Drs. Dennis Genovesi (seated at the laminar flow hood) and Milt Engelke observe an embryo rescue derived St. Augustinegrass plantlet growing in a test tube.

Interploid St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] Hybrids Recovered by Embryo Rescue

The research focus of the Turfgrass Breeding Program at the Urban Solutions Center in Dallas, Texas is to develop new and improved turfgrasses with superior biotic and abiotic stress resistance.  We are working on three different species simultaneously, St. Augustinegrass, zoysiagrass and hybrid bluegrass.  St. Augustinegrass is one of the most important warm season turfgrasses in the southern United States because of its shade tolerance.  Most cultivars are diploids (2n=2x=18) and are susceptible to various diseases and insects.   Polyploid cultivars in the species have some resistance to pests but most lack cold tolerance.  In this study, 8 polyploid genotypes were crossed with 6 diploid cultivars to transfer pest resistance to the diploids.  Because interploid crosses often result in aborted seed, it was necessary to use in vitro techniques.  Using embryo rescue, 268 plants were recovered from 2,463 emasculated and pollinated florets (10.88% crossability).  Because of the heterogeneous nature of the species, these purported hybrids could not be verified by phenotype.  DNA markers were used to identify the hybrids.  A subset of 25 plants from crosses between the aneuploid cultivar Floratam (2n=4x=32) and 5 diploid cultivars were analyzed using 144 EST-SSRs developed from buffelgrass cDNA sequence data.  Chi-square tests for paternal-specific markers revealed that all analyzed progeny were true F1 hybrids and none originated from self-fertilization or unintended out-crossing.  In addition to identifying DNA polymorphism, the EST-SSRs revealed that genetic variation exists among all analyzed cultivars and is not partitioned between ploidy levels.  These findings demonstrate that embryo rescue techniques enable the entire spectrum of St. Augustinegrass genetic variation to be better used through the recovery of interploid hybrids.

Anthony D. Genovesi, Russell W. Jessup, Milton C. Engelke and Byron L. Burson.  Interploid St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] hybrids recovered by embryo rescue.  In Vitro Cellular & Developmental Biology-Plant 45:659-666, 2009.

Victor Gaba and Sampath Amutha

Sima Singer

Benjamin Steinitz

Ilan Shomer

Liana Jashi

Krishnan Kathirava

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (leaving a single cotyledon) of young greenhouse-grown seedlings to test the hypothesis that abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. This adventitious shoot regeneration response occurs in a large number of dicotyledonous plant families. To understand why shoot regeneration starts with defective organogenesis the regeneration response was characterized by morphology, scanning electron and light microscopy in decapitated squash seedlings. Surprisingly, leaf primordia regenerated on the wound surface prior to differentiation of a shoot apical meristem. Early regenerating primordia have a greatly distorted structure with dramatically altered shape. Aberrant leaf morphogenesis in squashdisappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of dicotyledonous families stems are regenerated bearing abnormal leaves; normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is observed in exceptional circumstances such as during recovery from excision of all pre-formed seedling shoot meristems.

Sampath Amutha, Krishnan Kathiravan, Sima Singer, Liana Jashi, Ilan Shomer, Benjamin Steinitz, and Victor Gaba. Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem.  In Vitro Cellular & Developmental Biology-Plant 45:758-768, 2009. 

Featured in the photograph are the authors that contributed to the manuscript: Bernadette Marrero and Richard Heller. The Bioreactor pictured in the photo is manufactured by Synthecon Inc. http://www.synthecon.com/.

In Vitro 3-D Tumor Model Generated for Establishment of Treatment Parameters Against Melanoma.

Synthetic 3-D modeling is actively being explored in the laboratory as a means to establish an in vitro tumor model and to study gene transfer delivery techniques such as electroporation. It is a new addition to existing tissue culture practices in the laboratory. The 3D tumor model was generated in a microgravity environment using a bioreactor to mimic naturally occurring cells. The published manuscript describes the importance of the 3-D model and how a tumor spheroid was constructed by allowing two different cell types to interact.  The tumor spheroid consisted of mouse B16.F10 melanoma cells encased by human keratinocytes. We are interested in utilizing the established model to study cellular behavior and more importantly to shed light on the effects of cells when subjected to different types of stresses such as chemical or electrical. This is an important addition to laboratory techniques because it will provide critical information necessary to establish delivery conditions that will enable efficient delivery of plasmid DNA to cells.  It will also facilitate monitoring levels of protein produced by the delivered transgenes and most importantly will significantly reduce the need to perform this testing on animal subjects. The 3D model will provided a stepping stone to establish treatment parameters against tumors found in vivo.

B. Marrero, JL Messina, and R. Heller. Generation of a tumor spheroid in a microgravity environment as a 3D model of melanoma. In Vitro Cellular & Developmental Biology-Animal, 45:523-34, 2009.

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