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.