Isolation and Culture of Leaf Protoplasts from Ulmus sp.: Preliminary Report

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

“…A study using similar methods reported standard deviations of protoplast yields in U. minor were often greater than 50% of the mean [ 21 ], indicating that this approach was highly variable in elm or failed attempts were pooled in the data. Studies conducted in our lab using young American elm leaves as described by Dorion et al [ 18 , 19 ] concur with the findings of Conde and Santos [ 21 ] in that protoplast yields from young (1 st and 2 nd ) actively growing leaves were inconsistent regardless of the enzyme solution used, and in our experience isolations often fail completely. In order to develop protoplast regeneration and hybridization systems for American elm and other difficult woody plants it is imperative that the underlying biochemical mechanism preventing reproducible enzymatic degradation of source tissue is identified and that novel approaches are developed to facilitate reliable protoplast isolation.…”

“…During these initial trials a number of cell wall degrading enzymes were evaluated at different concentrations and combinations, including the reportedly more active mixture used successfully in several Ulmus spp. by Dorion et al [ 18 , 19 ] (data not shown). While protoplasts were occasionally obtained, the results were similar to what had been previously reported in that the yields were often very low [ 15 ] and the success rate was inconsistent regardless of composition of the enzyme solution.…”

“…While protoplasts were occasionally obtained, the results were similar to what had been previously reported in that the yields were often very low [ 15 ] and the success rate was inconsistent regardless of composition of the enzyme solution. Sometimes high yields as described by Dorion et al [ 18 , 19 ] were obtained, but this was not consistent even when the protocol was the same between isolation attempts and all reasonable precautions to use uniform plant material were taken. For example, a high yield of protoplasts was obtained from freshly emerged greenhouse leaves on March 30, 2011, but the isolation completely failed 5 days later on April 4, 2011 under same experimental conditions using fresh leaves from the same group of trees.…”

“…Interspecific hybridization using classical approaches has been for the most part unsuccessful because of the sexual incompatibility between American elm and other elms [ 14 ]. As such, attempts at protoplast isolation and regeneration with the ultimate goal of developing DED resistant somatic hybrids through somatic fusion have been attempted by various researchers as early as 1980 [ 12 , 15 - 19 ]. However, despite the repeated attempts by various researchers there have been no successful reports of protoplast regeneration in American elm.…”

“…The authors postulated that this recalcitrance may have been a consequence of toxic effects resulting from the long exposure to the enzyme solution. Preliminary studies conducted by Dorion et al [ 18 , 19 ] reported high protoplast yields from young greenhouse grown American elms using a 17 h incubation in a more active enzyme solution containing 0.2% Onozuka RS Cellulase, 0.05% Driselase, and 0.03 Pectolyase Y23. However, these reports do not provide any indication of variability or reproducibility of the protocol, and the isolated protoplasts did not display sustained cell division.…”