Ming Cheng, Brenda A. Lowe, Michael Spencer, Xudong Xudong Ye, and
Chuck L. Armstrong

Agrobacterium-mediated transformation of monocots

Since the success of Agrobacterium-mediated transformation of rice in the early 1990’s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocots. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems. Ming Cheng, Brenda A. Lowe, T. Michael Spencer, Xudong Ye, and Charles L. Armstrong. In Vitro Cellular and Developmental Biology – Plant, 40: 31 – 45, 2004.

14C-Enrichment in Plant Cell Suspension Cultures

Michael A. Grusak

Michael A. Grusak

Various plant secondary products have been implicated in the promotion of good health or the prevention of disease in humans, but little is known about the way they are absorbed in the gut, or in which tissues they are deposited throughout the body. While these issues could be studied if the phytochemicals were isotopically labeled, generating labeled molecules often is problematic because many compounds of interest can be synthesized only in planta at present. In order to generate 14C-labeled phytochemicals of high radioactive enrichment, we developed an enclosed-chamber labeling system in which cell suspension cultures can be safely and efficiently grown when supplied 14C-enriched precursors. The system is designed to hold culture flasks within a clear, polyacrylic compartment that is affixed to the top of a rotary shaker. The flow-through gas exchange nature of the system allows for O2 replenishment and complete capture of respired 14CO2 throughout the entire period of cell culture. Air is circulated internally with the aid of a small fan, and chamber air temperature is monitored continuously with an internal temperature probe and data logger. Production runs of 12-14 d with Vaccinium pahalae (ohelo berry) and Vitis vinifera (grape) suspension cultures, using [14C]sucrose as the carbon source, demonstrated a 20 to 23 % efficiency of 14C incorporation into the flavonoid-rich fractions. Further studies with ohelo cell cultures showed that flavonoids were produced with either sucrose or glucose as the carbohydrate source, although flavonoid productivity (measured as anthocyanins) was higher with sucrose. This comprehensive chamber system should have broad applicability with numerous cell types and can be used to generate a wide array of labeled phytochemicals. Michael A. Grusak, Randy B. Rogers, Gad G. Yousef, John W. Erdman, Jr., and Mary Ann Lila, An Enclosed-chamber Labeling System for the Safe 14C-enrichment of Phytochemicals in Plant Cell Suspension Cultures, In Vitro Cellular and Developmental Biology, 40: 80 – 85, 2004.

Glutathione in white spruce embryogenesis

Mark F. Belmonte and Edward C. Yeung

The glutathione-glutathione disulfide redox pair was utilized to improve white spruce somatic embryo development. Mature cotyledonary stage somatic embryos were divided into two groups (A and B) based on morphological normality and the ability of the mature somatic embryos to convert into plantlets. Group A embryos had four or more cotyledons and converted readily upon germination after a partial drying treatment. Group B embryos had three or fewer cotyledons with a low conversion frequency. The addition of reduced glutathione (GSH) at a concentration of 0.1 mM resulted in an increase in embryo production (total population) with a mean total number of 64 embryos per 100 mg embryogenic tissue as well as an increase in post-embryonic root growth. However, at a higher concentration (1 mM), GSH inhibited embryo formation. The manipulation of the tissue culture environment via the inclusion of glutathione disulfide (GSSG) at concentrations of 0.1 and 1.0 mM, enhanced the development of better quality embryos. This quality was best exemplified when embryos forming four or more cotyledons increased by at least 2 fold to 73.9 % when treated with 1.0 mM GSSG compared to 38 % in control. Furthermore, this improved quality was reflected by an increased conversion frequency. A 20 % increase in the ability of the somatic embryo to produce both root and shoot structures during post-embryonic development was noted when embryos were matured on maturation medium supplemented with 1.0 mM GSSG over the control. Mark F. Belmonte and Edward C. Yeung, The Effects of Reduced and Oxidized Gluthathione on White Spruce Somatic Embryogenesis, In Vitro Cellular and Developmental Biology – Plant, 40: 61 – 66, 2004.