Diva Maria de Alencar Dusi

Expressed sequence-tag analysis of ovaries of Brachiaria brizantha reveals genes associated with the early steps of embryo sac differentiation of apomictic plants

In apomixis, asexual mode of plant reproduction through seeds, an unreduced megagametophyte is formed due to circumvented or altered meiosis. The embryo develops autonomously from the unreduced egg cell, independently of fertilization. Brachiaria is a genus of tropical forage grasses that reproduces sexually or by apomixis. A limited number of studies have reported the sequencing of apomixis-related genes and a few Brachiaria sequences have been deposited at genebank databases. This work shows sequencing and expression analyses of expressed sequence-tags (ESTs) of Brachiaria genus and points to transcripts from ovaries with preferential expression at megasporogenesis in apomictic plants. From the 11 differentially expressed sequences from immature ovaries of sexual and apomictic Brachiaria brizantha obtained from macroarray analysis, 9 were preferentially detected in ovaries of apomicts, as confirmed by RT-qPCR. A putative involvement in early steps of Panicum-type embryo sac differentiation of four sequences from B. brizantha ovaries: BbrizHelic, BbrizRan, BbrizSec13 and BbrizSti1 is suggested. Two of these, BbrizSti1 and BbrizHelic, with similarity to a gene coding to stress induced protein and a helicase, respectively, are preferentially expressed in the early stages of apomictic ovaries development, especially in the nucellus, in a stage previous to the differentiation of aposporous initials, as verified by in situ hybridization.

Ectopic expression of a Meloidogyne incognita dorsal gland protein in tobacco accelerates the formation of the nematode feeding site

Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants.

Transgenic plants of ramie (Boehmeria nivea Gaud.) obtained by Agrobacterium mediated transformation

A regeneration and transformation protocol for ramie (Boehmeria nivea Gaud.) is presented. Regeneration was obtained from leaf discs placed on solid B-5 medium (Gamborg et al. 1968) containing adequate concentrations of auxin and cytokinin. Co-cultivation of leaf discs with Agrobacterium tumefaciens and subsequent regeneration resulted in transgenic plants as shown by Southern blot and analysis of expression of the GUS-marker gene.

In situ localization of three cDNA sequences associated with the later stages of aposporic embryo sac development of Brachiaria brizantha.

Summary.Brachiaria brizantha is a forage grass of African origin, highly cultivated in the Brazilian tropics for beef cattle production. We have analyzed the temporal and spatial expression of cDNA sequences by in situ hybridization in ovaries of apomictic and sexual plants. The studied sequences share molecular identity with myosin, aquaporin, and mitogen-activated protein kinase and were named BbrizMYO, BbrizAQP, and BbrizMAPK, respectively. BbrizMYO was expressed in apomictic and sexual embryo sacs, but somewhat later in the Polygonum type embryo sacs of sexual plants. BbrizAQP and BbrizMAPK transcripts were restricted to the Panicum type embryo sacs of apomictic plants; BbrizMAPK, in synergids; and BbrizAQP, also in different ovular cells during development. The common feature that arose from the analysis of the expression patterns of these three sequences was significant expression in the synergids. Their putative role in the maturation of Panicum type embryo sacs of apomictic plants and embryo development is discussed in view of the characteristics of apomictic reproduction.

Plant regeneration from embryogenic callus and cell suspensions of Brachiaria brizantha

In monocots, in vitro plant regeneration can be obtained through somatic embryogenesis (Vasil and Vasil 1980, 1982; Vasil 2005). Evidence of more than one morphogenic response in the same explant, under the control of different auxin/cytokinin ratios and concentrations, was observed in species of the Poaceae family such as sorghum (Sorghum bicolor, (L.) Moench.) , minor mil le t (Paspalum scrobiculatum L.), sugar cane (Saccharum officinarum L.), and baby bamboo (Pogonatherum paniceum Lam. Hack.) (reviewed by Wang et al. 2008). Brachiaria callus has been induced using seeds as explants from Brachiaria brizantha (Hochst. ex A. Rich.) Stapf, Brachiaria decumbens Stapf, Brachiaria ruziziensis Germain et Evrard and Brachiaria dictyoneura [Brachiaria humidicola (Rendle) Schweick vr. Lanero, ex B. dictyoneura] (Tohme et al. 1996). Subsequently, using the same methodology, calli were induced from 76% of isolated embryos from seeds of B. brizantha (Lenis-Manzano 1998). The formation of somatic embryos and multiple shoots from seedling apical meristems was demonstrated for B. ruziziensis (Ishigaki et al. 2009). In this system, mature seeds were used as source of explants and cultured on a medium containing 4 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Ishigaki et al. (2012) reported that embryogenic calli were more suitable than shoot apex explants for genetic transformation of ruzigrass. B. brizantha cv. Marandu is the most important and cultivated forage grass in Brazil. It is tolerant to spittlebugs (Homoptera: Cercopidae complex, mainly genera Deois and Zulia), an economically important pest for Brachiaria (Felismino et al. 2012). This cultivar is considered as the key forage in beef cattle production in Brazil. Breeding is hindered by its apomictic reproductive mode (the production of clones of the mother plant), which reduces the possibility of being used for hybridizations (Valle and Savidan 1996). Alternatives to conventional breeding of B. brizantha would include genetic modification via transformation. However, methods of transformation and tissue culture of these plants are not yet established. The development of embryogenic calli has been reported from 73% of the B. brizantha cv. Marandu isolated seed embryos in culture and 67% of calli regenerated plants (Silveira et al. 2003). More recently, the histology of embryos isolated from the in vitro cultivated seed was shown (Lenis-Manzano et al. 2010). The formation of multiple shoots was reported in cultured basal segments from micropropagated plantlets of this cultivar (Pinheiro et al. 2000), a system that was efficient for in vitro chromosome duplication in Brachiaria. Somatic embryogenesis and organogenesis in B. brizantha is influenced by several factors such as genotype, explant type, and culture conditions (Cabral et al. 2011). A high rate of cell division in explants used for genetic transformation ofmonocots is a prerequisite for the integration of exogenous DNA (Hiei et al. 1997; Vasil 2005). Rapid cell proliferation is a characteristic of embryogenic tissue. Cell suspension cultures (CS) are well known for their cell division capacity and the possibility of developmental-stage Electronic supplementary material The online version of this article (doi:10.1007/s11627-015-9690-0) contains supplementary material, which is available to authorized users. G. B. Cabral (*) :V. T. C. Carneiro : J. P. da Silva :D. M. A. Dusi Embrapa Genetic Resources and Biotechnology, Brasília, Brazil e-mail: glaucia.cabral@embrapa.br

BbrizAGL6 Is Differentially Expressed During Embryo Sac Formation of Apomictic and Sexual Brachiaria brizantha Plants

Species of the genus Brachiaria comprise plants with different modes of reproduction, sexual and apomictic. In apomixis, the embryo sac differentiates from an unreduced cell, and the embryo develops in the absence of egg cell fertilisation. In this work, the characterisation and expression analyses of a MADS-box gene from Brachiaria brizantha, named BbrizAGL6, was described in sexual and apomictic plants. Phylogenetic analyses indicated that BbrizAGL6 belongs to the AGL6-like subfamily of proteins and clusters together with the AGL6-like protein of other monocots. BbrizAGL6 and AGL6 show conservation of the protein complex. Furthermore, BbrizAGL6 expressed preferentially in reproductive tissues and corresponding transcripts were detected in anthers and ovules. In ovules of B. brizantha, where the main differences among sexual and apomictic reproduction occur, BbrizAGL6 was differentially modulated. Transcripts of BbrizAGL6 were localised in the megaspore mother cell of ovaries from apomictic and sexual plants and, additionally, in the region where aposporic initial cells differentiate, in the nucellus of apomictic plants. For the first time, a role of an AGL6-like gene in megasporogenesis of apomictic and sexual plants is suggested.

GID1 expression is associated with ovule development of sexual and apomictic plants

Key messageBbrizGID1is expressed in the nucellus of apomicticBrachiaria brizantha, previous to aposporous initial differentiation. AtGID1aoverexpression triggers differentiation ofArabidopsis thalianaMMC-like cells, suggesting its involvement in ovule development.AbstractGIBBERELLIN-INSENSITIVE DWARF1 (GID1) is a gibberellin receptor previously identified in plants and associated with reproductive development, including ovule formation. In this work, we characterized the Brachiaria brizantha GID1 gene (BbrizGID1). BbrizGID1 showed up to 92% similarity to GID1-like gibberellin receptors of other plants of the Poaceae family and around 58% to GID1-like gibberellin receptors of Arabidopsis thaliana. BbrizGID1 was more expressed in ovaries at megasporogenesis than in ovaries at megagametogenesis of both sexual and apomictic plants. In ovules, BbrizGID1 transcripts were detected in the megaspore mother cell (MMC) of sexual and apomictic B. brizantha. Only in the apomictic plants, expression was also observed in the surrounding nucellar cells, a region in which aposporous initial cells differentiate to form the aposporic embryo sac. AtGID1a ectopic expression in Arabidopsis determines the formation of MMC-like cells in the nucellus, close to the MMC, that did not own MMC identity. Our results suggest that GID1 might be involved in the proper differentiation of a single MMC during ovule development and provide valuable information on the role of GID1 in sexual and apomictic reproduction.

Toward in vitro fertilization in Brachiaria spp.

Brachiaria are forage grasses widely cultivated in tropical areas. In vitro pollination was applied to accessions of Brachiaria spp. by placing pollen of non-dehiscent anthers on a solid medium near isolated ovaries. Viability and in vitro germination were tested in order to establish good conditions for pollen development. Comparing sexual to apomictic plants, apomictic pollen has more abortion after meiosis during the microspore stage and a lower viability and, of both types, only some plants have sufficient germination in a high sugar concentration. Using in vitro pollination with the sexual plant, the pollen tube penetrates into the nucellus and micropyle, but the embryo sac degenerates and collapses. In the apomictic B.decumbens, in vitro pollination leads to the transfer of the sperm nuclei into the egg cell and the central cell. The results are discussed according to normal fertilization and barriers in sexual and apomictic plants.

The MAP3K-coding QUI-GON JINN (QGJ) Gene Is Essential To The Formation of Unreduced Embryo Sacs In Paspalum

Apomixis is a clonal mode of reproduction via seeds, which results from the failure of meiosis and fertilization in the sexual female reproductive pathway. In previous transcriptomic surveys, we identified a mitogen-activated protein kinase kinase kinase (N46) displaying differential representation in florets of sexual and apomictic Paspalum notatum genotypes. Here, we retrieved and characterized the N46 full cDNA sequence from sexual and apomictic floral transcriptomes. Phylogenetic analyses showed that N46 was a member of the YODA family, which was re-named QUI-GON JINN (QGJ). Differential expression in florets of sexual and apomictic plants was confirmed by qPCR. In situ hybridization experiments revealed expression in the nucellus of aposporous plants’ ovules, which was absent in sexual plants. RNAi inhibition of QGJ expression in two apomictic genotypes resulted in significantly reduced rates of aposporous embryo sac formation, with respect to the level detected in wild type aposporous plants and transformation controls. The QGJ locus segregated independently of apospory. However, a probe derived from a related long non-coding RNA sequence (PN_LNC_QGJ) revealed RFLP bands cosegregating with the Paspalum apospory-controlling region (ACR). PN_LNC_QGJ is expressed in florets of apomictic plants only. Our results indicate that the activity of QGJ in the nucellus of apomictic plants is necessary to form non-reduced embryo sacs and that a long non-coding sequence with regulatory potential is similar to sequences located within the ACR.

Genetic transformation of Brachiaria brizantha cv. Marandu by biolistics.

Brachiaria brizantha is a forage grass well adapted to tropical areas and cultivated in millions of hectares in Brazil. The apomictic mode of reproduction in this species, in addition to differences in ploidy between sexual and apomictic plants, impairs crossbreeding. The development of a methodology to transform apomictic cultivars will provide an option to introduce agronomic important traits to B. brizantha cv. Marandu. In addition, it will open the possibility to study in vivo the function of candidate genes involved in the apomictic reproduction. The objective of this work was to evaluate peeled seeds, isolated embryo from mature seeds, embryogenic calluses and embryogenic cell suspensions, as target explant for genetic transformation via biolistics. Plasmids bearing the marker genes gus and hptII under the control of the rice actin 1 promoter (pAct1-Os) or the maize ubiquitin 1 promoter (pUbi1Zm) were used. All the target-explants used were suitable for transient gene expression after bombardment, showing gus expression and resistance to hygromycin. Using embryogenic calluses and cell suspensions as target tissues, transgenic plants were regenerated and transgenes detected.