Regeneration of t Anthurium adventitious shoots using liquid or raft culture

This study was initiated to regenerate t Anthurium adventitious shoots, singly or in loose aggregates, by culturing homogenised inoculum in liquid or on the medium surface supported by a raft. The culture methods as compared to solid culture did not affect the time required for inoculum to regenerate. The regeneration rate was affected mainly by inoculum size, and it also influenced the regeneration frequency. All regenerated shoots were normal, without any sign of hyperhydricity. This revised version was published online in June 2006 with corrections to the Cover Date.     

Using human sequencing to guide craniofacial research

A recent convergence of technological innovations has re‐energized the ability to apply genetics to research in human craniofacial development. Next‐generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR‐Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.     

Molecular Analysis of In Vitro Shoot Organogenesis

Shoot organogenesis is one of the in vitro plant regeneration pathways. It has been widely employed in plant biotechnology for in vitro micropropagation and genetic transformation, as well as in study of plant development. Morphological and physiological aspects of in vitro shoot organogenesis have already been extensively studied in plant tissue culture for more than 50 years. Within the last ten years, given the research progress in plant genetics and molecular biology, our understanding of in vivo plant shoot meristem development, plant cell cycle, and cytokinin signal transduction has advanced significantly. These research advances have provided useful molecular tools and resources for the recent studies on the genetic and molecular aspects of in vitro shoot organogenesis. A few key molecular markers, genes, and probable pathways have been identified from these studies that are shown to be critically involved in in vitro shoot organogenesis. Furthermore, these studies have also indicated that in vitro shoot organogenesis, just as in in vivo shoot development, is a complex, well-coordinated developmental process, and induction of a single molecular event may not be sufficient to induce the occurrence of the entire process. Further study is needed to identify the early molecular event(s) that triggers dedifferentiation of somatic cells and serves as the developmental switch for de novo shoot development.     

Electroporation of intact embryonic leaflets of peanut: Gene transfer and stimulation of regeneration capacity

Summary Direct gene transfer into peanut intact embryonic leaflets was performed through electroporation. In transient β-glucuronidase expression assays, maximal expression was obtained by using pulses of 625 V cm⁻¹ in EPRm (modified electroporation) buffer supplemented with 75 μM NaCl. Kanamycin-resistant plants were obtained, and the presence of the nptII gene was demonstrated by PCR analysis. The positive effect of electroporation on the efficiency of in vitro regeneration was demonstrated. Explants submitted to field strengths between 500 and 625 V cm⁻¹ displayed a significantly increased number of shoots and originated faster growing calluses relative to control explants. Whereas in control explants callus formation occurred only at the petiolule, electroporated leaflets developed additional organogenic calluses on the foliar lamina. These authors have contributed equally to this work.     

Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids

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Generation of floxed Spag6l mice and disruption of the gene by crossing to a Hprt-Cre line

Mouse sperm-associated antigen 6 like (SPAG6L) is an axoneme central apparatus protein, essential for the normal function of the ependymal cell and lung cilia, and sperm flagella. Accumulated evidence has disclosed multiple biological functions of SPAG6L, including ciliary/flagellar biogenesis and polarization, neurogenesis, and neuronal migration. Conventional Spag6l knockout mice died of hydrocephalus, which impedes further investigation of the function of the gene in vivo. To overcome the limitation of the short lifespan of conventional knockout mice, we developed a conditional allele by inserting two loxP sites in the genome flanking exon 3 of the Spag6l gene. By crossing the floxed Spag6l mice to a Hrpt-Cre line which expresses Cre recombinase ubiquitously in vivo, mutant mice that are missing SPAG6L globally were obtained. Homozygous mutant Spag6l mice showed normal appearance within the first week after birth, but reduced body size was observed after 1 week, and all developed hydrocephalus and died within 4 weeks of age. The phenotype mirrored that of the conventional Spag6l knockout mice. The newly established floxed Spag6l model provides a powerful tool to further investigate the role of the Spag6l gene in individual cell types and tissues.