Failure of androgenesis in Miscanthus × giganteus in vitro culture of cytologically unbalanced microspores

Miscanthus × giganteus is a popular energy crop, which due to its hybrid origin is only vegetatively reproduced. Asexual embryogenesis in anther and microspore culture leading to double haploids production could allow to regain the ability for sexual reproduction and to increase the biodiversity of the species. Therefore, the goal of this paper was to investigate the requirements of androgenesis in Miscanthus. The standard protocols used for monocotyledonous plants were applied with many modifications regarding the developmental stage of the explants at the time of culture initiation, stress treatment applied to panicles and isolated anthers as well as various chemical and physical parameters of in vitro culture conditions. Our results indicated that the induction of androgenesis in M. × giganteus is possible. However, the very low efficiency of the process and the lack of regeneration ability of the androgenic structures presently prevent the use of this technique

Sterility of Miscanthus × giganteus results from hybrid incompatibility

Miscanthus ×giganteus Greef et Deu. (Poaceae), a hybrid of Miscanthus sinensis and M. sacchariflorus native to Japan, is an ornamental and a highly lignocellulosic bioenergy crop, cultivated in the European Union as an alternative source of energy. This grass reproduces exclusively vegetatively, by rhizomes or via expensive in vitro micropropagation. The present study was aimed at finding the barriers that prevent sexual seed production, based on detailed embryological analyses of the whole generative cycle, including microsporogenesis, pollen viability, megasporogenesis, female gametophyte development, and embryo and endosperm formation. Sterility of M. ×giganteus results from abnormal development of both male and female gametophytes. Disturbed microsporogenesis (laggard chromosomes, univalents, micronuclei) was further highlighted by low pollen staining. The frequency of stainable pollen ranged from 13.9% to 55.3% depending on the pollen staining test, and no pollen germination was observed either in vitro or in planta. The wide range of pollen sizes (25.5-47.6 μm) clearly indicated unbalanced pollen grain cytology, which evidently affected pollen germination. Only 9.7% of the ovules developed normally. No zygotes nor embryos were found in any analyzed ovules. Sexual reproduction of M. ×giganteus is severely hampered by its allotriploid (2n=3x=57) nature. Hybrid sterility, a strong postzygotic barrier, prevents sexual reproduction and, therefore, seed formation in this taxon

Glutathione provides antioxidative defence and promotes microspore-derived embryo development in isolated microspore cultures of triticale (xTriticosecale Wittm.)

The efficiency of microspore embryogenesis (ME) is determined by the complex network of internal and environmental factors. Among them, the efficient defence against oxidative stress seems to be one of the most important. The present study confirms this hypothesis showing the positive effect of glutathionethe most abundant cellular antioxidanton ME in isolated microspore cultures of triticale (xTriticosecale Wittm.). For the first time, low temperature (LT) pre-treatment of tillers was combined with the exogenous application of glutathione and associated with the total activity of low-molecular weight antioxidants, the endogenous content and redox status of glutathione, and the effectiveness of ME. The results indicate that efficient antioxidative defence is the first, although not the only, prerequisite for effective ME. In responsive genotypes, LT alone stimulated antioxidative defence and decreased cell redox status, which was associated with increased cell viability and high frequency (ca. 20%) of microspore reprogramming. Application of glutathione had no effect either on the microspore viability or on the initial number of embryogenic microspores. However, it increased the number of embryo-like structures, probably by stimulating the next phases of its development. In recalcitrant genotypes, the main role of glutathione seems to be its participation in cell protection from oxidative stress. However, even enhanced antioxidative activity, which sustained cell viability and increased the number of embryogenic microspores, was insufficient for efficient haploid/doubled haploid plant production. Evidently, there are still other defective elements in the complex network of factors that regulate the process of ME

Changes in gene expression patterns associated with microspore embryogenesis in hexaploid triticale (×Triticosecale Wittm.)

7 Págs., 2 Figs., 2 Tabls. Available online: 9 November 2013. This article is published with open access at Springerlink.com. This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.To gain a better understanding of the molecular mechanisms controlling microspore embryogenesis (ME) in triticale (×Triticosecale Wittm.), the expression patterns of 13 genes, previously identified in bread wheat to be associated with microspore-derived embryo development, were analysed. Four triticale doubled haploid (DH) lines, significantly different with respect to embryogenic potential, were studied. The gene expression profile was dissected at different points of the ME induction procedure up to the 8th day of in vitro culture (dc). RT-PCR revealed that these 13 genes were expressed during triticale ME. Variations in gene expression profiles were observed between the studied DH lines. DH28 (highly embryogenic) was the only one in which all analysed genes (Ta.TPD1-like, TAA1b, GSTF2, GSTA2, CHI3, Tad1, XIP-R1, TaAGL14, TaNF-YA7, SERK2, SERK1, TaEXPB4, TaME1) were up-regulated during the first 8dc. In the less embryogenic DH31, TAA1b, GSTA2 and TaEXPB4 were already induced on 4dc. In DH25, ME was initiated quite efficiently but soon inhibited, which coincided with the lack of gene expression (TaEXPB4, TaME1) or down-regulation (Tad1, XIP-R1, TaAGL14, TaNF-YA, SERK2, SERK1) on 8dc. In the recalcitrant DH50 line, the majority of genes were expressed at a lower level or not at all, indicating disturbances in ME initiation. In this study, the molecular mechanisms involved in triticale ME induction were analysed for the first time, laying the foundation for further characterisation of specific genes controlling microspore-derived embryo development.The work was supported by Project AGL2010-17509 from ‘Plan Nacional de Recursos y Tecnologías Agroalimentarias’ of Spain, the Bilateral Project CSIC (Spain)-PAS (Poland) 2010PL0006 and by COST Action FA0903 ‘Harnessing of Reproduction for Plant Improvement’ (HAPRECI). Dr. Ewa Dubas was the recipient of a STSM fellowship from COST Action FA0903 ‘Harnessing of Repro- duction for Plant Improvement’. RA Sánchez-Díaz was the recipient of a predoctoral fellowship, from Junta Ampliación de Estudios, Consejo Superior de Investigaciones Científicas (JAE-CSIC) of Spain.Peer reviewe

ROS-scavengers, osmoprotectants and violaxanthin de-epoxidation in salt-stressed Arabidopsis thaliana with different tocopherol composition

To determine the role of α- and γ-tocopherol (TC), this study compared the response to salt stress (200 mM NaCl) in wild type (WT) Arabidopsis thaliana (L.) Heynh. And its two mutants: (1) totally TC-deficient vte1; (2) vte4 accumulating γ-TC instead of α-TC; and (3) tmt transgenic line overaccumulating α-TC. Raman spectra revealed that salt-exposed α-TC accumulating plants were more flexible in regulating chlorophyll, carotenoid and polysaccharide levels than TC deficient mutants, while the plants overaccumulating γ-TC had the lowest levels of these biocompounds. Tocopherol composition and NaCl concentration affected xanthophyll cycle by changing the rate of violaxanthin de-epoxidation and zeaxanthin formation. NaCl treated plants with altered TC composition accumulated less oligosaccharides than WT plants. α-TC deficient plants increased their oligosaccharide levels and reduced maltose amount, while excessive accumulation of α-TC corresponded with enhanced amounts of maltose. Salt-stressed TC-deficient mutants and tmt transgenic line exhibited greater proline levels than WT plants, lower chlorogenic acid levels, and lower activity of catalase and peroxidases. α-TC accumulating plants produced more methylated proline- and glycine- betaines, and showed greater activity of superoxide dismutase than γ-TC deficient plants. Under salt stress, α-TC demonstrated a stronger regulatory effect on carbon- and nitrogen-related metabolites reorganization and modulation of antioxidant patterns than γ-TC. This suggested different links of α- and γ-TCs with various metabolic pathways via various functions and metabolic loops

The influence of heat stress on auxin distribution in transgenic B-napus microspores and microspore-derived embryos

Plant embryogenesis is regulated by differential distribution of the plant hormone auxin. However, the cells establishing these gradients during microspore embryogenesis remain to be identified. For the first time, we describe, using the DR5 or DR5rev reporter gene systems, the GFP- and GUS-based auxin biosensors to monitor auxin during Brassica napus androgenesis at cellular resolution in the initial stages. Our study provides evidence that the distribution of auxin changes during embryo development and depends on the temperature-inducible in vitro culture conditions. For this, microspores (mcs) were induced to embryogenesis by heat treatment and then subjected to genetic modification via Agrobacterium tumefaciens. The duration of high temperature treatment had a significant influence on auxin distribution in isolated and in vitro-cultured microspores and on microspore-derived embryo development. In the "mild" heat-treated (1 day at 32 A degrees C) mcs, auxin localized in a polar way already at the uni-nucleate microspore, which was critical for the initiation of embryos with suspensor-like structure. Assuming a mean mcs radius of 20 mu m, endogenous auxin content in a single cell corresponded to concentration of 1.01 mu M. In mcs subjected to a prolonged heat (5 days at 32 A degrees C), although auxin concentration increased dozen times, auxin polarization was set up at a few-celled pro-embryos without suspensor. Those embryos were enclosed in the outer wall called the exine. The exine rupture was accompanied by the auxin gradient polarization. Relative quantitative estimation of auxin, using time-lapse imaging, revealed that primordia possess up to 1.3-fold higher amounts than those found in the root apices of transgenic MDEs in the presence of exogenous auxin. Our results show, for the first time, which concentration of endogenous auxin coincides with the first cell division and how the high temperature interplays with auxin, by what affects delay early establishing microspore polarity. Moreover, we present how the local auxin accumulation demonstrates the apical-basal axis formation of the androgenic embryo and directs the axiality of the adult haploid plant