12.  How to analyze the phenotype of a mutant

12.1 Observation of protonema colony morphology

Yuji Hiwatashi



Growth of protonemata is regulated by various environmental signals (light, temperature, etc).  Thus, to examine the phenotype of transformants, transformants should be incubated under the same conditions as wild type plants to be compared. 



1. Sub-culture protonemata of wild type and transformants every 5-7 days on BCDATG plates in order to obtain chloronema-rich protonemata. Sub-culture at least twice. 

2. Inoculate a small protonemal colony (~1 mm in diameter) of each wild type and transformant line on a BCDATG, BCDAT, or BCD plate. Do not forget to cultivate the wild type in the same plate as a control.  Inoculate each line at the same distance apart on the agar plates (see below,WT; wild type, d1-d5: disruptants). 



3. Incubate the plates under standard conditions (25˚C, continuous white light) and observe them every week with stereomicroscopes. 



Key points

Polyploids are sometimes formed by protoplast fusion during the transformation procedure, and have a different morphology from that of wild type. Be careful not to confuse the polyploidy phenotype with the transformant phenotype. A polyploid colony contains more caulonemata and fewer gametophores than that of wild type and the colony appears “fluffy” (see figures below). Polyploid gametophores are often twisted. PCR analysis usually shows the presence of a wild type locus as well as a targeted locus.

(Figure) Wild type (left) and diploid (right) colonies cultivated on BCDAT for 3 weeks.