Morphohistological and flow cytometric analyses of somatic embryogenesis in Trifolium nigrescens Viv.

Abstract

Microscopy and flow cytometry (FCM) were used to study somatic embryogenesis (SE) from zygotic embryos of Trifolium nigrescens Viv. to determine if there were any relationships between characteristics of somatic embryos (morphology, anatomy, genome size stability) and their regenerability. Embryoids were induced on Murashige and Skoog (MS) medium containing 4 mg l -1 2,4-dichlorophenoxyacetic acid (2,4-D) and 2 mg l -1 N 6 -[2-isopentenyl]-adenine (2iP) either directly from hypocotyls or via an intervening callus, depending on the duration of culture. The morphology of somatic embryos varied from zygoticlike structures to abnormal structures including hornshaped, polycotyledonary, and fused embryoids. The incidence of abnormalities was higher in callus cultures than in direct regeneration. Horn-shaped embryoids were the most frequent type of abnormal embryos. Only embryoids having zygotic-like morphology regenerated into plantlets. Histological observations revealed that the absence of shoot and root apical meristems along with parenchymatization of embryos were major obstacles to conversion of horn-shaped embryoids. The estimated 2C value for T. nigrescens was 0.9 pg. FCM analysis revealed differences in DNA content between embryoids induced via an intervening callus and those produced directly from explants. Individuals with species-specific as well as increased DNA content were detected among those zygotic-like embryos derived from callus, but all horn-shaped embryoids had increased genome sizes. The observed lack of differences in DNA content between zygotic-like and horn-shaped embryoids, from direct SE, indicated that these phenotypic abnormalities were of physiological origin. The mean DNA content of regenerants was species-specific, suggesting that only diploid embryoids were capable for regeneration into plantlets. Keywords Auxin Á Clover Á Flow cytometry Á Histology Á Nuclear DNA content Á Somatic embryo morphology Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid 2iP N 6 -[2-isopentenyl]-adenine FCM Flow cytometry MS Murashige and Skoog (1962)

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

“…However, two distinct groups of cells were observed: one was formed of large, non-embryonic cells at the periphery of the explants; the other group was localized more centrally and composed of small cells with a meristematic appearance. The same structural pattern was observed by Konieczny et al (2012) during the somatic embryogenesis of Trifolium nigrescens Viv.…”

“…The occurrence of abnormalities during the embryogenic process is common (Fernando et al 2002, Konieczny et al 2012. It has been suggested that these abnormalities are associated with the culture duration and the use of high auxin concentrations (Pescador et al 2008).…”

Arabinogalactan proteins improve plant regeneration in barley (Hordeum vulgare L.) anther culture

“…Conversely, branched and mostly parenchymatous outgrowths were prevalent in the control culture. Recently, we have observed that aberrant embryo morphology may be the prime cue for low conversion rate or even total inability to develop into plants after subculture on the regeneration media (Konieczny et al 2012). Interestingly, the anthers-derived calli of barley cultured in the presence of gum arabic showed a significantly higher ability to regenerate plants relative to calli maintained without gum arabic.…”

Arabinogalactan-protein and pectin epitopes in relation to an extracellular matrix surface network and somatic embryogenesis and callogenesis in Trifolium nigrescens Viv.

“…Š amaj et al (1995) and Bobák et al (2003/4) suggested that the formation of an ECMSN can be a stress response of the explants imposed by specific in vitro conditions. Previously, we found that the incidence of both morphological abnormalities and genetic instability in SE of T. nigrescens increased with time of culture (Konieczny et al 2012). It may not be ruled out, that occurrence of ECMSN at relatively late stages of culture (around 15 days after explantation), could be a result of prolonged exposure to unfavorable culture conditions.…”