THE INFLUENCE OF fie AND met1 MUTATIONS AND IN VITRO CULTURE CONDITIONS ON AUTONOMOUS ENDOSPERM DEVELOPMENT IN UNFERTILIZED OVULES OF ARABIDOPSIS THALIANA

Abstract

In flowering plants, seeds are produced both sexually (double fertilization is required) and asexually via apomixis (meiotic reduction and egg fertilization are omitted). An apomictic-like pattern of endosperm development in planta is followed by fis mutants of sexual Arabidopsis thaliana. In our experiments in planta, autonomous endosperm (AE) developed in met1 mutants. Furthermore we obtained autonomous endosperm formation in vitro not only in unfertilized ovules of fie mutants but also in wild genotypes (Col-0, MET1/MET1, FIE/FIE) and met1 mutants. AE induction and development occurred in all genotypes on the each of the media used and in every trial. The frequency of AE was relatively high (51.2% ovaries) and genotype-dependent. AE induced in vitro represents a more advanced stage of development than AE induced in fie mutants in planta. This was manifested by a high number of nuclei surrounded by cytoplasm and organized in nuclear cytoplasmic domains (NCDs), nodule formation, division into characteristic regions, and cellularization. The high frequency of AE observed in homozygous met1 (met1/met1) mutants probably is due to accumulation of hypomethylation as an effect of the met1 mutation and the in vitro conditions. AE development was most advanced in FIE/fie mutants. We suggest that changes in the methylation of one or several genes in the DNA of Arabidopsis genotypes caused by in vitro conditions resulted in AE induction and/or further AE development. K Ke ey y w wo or rd ds s: : Arabidopsis, fie and met1 mutants, apomixis, autonomous endosperm, in vitro culture, methylation.

Exogenous steroid hormones stimulate full development of autonomous endosperm in Arabidopsis thaliana

“…The mature FG consisted of an egg apparatus on micropylar pole, antipodals at chalazal pole and central cell with 2 polar nuclei or secondary nucleus formed after fusion of polar nuclei similarly as previously described by Rojek et al [10]. In analyzed ovules, mature female gametophytes occurred with highest frequency (~80%), confirming that the mature FG was the best stage for autonomous endosperm induction.…”

“…In addition, a combination of fis and met1 mutations allows, in the absence of fertilization, further (more advanced) development of autonomous endosperm in planta and more frequent occurrence of autonomous endosperm (fie-1 mutant plants carrying an MET1 antisense silencing construct [7]; mea-1/MEA; met1-3/MET1 mutant [6]). A similar phenomenon was observed in in vitro cultured unfertilized ovules (inside ovaries) of wild Arabidopsis genotypes [8,9] and in met1 mutant, in which AE induction does not or was very sporadically noted in planta [10]. AE development was studied on modified MS medium (with 6% sucrose) supplied with auxin and cytokinin [10].…”

“…A similar phenomenon was observed in in vitro cultured unfertilized ovules (inside ovaries) of wild Arabidopsis genotypes [8,9] and in met1 mutant, in which AE induction does not or was very sporadically noted in planta [10]. AE development was studied on modified MS medium (with 6% sucrose) supplied with auxin and cytokinin [10].…”

“…1a,b). In this stage anthers are before anthesis preventing bud pollination [10]. Buds were sterilized in 3% hydrogen peroxide and 95% ethanol (1:1 v/v) for 5 min, then rinsed in distilled water 3 times for 5 min each.…”

“…1) were fixed on the 3rd, 5th, and 7th day of culture in either acetic alcohol (glacial acetic acid : 96% ethanol, 1:3, v/v), or FAA (40% formalin : glacial acetic acid : 70% ethanol, 5:5:90, v/v/v). Fixed material was prepared and analyzed as described by Rojek et al [10].…”