Acclimation of in vitro grown individual lines of big sagebrush (Artemisia tridentata) to ex vitro conditions in support of genotype-by-environment experiments and restoration v2

Abstract

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. When citing this protocol, please also cite the full paper.

Acclimation and hardening of a slow‐growing woody species emblematic to western North America from in vitro plantlets

“…Ψ soil was used to guide the watering regime (using TEROS‐21 probes; METER Group, Pullman, Washington, USA) during in vitro to ex vitro acclimation and hardening to reduce mortality at this critical step of our protocol. Ψ soil should be maintained above −0.1 MPa for well‐watered conditions and between −0.2 and −0.9 MPa for hardening purposes (Drivas and Everett, 1988; see Figure 8 in Martinez et al, 2022). In addition, we found that when the plantlets in silty soil (see below for more details) were exposed to soil water potentials of −1 MPa (see Figure 8 in Martinez et al, 2022), the Ψ shoot declined to about −2.2 MPa, which based on preliminary work in our lab, could lead to stomatal closure in sagebrush seedlings and mortality by hydraulic failure.…”

“…Ψ soil should be maintained above −0.1 MPa for well‐watered conditions and between −0.2 and −0.9 MPa for hardening purposes (Drivas and Everett, 1988; see Figure 8 in Martinez et al, 2022). In addition, we found that when the plantlets in silty soil (see below for more details) were exposed to soil water potentials of −1 MPa (see Figure 8 in Martinez et al, 2022), the Ψ shoot declined to about −2.2 MPa, which based on preliminary work in our lab, could lead to stomatal closure in sagebrush seedlings and mortality by hydraulic failure. Thus, the watering regime recommended in our protocol is more conducive to optimal growth in preparation for G×E experiments.…”

“…We anticipate that this protocol will serve as a benchmark for slow‐growing, woody species occurring in dry ecosystems worldwide. We describe and demonstrate the efficacy of our protocol here; the detailed, step‐by‐step protocol and materials list are available at protocols.io (http://doi.org/10.17504/protocols.io.j8nlk4zpxg5r/v2; Martinez et al, 2022). The overall protocol is subdivided into two major steps: (i) in vitro to ex vitro plantlet acclimation under controlled laboratory conditions (16 weeks), and (ii) preparing acclimated plantlets for G×E experiments (plantlets are usually kept in a greenhouse setting up to the age of six months).…”

“…The data for survival, shoot water potentials, volumetric water content, and soil matrix potential (sandy and silty soils) can be found in Appendix . The detailed, step‐by‐step protocol is available at protocols.io (https://www.protocols.io/view/acclimation-of-in-vitro-grown-individual-lines-of-j8nlk4zpxg5r/v2; Martinez et al, 2022).…”