RESUMO
Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.
Assuntos
Hordeum/genética , Plantas Geneticamente Modificadas/genética , Plântula/genética , Transformação Genética , Agrobacterium tumefaciens/genética , Vetores Genéticos , Genoma de Planta , Genômica , Germinação/genética , Hordeum/embriologia , Hordeum/crescimento & desenvolvimento , Canamicina Quinase/genética , Meristema/embriologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/embriologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plântula/embriologia , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , TransgenesRESUMO
SWI2/SNF2 chromatin remodeling ATPases play important roles in plant and metazoan development. Whereas metazoans generally encode one or two SWI2/SNF2 ATPase genes, Arabidopsis encodes four such chromatin regulators: the well-studied BRAHMA and SPLAYED ATPases, as well as two closely related non-canonical SWI2/SNF2 ATPases, CHR12 and CHR23. No developmental role has as yet been described for CHR12 and CHR23. Here, we show that although strong single chr12 or chr23 mutants are morphologically indistinguishable from the wild type, chr12 chr23 double mutants cause embryonic lethality. The double mutant embryos fail to initiate root and shoot meristems, and display few and aberrant cell divisions. Weak double mutant embryos give rise to viable seedlings with dramatic defects in the maintenance of both the shoot and the root stem cell populations. Paradoxically, the stem cell defects are correlated with increased expression of the stem cell markers WUSCHEL and WOX5. During subsequent development, the meristem defects are partially overcome to allow for the formation of very small, bushy adult plants. Based on the observed morphological defects, we named the two chromatin remodelers MINUSCULE 1 and 2. Possible links between minu1 minu2 defects and defects in hormone signaling and replication-coupled chromatin assembly are discussed.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Fatores de Transcrição/genética , Adenosina Trifosfatases/metabolismo , Alelos , Arabidopsis/citologia , Arabidopsis/embriologia , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Genes Reporter , Meristema/citologia , Meristema/embriologia , Meristema/genética , Mutação , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/embriologia , Raízes de Plantas/genética , Células-Tronco , Fatores de Transcrição/metabolismoRESUMO
Stamens undergo a very elaborate development program that gives rise not only to many specific tissue types, but also to the male gametes. The specification of stamen identity is coordinated by a group of homeotic genes such as APETALA3 (AP3) and PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA (SEP1-4) genes. Genome-wide transcriptomic comparisons between floral buds of wild-type and ap3 mutants led to the identification of the REM22 gene, which is expressed in the early stages of stamen development. This gene is member of the plant-specific B3 DNA-binding superfamily. In this work, we dissect the spatio-temporal expression pattern of REM22 during the early stages of stamen development. To this end, both in situ hybridization analyses as well as in vivo fluorescence strategies were employed. At stage 4 of flower development, REM22 is expressed exclusively in those undifferentiated cells of the floral meristem that will give rise to the stamen primordia. At stage 5, REM22 expression is restricted to the epidermal and the subepidermal layers of anther primordia. Later, this expression is confined to the middle layer and the differentiating tapetal cells. After stage 10 when all the tissues of the anther have differentiated, REM22 expression is no longer detectable. Furthermore, we examined the pREM22::GUS-GFP marker line in an inducible system where the ectopic AG function is used to promote microsporogenesis. The data support the idea that REM22 expression is a useful marker to study the early stages of stamen development.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Flores/genética , Meristema/embriologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular , Flores/crescimento & desenvolvimento , Gametogênese Vegetal , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genótipo , Hibridização In Situ , Epiderme Vegetal , Plantas Geneticamente ModificadasRESUMO
BACKGROUND AND AIMS: The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. METHODS: TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0-600 microM Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. KEY RESULTS: Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150-600 microM Picloram (83-97%, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43% embryogenic callus production from shoot meristem TCL on 300 microM Picloram. In maturation conditions, 34+/-4 somatic embryos per embryogenic callus were obtained, and 45.0+/-3.4% of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80% survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92% of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. CONCLUSIONS: The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees.
Assuntos
Arecaceae/embriologia , Clonagem de Organismos/métodos , Desenvolvimento Embrionário/fisiologia , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Arecaceae/citologia , Arecaceae/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Meristema/citologia , Meristema/embriologia , Meristema/genética , Picloram/administração & dosagem , Picloram/farmacologia , Brotos de Planta/citologia , Brotos de Planta/embriologia , Brotos de Planta/genéticaRESUMO
A series of experiments were carried out in order to optimize a protocol for the direct organogenesis of Chilean red clover germplasm. A range of cultivars were used to analyze the effect of explant source (crown or stem meristems of vegetative plants), culture media and plant growth regulators. Our findings showed that stem meristems were easier to obtain, presented lower levels of contamination and a better development than crown meristems. The L2 medium showed better results than B5 and MS media for the cultivars and experimental lines studied. L2 medium supplemented with 0.003 mg/l of 4-amino-3,5,6-trichloropicolinic acid and 1.0 mg/l of 6-benzylaminopurine gave consistently better results and will be applied in our breeding program to propagate, maintain and eliminate viruses from elite red clover clones.