Novel genetic engineering technology which increases leaf lipid content modifies the ensiling properties of perennial ryegrass : a thesis presented in partial fulfillment of the requirements for the degree of Masters in Agricultural Science at Massey University, Palmerston North

A novel strategy to increase the metabolisable energy (ME) yield of pastures has been the development of a genetic engineering technology which increases the leaf lipid content and biomass production of perennial ryegrass (PRG). Outdoor plot/feeding trials of genetically engineered crops are prohibited under the current New Zealand (NZ) regulatory framework. However, this high lipid PRG may become available to farmers and eventually be used to make silage, which could fulfill an important role as a high ME, inexpensive, supplementary feed for livestock. Ensiling preserves a crop’s nutrients at a high moisture content and at a low pH, by microbial fermentation of plant sugars into lactic acid under anaerobic conditions. In a preliminary investigation into the ensiling biochemistry of this high lipid PRG, glasshouse-grown materials were wilted and inoculated, and then ensiled on a miniature scale. A series of method development ensiling experiments revealed that non-transgenic PRG grown in glasshouse conditions during the NZ spring/summer was very difficult to ensile naturally, due to its low water soluble sugar to buffering capacity ratio. In order to generate well-preserved silage in the main experiment, glucose was added (post-harvest) to a non-transgenic PRG genotype (WT) and two transgenic PRG genotypes containing ‘medium’ and ‘high’ leaf lipid levels (ML and HL). The HL plants produced 51% more dry biomass than WT during the regrowth period. Pre-ensiled HL had 31% higher fatty acid content, 70% higher nitrate content and a 17% lower water soluble sugar to crude protein ratio than WT. ML was intermediate. The glasshouse growth environment resulted in an atypical overall PRG nutritional composition. WT, ML and HL underwent a similar fermentation, and nutrients were well-preserved. The nutritional differences in the ensiled material largely reflected those in their fresh counterparts, although a longer wilt caused greater overall digestible nutrient losses in HL. In an in vitro rumen incubation experiment the fatty acids in HL silage exhibited less complete biohydrogenation than in fresh and ensiled WT. Experiments using a range of high lipid PRG lines grown in a range of environments will be needed to validate these results

Storing carbon in leaf lipid sinks enhances perennial ryegrass carbon capture especially under high N and elevated CO₂

By modifying two genes involved in lipid biosynthesis and storage [cysteine oleosin (cys-OLE)/diacylglycerol O-acyltransferase (DGAT)], the accumulation of stable lipid droplets in perennial ryegrass (Lolium perenne) leaves was achieved. Growth, biomass allocation, leaf structure, gas exchange parameters, fatty acids, and water-soluble carbohydrates were quantified for a high-expressing cys-OLE/DGAT ryegrass transformant (HL) and a wild-type (WT) control grown under controlled conditions with 1–10 mM nitrogen (N) supply at ambient and elevated atmospheric CO₂. A dramatic shift in leaf carbon (C) storage occurred in HL leaves, away from readily mobilizable carbohydrates and towards stable lipid droplets. HL exhibited an increased growth rate, mainly in non-photosynthetic organs, leading to a decreased leaf mass fraction. HL leaves, however, displayed an increased specific leaf area and photosynthetic rate per unit leaf area, delivering greater overall C capture and leaf growth at high N supply. HL also exhibited a greater photosynthesis response to elevated atmospheric CO₂. We speculate that by behaving as uniquely stable microsinks for C, cys-OLE-encapsulated lipid droplets can reduce feedback inhibition of photosynthesis and drive greater C capture. Manipulation of many genes and gene combinations has been used to increase non-seed lipid content. However, the cys-OLE/DGAT technology remains the only reported case that increases plant biomass. We contrast cys-OLE/DGAT with other lipid accumulation strategies and discuss the implications of introducing lipid sinks into non-seed organs for plant energy homeostasis and growth