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.