Mikihisa Umehara

Benzyl Ether-type Compounds Inhibit Somatic Embryogenesis in Japanese Larch

In seed plants, a fertilized cell divides transversely and asymmetrically to form both a terminal cell, which gives rise to the embryo proper, and a basal cell, which forms the suspensor. The mechanisms of embryogenesis have often been investigated using somatic embryos. However, the suspensor usually fails to develop during the culture in somatic embryogenesis of angiosperms. In Japanese larch (Larix leptolepis Gordon) a well-developed suspensor forms during somatic embryogenesis. The suspensor is the essential tissue for development of the embryo proper. In high-cell-density culture, the embryogenic cells proliferate but no somatic embryos form because suspensor development is suppressed. Previously, we identified vanillyl benzyl ether (VBE) as a novel factor suppressing suspensor development from the high-cell-density conditioned medium (HCM), but the inhibitory effect of VBE was weaker than that of HCM added. Therefore, we attempted to identify another inhibitory factor in the culture medium. Induction of somatic embryos was performed in a medium containing both VBE and a fraction of each chromatogram extracted from the culture medium. Results of the bioassay showed that a fraction had strong inhibitory activity with VBE, but weak activity without it. By physicochemical analyses of the fraction, 4-[(phenylmethoxy)methyl]phenol (4PMP), whose chemical structure was very similar to VBE, was identified as a complementarily inhibitory factor of larch somatic embryogenesis. VBE and 4PMP are novel compounds as bioactive substances. Although VBE and 4PMP have not been quantified in immature and mature seed, at least, elimination or dilution of these benzyl ether-type compounds in the medium will be required to improve the efficiency of somatic embryos formation in Japanese larch and some conifer tissue culture. Mikihisa Umehara, Shinjiro Ogita, Hamako Sasamoto, Hiroyuki Koshino, Takemichi Nakamura, Tadao Asami, Shigeo Yoshida and Hiroshi Kamada. Identification of a factor that complementarily inhibits somatic embryogenesis with vanillyl benzyl ether in Japanse larch, In Vitro Cellular and Developmental Biology – Plant, 43:203-208, 2007.

Left to right: (back row) Michael Kane, Ph.D., Philip Kauth, Scott Stewart, Ph.D., Timothy Johnson; (front row) Daniela Dutra, Nancy Philman

Symbiotic Seed Germination and Evidence for In Vitro Mycobiont Specificity in Spiranthes Brevilabris (Orchidaceae) and Its Implications for Species-level Conservation

In nature, orchids digest endomycorrhizal fungi as sources of minerals, carbohydrates, water, and vitamins in an action termed mycotrophy. This orchid-fungal parasitic association can be replicated under in vitro conditions through the co-culture of orchid seed and appropriate fungi. These co-culture seed germination methods represent a physiologically- and ecologically-sound manner in which to produce orchid plants for conservation and restoration purposes. However, orchid-mycobiont preference has been considered controversial and not well understood for over a century. Differences in mycobiont preference during germination in vitro versus in situ have lead some researchers to consider orchid-mycobiont preference as being generally low; however, others have suggested that preference, especially under in vitro conditions, is surprisingly high. The degree of preference may be genus or species specific. To study this mycobiont preference phenomenon, an in vitro co-culture seed germination experiment was designed using seed and mycobionts from the endangered Florida terrestrial orchid Spiranthes brevilabris and mycobionts from the endemic congener S. floridana. In a screen of three S. floridana and one S. brevilabris mycobiont, the mycobionts originating from the roots of S. floridana supported high initial seed germination (i.e., rupture of seed coat). However, only the mycobiont originating from S. brevilabris supported advanced germination and seedling development of S. brevilabris seed. These findings suggest that S. brevilabris maintains a high degree of mycobiont preference under in vitro symbiotic co-culture conditions. High orchid-mycobiont preference in S. brevilabris may be indicative of the rare status and limited distribution of this orchid in Florida and throughout the southeastern coastal plain of the United States. These data are now being incorporated into a state-wide integrated conservation plan for S. brevilabris and S. floridana, which also includes research focused on the ecology, pollination biology, and population genetic diversity of these species. Furthermore, similar techniques for studying the mycological associations and propagation science of other native Florida terrestrial and epiphytic orchids are pursued in partnership with the U.S. Fish and Wildlife Service. Scott L. Stewart and Michael E. Kane. Symbiotic seed germination and evidence for in vitro mycobiont specificity in Spiranthes brevilabris (Orchidaceae) and its implications for species-level conservation, In Vitro Cellular & Developmental Biology-Plant, 43:178-186, 2007.

Paula M. Pijut

Technological Advances in Temperate Hardwood Tree Improvement Including Breeding and Molecular Marker Applications

Hardwood forests and plantations are an important economic resource for the forest products industry worldwide and to the international trade of lumber and logs. Hardwood trees are also planted for ecological reasons, for example, wildlife habitat, native woodland restoration, and
riparian buffers. The demand for quality hardwood from tree plantations will continue to rise as the worldwide consumption of forest products increases. Tree improvement of temperate hardwoods has lagged behind that of coniferous species and hardwoods of the genera Populus and Eucalyptus. The development of marker systems has become an almost necessary complement to the classical breeding and improvement of hardwood tree populations for superior growth, form, and timber characteristics. Molecular markers are especially valuable for determining the reproductive biology and population structure of natural forests and plantations, and the identity of genes affecting quantitative traits. Clonal reproduction of commercially important hardwood tree species provides improved planting stock for use in field testing and production forestry. Development of in vitro and conventional vegetative propagation methods allows mass production of clones of mature, elite genotypes or genetically improved genotypes. Genetic modification of hardwood tree species could potentially produce trees with herbicide tolerance, disease and pest resistance, improved wood quality, and reproductive manipulations for commercial plantations. The Hardwood Tree Improvement and Regeneration Center (HTIRC) located at Purdue University is a collaborative regional research, development, and technology transfer effort between industry, university, private, state and federal entities to advance tree improvement of central hardwoods for increased forest productivity in hardwood restoration and reforestation programs. The national mission of the HTIRC is to advance the science of hardwood tree improvement, genomics, physiology, protection, and utilization in the hardwood region of the U.S. by: developing and disseminating knowledge on improving the genetic quality of hardwood tree species and conserving fine hardwood germ plasm; developing elite hardwood trees for restoration and regeneration of sustainable hardwood forests and riparian zones for production of forest products and maintenance of genetically diverse ecosystems; increasing knowledge and developing systems for nursery production and plantation establishment; increasing knowledge and developing strategies for protection, utilization, and marketing of the hardwood resource; and developing recognized and respected science leaders in forest genetics, physiology, regeneration, protection, and utilization. Partners include the USDA Forest Service Northern Research Station, National Seed Laboratory, and Northeastern Area State and Private Forestry; Purdue University Dept. of Forestry and Natural Resources, Indiana Dept. of Natural Resources Division of Forestry, Indiana Hardwood Lumbermen’s Association, National Hardwood Lumber Association, ArborAmerica, Indiana Forestry and Woodland Owners Association, Walnut Council, American Chestnut Foundation, and the Fred M. van Eck Forest Foundation. In addition, the HTIRC is the lead center in the National Science Foundation Industry/University Cooperative Research Center program called the Center for Tree Genetics, a cooperative program with Oregon State University. Paula M. Pijut, Keith E. Woeste, G. Vengadesan, and Charles H. Michler. Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications, In Vitro Cellular and Developmental Biology – Plant 43: 283-303, 2007.

left to right: Masao Takeuchi, Arihiro Kohara, Hiroshi Mizusawa, Azusa Ohtani, Setsuko Shioda and Yutaka Ozawa.

Chromosomal Instability in Human Mesenchymal Stem Cells Immortalized with Human Papilloma Virus E6, E7 and hTERT Genes

Human mesenchymal stem cells (hMSCs) are expected to be an enormous potential source for future cell therapy, because of their self-renewing divisions and also because of their multiple- lineage differentiation. The finite lifespan of these cells, however, is a hurdle for clinical application. Recently, several hMSC lines have been established by immortalized human telomerase reverse transcriptase gene (hTERT) alone or with hTERT in combination with human papillomavirus type 16 E6/E7 genes (E6/E7) and human proto-oncogene, Bmi-1, but have not so much been characterized their karyotypic stability in detail during extended lifespan under in vitro conditions. In this report, the cells immortalized with the hTERT gene alone exhibited little change in karyotype, whereas the cells immortalized with E6/E7 plus hTERT genes or Bmi-1, E6 plus hTERT genes were unstable regarding chromosome numbers, which altered markedly during prolonged culture. Interestingly, one unique chromosomal alteration was the preferential loss of chromosome 13 in three cell lines, observed by fluorescence in situ hybridization (FISH) and comparative-genomic hybridization (CGH) analysis. The four cell lines all maintained the ability to differentiate into both osteogenic and adipogenic lineages, and two cell lines underwent neuroblastic differentiation. Thus, our results were able to provide a step forward toward fulfilling the need for a sufficient number of cells for new therapeutic applications, and substantiate that these cell lines are a useful model for understanding the mechanisms of chromosomal instability and differentiation of hMSCs. Masao Takeuchi, Kikuko Takeuchi, Arihiro Kohara, Motonobu Satoh, Setsuko Shioda, Yutaka Ozawa, Azusa Ohtani, Keiko Morita, Takashi Hirano, Masanor Terai, Akihiro Umezawa, and Hiroshi Mizusawa. Chromosomal Instability in Human Mesenchymal Stem Cells Immortalized with Human Papilloma Virus E6, E7 and hTERT Genes, In Vitro Cellular and Developmental Biology – Animal 43: 129-138, 2007.