Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation

Background Land plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium ( Physcomitrella ) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. paten s, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. paten s. Results Here, we investigated the effect of nitrogen conditions on the growth and development of P. patens . We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space. Conclusions Our more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens .