In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Abstract

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormonefree Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l À1 ) combined with naphthylacetic acid (NAA; 0.1 mg l À1 ), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l À1 , and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to $50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.

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

“…To determine the most favorable dose of particular MSH for the AE induction and development, other concentrations of hormones should be investigated. Similar to our previous studies on unfertilized ovules of Arabidopsis in cultures, AE development was delayed as compared to findings in planta [8,10] but the successive stages from 2-nucleate to cellular AE were observed in vitro. The pattern of in vitro-induced AE development was similar to that of endosperm of fis mutant in planta [7] and also to AE produced in unfertilized ovules of met1 mutants in vitro [10].…”

“…The overthrow of the general phenomenon occurring in fis mutants [1] -the lack of cellularization of autonomous endosperm -gives hope for finding the key which allows switching the AE development from nuclear to cellular. All tested hormones in the present study and in previous experiments induced central cell division and AE development in unfertilized ovules of Arabidopsis genotypes [8][9][10]. They did not stimulate divisions of egg cell and haploid embryo formation although embryo-like structures of unknown origin were observed on medium with epiBL and in fie-1/FIE Arabidopsis mutant cultured on M1 (data not shown).…”

“…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].…”

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

“…Autonomous endosperm development has also been induced in culture of unpollinated flowers, pistils, ovaries or isolated ovules of several wild, ornamental and cultivated plant species (Wijowska et al, 1999a,b;Rojek et al, 2005). Embryogeny failed but autonomous endosperm developed in culture of unpollinated ovaries of two wild-type Arabidopsis thaliana L. genotypes: Columbia and Lansberg erecta (Rojek et al, 2005;Kapusta et al, 2007).…”

Arabidopsis Cyclin-Dependent Kinase Gene CDKG;2 is Involved in Organogenic Responses Induced in Vitro

“…More recently, in vitro technique has been applied to study somatic embryogenesis in Arabidopsis ecotype Columbia and mutants (Gaj 2001a(Gaj ,b, 2004Gaj et al, 2005Gaj et al, , 2006Kurczyńska et al, 2007;Fraś et al, 2008;Baster et al, 2009;Kraut et al, 2011;Nowak et al, 2012). Studies of the effect of in vitro culture conditions on MEA and FIE/FIS gene expression analyzed induction of egg cell and central cell divisions without fertilization in cultures of unfertilized ovules of Arabidopsis ecotypes (Columbia, Landsberg) (Rojek et al, 2005;Kapusta et al, 2007).…”