Induction of Hairy Roots on Somatic Embryos of Rhizoclones from Typha domingensis Seedlings.

A protocol for the induction of hairy roots on somatic embryos of rhizoclones from Typha domingensis seedlings grown in hydroponic rhizotron systems was established for the first time. Rhizogenesis was induced through the agrotransformation of somatic embryos in oblong and scutellar states of development using the K599, LBA9402, and A4 strains of Agrobacterium rhizogenes. The transfection to the embryos was performed by cocultivation of rhizoclones on a Murashige and Skoog mineral medium at 50% strength (MS0.5), in the dark, at 28 ± 2 °C for 72 h. In contrast to nontransformed embryos that did not exhibit any root tissue, transformed embryos presented hairy roots that varied in number, length, and density depending on the bacterial strain, and K599 was the most effective strain. After analysis via optical microscopy, the transformed embryos were collected and transferred to fresh culture media supplemented with 400 mg mL-1 cefotaxime and 10 mg L-1 ascorbic acid. The efficiency of transformation and survival of the oblong and scutellar embryos were similar among the three bacterial strains. The results show that agrotransformation of somatic embryos of rhizoclones from T. domingensis is a novel and viable strategy for the generation of genetic transformants of Typha that have potential applications in bioremediation technologies.

Morpho-histological development of the somatic embryos of Typha domingensis.

BACKGROUND: Sustainable methods of propagation of Typha domingensis through somatic embryogenesis can help mitigate its current condition of ecological marginalization and overexploitation. This study examined whether differentiation up to coleoptilar embryos could be obtained in an embryogenic line proliferated with light and high auxin concentration. METHODS: Murashige and Skoog medium at half ionic strength and containing 3% sucrose and 0.1% ascorbic acid was used for the three embryogenic phases. Induction started with aseptic 9-day-old germinated seeds cultured in 0.5 mg L-1 2,4-dichlorophenoxyacetic (2,4-D). Proliferation of the embryogenic callus was evaluated at 2,4-D concentrations ranging from 0 to 2 mg L-1 in cultures maintained in the dark. The dominant embryogenic products obtained in each treatment were used as embryogenic lines in the third phase. Thus, maturation of the somatic embryos (SEs) was analyzed using four embryogenic lines and under light vs. dark conditions. Embryogenic differentiation was also monitored histologically. RESULTS: Proliferation of the nine morphogenetic products was greater in the presence of 2,4-D, regardless of the concentration, than in the absence of auxin. Among the products, a yellow callus was invariably associated with the presence of an oblong SE and suspended cells in the 2,4-D treatments, and a brown callus with scutellar somatic embryos (scSEs) in the treatment without 2,4-D. During the maturation phase, especially the embryogenic line but also the light condition resulted in significant differences, with the highest averages of the nine morphogenetic products obtained under light conditions and the maximum concentration of auxin (YC3 embryogenic line). Only this line achieved scSE growth, under both light and dark conditions. Structurally complete coleoptilar somatic embryos (colSEs) could be anatomically confirmed only during the maturation phase. DISCUSSION: In the embryogenic line cultured with the highest auxin concentration, light exposure favored the transdifferentiation from embryogenic callus to scSE or colSE, although growth was asynchronous with respect to the three embryogenic phases. The differentiation and cellular organization of the embryos were compatible with all stages of embryogenic development in other monocotyledons. The growth of colSEs under light conditions in the YC3 embryogenic line and the structurally complete anatomic description of colSEs demonstrated that differentiation up to coleoptilar embryos could be obtained. The diversity of embryogenic products obtained in the YC3 embryogenic line opens up the opportunity to synchronize histological descriptions with the molecules associated with the somatic embryogenesis of Typha spp.