![]() Field or greenhouse-grown Populus under adequate irradiation conditions usually produce leaves with a thick cuticle and recalcitrant cell walls, which are likely to be the major factors limiting protoplast isolation. No or very few protoplasts could be isolated from these plants (data not shown). tremula X alba clone 717-1B4) were used for the study. Newly emerged leaves from two different Populus clones ( Populus trichocarpa clone Nisqually-1 and P. Initially, we tried to isolate protoplasts from leaves of soil-grown Populus plants cultivated in a greenhouse. Because this is no Populus cell suspension culture available, we tried to establish a robust protocol for isolating protoplasts from Populus leaves. In the latter study, it was briefly mentioned that an essentially same procedure was used as that in Arabidopsis. Two of them reported the isolation of protoplasts from Populus cell suspension culture or leaves, and the other used Populus protoplasts to study protein subcellular localization of Populus salt overly sensitive genes. There have been three reports on isolation of protoplasts from Populus. Isolation of Protoplasts from Populus LeavesĪ protocol for the isolation of Arabidopsis mesophyll protoplasts has been well established. We then established an efficient Populus protoplast transient expression system and demonstrated the efficacy of using this in vivo system to study the activity of Populus genes. We also optimized enzyme concentrations and digestion time for efficient protoplast isolation. In this paper, we described the micro-propagated Populus grown on MS media as an excellent alternative starting material for the efficient isolation of leaf mesophyll protoplasts. However, the inherently different genomic, physiological and developmental characteristics between a herbaceous plant and a perennial tree species may render Arabidopsis protoplasts a less ideal system for dissecting the signaling events that regulate the development of Populus. Arabidopsis leaf mesophyll protoplasts have also been used to study the function of Populus genes. Leaf mesophyll protoplasts have been developed as an informative system in Arabidopsis and a few other plant species for rapid dissection of signaling events that lead to the regulation of gene expression, as well as for many other molecular characterizations, such as protein-protein interaction, subcellular localization, and protein-DNA interaction. This limitation is more pronounced when dealing with cellular signaling events that often involve multiple gene products. Therefore, the capacity of conventional genetic manipulation in Populus, one of the most commonly used approaches for studying gene function, is largely limited by the lengthy time required to obtain suitable genetic materials. Furthermore, it typically takes four to eight months from inoculation to generate rooted seedlings via Agrobacterium-mediated transformation approach. ![]() One of the rate-limiting steps in studying the function of genes in Populus is its long life cycle (e.g., it takes a minimum 6 to 12 years to reach reproductive maturity ). ![]() With the availability of its full genome sequence and the establishment of transgenic techniques, ,, Populus is also emerging as a model organism for studying woody plants. Populus is characterized by a rapid growth rate, high productivity and wide adaption to environment and therefore, has become one of the promising bioenergy feedstocks. Among all of the options, production of lignocellulosic ethanol from plant feedstock has become one of the favorable approaches,. The establishment of renewable energy sources is essential for energy security and reduction of CO 2 emissions.
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