Generation of Tnt1 insertional mutants in moss (P. patens) and their molecular characterization

Abstract

Morphogenesis in plant development has been of great interest for researchers engaged in plant physiological studies. Complex tissue types of higher plants make these studies difficult. A lot of work has been done on the development of plants but, we still lack crucial information on gene regulatory networks (GRN) involved in gametophytic development. Physcomitrella patens (moss) is a simple, non-vascular plant belonging to bryophyte family shows relatively simple tissue types but still reflects all major developmental patterns of higher plants. We focused on the GRN involved in bud formation and its transition to gametophore in moss. In this regard, tobacco retrotransposon (Tnt1) and T-DNA was chosen as insertional mutagenesis tool to develop mutants in moss. At the time we started our study, there was no literature available on the protocol for Tnt1 retrotransposon use in moss. We established Tnt1 as mutagenesis tool in moss. In our study, TAIL-PCR analysis showed that Tnt1 retrotransposon has specificity to the gene rich region. A recent study on the use of Tnt1 as mutagenesis tool in moss, supports our data (Vives et al., 2016). We found that LTR promoter of (Tnt1) retrotransposon and modified LTR promoter (mLTR) were inducible by CuCl2, Auxin (IAA), Salicylic acid treatments and temperature gradient. Our forward genetic screen yielded a mutant deficient in bud development (LTR-GUS line 7) and two mutants showing less gametophore formation (LTR-GUS line 4 and Tnt1 line 13). In our analysis, Tnt1 line 13 was found to be deficient in sensing gravity. Overall, this study shows that Tnt1 can be efficiently used as a mutagenesis tool in moss. Further study on these mutant lines will throw light on genes involved in bud and gametophore development.