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We report functional genomics studies of a CYP74 rubber particle protein from Parthenium argentatum, commonly called guayule. Previously identified as an allene oxide synthase (AOS), this CYP74 constitutes the most abundant protein found in guayule rubber particles. Transgenic guayule lines with AOS gene expression down-regulated by RNAi (AOSi) exhibited strong phenotypes that included agricultural traits conducive to enhancing rubber yield. AOSi lines had higher leaf and stem biomass, thicker stembark tissues, increased stem branching and improved net photosynthetic rate. Importantly, the rubber content was significantly increased in AOSi lines compared to the wild-type (WT), vector control and AOS overexpressing (AOSoe) lines, when grown in controlled environments both in tissue-culture media and in greenhouse/growth chambers. Rubber particles from AOSi plants consistently had less AOS particle-associated protein, and lower activity (for conversion of 13-HPOT to allene oxide). Yet plants with downregulated AOS showed higher rubber transferase enzyme activity. The increase in biomass in AOSi lines was associated with not only increases in the rate of photosynthesis and non-photochemical quenching (NPQ), in the cold, but also in the content of the phytohormone SA, along with a decrease in JA, GAs, and ABA. The increase in biosynthetic activity and rubber content could further result from the negative regulation of AOS expression by high levels of salicylic acid in AOSi lines and when introduced exogenously. It is apparent that AOS in guayule plays a pivotal role in rubber production and plant growth.

Basal media including Murashige and Skoog (MS), Woody Plant Medium (WPM), and Driver and Kuniyuki (DKW) media were compared for use in guayule regeneration and propagation. Explants grown on MS basal medium showed large size calli with multiple shoots, but the calli produced on DKW medium were of higher quality, and longer true shoots were produced. Adventitious root formation was best in WPM basal medium. A comparison of macro and micro nutrient levels in MS, DKW, and WPM media revealed significant differences in the calcium levels. Under controlled conditions, guayule plants in vitro incorporated calcium into plant tissues in proportion to the amount available. Moreover, the addition of calcium nitrate tetrahydrate at moderate levels into MS medium significantly improved the emergence of single, elongated true shoots, suggesting a requirement for calcium in guayule tissue culture. We have further explored selection agent (antibiotic) concentration for the identification of transgenic plants using a spectinomycin resistance gene (aadA). Based on the results, we have improved tissue culture protocols for guayule including spectinomycin as the selection agent.

Populus serves as a model tree for biotechnology and molecular biology research due to the availability of the reference genome sequence of Populus trichocarpa (Torr. & Gray) genotype ‘Nisqually-1’. However, ‘Nisqually-1’ has been shown to be very recalcitrant to micropropagation, regeneration and transformation. In this study, a highly efficient micropropagation protocol from greenhouse-grown shoot tips of ‘Nisqually-1’ was established. The optimal micropropagation protocol involves growing in vitro shoots in plant growth regulator-free Murashige and Skoog (MS) basal medium supplemented with 3% sucrose, 0.3% Gelrite® and 5–10 g L−1 of activated charcoal. Plants grown on this medium were significantly longer, and contained significantly higher concentrations of chlorophyll. This highly effective protocol provides a consistent supply of quality leaf and stem materials throughout the year for transformation experiments and other in vitro manipulations, therefore eliminating inconsistency due to seasonal and greenhouse environmental variations and the need for repetitive tissue sterilization.