Stress-inducible Arabidopsis thaliana RD29A promoter constitutively drives Citrus sinensis APETALA1 and LEAFY expression and precocious flowering in transgenic Citrus spp.

Transgenic 'Duncan' grapefruit (Citrus paradisi Macf.) and 'Valencia' sweet orange (Citrus sinensis [L.] Osbeck) plants ectopically expressing C. sinensis (cv. Washington navel orange) APETALA1 (CsAP1) or LEAFY (CsLFY) genes under control of the Arabidopsis thaliana stress-inducible promoter AtRD29A flowered under non-inductive (warm temperature, well-watered) greenhouse conditions, whereas their wild-type (WT) counterparts did not. The transgenic plants that flowered exhibited no altered morphological features, except the lack of thorns characteristic of juvenile WT plants. The most precocious T0 line, 'Duncan' grapefruit (Dun134-3) expressing the CsAP1 gene, flowered and fruited when it was 4.5 years old and the T1 siblings from this line flowered and fruited when they were just over 18 months old. In contrast, T1 seedlings from three lines of 'Duncan' grapefruit expressing the CsLFY gene flowered within 3 months after germination, but were unable to support fruit development. Transcript levels of corresponding transgenes in leaves were not correlated with earliness of flowering. To further study the activity of AtRD29A, leaves from three 'Carrizo' citrange (C. sinensis × Poncirus trifoliata) rootstock seedlings transformed with the green fluorescent protein (GFP) gene under regulation of the AtRD29A promoter were subjected to drought stress or well-watered conditions. Expression of GFP was not stress-dependent, consistent with the observation of flowering of CsAP1 and CsLFY transgenic plants under non-inductive conditions. Taken together, the results suggest that AtRD29A is constitutively expressed in a citrus background. Despite the loss of control over flowering time, transgenic citrus lines ectopically expressing C. sinensis AP1 or LFY genes under control of the A. thaliana RD29A promoter exhibit precocious flowering, fruit development and viable transgenic seed formation. These transformed lines can be useful tools to reduce the time between generations to accelerate breeding.

A simple and efficient agroinfiltration method for transient gene expression in Citrus.

KEY MESSAGE: Microwounding pre-treatment facilitates agroinfiltration and transient gene expression in hard-to-agroinfiltrate citrus varieties. Agrobacterium infiltration is a widely used method for transient expression studies in plants, but this method is not used extensively in citrus because of its low efficiency. In this study, we developed an easy, cheap, and reliable agroinfiltration method for transient gene expression in citrus. A microneedle roller was used to create microscopic wounds in the leaf epidermis to facilitate agroinfiltration. Several optimization parameters were explored in this study, including the density of wounds per cm2 of abaxial leaf area, the leaf maturity grade, the effect of the Agrobacterium strain, and the length of the incubation period. Increasing the density of wounds on the leaf surface had a positive effect on transient expression. Higher transient expression levels were observed in well-expanded young leaves in comparison with older leaves. The Agrobacterium strain GV2260 was the most suitable to express a large amount of recombinant protein, and an eight- to ten-day incubation period resulted in the highest expression. Endoplasmic reticulum and cytoskeleton-targeted GFP were both successfully localized, confirming that this protocol can be used for protein subcellular localization in citrus. Finally, up to 100 ng of GFP per milligram of agroinfiltrated leaf tissue was estimated to be expressed using this method. This protocol was tested for GFP expression in five different citrus varieties with no significant statistical differences among them. This simple and easy method can speed up functional genomic studies in citrus and may be applied to other recalcitrant species with extensive epidermal cuticular wax.

Changes in abscisic acid metabolism in relation to the maturation of grapevine (Vitis vinifera L., cv. Mencía) somatic embryos.

BACKGROUND: Somatic embryogenesis in grapevines is a complex process that depends on many physiological and genetic factors. One of its main limitations is the process of precocious germination of the somatic embryos in differentiation medium. This process lowers plant conversion rates from the somatic embryos, and it is probably caused by a low endogenous abscisic acid (ABA) content. RESULTS: Precocious germination of the somatic embryos was successfully avoided by culturing grapevine cv. Mencía embryogenic aggregates over a semipermeable membrane extended on top of the differentiation medium. The weekly analysis of the endogenous ABA and ABA-glucosyl ester (ABA-GE) contents in the aggregates showed their rapid accumulation. The expression profiles of 9-cis-epoxycarotenoid dioxygenase (VvNCED1), 8'-hydroxylase (VvHyd2), UDP-glucosyltransferase (VvUGT) and ß-glucosidase (VvBG2) genes in grapevine revealed that the occurrence of a first accumulation peak of endogenous ABA in the second week of culture over the semipermeable membrane was mainly dependent on the expression of the VvNCED1 gene. A second increase in the endogenous ABA content was observed in the fourth week of culture. At this point in the culture, our results suggest that of those genes involved in ABA accumulation, one (VvNCED1) was repressed, while another (VvBG2) was activated. Similarly, of those genes related to a reduction in ABA levels, one (VvUGT) was repressed while another (VvHyd2) was activated. The relative expression level of the VvNCED1 gene in embryogenic aggregates cultured under the same conditions and treated with exogenous ABA revealed the significant downregulation of this gene. CONCLUSIONS: Our results demonstrated the involvement of ABA metabolism in the control of the maturation of grapevine somatic embryos cultured over a semipermeable membrane and two important control points for their endogenous ABA levels. Thus, subtle differences in the expression of the antagonistic genes that control ABA synthesis and degradation could be responsible for the final level of ABA during the maturation of grapevine somatic embryos in vitro. In addition, the treatment of somatic embryos with exogenous ABA suggested the feedback-based control of the expression of the VvNCED1 gene by ABA during the maturation of grapevine somatic embryos.

Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.).

KEY MESSAGE: The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

EMS mutagenesis and qPCR-HRM prescreening for point mutations in an embryogenic cell suspension of grapevine.

KEY MESSAGE: Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. ABSTRACT: Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.

Gene Expression in Citrus Plant Cells Using Helios® Gene Gun System for Particle Bombardment.

To understand how Citrus tristeza virus (CTV) replicates and moves inside the plant, it is critical to study the cellular interactions and localization of its encoded proteins. However, due to technical limitations, so far these studies have been limited to the nonnatural host Nicotiana benthamiana.Particle bombardment is a physical method to deliver nucleic acid and other biomolecules into the cells directly. The Helios® gene gun (Bio-Rad, Hercules, CA) is a handheld device that uses a low-pressure helium pulse to accelerate high-density, subcellular-sized particles into a wide variety of targets for in vivo and in vitro applications. Here, we describe a detail protocol for either transient or stable gene expression in citrus leaf cells using this gene gun. This protocol can be used to study protein-protein interactions and subcellular localization in different kinds of plant cells.

Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection.

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.