Lipid profiles of Cuphea PSR23 leaf, callus and seed and in vitro calli optimization of NA and BAP concentrations for four Cuphea species.

摘要

Cuphea is a potential oilseed crop that is rich in medium chain fatty acids, which are currently available only from imported coconut and palm kernel oils. These oils are used in the manufacture of detergents, personal care products, feedstocks, industrial lubricants and possibly even as an additive for biodiesel. Knowledge of the biosynthetic mechanism controlling the lipids produced by Cuphea is limited. We are interested to know how oil composition differs between the leaf, callus and mature seed. We are especially interested in the oil composition of the callus tissue, because the callus can be used for genetic manipulation in order to understand the biosynthetic pathway with the goal of increasing the oil content. Cuphea PSR23 was analyzed for lipid composition in the mature seed, leaf and callus. Lipids were extracted using organic solvents and in addition seed oils were separated using solid phase extraction columns. Lipid composition was analyzed using electrospray ionization tandem mass spectrometry. The lipid profile of the leaf resembled that of previous studies of leaf membranes. The callus culture resembled the polar seed fraction more than the leaf in terms of lipid distribution. In the neutral portion of seed high amounts of phosphatidylglycerol/triacylglycerol were detected but with low capric acid (C10:0) levels. Trace amounts of capric acid were also identified in the phosphatidylcholine and phosphatidylethanolamine fractions. The lower than expected capric acid (C10:0) levels in the seed and the trace amounts in the polar lipid fraction indicate that there is a loss of capric acid (C10:0) during extraction. The relative quantities of phosphatidylglycerol and triacylglycerol in the mixture require further analysis as they shared a common mass to charge ratio on the product ion scan.;We are particularly interested in the feasibility of using the callus as a platform for metabolic engineering to determine the regulatory and metabolic pathways to develop biosynthesis of desired lipids. The establishment of an efficient and reliable calli generation system is necessary to enable transformation procedures. Leaf explants of Cuphea PSR23, its parents, Cuphea lanceolata and Cuphea viscossissima, and a high capric acid yielding species, Cuphea koehneana, were induced in basal media containing 1.8 mg L-1 1--naphthalene acetic acid (NAA) and 2.0 mg L-1 6-benzylaminopurine (BAP). Calli were cultured on basal media supplemented with 0, 1, 5 and 10 mg L -1 NAA and BAP in a 4x4 matrix combination. The calli showed a range of growth and morphological variation dependent upon the species and growth regulator applied. Optimal conditions were based not solely on maximum growth but also on the quality of the calli. Optimal calli growth conditions for C. viscossissima were 1 mg L-1 NAA and 5 mg L -1 BAP; C. lanceolata grew optimally at 1 mg L -1 NAA and 1 mg L-1 BAP; C. PSR23 grew optimally in media containing 1.8 mg L-1 NAA and 2.0 mg L-1 BAP and C. koehneana had optimal growth at 1 mg L-1 NAA and 10 mg L-1 BAP.