Tata Institute of Fundamental Research, Department of Biological Sciences, Mumbai, India
The strategies and experimental approaches that led the author to demonstrate the role of auxin in caulonema differentiation in the protonema of the moss Funaria hygrometrica are discussed. In stationary suspension cultures, the status of cell differentiation is regulated by inoculum cell density and auxin level. At low inoculum cell densities, 2-5 µM indole acetic acid (IAA) led to the differentiation of 65-70% caulonema filaments in 5-6 days. Caulonema can also differentiate in auxin-free medium if buffered at pH 5.0 after a lag of 6±1 days. The duration of lag can be manipulated and the cells are capable of responding to auxin at a higher level (3-10 µM) and produce about 20% caulonema after 3 days. This responsiveness or sensitivity to auxin can be enhanced further by growing cells in a nutrient-limited medium buffered at pH 5.0. In this medium, addition of 3 µM IAA led to the differentiation of 75-80% caulonema and rhizoids within 3 to 4 days. Work done in other laboratories has shown that auxin promotes caulonema differentiation in the moss Physcomitrella patens by positively regulating two basic helix-loop-helix type of transcription factor genes namely root hair defective six-like1 (PpRSL1) and PpRSL2 (Jang and Dolan 2011, New Phytologist 192: 319-327).