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Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C3-C4 intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel™. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel™ and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots. [ABSTRACT FROM AUTHOR]

A wide range of Sitka spruce breeding material ( Picea sitchensis (Bong.) Carr.) was assessed for its ability to respond to somatic embryogenesis (SE) and cryo-preservation methods, with 765 cell lines being established from seeds taken from the UK's breeding population for testing with the relevant methodologies. Cell lines were most readily established from immature seeds (63%) on a proliferation medium originally developed for white spruce ( P. glauca) with agar as the gelling agent. However, it was also found that 48% of the zygotic embryos taken from freshly harvested mature seed were also able to establish cell lines on the same medium, as were 20% of seeds that had been stored at −20 °C for up to 8 years. Most of the cell lines tested (57%) produced significant numbers of somatic embryos, but the rate at which they could be germinated into plants capable of surviving the transfer to nursery conditions varied considerably from 67 to 8% between years for reasons that were not clear. The ability of the cell lines to survive cryo-preservation was also assessed, and out of 199 cell lines tested, 45 cell lines from 21 breeding families were successfully established in a cryo-storage facility. The best rates of recovery were obtained when Phytagel was used in the medium for growing the samples before and after they were cryo-preserved. [ABSTRACT FROM AUTHOR]

Gram's Iodine Assay is currently used to detect endoglucanase, a cellulase enzyme which cleaves β-1,4-glycosidic bonds in carboxymethylcellulose (CMC). When a zone of degradation was observed around non-cellulolytic Escherichia coli JM109 with the use of Gram's Iodine, the assay was further examined. Additional cellulolytic bacteria were chosen to display these inconsistencies. Bacillus thuringiensis subsp. Kurstaki ( Btk) and Cellulomonas persica, known to possess endoglucanase, were incubated at 30 °C for 48 h on several different media; 1.5 % agar only, Davis minimal media, CMC agar, non-CMC agar, and Phytagel CMC. After the incubation period all plates were flooded with Gram's Iodine for 3-5 min. A clear ring of degradation was identified around colonies in plates containing no cellulose and plates containing agar only. When agar was removed as the solidifying agent and substituted with Phytagel, zones of clearing around cellulolytic organisms were altered. These findings indicate that agar alone can cause additional artifacts and should be replaced by other solidifying agents as well as supplementation of the Congo Red Assay. Taking these precautions can overcome inconsistency problems for future cellulolytic screenings. [ABSTRACT FROM AUTHOR]

Tissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased hardness of Gelrite gels; NHNO had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their effects being more pronounced on Gelrite than on agar gels. [ABSTRACT FROM AUTHOR]