Morphological Analysis of Cell Growth Mutants in Physcomitrella

Abstract

This protocol describes a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. The protocol can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Information is presented to guide the investigator in the choice of vectors and basic conditions to perform transient RNA interference in moss. Detailed directions and examples for fluorescence image acquisition of small regenerating moss plants are provided. Instructions for the use of an ImageJ-based macro for quantitative morphological analysis of these plants are also provided.

The kinesin‐like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light‐avoidance in Physcomitrella patens

“…The aggregated-chloroplast phenotype scarcely affects the growth or morphology of the one-week-old plants, as determined by computer-assisted morphology analysis ( Figure 1F, G) (Vidali et al 2007;Bibeau and Vidali 2014). We used calcofluor staining to estimate the overall size of plants (Vidali et al 2009), instead of direct chlorophyll imaging (Vidali et al 2007), since cell wall staining allows a more precise estimate of growth, in particular for plants with clustered chloroplasts.…”

Myosin XI Interacting with a RabE GTPase Is Required for Polarized Growth

“…The myosin XI-CBD expression constructs were co-transformed (Liu and Vidali, 2011) with a previously developed myosin XI 5'UTR silencing construct (Vidali et al, 2010) to evaluate the capacity of the mutant MyoXI-CBDs to rescue polarized growth. Plants were imaged 1-week post-transformation for polarized growth and size defects (Vidali et al, 2007;Bibeau and Vidali, 2014).…”

“…Approximately 65 µl of protoplasts were transferred to the circle bounded by Parafilm on the slide, then sealed with a 22x22mm glass coverslip. Images of fluorescent protoplasts were acquired using the same equipment described by Bibeau and Vidali (Bibeau and Vidali, 2014).…”

The Motor Kinesin 4II Is Important for Growth and Chloroplast Light Avoidance in the Moss <i>Physcomitrella patens</i>

“…Protonemal growth was evaluated from regenerating protoplasts using previously published conditions [45] [46]. In order to test the effects of knocking down kinesin 4II the following modifications were made: Protoplast were regenerated for 4 days with no estradiol, then moved to plates with 1 µM estradiol for 4 days while controls were not treated with estradiol.…”

“…Cells were transferred to agar pads and stained with 10 µg/ml calcoflour and imaged using fluorescence microscopy. Area and solidity values per plant were measured using an ImageJ macro as previously described [46].…”