In phase I, the algorithm quickly builds a likely suboptimal initial species tree using a greedy leaf adding procedure.
For each tree size (4 60 leafs) this procedure was repeated 100 times to assess the average relative performance of each program.
For resolving stem apoplast proteins, the leaf apoplast protein procedure was followed.
Time to flowering was estimated using the final leaf number (FLN) procedure previously described [ 16].
An existing DNA extraction method was converted into a high-throughput plant leaf DNA extraction procedure yielding DNA suitable for real-time PCR analyses.
With our procedure, leaf tissue (~0.5 cm) is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction.
In this procedure, leaf tips were transferred to tubes (Costar 1.2 ml tubes 4408 and caps 4418) that contained two steel balls (bicycle, 1/8" in 60 gross, Tiangin, China) and were placed in racks (Costar) on ice.
In the usual culture procedure, leaf explants were plated onto P4 medium containing 10 μM NAA (1-naphthaleneacetic acid, Sigma-Aldrich, St . Louis MO, USA) and 4 ╀M BAP (6-benzylaminopurine, Sigma-Aldrich).
Following an intensive rinsing procedure, leaf pieces stained via this method displayed intense mitochondrial staining with little background staining, although it can be noted that a small amount of CMXRos dye is sequestered to the cell wall despite the presence or absence of mitochondria.
In a typical reaction procedure, ZM leaf extract was prepared by taking 2 g of dry leaf powder with 25 mL of distilled water in a conical flask along with 2 mL of methanol (minimum methanol was added to initiate the isolation of compounds).
