Morpho-histological development of the somatic embryos of Typha domingensis

Abstract

BackgroundSustainable 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.MethodsMurashige 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.ResultsProliferation 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.DiscussionIn 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.

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

“…These treatments included growth on MS0.1 semisolid culture medium (Figure 2A), growth in liquid MS0.1 medium without growth regulators (Figure 2B), or growth in purified sterile water alone (Figure 2C); in addition, all the treatments included ascorbic acid. Both liquid systems produced friable tissue (Figure 2B,C) with crumbly rhizoclones suitable for the induction of embryogenic callus with embryos in several developmental stages (Figure 2D), as previously reported [33]. Moreover, further experiments revealed that the rhizoclones induced in rhizotron systems, incubated in the dark, yielded a large amount of unpigmented embryos in the scutellar and oblong morphological stages of development (Figure 2D-F), which were suitable for studying the induction of hairy roots by cocultivation with A. rhizogenes (Figure 2H).…”

“…As previously reported [48], the adherence of Agrobacterium to the surface of embryos might have been improved by the acid pH (5.5) of the culture medium used in the coculture, the absence of accessory structures such as cutin in this type of tissue [47], or the addition of ascorbic acid to the culture system [49]. In addition, Agrobacterium has been reported to show enhanced virulence in the presence of phenolic compounds [50], such as those produced by T. domingensis embryos [33]. Thus, consistent with these results, the acid pH, addition of ascorbic acid, and liquid state of the coculture medium all favored biochemical adherence of the bacteria to the embryo surface, thereby enhancing the agrotransformation process.…”

“…After 15 days germination, the root system of seedlings with 1 cm length and with similar development (pair of leaves, radicle with adventitious root) were separated from the caulinar base and transferred into a new sRU for the establishment of rhizoclones (Figure 2). Individual roots were positioned vertically on the surface of the semisolid culture medium, avoiding injuries, and were incubated under photoperiod (16/8 h light/darkness) by using cold white light (Philips, USA), at a photon flux density of 20 µmol photons m −2 s −1 (Quantum Light Meter; Spectrum Technologies, Aurora, IL, USA), and a temperature of 28 ± 2 • C. The rhizoclones were used for the induction of SEs by incubation in the dark in MS culture medium at half ionic strength (MS0.5), and supplemented with vitamins, sucrose (3% w/v), ascorbic acid (10 mg L −1 ), and 0.5 mg L −1 2,4-D, as described previously [33]. The SEs in the oblong (SEo) and scutellar (SEsc) stages of development were isolated from the embryogenic line YC1 of T. domingensis that was established and maintained as described above.…”

“…The observational unit was the viable SEs (n = 40 per treatment), which were randomly sampled from the MS medium with cefotaxime. The transformed embryos were separated following a previously reported protocol [33]. The survival, hairy morphology, and transformation frequency of all viable SEs, SEos, and SEscs were analyzed at the end of the experiment.…”

“…Additionally, in Mexico, T. domingensis is known as a cosmopolitan species that can be found in tropical wetlands [8,32] and has expanded its domain into eutrophic areas [16]. In a previous work, we reported that a callus tissue embryogenic line (YC1, yellow callus obtained with 0.5 mg L −1 2,4-D) of T. domingensis was able to induce high proliferation of somatic embryos (SEs) in the oblong (SEo) and scutellar (SEsc) states of development [33]. In the present work, genetic transformation for the induction of hairy roots on SE of rhizoclones from T. domingensis seedlings by cocultivation with A. rhizogenes was investigated.…”