Overexpression of PaHAP3A stimulates differentiation of ectopic embryos from maturing somatic embryos of Norway spruce

Abstract

Somatic embryogenesis can be used for large-scale propagation of plants. In most conifers, it is only possible to establish embryogenic cultures from zygotic embryos or young seedlings. There is, however, a great interest to propagate selected trees with valuable traits via somatic embryos. To be able to establish embryogenic cultures from adult tissues, more knowledge about the molecular regulation of totipotency and embryogenic potential is needed. In Arabidopsis (Arabidopsis thaliana), LEAFY COTYLEDON1 (LEC1) is required for somatic embryogenesis, and overexpression of LEC1 can stimulate the formation of embryo-like structures from vegetative tissues. We have previously characterized a conifer LEC1-type gene, PaHAP3A, which is preferentially active during embryo development in Norway spruce (Picea abies). In this work, we show, by using histochemical GUS assays, that PaHAP3A is expressed in germinated embryos at presumptive sites from which embryogenic tissue differentiate during initiation of embryogenic cultures. Furthermore, we have overexpressed PaHAP3A, using both constitutive and inducible promoters, in order to elucidate whether elevated transcript levels of PaHAP3A are sufficient to induce embryonic properties after germination. In contrast to its angiosperm counterpart, PaHAP3A does not stimulate embryonic features in vegetative tissues. However, overexpression of PaHAP3A during the maturation stage leads to the differentiation of ectopic embryos from maturing somatic embryos. Our results not only indicate at least partial conservation between the conifer and angiosperm LEC1-type genes but also suggest that PaHAP3A is dependent of the cellular status to confer its presumptive role as an important factor influencing the competence of the tissue to initiate somatic embryos.

Identification of molecular processes that differ among Scots pine somatic embryogenic cell lines leading to the development of normal or abnormal cotyledonary embryos

“…Furthermore, when embryogenic cultures of Norway spruce were treated with the HDAC inhibitor trichostatin A (TSA) during maturation, the process was arrested and the expression level of PaHAP3A remained high while the expression of PaVP1 remained low. The development of cotyledonary embryos was not affected when overexpressing PaHAP3A in somatic embryos of Norway spruce, but overexpression stimulated differentiation of ectopic embryos on cotyledonary somatic embryos (Uddenberg et al 2016). In this study, we show that the expression level of a HAP3A-related transcript decreased after stage 1b in both the normal and the abnormal cell lines of Scots pine (Table S7).…”

“…T-controls were selected by incubating approximately 100 mg tissue from stage 1 in GUS buffer containing 1 mM 5-bromo-4-chloro-3-indole β-D-glucuronic acid in the darkness at 37°C for up to 5 h (Uddenberg et al 2016). Based on high GUS activity, sub-lines 35S:GUS.1-1, 35S:GUS.1-2, and 35S:GUS.1-3 derived from cell line 61:21, and sub lines 35S:GUS.1-4 and 35S:GUS.1-5 derived from cell line 11:18, were selected.…”

“…It has so far been difficult to transform embryogenic cultures of Scots pine (Abrahamsson, unpublished), while embryogenic cultures of Norway spruce are routinely transformed (Zhu et al 2014;Uddenberg et al 2016). We therefore performed these studies in Norway spruce.…”

Coexpression of <i>WUSCHEL related homeobox</i> (<i>WOX</i>) <i>2</i> with <i>WOX8</i> or <i>WOX9</i> promotes regeneration from leaf segments and free cells in <i>Nicotiana tabacum</i> L.

“…As described in the Introduction, WUS may be a prime candidate for an RP gene applicable to a wide range of species. Several attempts have been reported for genes other than WUS using BABY BOOM in tobacco (Srinivasan et al 2007), pepper (Heidmann et al 2011) and cacao (Florez et al 2015); LEAFY COTYLEDON 1 (LEC1) and LEC2 in tobacco (Guo et al 2013); LEC1 homolog in conifer (Klimaszewska et al 2010;Uddenberg et al 2016); AGAMOUS-LIKE15 in soybean (Harding et al 2003). However, whether these results are widely applicable to other cultivars or relative species remains unclear and the number of RP genes is still limited.…”

Genome-wide analysis of the citrus B3 superfamily and their association with somatic embryogenesis

“…Based on the fact that sweet orange and grapefruit were derived from hybridizations among mandarin and pummelo [43,72,73], we suggest that CsARF19 was originated in mandarin, and has introgression into the hybrids pool. SE initiation is believed to require an induction signal that causes somatic cells to change identity [74]. Previous study showed that two ARF genes (AtARF7 and AtARF19) directly or indirectly target four auxin-responsive LBD (LATERAL ORGAN BOUNDARIES DOMAIN) genes to regulate callus formation in Arabidopsis regeneration [75].…”