RESUMO
Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the ß-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.
Assuntos
Citrus sinensis/genética , Glucuronidase/metabolismo , Floema/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Citrus sinensis/citologia , Citrus sinensis/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Glucuronidase/genética , Proteínas de Membrana Transportadoras/genética , Especificidade de Órgãos , Floema/metabolismo , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Lectinas de Plantas/genética , Caules de Planta/genética , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Regeneração , Rhizobiaceae/fisiologia , Transformação GenéticaRESUMO
A regeneração de plantas, por organogênese ou embriogênese somática, a partir do cultivo de células e tecidos vegetais in vitro, é a base para a utilização da biotecnologia no melhoramento. O objetivo deste trabalho foi avaliar a embriogênese somática a partir de calos embriogênicos das cultivares de laranjeiras doces 'Hamlin', 'Pêra', 'Natal', 'Lima Verde' e 'Westin', em função da composição dos meios de cultura relacionada à fonte e concentração de diferentes carboidratos, utilizando-se meio de cultura MT modificado com 500mg L-1 de extrato de malte, acrescido de sacarose, galactose, glicose, sorbitol, lactose ou maltose, nas concentrações de 18, 37, 75, 110 ou 150mM à 27°C. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial cinco (cultivares) x seis (fontes de carboidratos) x cinco (concentrações das fontes de carboidratos no meio de cultura), com cinco repetições. A formação de embriões somáticos variou conforme a cultivar, e as laranjeiras 'Hamlin' e 'Natal' registraram o maior número de embriões, enquanto 'Lima Verde' e 'Westin' apresentaram menores números. A melhor fonte de carboidratos para indução de embriogênese somática foi a maltose, seguida pela lactose, nas concentrações de 37 e 75mM. Embriogênese somática não foi observada nos meios de cultura contendo galactose, glicose ou sorbitol para nenhuma cultivar estudada.
Plant regeneration, by organogenesis or somatic embryogenesis, from cell cultures and in vitro plant tissue culture is the basis for biotechnology usage in plant breeding. This research aimed to evaluate somatic embryogenesis from embryogenic calli of 'Hamlin', 'Pêra', 'Natal', 'Lima Verde', and 'Westin' sweet orange cultivars related to culture medium composition as source and concentration of carbohydrates with the use of MT culture medium modified with 500mg L-1 of malt extract, supplemented with sucrose, galactose, glucose, maltose, lactose, or sorbitol at concentrations of 18, 37, 75, 110 or 150mM at 27°C. Statistical design were complete randomized, in a factorial five (cultivars) x six (carbohydrate source) x six (carbohydrate concentration in culture medium) with five replicates. The production of somatic embryos varied with the genotype, as 'Hamlin' and 'Natal' cultivars registered higher number of embryos, whereas 'Lima Verde' and 'Westin' showed lower numbers. The best carbohydrate source was maltose, followed by lactose at concentrations of 37 and 75mM. Somatic embryogenesis was not observed on culture media supplemented with galatose, glucose or sorbitol in any studied cultivar.