Acclimation of in vitro clonal lines of big sagebrush (Artemisia tridentata) to ex vitro conditions in support of genotype-by-environment experiments and restoration v1

The following protocol describes the ex vitro, hardening and acclimation of in vitro grown genetically identical plantlets (i.e. clones) of Artemisia tridentata ssp. tridentata (Asteraceae). The goal of this protocol is to create functional clonal lines of Artemisia tridentata to be used in genotype-by-environment (GxE) experiments. Overall, the protocol consists of the following four major steps and takes 16 weeks to complete on 11-16-week old in vitro plantlets: i) transfer in vitro plantlets from modified Murashige & Skoog (MMS) media to a sand and vermiculite soil mixture (4:1 ratio; hereafter referred to as sandy soil) in an enclosed, high humidity vessel to initiate plantlet establishment and root growth (four weeks); ii) gradually open vessels to initiate dropping of in vitro leaves and growth of functional leaves (four weeks) associated with decreased of humidity and increased gas exchange; iii) establishing a watering regime to promote and maintain growth of functional plantlets (six weeks) and iv) transfer plantlets into a more complex soil mixture (similar to natural conditions composed of sand, silt and vermiculite at 2:1.5:0.5 ratio; referred to as silt soil) in an open vessel to complete acclimation; especially hardening of the root system (two weeks or more depending on specific needs). Upon completion, the sagebrush plantlets will be exhibiting a similar phenotype as sagebrush seedlings. Finally, although optional, we are encouraging users to conduct stem xylem pressure measurements on acclimatized and well-watered plantlets prior to starting GxE experiments to evaluate their hydraulic conductivity and overall level of stress.