The expression pattern of the LEC2 gene during somatic embryogenesis (SE) in Arabidopsis explants (immature zygotic embryos) induced in vitro was followed, using real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC2 transcripts within a 30 days time course of somatic embryo development. A significant auxin-dependent upregulation of the LEC2 gene was found to be associated with the induction phase of SE. In contrast to embryogenic culture the level of LEC2 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. The study with 35S::LEC2-GR transgenic plants showed that overexpression of LEC2 can compensate for the auxin requirement, and that transgenic explants formed somatic embryos when cultured in vitro under auxin-free conditions. However, unlike in auxin-induced SE, intense callus formation preceded the embryogenic response triggered via LEC2 overexpression, suggesting an indirect pathway of morphogenesis. Moreover, a negative interaction between auxin treatment and LEC2 overexpression in terms of SE efficiency was observed, as transgenic explants cultured on auxin medium displayed a significantly reduced level of embryogenic potential. The study provides further experimental evidence that in the determination of the embryogenic response in Arabidopsis somatic cells, a close link exists between auxin and the LEC2 activity.

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.

To understand the molecular mechanism controlling in vitro plant morphogenesis, a culture system enabling induction of alternative morphogenic pathways (somatic embryogenesis, SE; shoot organogenesis, ORG) in a well defined population of somatic cells is needed. Arabidopsis is the most useful model plant for genomic studies, but a system in which SE or ORG can be induced alternatively in the same type of explant has not been proposed. Immature zygotic embryos (IZEs) of Arabidopsis provide the only explants with embryogenic potential, and have been recommended for studying mechanisms of SE induced in vitro. This study was aimed at defining culture conditions promoting induction of alternative morphogenic pathways: shoot ORG in IZE explants. The established protocol involves pretreatment of IZE explants with liquid auxin-rich callus induction (CIM) medium, followed by subculture on solid cytokinin-rich shoot induction medium (SIM). The method enables efficient shoot induction in Columbia (Col-0) and Wassilewskija (Ws), genotypes commonly used in molecular studies. During 3 weeks of culture up to 90% of Col-0 and 70% of Ws explants regenerated shoots via an indirect morphogenic pathway. We analyzed the qRT-PCR expression patterns of the LEC (LEC1, LEC2 and FUS3) genes, the key regulators of Arabidopsis embryogenesis, in the IZE explants induced to promote shoot ORG. The sharp decline of LEC expression on SIM medium confirmed that culture of Arabidopsis IZE explants enables experimental manipulation of the morphogenic response of somatic cells. A scheme illustrating various in vitro morphogenic responses of IZEs in relation to hormonal treatment is presented.K Ke ey y w wo or rd ds s: : Arabidopsis, immature zygotic embryos, in vitro morphogenesis, shoot regeneration.

A mutation in the Arabidopsis TANMEI/ EMB2757 (TAN) gene with an embryo defective phenotype was analysed for its effect on the morphogenic potential of somatic tissue cultured under in vitro conditions. The capacity for in vitro morphogenesis was evaluated using cultures of immature zygotic embryos, and seedling explants of the tan mutant and the parental Col-0 genotype. The explants were cultured on media supplemented with different plant growth regulators, and the capacity for two alternative pathways of morphogenesis, somatic embryogenesis (SE) and shoot organogenesis, was evaluated. Reporter genes (GUS, GFP) were used to monitor auxin and LEC2 and FUS3 gene activity in the tan explants. Moreover, the expression pattern of the TAN gene was analyzed during SE and in callus tissue of Col-0. It was indicated that the tan mutation resulted in a total lost of embryogenic and organogenic capacity of cultured tissues, suggesting the involvement of the TAN gene in basic cellular processes related to cell growth and differentiation. However, differential expression of the TAN gene during SE, and its increased activity at advanced stages of embryogenesis, implicate a specific role for the gene in the development of somatic embryos.