3.2  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 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 1 week with stereomicroscopes. 



Key points

Polyplods are sometimes formed by protoplast fusion during transformation procedure, and have different morphology from that of wild type. Be careful not to misunderstand the polyploidy phenotype as tansformant phenotype. A colony of polyploids contains more caulonemata and fewer gametophores than that of wild type (see figures below). Polyploid gametophores are often twisted.

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