grass. Subsequent sequencing and analyses revealed that the ten fragments from buffalo grass carried a similar genetic element with no significant homology to any previously characterized gene. The deduced protein sequence was rich in acidic amino acid residues in the C-terminal half, and two of the glutamic acid residues in the C-terminal half were shown to play an important role in drought tolerance. The results demonstrate that an open-ended screening approach using random genomic fragments could discover trait genes distinct from gene discovery based on known pathways or biased toward coding sequence over-expression.

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

Establishment of an In Vitro Fertilization System in Wheat (Triticum aestivum L.).

In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect post-fertilization events in angiosperms, since the female gametophytes of plants are deeply embedded within ovaries. In addition, IVF systems have been used to produce hybrid and polyploid zygotes. Complete IVF systems have been established in maize and rice, two of three major crop species providing the majority of human energy intake. Among those crop species, gametes of wheat have not been used to establish a complete IVF system successfully. In this study, a wheat IVF system was developed to introduce the advantages of this technology to wheat research. Fusion of gametes was performed via a modified electrofusion method, and the fusion product, a zygote, formed a cell wall and two nucleoli. The first division of zygotes was observed 19-27 h after fusion, and the resulting two-celled embryo developed into 10-20-celled globular-like embryos and multicellular club-shaped embryos by 3 and 7-10 d after fusion, respectively. Such zygotic division profiles were mostly consistent with those in the embryo sac, suggesting that the division profile of IVF-produced early embryos reflects that of early embryos in planta. Although the IVF-produced club-shaped embryos did not develop into differentiated embryos but into compact embryonic calli, fertile plants could be regenerated from the embryonic calli, and the seeds harvested from those plants grew normally into seedlings. The IVF system described here might become an important technique for generating new genotypes of wheat and/or new hybrids as well as for investigating fertilization-induced events in wheat.

Development of gene expression system in egg cells and zygotes isolated from rice and maize.

Polyethylene glycol calcium (PEG-Ca2+) transfection-mediated analysis allows rapid and efficient examination of gene function. To investigate the diverse cellular functions of genes of interest in plant cells, macromolecules, such as DNA, RNA, and proteins, are delivered into protoplasts prepared from somatic tissues or calli using a PEG-Ca2+ transfection procedure. To take advantage of this macromolecule delivery system in the reproductive and developmental biology of angiosperms, this study established a PEG-Ca2+ transfection system with isolated egg cells and zygotes. The conditions for PEG and plasmid DNA concentrations for transfection of rice egg cells were first addressed, and ~30% of PEG-Ca2+-transfected egg cells showed exogenous and transient expressions of fluorescent proteins from plasmid DNA delivered into the cells. Interestingly, a dual expression of two different fluorescent proteins in the same egg cell using two kinds of plasmid DNAs was also observed. For PEG-Ca2+ transfection with maize zygotes, ~80% of zygotes showed expression of GFP proteins from plasmid DNA. Importantly, PEG-transfected zygotes developed normally into cell masses and mature plants. These results suggest that the present PEG-Ca2+-mediated transient expression system provides a novel and effective platform for expressing and analyzing genes of interest in egg cells and zygotes. Moreover, combined with the CRISPR/Cas9 approach, the present transient expression system in zygotes will become a powerful and alternative tool for the preparation of gene-edited plants.

A study of the effect of visual depth information on upper limb movement by use of measurement of smoothness.

[Purpose] This study verified that the smoothness of reaching movements is able to quantitatively evaluate the effects of two- and three-dimensional images on movement in healthy people. In addition, clinical data of cerebrovascular accident patients were also analyzed by the same method. [Subjects] Ten healthy adult volunteers and two male patients with previous cerebrovascular accidents participated. [Methods] The subjects were tasked with reaching for objects shown on a display. The target and virtual limb, rendered with computer graphics, were shown on the display. Movements of the virtual limb were synchronized with those of the subject. Healthy subjects reached for targets with their dominant arm, and cerebrovascular accident patients used their paretic arm. A polarized display and polarized glasses were used when the subjects were shown three-dimensional images. In the present study, jerk cost was used to quantify the smoothness of movement. [Results] Six of the 10 healthy subjects had significantly smoother reaching movements when viewing the three-dimensional images. The two cerebrovascular accident patients tended to have smoother movements in response to the three-dimensional images. [Conclusion] Analysis of the smoothness of movement was able to detect the influence of the depth cue in vision on movement quantitatively for the healthy subjects and cerebrovascular accident patients.

Development and evaluation of a new telerehabilitation system based on VR technology using multisensory feedback for patients with stroke.

[Purpose] The purpose of this study was to develop a new telerehabilitation system based on VR technology for training of paralyzed upper and lower extremities and poor balance in patients with stroke. Moreover, the effectiveness of the system was verified by analysis of the recovery of these patients. [Subjects] Five healthy persons and five people with motor paralysis, caused by cerebrovascular disease, participated. [Methods] The features of our system are as follows: (1) Our system can train upper and lower limbs and balancing with 3D images. (2) A Kinect(®) is used for user posture detection. (3) A vibrator is used for feedback to a sensory receptor in order to promote the learning effect of motion. Upper limb and balance training were conducted in this study. [Results] The time necessary for the upper limb and balance training tasks was shortened for the participants with disabilities. The joint angle for the participants with disabilities tended to equate to that of the healthy participants over time. Moreover, our system had no side effects. [Conclusion] These points suggest that our system is effective and safe. The user interface and assessment of the conditions of patients from a distance should be studied in the future.

A study of upper extremity training for patients with stroke using a virtual environment system.

[Purpose] A virtual environment (VE) system was designed to facilitate the retraining of motor control by feedback of movement trajectory to patients with neurological impairments, such as stroke victims or those with an acquired brain injury. In this study, we quantitatively assessed motion trajectory of the upper extremity during VE in order to further understand the effect of paralyzed upper extremity movement in VE for each patient as well as the functional clinical evaluations. [Subjects and Methods] Six patients with stroke were participated in this study. The VE system consisted of a computer, an electromagnetic motion tracking device, which monitored and displayed patient movement on the computer, and the VE software, which provided the tools for creating training scenes. This system was used to facilitate motor relearning of the upper extremity for six patients with stroke. [Results] The results showed there were improvements in variability and accuracy of the arm movement in motion trajectory. In addition, the scores of clinical evaluations improved, and VE did not hinder motor relearning of the upper extremity, which is necessary for activities of daily living. [Conclusion] This VE system might be effective at facilitating motor relearning of the upper extremity for stroke patients.

Neurocognitive function of patients with brain metastasis who received either whole brain radiotherapy plus stereotactic radiosurgery or radiosurgery alone.

PURPOSE: To determine how the omission of whole brain radiotherapy (WBRT) affects the neurocognitive function of patients with one to four brain metastases who have been treated with stereotactic radiosurgery (SRS). METHODS AND MATERIALS: In a prospective randomized trial between WBRT+SRS and SRS alone for patients with one to four brain metastases, we assessed the neurocognitive function using the Mini-Mental State Examination (MMSE). Of the 132 enrolled patients, MMSE scores were available for 110. RESULTS: In the baseline MMSE analyses, statistically significant differences were observed for total tumor volume, extent of tumor edema, age, and Karnofsky performance status. Of the 92 patients who underwent the follow-up MMSE, 39 had a baseline MMSE score of < or =27 (17 in the WBRT+SRS group and 22 in the SRS-alone group). Improvements of > or =3 points in the MMSEs of 9 WBRT+SRS patients and 11 SRS-alone patients (p = 0.85) were observed. Of the 82 patients with a baseline MMSE score of > or =27 or whose baseline MMSE score was < or =26 but had improved to > or =27 after the initial brain treatment, the 12-, 24-, and 36-month actuarial free rate of the 3-point drop in the MMSE was 76.1%, 68.5%, and 14.7% in the WBRT+SRS group and 59.3%, 51.9%, and 51.9% in the SRS-alone group, respectively. The average duration until deterioration was 16.5 months in the WBRT+SRS group and 7.6 months in the SRS-alone group (p = 0.05). CONCLUSION: The results of the present study have revealed that, for most brain metastatic patients, control of the brain tumor is the most important factor for stabilizing neurocognitive function. However, the long-term adverse effects of WBRT on neurocognitive function might not be negligible.

Binary vectors and super-binary vectors.

A binary vector is a standard tool in the transformation of higher plants mediated by Agrobacterium tumefaciens. It is composed of the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, selectable marker genes, reporter genes, and other accessory elements that can improve the efficiency of and/or give further capability to the system. A super-binary vector carries additional virulence genes from a Ti plasmid, and exhibits very high frequency of transformation, which is valuable for recalcitrant plants such as cereals. A number of useful vectors are widely circulated. Whereas vectors with compatible selectable markers and convenient cloning sites are usually the top criteria when inserting gene fragments shorter than 15 kb, the capability of maintaining a large DNA piece is more important for consideration when introducing DNA fragments larger than 15 kb. Because no vector is perfect for every project, it is recommended that modification or construction of vectors should be made according to the objective of the experiments. Existing vectors serve as good sources of components.

Speed and amplitude of lung tumor motion precisely detected in four-dimensional setup and in real-time tumor-tracking radiotherapy.

BACKGROUND: To reduce the uncertainty of registration for lung tumors, we have developed a four-dimensional (4D) setup system using a real-time tumor-tracking radiotherapy system. METHODS AND MATERIALS: During treatment planning and daily setup in the treatment room, the trajectory of the internal fiducial marker was recorded for 1 to 2 min at the rate of 30 times per second by the real-time tumor-tracking radiotherapy system. To maximize gating efficiency, the patient's position on the treatment couch was adjusted using the 4D setup system with fine on-line remote control of the treatment couch. RESULTS: The trajectory of the marker detected in the 4D setup system was well visualized and used for daily setup. Various degrees of interfractional and intrafractional changes in the absolute amplitude and speed of the internal marker were detected. Readjustments were necessary during each treatment session, prompted by baseline shifting of the tumor position. CONCLUSION: The 4D setup system was shown to be useful for reducing the uncertainty of tumor motion and for increasing the efficiency of gated irradiation. Considering the interfractional and intrafractional changes in speed and amplitude detected in this study, intercepting radiotherapy is the safe and cost-effective method for 4D radiotherapy using real-time tracking technology.